KR20230078657A - Methods and compositions for treating autoimmune diseases and cancer - Google Patents

Abstract

Molecular constructs that target tumor necrosis factor receptor 1 (TNFR1) and/or tumor necrosis factor receptor 2 (TNFR2) are provided. The constructs are intended to treat diseases, disorders and conditions in which these receptors and/or TNF are involved in pathogenesis or inhibition or activation thereof can ameliorate the disease, disorder and condition or symptoms thereof. Among the constructs provided herein are TNFR1 antagonist constructs engineered to inhibit TNFR1 function and eliminate any TNFR1 agonistic activity. Constructs provided herein include agonists and antagonists of TNFR1 and TNFR2. TNFR1 antagonist constructs are engineered to inhibit TNFR1 function and, in some embodiments, to avoid agonistic activity. Also included are agonists and antagonists of TNFR2. Agonists of TNFR2 control acute or chronic inflammation by increasing regulatory T-cell function. Antagonists of TNFR2 are intended to increase immunity and treat cancer and certain immunodeficiency diseases by reducing regulatory T-cell function. Methods of treating various diseases in which TNF and its receptors play a role are also provided.

Description

Methods and compositions for treating autoimmune diseases and cancer

related application

Priority is claimed on U.S. Provisional Application Serial No. 63/071,313, filed August 27, 2020, entitled "METHODS AND COMPOSITIONS TO TREAT AUTOIMUNE DEASESES AND CANCER" by inventor H. Michael Shepard and applicant Enosi Life Sciences Corp. .

This application is filed on February 19, 2020 and published on August 27, 2020 under International PCT Publication No. WO 2020/172218 entitled "ANTIBODIES AND ENONOMERS" by inventor H. Michael Shepard and applicant Enosi Life Sciences Corp. Published International PCT Application No. PCT/US2020/018739. This application also claims the benefit of Provisional Application Serial No. 62/808,635, filed on February 21, 2019, filed on February 19, 2020, and filed on August 20, 2021 in the United States National Phase of PCT/US2020/018739. It is related to US Application Serial No. 17/432,720, filed on

Where permitted, the summary of each such application is incorporated by reference in its entirety.

Incorporation by reference of an electronically provided sequence listing

An electronic version of the sequence listing is filed with this application, the contents of which are incorporated by reference in their entirety. The electronic file created on August 26, 2021 is 1.69 MB in size and is named 5301SEQPC1.txt.

Field

This application relates to nucleic acid constructs and encoded products for use in anti-TNF therapy. The disease being treated is a disease in which the TNF receptor and/or the TNF or TNF/TNF receptor(s) pathway is involved or plays a role in its pathogenesis.

Anti-TNF therapy/TNF-blockers (a type of biological disease modifying anti-rheumatic drug; DMARD) are typically prescribed after conventional DMARDs have failed. Such therapies include monoclonal antibodies (mAbs) such as the chimeric mAb infliximab ( ^Remicade® ); contains a murine variable region and a human IgG1 constant region, and fully humanized mAbs (IgG1s) adalimumab (eg sold under the trade names ^Humira® ) and golimumab ( ^Simponi® antibody); PEGylated humanized Fab' fragment of a mAb targeting TNF, certolizumab pegol ( ^Cimzia® antibody); and TNFR2 fusion proteins such as the TNFR2-Fc fusion protein etanercept (sold under the trademark Enbrel ^® ), which contains an extracellular receptor region containing the binding site of human TNFR2 fused to the Fc of human IgG1. The drug marketed under the trademarks Remsima ^® and Inflectra ^® are biosimilars of infliximab approved for use in the European Union for the treatment of a variety of autoimmune and chronic inflammatory diseases and disorders. Such TNF inhibitors that sequester TNF include, for example, RA, psoriasis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis (JIA), and/or inflammatory bowel disease (IBD; such as Crohn's disease and ulcerative colitis). It is used in the treatment of various diseases and conditions.

However, such therapies are, for example, septicemia and serious infections such as listeriosis, reactivation of tuberculosis, reactivation of hepatitis B/C, herpes zoster, and reactivation of invasive fungi and M. tuberculosis ( M. tuberculosis ) is associated with severe side effects including an increased risk of other opportunistic infections, including reactivation of the infection. This therapy has been shown to induce macrophage apoptosis in rheumatoid synovial membranes. Infliximab is associated with increased apoptosis of inflammatory cells infiltrating the digestive tract of patients with Crohn's disease. Other anti-rheumatic drugs such as methotrexate and glucocorticoids can also induce apoptosis in immune cells (see, eg, Vigna-Pιrez et al. (2005) Clin. Exp. Immunol. 141(2):372-380). These treatments may also cause exacerbation of severe congestive heart failure, drug-induced lupus, demyelinated central nervous system (CNS) disease, as well as lymphomas and non-melanoma skin cancers (see, e.g., Benjamin et al., Disease Modifying Anti- Rheumatic Drugs (DMARDs) [updated 27 February 2020].In: StatPearls [Internet].Treasure Island (FL): StatPearls Publishing;2020 Jan. Source: (ncbi.nlm.nih.gov/books/NBK507863/] Other adverse side effects include increased susceptibility to liver damage, demyelinating disease/CNS disorders, cancer including lupus, psoriasis, sarcoidosis, lymphoma and solid malignancies, as well as further autoimmune diseases (e.g. , Dong et al. (2016) Proc . Natl . Acad . Sci . USA . 113(43):12304-12309; Zalevsky et al. (2007) J. Immunol . 179:1872-1883; Zoran et al. (2019) Sci . Rep. 9:17231]). Thus, the use of these therapeutics is limited, especially for chronic diseases/conditions requiring long-term administration, such as arthritis and inflammatory bowel disease (IBD). About 30% of RA patients are unresponsive or do not receive sustained therapeutic benefit with anti-TNF therapy (see, e.g., McCann et al. (2014) Arthritis & Rheumatology 66(10):2728-2738). Failure to respond also occurs in non-RA patients receiving anti-TNF therapy. Depending on the anti-TNF agent, 13-33% of treated patients do not respond to treatment, and up to 46% stop responding, prompting treatment to be discontinued or dose increased (see, e.g., Richter et al. (2019) MABS 11(4):653-665). Therefore, there is a need for therapies with improved therapeutic efficacy and safety.

Molecular constructs targeting tumor necrosis factor receptor 1 (TNFR1) and/or tumor necrosis factor receptor 2 (TNFR2) and nucleic acids encoding the same are provided. The constructs are intended to treat diseases, disorders and/or conditions in which these receptors and/or TNF are involved in pathogenesis or inhibition or activation can ameliorate the disease, disorder and condition or symptoms thereof. Constructs provided herein include agonists and antagonists of TNFR1 and TNFR2. TNFR1 antagonist constructs are engineered to inhibit TNFR1 function and avoid TNFR1 agonistic activity. Also included are agonists and antagonists of TNFR2. Agonists of TNFR2 control acute or chronic inflammation by increasing regulatory T-cell function. Antagonists of TNFR2 are intended to increase immunity and treat cancer and certain immunodeficiency diseases by reducing regulatory T-cell function.

Cells have two TNF receptors, TNFR1 and TNFR2. These pathways balance each other in normal physiology. TNF/TNFR1 causes inflammation and TNF/TNFR2 is anti-inflammatory. TNFR2 is usually activated later than TNFR1, so it does not immediately affect useful TNF-induced inflammation, but it is activated later to suppress overactivation of inflammatory pathways. Simultaneous inhibition of both pathways abolishes the inflammatory attenuating effects of TNFR2. Existing TNF blockers have limited efficacy because they turn off the anti-inflammatory Treg generator (TNFR2).

Among other properties that differ from prior therapies targeting TNF/TNFR, the constructs provided herein inhibit TNFR1 signaling or activity without compromising the ability of the treated subject to fight opportunistic infections. Among the constructs provided herein is one type, a modified single chain antibody that specifically targets and inhibits TNFR1 but does not antagonize TNFR2, thereby preventing transient activation of TNFR1 through receptor clustering. Constructs provided herein silence the TNF inflammatory pathway mediated by TNFR1, but retain and, in some embodiments, enhance the healing pathway of TNFR2. Such constructs can be administered to treat indications in which TNF blockers have failed. Among the constructs provided herein are constructs that specifically inhibit tumor necrosis factor receptor type 1; Methods and uses of the constructs are provided for treating diseases, disorders and conditions in which TNF or its receptors play a role in pathogenesis or symptoms.

Existing anti-TNF drugs are used to treat excessive use in autoimmune diseases, including rheumatoid arthritis, polyarticular juvenile idiopathic arthritis, axial spondyloarthritis, ankylosing spondylitis, psoriatic arthritis, psoriasis, Crohn's disease, juvenile Crohn's disease, and ulcerative colitis. block inflammation; The constructs of the present invention can be used to treat the same disease but avoid harmful or adverse side effects. Constructs provided herein are more effective at inhibiting inflammatory cytokines in vivo than previous therapeutic agents such as the TNFR2-Fc fusion protein etanercept (sold under the trademark ^Enbrel® ) and preserve regulatory T-cell function. . The constructs may include activity modifiers or property modifiers to increase serum half-life and have demonstrated activity in blocking TNFR1 signaling as in a TNF assay comparing activity with adalimumab and/or etanercept .

As established in the mouse model, the construct shows that it preserves macrophage function better than adalimumab and does not lead to opportunistic infection; It also preserves Treg function substantially better than adalimumab or etanercept and is therapeutically effective in treating diseases, disorders and conditions such as rheumatoid arthritis. In some embodiments, the Kd is less than 1 nM and the in vivo t _1/2 is about 10-12 days. The constructs may be administered by any suitable route for a particular indication. Routes include, but are not limited to, subcutaneous, intravenous, intratumoral, intrahepatic, topical, mucosal, intradermal and any other suitable route.

The constructs provided herein are as follows. Provided are constructs that are tumor necrosis factor receptor 1 (TNFR1) antagonist constructs of formula 1: (TNFR1 inhibitor) _n -linker _p- (active modifier) _q , wherein: each n and q is an integer, and each independently is 1, 2 or 3; p is 0, 1, 2 or 3; A TNFR1 inhibitor is a molecule that binds to and inhibits (antagonizes) the activity of TNFR1; An active modifier is a moiety that modulates or alters the activity or pharmacological properties of a construct compared to a construct without the active modifier; The linker increases the flexibility of the construct, modulates or reduces the steric effect or interaction of the construct with a receptor, and/or increases the solubility of the construct in aqueous media. A linker may contain multiple components. Linkers include chemical linkers, polypeptide linkers, and combinations thereof. The constructs may be linked through chemical and/or physical bonds. The construct may be a fusion protein.

TNFR1 inhibitors may include domain antibodies (dAbs) or single chain antibodies. The construct includes an active modifier that is not a single unmodified Fc region or human serum albumin antibody, where the TNFR1 inhibitor is a domain antibody (dAb). For example, the activity modifier (or property modifier) is a modified Fc region or human serum albumin. In the construct, the TNFR1 inhibitor can be one that inhibits TNFR1 signaling and/or the active modifier increases the serum half-life of the construct. For example, the constructs include those in which the activity modifier is an Fc or albumin that has been modified with reduced or no ADCC (antibody dependent cytotoxicity) activity and/or reduced or no CDC (complement dependent cytotoxicity) activity. A TNFR1 inhibitor may be one that inhibits TNFR1 activity but does not antagonize tumor necrosis factor receptor 2 (TNFR2) activity. A TNFR1 inhibitor may be one that inhibits TNFR1 signaling.

Multispecific constructs are also provided. For example, constructs are provided that are multispecific constructs comprising a TNFR1 inhibitor and a Treg expander, the bispecific constructs interacting with two different target receptors or antigens or epitopes on the receptors. Among the multispecific constructs are bispecific for TFNR1 and Treg expanders. Treg expanders can be TNFR2 agonists.

The construct may include linkers to provide flexibility, increase solubility, mitigate and/or reduce steric hindrance and/or van der Waals interactions. The construct optionally but generally includes active modifiers to alter or modulate the activity or properties of the construct. A construct having formula 2 is provided: (TNFR1 inhibitor) _n - (active modifier) _r1 - (linker (L)) _p - (active modifier) _r2 - (TNFR2 agonist) _q or (TNFR1 inhibitor) _n - (active modifier) _r1 - (linker (L)) _p - (active modifier) _r2 - (Treg extender) _q where: n = 1, 2 or 3, p = 1, 2 or 3, q = 0 , 1 or 2, and r1 and r2 are each independently 0, 1 or 2; The components may be in the order specified or in any other order as long as the construct interacts with and antagonizes TNFR1 and TNFR2, agonizes TNFR2, or has Treg expander activity. For example, constructs from any of those provided herein that inhibit the binding and/or signal transduction of TNFα to which the TNFR1 inhibitor moiety binds to TNFR1 are included.

Also provided are constructs of formula 3a or 3b: (TNFR2 agonist or Treg expander) _n - linkerp - (active modifier)q, formula 3a, or (active modifier) _q - linker _p - (TNFR2 agonist or a Treg expander) _n , formula 3b, wherein: each n and q is an integer, and each is independently 1, 2, or 3; p is 0, 1, 2, or 3; Active modifiers are moieties that alter the pharmacological properties or activity of a construct; TNFR2 agonists interact with TNRFR2 to result in TNFR2 activity; Treg expanders are molecules that include TNFR2 agonists and increase Treg cells; The linker increases flexibility and/or modulates or reduces the steric effect of the construct or its interaction with a receptor; alter the solubility of the construct. In some embodiments, the active modifier is an Fc region or modified Fc region or short FcRnBP; A linker is a linker comprising a hinge region or comprising G and S residues. Exemplary linkers are those that increase the serum half-life of the construct. For example, the linker has a sequence set forth in any one of SEQ ID NOs: 812-834 or is a PEG moiety linker. In some embodiments, the construct comprises an active modifier, which is a modified Fc region or peptide that increases the serum half-life of the construct. The Fc region may be an Fc dimer; An Fc region may be modified to have reduced ADCC and/or CDC activity, such as an Fc modified to have reduced or no ADCC activity.

In the constructs provided herein, the TNFR1 inhibitor is any defined in the sequence listing listed below or known in the art; The Treg expander is any known in the art, a TNFR2 agonist, or any Treg expander shown in the Sequence Listing or known in the art; The linker is any listed in the Sequence Listing or Sequence Listing below or known in the art; Active modifiers are any known in the art and/or set forth in the Sequence Listing provided below.

Constructs are provided that are TNFR1 antagonist constructs, including single chain antibodies, or antigen-binding portions thereof, TNFR1 inhibitors that specifically target and inhibit TNFR1 but do not antagonize TNFR2, thereby preventing transient activation of TNFR1 through receptor clustering. The antibody or antigen-binding portion thereof in such constructs contains modifications that improve the pharmacological properties and/or structure of the construct.

In any of the constructs provided herein, the construct agonizes TNFR2 signaling to increase expression of regulatory T cells (Treg), thereby providing TNFR1 antagonism and concomitant (or substantially concomitant) increase in Treg expression. contains the ingredient(s). In the constructs provided herein, the TNFR1 inhibitor is a single chain antibody that inhibits TNFR1 by inhibiting TNFR1 signaling, such as when the antibody portion or antigen binding portion of the construct inhibits binding of TNFα to TNFR1. can Among the constructs, the TNFR1 inhibitor is an antibody or antigen binding portion that does not inhibit the binding of TNFα to TNFR1 but does inhibit TNFR1 signaling. A property or activity that can be modulated/altered may be serum half-life.

The construct may include an Fc modified to eliminate ADCC and/or CDC activity. The construct may contain Fc dimers, such that one Fc monomer contains a hole and the other contains a knob to form a heterodimer. For example the knob mutation(s) is selected from among S354C, T366Y, T366W and T394W by EU numbering; The hole mutation(s) are selected from among Y349C, T366S, L368A, F405A, Y407T, Y407A and Y407V by EU numbering, and the Fc monomers form heterodimers. In some embodiments where the construct comprises an Fc, the Fc is from Trastuzumab. The construct can be dimerized by fusion of the C-terminus and the N-terminus of trastuzumab.

In some embodiments where the construct comprises a linker, the linker is or comprises a hinge region from an Fc region. For example, the hinge region is from trastuzumab and connects to the Fc region. Constructs include those comprising a linker linked to an anti-TNFR1 antagonist antibody or antigen binding portion thereof. The linker may be linked to the anti-TNFR1 antagonist antibody or antigen-binding portion thereof and may be linked to the Fc region directly or through a hinge region. For example, the Fc region or modified Fc region comprises the amino acid sequence set forth in any of SEQ ID NOs: 10, 12, 14, 16, 27, 30, 1469 and 1470.

Also provided are constructs that bind to the neonatal Fc receptor (FcRn). For example, TNFR1 constructs are provided that include short FcRn binding peptides (FcRnBPs), wherein the short FcRn binding peptides (FcRnBPs) provide interaction of the construct with FcRn and are 6-25 or 10-20 amino acid residues. contains For example, FcRnBP contains 12-20 residues or 15 residues or 16 residues. Examples of these are TNFR1 antagonist constructs wherein the FcRn binding peptide (FcRnBP) comprises or consists of a peptide of any of SEQ ID NOs:48-51. The construct includes a TNFR1 construct comprising an Fc heterodimer, wherein one Fc monomer comprises a hole and the other comprises a knob, whereby the resulting Fc dimer is a heterodimer.

TNFR1 inhibitors; Fc dimer; and a TNFR1 antagonist construct comprising a Treg expander, wherein: the Fc dimer comprises two complementary Fc monomers; The TNFR1 inhibitor is linked to one Fc monomer and the Treg expander is linked to the other Fc monomer. The Treg expander in such a construct may be a TNFR2 agonist. The construct further comprises a second Treg expander linked to the same Fc monomer as the TNFR1 inhibitor, wherein the first and second Treg expanders are the same or different. The second Treg expander may be a TNFR2 agonist. In some embodiments, the Treg expanders are the same. A TNFR1 inhibitor can be one that inhibits or blocks TNFR1 signaling. In some embodiments, a TNFR1 inhibitor binds TNFR1 and blocks or inhibits TNFα binding and TNFR1 signaling. In some embodiments, a TNFR1 inhibitor binds TNFR1, does not interfere with or interferes with TNFα binding, and blocks or inhibits TNFR1 signaling. In some embodiments of these constructs, the Treg expander is a TNFR2 agonist. A TNRF2 agonist can be one that stimulates or induces TNFR2 signaling. An example of a Treg expander is a scFv, a VHH single domain antibody, or a TNFR2 agonist that is a Fab of a TNFR2 agonist monoclonal antibody. In these constructs, the Treg expander can be a TNFR2 agonist that is a small molecule, or a nucleic acid aptamer, or a peptide aptamer.

It is also a TNFR2 agonist or any of such constructs are provided. A TNFR2 agonist is a construct of formula 3a or 3b, where formula 3a is (Treg extender) _n - linker _p - ring active modifier) _q and formula 3b is (active modifier) _q - linker _p - ring Treg extender) is _n . In these formulas, n and q are each an integer and are each independently 1, 2 or 3; p is 0, 1, 2 or 3; An active modifier is a moiety that modulates or alters the activity or pharmacological properties of a construct compared to a construct without the active modifier; The linker increases the flexibility of the construct, modulates or reduces the steric effect or interaction of the construct with a receptor, and/or increases the solubility of the construct in aqueous media. In any of these constructs, the Treg expander of the construct is a TNFR2 agonist. For example, TNFR2 agonists stimulate or induce TNFR2 signaling. In another example, the Treg expander is a scFv, a VHH single domain antibody, or a TNFR2 agonist that is a Fab of a TNFR2 agonist monoclonal antibody. Treg expanders can be TNFR2 agonists that are small molecules or nucleic acids or peptide aptamers. In constructs comprising all or part of Trastuzumab, such as the Fc portion and/or Fc and hinge region or modified forms thereof, the construct dimerizes by N-terminal fusion with the C-terminus of Trastuzumab It can be.

Constructs comprising a TNFR1 inhibitor moiety linked to one or more Treg expanders via a central PEG linker, comprising at least two identical or different TNFR1 inhibitors, or comprising two identical or different Treg expanders are provided. A construct comprising a PEG moiety, such as a central PEG linker, may include a branched PEG moiety linking a TNFR1 inhibitor and one or more Treg expanders. Examples are those having a structure selected from formulas 4A-4D:

Equation 4A:

, 또는

, or

n is 1 to 5;

R ¹ is H or CH ₃ , or CH ₂ CH ₃ or another C ₁ -C ₅ alkyl;

는 TNFR1 억제제(TNFR1 길항제)이고;

is a TNFR1 inhibitor (TNFR1 antagonist);

는 Treg 확장제이거나; 또는

is a Treg expander; or

Equation 4B:

는 TNFR1 억제제(TNFR1 길항제)이고

is a TNFR1 inhibitor (TNFR1 antagonist);

는 Treg 확장제이고;

is a Treg expander;

n is 1 to 5; or

Equation 4C:

는 TNFR1 억제제(TNFR1 길항제) 또는 Treg 확장제이고; 그리고

is a TNFR1 inhibitor (TNFR1 antagonist) or Treg expander; and

n is 1 to 5; or

Equation 4D:

또는

or

, 여기서

, here

는 동일하거나 상이하고 각각 독립적으로 TNFR1 억제제(TNFR1 길항제) 및 TNFR2 효능제로부터 선택되며; Each

are the same or different and are each independently selected from TNFR1 inhibitors (TNFR1 antagonists) and TNFR2 agonists;

Active modifiers are optional and may be linked to any suitable locus in the molecule; and n is 1 to 5.

In the TNFR1 antagonist constructs and other constructs provided herein, the Treg expander may be a TNFR2 agonist. Such constructs may include an active modifier, such as where the active modifier is an Fc region or an Fc region comprising a hinge region or other linker; An Fc region or Fc region having a hinge region is an Fc that has been modified to reduce or eliminate ADCC and/or CDC activity. Examples are constructs where the Fc or modified Fc is an IgG Fc or an IgG1 or IgG4 Fc and/or a construct that binds to the neonatal Fc receptor (FcRn). Examples of these constructs are the following: the construct comprises short FcRn binding peptides (FcRnBPs), wherein the short FcRn binding peptides (FcRnBPs) provide for interaction of the construct with FcRn, 6-25; For example, it contains 10-20 amino acid residues; wherein the FcRnBP contains 12-20 residues or 15 residues or 16 residues, for example the FcRn binding peptide (FcRnBP) comprises or consists of any of SEQ ID NOs:48-51 peptides.

In addition, also a) a TNFR1 selective TNFR1 inhibitor moiety; b) optionally one or more linkers; and c) optionally comprising a half-life extending moiety. In this construct, the TNFR1 selective antagonist is one that selectively binds and inhibits TNFR1 signaling but not TNFR2 signaling. As described for the constructs above, the TNFR1 inhibitor, linker and other components may be those previously described. These include constructs comprising antigen-binding fragments in which a selective antagonist, a TNFR1 inhibitor, selectively binds to and inhibits TNFR1 signaling, but not TNFR2 signaling. For example, an antigen-binding fragment that selectively binds to and inhibits TNFR1 signaling but not TNFR2 signaling may include a domain antibody (dAb), scFv or Fab fragment. In any of the constructs described herein, the TNFR1 inhibitor comprises an antigen-binding fragment of a human anti-TNFR1 antagonist monoclonal antibody. For example, a human anti-TNFR1 antagonist monoclonal antibody may comprise H398 comprising SEQ ID NO: 678, ATROSAB, or an antigen binding portion thereof, a sequence having at least 95% sequence identity to SEQ ID NO: 31 or 32 or 673 or 678, or It is an antigen binding portion thereof that binds to TNFR1. An exemplary TNFR1 inhibitor comprises a domain antibody (dAb) or antigen binding portion thereof or comprises a sequence of amino acids set forth in any of SEQ ID NOs: 52-672 or a sequence having at least 95% sequence identity to a sequence that retains TNFR1 inhibitor activity. and/or; and/or comprises a scFv set forth in any of SEQ ID NOs:673-678 or variants of these polypeptides that have at least 90% or 95% sequence identity to those that retain TNFR1 inhibitor activity; and/or comprises a sequence having at least 90% or 95% sequence identity to a Fab or TNFR1 inhibitor set forth in any of SEQ ID NOs:679-682 or that retains binding activity; The sequence is set forth in SEQ ID NO: 683 or 684 or comprises a Nanobody having at least 90% or 95% sequence identity that retains a TNFR1 inhibitor or binding activity. Among the TNFR1 inhibitors are those comprising, for example, dominant-negative tumor necrosis factor (DN-TNF) or TNF muteins, for example, DN-TNF or TNF muteins confer selective inhibition of TNFR1 and are selected from one of the following: A soluble TNF molecule comprising the above amino acid substitutions:

V1M, L29S, L29G, L29Y, R31C, R31E, R31N, R32Y, R32W, C69V, A84S, V85T, S86T, Y87H, Q88N, T89Q, I97T, C101A, A145R, E146R, L29S/R32W, L29S/S86T, R32W/ S86T, L29S/R32W/S86T, R31N/R32T, R31E/S86T, R31N/R32T/S86T, I97T/A145R, V1M/R31C/C69V/Y87H/C101A/A145R, and A84S/V85T/S86T/Y87H/Q88N /T89Q . For example, the TNFR1 inhibitor has at least or at least about 90% or 95% sequence identity to the sequence of residues set forth in any one of SEQ ID NOs: 701-703, or to the sequence of residues set forth in any one of SEQ ID NOs: 701-703 It is a TNF mutein comprising a sequence having or a fragment thereof that retains TNFR1 inhibitory activity.

Any of the foregoing constructs provided herein may include a linker, wherein the linker comprises all or part of the hinge sequence of trastuzumab, SCDKTH corresponding to residues 222-227 of SEQ ID NO: 26 or the sequence EPKSCDKTHTCPPCP (sequence corresponding to residues 219-233 of SEQ ID NO: 26), or at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues thereof, or residues ESKYGPPCPPCP residues 212-223 of SEQ ID NO: 29, or a linker up to the entire sequence of the hinge region of trastuzumab that contains or has a sequence having at least 98% or 99% sequence identity thereto. For example, the construct can include a linker, wherein the linker includes the sequence SCDKTH corresponding to residues 222-227 of SEQ ID NO:26. The construct may include a linker comprising glycine and serine (GS) residues, a GS linker, in place of or in addition to other linkers. Exemplary GS linkers for any of the constructs provided herein include (GlySer) _n where n=1-10; (GlySer ₂ ); (Gly ₄ Ser) _n (where n=1-10); (Gly ₃ Ser) _n (where n= 1-5); (SerGly ₄ ) _n (where n=1-5); (GlySerSerGly) _n (where n= 1-5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS. Also included are GS linkers corresponding to residues EPKSCDKTHTCPPCP (residues 219-233 of SEQ ID NO: 26) and linkers comprising all or part of the hinge sequence of Trastuzumab, for example the linker may comprise a GS linker. or contains or contains only the sequence SCDKTH corresponding to residues 217-222 of SEQ ID NO: 31 from the hinge sequence. Such linkers include, for example, those comprising the GS linker and all or part of the hinge sequence of nivolumab corresponding to residues 212-223 of SEQ ID NO:29.

The constructs herein may include active modifiers. Active modifiers include any described herein, including those described above and below, and others known to those skilled in the art; Active modifiers alter the activity or properties of a construct. The modified activity can be an IgG Fc, a polyethylene glycol (PEG) molecule or a half-life extending moiety that is human serum albumin (HSA). Examples of IgG Fc are IgG1 or IgG4 Fc. The IgG1 Fc may be the Fc of Trastuzumab set forth in SEQ ID NO: 27 or a sequence of amino acids having at least 95% sequence identity therewith; The IgG4 Fc may be the Fc of nivolumab set forth in SEQ ID NO: 30 or an amino acid sequence having at least 95% sequence identity therewith. For example, IgG1 Fc is the Fc of human IgG1 as shown in SEQ ID NO: 10, and IgG4 Fc is the Fc of human IgG4 as shown in SEQ ID NO: 16.

Constructs described herein include those that are or include TNFR1 inhibitor(s). These include constructs in which the TNFR1 inhibitor is monovalent. These include linkers such as those comprising (Gly ₄ Ser) ₃ and/or linkers comprising (Gly ₄ Ser) ₃ and SCDKTH (residues 217-222 of SEQ ID NO:31); and/or (Gly ₄ Ser) ₃ and a linker comprising the hinge sequence of Trastuzumab corresponding to residues 219-233 of SEQ ID NO: 26; and/or (Gly ₄ Ser) ₃ and the hinge sequence of nivolumab corresponding to residues 212-223 of SEQ ID NO:29. Exemplary constructs provided herein that inhibit TNFR1 are directed to the sequence of residues set forth in any one of SEQ ID NOs: 704-764, or to the sequence of residues set forth in any one of SEQ ID NOs: 704-764 that inhibits TNFR1. and constructs having sequences having at least or at least about 95% sequence identity.

TNFR1 antagonist constructs are provided herein. These include an FcRn binding peptide (FcRnBP) in which the TNFR1 construct is short; Short FcRn binding peptides (FcRnBPs) provide for the interaction of the construct with FcRn and contain 6-25, such as 10-20 amino acid residues, such as FcRnBPs of 12-20 residues or 15 residues or 16 residues. residues, such as, for example, an FcRn binding peptide (FcRnBP) is any of SEQ ID NOs: 48-51 or a peptide having at least about 95% sequence identity thereto, or any SEQ ID NO: 48- including the FcRn-binding peptide (FcRnBP) composed of 51 peptides.

Other exemplary TNFR1 inhibitory constructs provided herein include constructs comprising: a) a domain antibody that inhibits TNFR1; b) increase flexibility; linkers that reduce steric effects or increase solubility; and c) a half-life extending moiety. Constructs in which the half-life extending moiety is not a human serum albumin antibody or an unmodified Fc are included. These constructs include those that are TNFR1 antagonists comprising: a) a domain antibody (dAb) of any of SEQ ID NOs:52-672, or a scFv of any of SEQ ID NOs:673-678 or SEQ ID NO:679 a Fab of any of -682, or a Nanobody of SEQ ID NO: 683 or 684, or a TNF mutein of any of SEQ ID NOs: 685-703; b) (GlySer) _n (where n=1-10); (GlySer ₂ ); (Gly ₄ Ser) _n (where n=1-10); (Gly ₃ Ser) _n (where n= 1-5); (SerGly ₄ ) _n (where n=1-5); (GlySerSerGly) _n (where n= 1-5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and a GS linker selected from among GSSSGS; and c) a half-life extending moiety that is an IgG Fc. In any of these constructs or anything provided herein that includes one or more of these components, the GS linker can be (GGGGS) ₃ ; The IgG Fc may be the Fc of Trastuzumab or the Fc of Nivolumab.

Other constructs provided herein that are TNFR1 antagonist constructs include constructs comprising: a) a domain antibody (dAb) of any of SEQ ID NOs:52-672, or any of SEQ ID NOs:673-678 ?? a scFv of or a Fab of any of SEQ ID NOs: 679-682, or a nanobody of SEQ ID NOs: 683 or 684, or a TNF mutein of any of SEQ ID NOs: 685-703; b) a linker selected from all or part of the hinge sequence of Trastuzumab and all or part of the hinge sequence of Nivolumab; and c) a half-life extending moiety that is an IgG Fc. In such constructs, the linker may include all or part of the hinge sequence of Trastuzumab, wherein the IgG Fc is the Fc of Trastuzumab. In another embodiment, the linker can include all or part of the hinge sequence of nivolumab, wherein the IgG Fc is the Fc of nivolumab.

Provided are any constructs provided herein that are TNFR1 antagonist constructs comprising:

a) a domain antibody (dAb) of any one of SEQ ID NOs: 52-672, or a scFv of any one of SEQ ID NOs: 673-678 or a Fab of any one of SEQ ID NOs: 679-682, or SEQ ID NOs: 683 or 684 or a TNF mutein of any one of SEQ ID NOs: 685-703;

b) (GlySer) _n (where n=1-10); (GlySer ₂ ); (Gly ₄ Ser) _n (where n=1-10); (Gly ₃ Ser) _n (where n= 1-5); (SerGly ₄ ) _n (where n=1-5); (GlySerSerGly) _n (where n= 1-5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; And a first linker that is a GS linker selected from GSSSGS;

c) a second linker selected from all or part of the hinge sequence of Trastuzumab and all or part of the hinge sequence of Nivolumab; and

d) a half-life extending moiety that is an IgG Fc.

In some embodiments, these constructs are GS linkers, wherein the first linker is (GGGGS) ₃ ; the second linker comprises the sequence SCDKTH (residues 217-222 of SEQ ID NO: 31); IgG Fc is the Fc of Trastuzumab. In another embodiment, the first linker is a GS linker and is (GGGGS) ₃ ; the second linker comprises all or part of the hinge sequence of nivolumab; IgG Fc is the Fc of nivolumab.

A construct is provided that is a TNFR1 agonist comprising:

a) a domain antibody (dAb) of any one of SEQ ID NOs: 52-672, or a scFv of any one of SEQ ID NOs: 673-678 or a Fab of any one of SEQ ID NOs: 679-682, or SEQ ID NOs: 683 or 684 or a TNF mutein of any one of SEQ ID NOs: 685-703;

b) (GlySer) _n (where n=1-10); (GlySer ₂ ); (Gly ₄ Ser) _n (where n=1-10); (Gly ₃ Ser) _n (where n= 1-5); (SerGly ₄ ) _n (where n=1-5); (GlySerSerGly) _n (where n= 1-5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and a GS linker selected from GSSSGS; and

c) a half-life extending moiety that is a PEG molecule. The GS linker can be any described herein or known to those skilled in the art, such as (GGGGS) ₃ . The PEG molecule may have a molecular weight of at least 25 kDa, typically at least 30 kDa or greater, such as at least 40 kDa or 50 kDa, or 60 kDa, or 80 kDa, or greater.

A construct is provided that is a TNFR1 agonist construct comprising:

a) a domain antibody (dAb) of any one of SEQ ID NOs: 52-672, or a scFv of any one of SEQ ID NOs: 673-678 or a Fab of any one of SEQ ID NOs: 679-682, or SEQ ID NOs: 683 or 684 or a TNF mutein of any one of SEQ ID NOs: 685-703;

b) (GlySer) _n (where n=1-10); (GlySer ₂ ); (Gly ₄ Ser) _n (where n=1-10); (Gly ₃ Ser) _n (where n= 1-5); (SerGly ₄ ) _n (where n=1-5); (GlySerSerGly) _n (where n= 1-5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and a GS linker selected from GSSSGS; and

c) a half-life extending moiety that is human serum albumin. Exemplary linkers are any described herein, such as where the GS linker is (GGGGS) ₃ .

The primary amino acid sequence of any construct provided herein (as described above and below) may be optimized or modified to remove immunogenic sequences or immunogenic epitopes. For example, in a construct containing an IgG Fc, the IgG Fc may be modified to include one or more of the following modifications: a) modification(s) to introduce a knob-into-hole; b) modification(s) to increase or enhance neonatal Fc receptor (FcRn) recycling; and c) modification(s) to reduce or eliminate immune effector function. In such constructs and in any containing an IgG Fc, the knob mutation may be selected from among S354C, T366Y, T366W and T394W by EU numbering; The hole mutation is selected from among Y349C, T366S, L368A, F405A, Y407T, Y407A and Y407V by EU numbering. These TNFR1 antagonist constructs may have the modification(s) to increase or enhance FcRn recycling selected from one or more of the following: T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E by EU numbering. , T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M252Y/T256Q, M252F/T256D, M252Y/S254T/T256E, H433K/ N434F/Y436H, N434F/Y436H, T250Q /M428L, T250R/M428L, M428L/N434S, V259I/V308F, V259I/V308F/M428L, E294del/T307P/N434Y, and T256N/A378V/S383N/N434Y. Decrease immune effector function(s), such as immune effector function(s) selected from one or more of complement dependent cytotoxicity (CDC), antibody dependent cell mediated cytotoxicity (ADCC), and antibody dependent cell mediated phagocytosis (ADCP) A TNFR1 antagonist construct that can be modified to eliminate or eliminate. For example, in these TNFR1 antagonist constructs, the modification(s) to reduce or eliminate immune effector function is selected from one or more of the following:

In IgG1: L235E, L234A/L235A, L234E/L235F/P331S, L234F/L235E/P331S, L234A/L235A/P329G, L234A/L235A/G237A/P238S/H268A/A330S/P331 by EU numbering S, G236R/L328R, G237A , E318A, D265A, E233P, N297A, N297Q, N297D, N297G, N297G/D265A, A330L, D270A, P329A, P331A, K322A, V264A, and F241A; and

In IgG4: L235E, F234A/L235A, S228P/L235E, and S228P/F234A/L235A by EU numbering.

A TNFR1 antagonist or multispecific construct may include a central PEG linker moiety; The construct may comprise a modified Fc region as described above, wherein the Fc region is a modified IgG Fc and the modified IgG Fc comprises one or more of the following modifications:

a) deformation(s) to introduce a knob-into-hole; here

Knob mutations are selected from among S354C, T366Y, T366W and T394W by EU numbering; and

Hall mutations are variants selected from among Y349C, T366S, L368A, F405A, Y407T, Y407A and Y407V by EU numbering;

b) modification(s) to increase or enhance neonatal Fc receptor (FcRn) recycling, selected from one or more of the following:

T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M2 52Y/T256Q, M252F/T256D, M252Y/ S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F, V259I/V308F/M428L, E294del/T307P/N4 34Y, and T256N/A378V/S383N/N434Y ; and

c) modification(s) to increase or enhance one or more immune effector functions,

the immune effector function(s) is selected from one or more of CDC, ADCC and ADCP; and

Modification(s) selected from one or more of the following to increase or enhance immune effector function:

In IgG1: S239D, I332E, S239D/I332E, S239D/A330L/I332E, S298A/E333A/K334A in the first heavy chain; F243L/R292P/Y300L/V305I/P396L; L235V/F243L/R292P/Y300L/P396L; F243L/R292P/Y300L; L234Y/G236W/S298A and S239D/A330L/I332E in the second heavy chain; L234Y/L235Q/G236W/S239M/H268D/D270E/S298A in the first heavy chain and D270E/K326D/A330M/K334E in the second heavy chain; A327Q/P329A; D265A/S267A/H268A/D270A/K326A/S337A; T256A/K290A/S298A/E333A/K334A; G236A; G236A/I332E; G236A/S239D/I332E; G236A/S239D/A330L/I332E; introduction of a biantennary glycan at residue N297; residue N297; K326W; K326A; E333A; K326A/E333A; K326W/E333S; K326M/E333S; K222W/T223W; K222W/T223W/H224W; D221W/K222W; C220D/D221C; C220D/D221C/K222W/T223W; H268F/S324T; S267E; H268F; S324T; S267E/H268F/S324T; G236A/I332E/S267E/H268F/S324T; E345R; and introduction of afucosylated glycans at E345R/E430G/S440Y; by EU numbering.

In some embodiments, of any construct comprising an Fc region, the construct may comprise an IgG1 Fc comprising one or more modifications to increase binding to the inhibitory Fcγ receptor (FcγR) FcγRIIb. For example, the modification or modifications that increase binding to FcγRIIb are selected from one or more of S267E, N297A, L328F, L351S, T366R, L368H, P395K, S267E/L328F and L351S/T366R/L368H/P395K by EU numbering .

Constructs that are Treg expander constructs are also provided. Such constructs include those containing: a) Treg expanders; b) a linker that increases the flexibility of the construct and/or modulates or reduces the steric effect or interaction of the construct with a receptor and/or increases the solubility of the construct in an aqueous medium; and c) an active modifier, wherein the moiety modulates or alters an activity or pharmacological property of the construct compared to a construct without the active modifier. Treg expanders can be TNFR2 agonists. These constructs may further include a TNFR1 inhibitor. In some embodiments, the TNFR2 agonist is a TNFR2 selective agonist.

Provided are constructs described herein that are TNFR2 agonist constructs comprising: a) a Treg agonist; b) a linker that increases the flexibility of the construct and/or modulates or reduces the steric effect or interaction of the construct with a receptor and/or increases the solubility of the construct in an aqueous medium; and c) an active modifier, wherein the moiety modulates or alters an activity or pharmacological property of the construct compared to a construct without the active modifier. In a TNFR2 agonist construct, the TNFR2 agonist may be a TNFR2 selective agonist. The construct may include an active modifier, such as an active modifier that is a half-life extending moiety. The construct may be a TNFR2 agonist construct that selectively activates or antagonizes TNFR2 without activating or antagonizing TNFR1. Included are TNFR2 agonist constructs wherein the TNFR2 agonist binds to one or more epitopes within TNFR2. Included herein is human TNFR2. Such epitopes include, for example, epitopes selected from one or more epitopes comprising or consisting of the sequences of amino acids set forth in SEQ ID NOs: 839-865, 1202 and 1204.

A TNFR2 agonist construct is provided, wherein the TNFR2 agonist comprises an agonist human anti-TNFR2 antibody or an antigen-binding fragment of a humanized anti-TNFR2 antibody, or an antigen-binding portion thereof, or a single chain form thereof. An example of such an antibody is an agonist anti-TNFR2 antibody selected from MR2-1 (also designated ab8161; US Pat. No. 9,821,010) or MAB2261 (US Pat. No. 9,821,010). The TNFR2 agonist can be an antigen binding fragment selected from dAb, scFv or Fab fragments. In some embodiments, the TNFR2 agonist is a TNFR2 selective agonist. Selective agonists can include TNFR2 agonist TNF muteins. Exemplary TNFR2 selective effective mutations are all K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146H, E146N, E146N D143N /A145R, A145R/S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D , A145N/E146D/S147D, A145T /E146S/S147D, A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S 147T, E146D/S147D, D143V/F144L /A145S, S95C/G148C, and D143V/A145S, and soluble TNF variants comprising one or more TNFR2 selective mutations selected from combinations of the foregoing. For example, a TNFR2 agonist is a TNF mutein comprising the mutations D143N/A145R.

In TNFR2 agonist constructs, the linker includes any described herein or known to those skilled in the art for use as a linker. Exemplary linkers include all or part of the hinge sequence of trastuzumab corresponding to residues 219-233 of SEQ ID NO: 26, or all or part of the hinge sequence of nivolumab corresponding to residues 212-223 of SEQ ID NO: 29 part, or sequences with at least 95% sequence identity thereto. Another exemplary linker comprises or consists of the sequence SCDKTH corresponding to residues 217-222 of SEQ ID NO:31. Linkers include glycine-serine (GS) linkers such as (GlySer) _n where n=1-10; (GlySer ₂ ); (Gly ₄ Ser) _n (where n=1-10); (Gly ₃ Ser) _n (where n= 1-5); (SerGly ₄ ) _n (where n=1-5); (GlySerSerGly) _n (where n= 1-5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; And it may be not limited to a GS linker selected from GSSSGS. The linker may be, for example, all or part of the hinge sequence of trastuzumab corresponding to the GS linker and residues 219-233 of SEQ ID NO: 26, or the GS linker and the sequence corresponding to residues 217-222 of SEQ ID NO: 31 SCDKTH , or a combination of linkers, such as a GS linker and a linker comprising all or part of the hinge sequence of nivolumab corresponding to residues 212-223 of SEQ ID NO: 29.

Any construct provided herein may contain active modifiers that alter or modulate the properties or activity of the construct. For example, half-life extending moieties are activity or property modifiers. Examples of those discussed above and also below are portions or derivatives of IgG Fc, polyethylene glycol (PEG) molecules, and human serum albumin (HSA), or variants thereof. For example, in some cases the IgG Fc is an IgG1 or IgG4 Fc. An example of an IgG1 Fc is the Fc of Trastuzumab set forth in SEQ ID NO: 27; The Fc of IgG4 Fc is the Fc of nivolumab as set forth in SEQ ID NO: 30, human version, wherein the IgG1 Fc is the Fc of human IgG1 as set forth in SEQ ID NO: 10, and the IgG4 Fc is the Fc of human IgG4 as set forth in SEQ ID NO: 16 am.

In some embodiments of the TNFR2 agonist construct, the TNFR2 agonist is monovalent; In other cases it is polyvalent, such as divalent or trivalent. TNFR2 constructs may contain linkers as described herein. For example, the linker may be a Gly-Ser, such as (Gly ₄ Ser) ₃ , or (Gly ₄ Ser) ₃ and SCDKTH (residues 217-222 of SEQ ID NO:31), or (Gly ₄ Ser) ₃ and SEQ ID NO: the hinge sequence of Trastuzumab corresponding to residues 219-233 of 26, or the hinge sequence of Nivolumab corresponding to (Gly ₄ Ser) ₃ and residues 212-223 of SEQ ID NO: 29, or at least 95% sequence identity thereto It may include variants of any of the foregoing with These constructs may also contain an active modifier, for example a modifier that is a half-life extending moiety such as PEG or HSA as described above. The PEG moiety has a size of at least 20 kDa, typically at least 30 kDa or greater (as described above and below).

Also provided are TNFR2 agonist constructs comprising:

a) a TNFR2 agonist that binds to one or more epitopes in human TNFR2 selected from among the epitopes set forth in SEQ ID NOs: 839-865, 1202 and 1204;

b) (GlySer) _n (where n=1-10); (GlySer ₂ ); (Gly ₄ Ser) _n (where n=1-10); (Gly ₃ Ser) _n (where n= 1-5); (SerGly ₄ ) _n (where n=1-5); (GlySerSerGly) _n (where n= 1-5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and a GS linker selected from GSSSGS; and

c) an activity modifier that is a half-life extending moiety that is an IgG Fc.

As above, in an exemplary embodiment, the GS linker can be (GGGGS) ₃ ; IgG Fc is the Fc of Trastuzumab or the Fc of Nivolumab.

Other TNFR2 agonist constructs include:

a) a TNFR2 agonist that binds to one or more epitopes in human TNFR2 selected from among the epitopes set forth in SEQ ID NOs: 839-865, 1202 and 1204;

b) a linker selected from all or part of the hinge sequence of Trastuzumab and all or part of the hinge sequence of Nivolumab; and

c) an activity modifier that is a half-life extending moiety that is an IgG Fc. Examples of linkers and active modifiers are the hinge sequences of trastuzumab; IgG Fc is all or part of the Fc of Trastuzumab, or the hinge sequence of Nivolumab; IgG Fc is the Fc of nivolumab.

In another embodiment, the TNFR2 construct comprises:

a) a TNFR2 agonist that binds to one or more epitopes in human TNFR2 selected from among the epitopes set forth in SEQ ID NOs: 839-865, 1202 and 1204;

b) (GlySer) _n (where n=1-10); (GlySer ₂ ); (Gly ₄ Ser) _n (where n=1-10); (Gly ₃ Ser) _n (where n= 1-5); (SerGly ₄ ) _n (where n=1-5); (GlySerSerGly) _n (where n= 1-5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; And a first linker that is a GS linker selected from GSSSGS;

c) a second linker selected from all or part of the hinge sequence of Trastuzumab and all or part of the hinge sequence of Nivolumab; and

d) an activity modifier that is a half-life extending moiety that is an IgG Fc. An example of such a construct is one in which the first GS linker is (GGGGS)3 and the second linker comprises the sequence SCDKTH (residues 217-222 of SEQ ID NO: 31); IgG Fc is the Fc of Trastuzumab. In another embodiment, the first linker is (GGGGS) ₃ ; the second linker comprises all or part of the hinge sequence of nivolumab; IgG Fc is the Fc of nivolumab.

In some embodiments, the construct is a TNFR2 agonist construct comprising:

a) a TNFR2 agonist comprising an antigen-binding fragment of an agonist human anti-TNFR2 antibody selected from MR2-1 or MAB2261;

b) a linker comprising:

i) (GlySer) _n (where n = 1-10); (GlySer ₂ ); (Gly ₄ Ser) _n (where n=1-10); (Gly ₃ Ser) _n (where n= 1-5); (SerGly ₄ ) _n (where n=1-5); (GlySerSerGly) _n (where n= 1-5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and a GS linker selected from GSSSGS; and/or

ii) all or part of the hinge sequence of trastuzumab or all or part of the hinge sequence of nivolumab; and

c) an active modifier which is a half-life extending moiety selected from an IgG1 or IgG4 Fc, a PEG molecule and human serum albumin (HSA);

IgG1 Fc is the Fc of human IgG1 as shown in SEQ ID NO: 10 or the Fc of Trastuzumab as shown in SEQ ID NO: 27; and

wherein the PEG molecule has a molecular weight of at least or at least about 30 kDa.

In some embodiments, the construct is a TNFR2 agonist construct comprising:

a) K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R with reference to SEQ ID NO: 2 /S147T, Q88N /T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145 T/E146S/S147D, A145Q/E146D /S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/S147D, D143V/F 144L/A145S, S95C/G148C, and a TNFR2 selective TNF mutein, which is a soluble TNF variant comprising one or more TNFR2 selective mutations selected from D143V/A145S;

b) a linker comprising:

i) (GlySer) _n (where n = 1-10); (GlySer ₂ ); (Gly ₄ Ser) _n (where n=1-10); (Gly ₃ Ser) _n (where n= 1-5); (SerGly ₄ ) _n (where n=1-5); (GlySerSerGly) _n (where n= 1-5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and a GS linker selected from GSSSGS; and/or

ii) all or part of the hinge sequence of trastuzumab or all or part of the hinge sequence of nivolumab; and

c) an active modifier which is a half-life extending moiety selected from an IgG1 or IgG4 Fc, a PEG molecule and human serum albumin (HSA);

IgG1 Fc is the Fc of human IgG1 as shown in SEQ ID NO: 10 or the Fc of Trastuzumab as shown in SEQ ID NO: 27; and

The PEG molecule has a molecular weight of at least or at least about 30 kDa.

In some embodiments, the construct is a TNFR2 agonist construct comprising:

a) a TNFR2 TNF mutein comprising the mutations D143N/A145R;

b) (GGGGS) ₃ linker; and

c) an active modifier that is a half-life extending moiety that is the Fc of Trastuzumab or the Fc of Nivolumab.

In some embodiments, the construct is a TNFR2 agonist construct comprising:

a) a TNFR2 TNF selective mutein comprising the mutations D143N/A145R;

b) a second linker comprising the (GGGGS) ₃ linker and the sequence SCDKTH (residues 217-222 of SEQ ID NO: 31); and

c) an active modifier that is the Fc half-life extending moiety of Trastuzumab.

In some embodiments, the construct is a TNFR2 agonist construct comprising:

a) a TNFR2 TNF selective mutein comprising the mutations D143N/A145R;

b) a (GGGGS) ₃ linker and a second linker comprising all or part of the hinge sequence of nivolumab; and

c) an active modifier that is a half-life extending moiety that is the Fc of nivolumab.

In some embodiments, the construct is a TNFR2 agonist construct comprising:

a) a TNFR2 TNF selective mutein comprising the mutations D143N/A145R;

b) a linker comprising all or part of the hinge sequence of Trastuzumab corresponding to residues 219-233 of SEQ ID NO:26; and

c) a half-life extending moiety that is the Fc of Trastuzumab.

In some embodiments, the construct is a TNFR2 agonist construct comprising:

a) a TNFR2 TNF selective mutein comprising the mutations D143N/A145R;

b) a linker comprising all or part of the hinge sequence of nivolumab corresponding to residues 212-223 of SEQ ID NO:29; and

c) an active modifier that is a half-life extending moiety that is the Fc of nivolumab.

A TNFR1 antagonist construct, a TNFR2 agonist construct, and an IgG Fc are both monomeric or dimer are provided. Constructs provided herein may include dAb (or Vhh). The constructs may include single or double chain Vhh containing dAbs. Such constructs may contain HSA linked to the dAb either directly or through a linker. These HSAs and dAbs can be linked in any order, such as the C-terminus of the dAb linked directly to the N-terminus of the HSA or via any of the linkers above. Examples of such constructs are those including:

a) dAb Dom1h-131-206 of SEQ ID NO: 59 linked via a linker to the HSA set forth in SEQ ID NO: 1475, comprising residues 20-732, or a construct of SEQ ID NO: 1475 or residues 20-73 of SEQ ID NO: 1475 A construct having at least 95%, 96%, 97%, 98%, 99% sequence identity to 732 and having TNFR1 antagonist activity.

b) the dAb set forth in any one of SEQ ID NOs: 53-83 and 503-671, and variants thereof having at least 95%, 96%, 97%, 98%, 99% sequence identity therewith, wherein the construct is a TNFR1 antagonist have activity; or

c) a dAb having the sequence set forth in any one of SEQ ID NOs: 57-59, variants thereof having at least 95% sequence identity therewith, whereby the construct has TNFR1 antagonist activity; or

d) the dAb is designated as DOM1h-131-206 of SEQ ID NO: 59 and has at least 95%, 96%, 97%, 98%, 99% sequence identity therewith and a variant thereof having TNFR1 antagonist activity; or

e) any combination of a)-d); or

f) where a sufficient portion of the humanized sequence of any of a) to f) or construct for administration to a human is humanized, the sufficient portion is sufficient to eliminate or reduce any immune response to the construct when administered to a human. enough for

A construct provided herein that is a TNFR1 construct may further comprise a TNFR2 agonist or the construct may be a TNFR2 agonist construct. In a construct comprising a TNFR2 agonist, the TNFR2 agonist can be modified to remove sequences of amino acids or epitopes that are immunogenic in the subject to be treated, for example for administration to a human subject. In constructs containing a TNFR2 agonist it may be a TNFR2 selective agonist. These constructs may include modified IgG Fc. For example, an IgG Fc can include one or more of the following modifications:

a) deformation(s) to introduce a knob-into-hole;

b) modification(s) to increase or enhance neonatal Fc receptor (FcRn) recycling; and

c) modification(s) to reduce or eliminate an immune effector function selected from one or more of complement dependent cytotoxicity (CDC), antibody dependent cell mediated cytotoxicity (ADCC) and antibody dependent cell mediated phagocytosis (ADCP). Examples of such transformations are:

a) the modification(s) to introduce a knob-into-hole are selected from:

at least one knob mutation selected from among S354C, T366Y, T366W and T394W by EU numbering; and

at least one hole mutation selected from Y349C, T366S, L368A, F405A, Y407T, Y407A and Y407V by EU numbering, wherein Fc forms a dimer;

b) the variant(s) to increase or enhance FcRn recycling are, by EU numbering: N434A, N434W, N434S, N434Y, Y436H, M252Y/T256Q, M252F/T256D, M252Y/S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M4 28L, M428L/N434S, V259I/V308F, selected from one or more of V259I/V308F/M428L, E294del/T307P/N434Y, and T256N/A378V/S383N/N434Y; and

c) the modification(s) to reduce or eliminate immune effector function is selected from one or more of the following:

In IgG1: L235E, L234A/L235A, L234E/L235F/P331S, L234F/L235E/P331S, L234A/L235A/P329G, L234A/L235A/G237A/P238S/H268A/A330S/P331 by EU numbering S, G236R/L328R, G237A , E318A, D265A, E233P, N297A, N297Q, N297D, N297G, N297G/D265A, A330L, D270A, P329A, P331A, K322A, V264A, and F241A; and

In IgG4: L235E, F234A/L235A, S228P/L235E, and S228P/F234A/L235A by EU numbering.

Constructs provided herein include TNFR2 agonist constructs comprising a modified IgG Fc, wherein the IgG Fc comprises one or more of the following modifications:

a) deformation(s) to introduce a knob-into-hole; here

Knob mutations are selected from among S354C, T366Y, T366W and T394W by EU numbering; and

Hall mutations are variants selected from among Y349C, T366S, L368A, F405A, Y407T, Y407A and Y407V by EU numbering;

b) modification(s) to increase or enhance neonatal Fc receptor (FcRn) recycling, selected from one or more of the following:

T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M2 52Y/T256Q, M252F/T256D, M252Y/ S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F, V259I/V308F/M428L, E294del/T307P/N4 34Y, and T256N/A378V/S383N/N434Y ; and

c) as modification(s) to increase or enhance immune effector function;

the immune effector function(s) is selected from one or more of CDC, ADCC and ADCP; and

The modification(s) to increase or enhance immune effector function are selected from one or more of the following:

In IgG1: S239D, I332E, S239D/I332E, S239D/A330L/I332E, S298A/E333A/K334A in the first heavy chain; F243L/R292P/Y300L/V305I/P396L; L235V/F243L/R292P/Y300L/P396L; F243L/R292P/Y300L; L234Y/G236W/S298A and S239D/A330L/I332E in the second heavy chain; L234Y/L235Q/G236W/S239M/H268D/D270E/S298A in the first heavy chain and D270E/K326D/A330M/K334E in the second heavy chain; A327Q/P329A; D265A/S267A/H268A/D270A/K326A/S337A; T256A/K290A/S298A/E333A/K334A; G236A; G236A/I332E; G236A/S239D/I332E; G236A/S239D/A330L/I332E; introduction of a biantennary glycan at residue N297; residue N297; K326W; K326A; E333A; K326A/E333A; K326W/E333S; K326M/E333S; K222W/T223W; K222W/T223W/H224W; D221W/K222W; C220D/D221C; C220D/D221C/K222W/T223W; H268F/S324T; S267E; H268F; S324T; S267E/H268F/S324T; G236A/I332E/S267E/H268F/S324T; E345R; and introduction of afucosylated glycans at E345R/E430G/S440Y; by EU numbering.

Constructs provided herein, which are TNFR2 agonist constructs, can include modifications such as when the Fc is modified to increase binding to the inhibitory Fcγ receptor (FcγR) FcγRIIb, which may include modifications that increase binding to FcγRIIb. IgG1 Fc may be included. Examples of such variants are those selected from one or more of S267E, N297A, L328F, L351S, T366R, L368H, P395K, S267E/L328F and L351S/T366R/L368H/P395K by EU numbering.

Constructs that are or include TNFR2 agonist constructs that selectively activate or agonize TNFR2 without activating or antagonizing TNFR1 are provided. These constructs are a) TNFR2 agonists; b) one or more linkers; and c) an active modifier that is a half-life extending moiety, wherein:

The TNFR2 agonist construct is a fusion protein comprising a single chain TNFR2 selective TNF mutein trimer fused with a multimerization domain, comprising the formula:

MD-L1-TNFmut-L2-TNFmut-L3-TNFmut (formula II); or

TNFmut-L1-TNFmut-L2-TNFmut-L3-MD (formula III);

MD is a multimerization domain, each identical or different; TNFmut is a TNFR2 selective TNF mutein; and L1, L2 and L3 are linkers which may be the same or different. TNF muteins are K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R/S147T , Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147 D, A145T/E146S/S147D, A145Q /E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/S147D, D 143V/F144L/A145S, S95C/G148C , and D143V/A145S, eg, the TNFR2 selective mutations D143N/A145R. In these constructs, the multimerization domains are EHD2 (SEQ ID NO: 808), MHD2 (SEQ ID NO: 811), the trimerization domain of chicken tenascin C (TNC) (residues 110-139 of SEQ ID NO: 804; SEQ ID NO: 804). : 805), or the trimerization domain of human TNC (residues 110-139 of SEQ ID NO: 806, SEQ ID NO: 807), or having at least 95%, 96%, 97%, 98%, 99% sequence identity therewith. variants thereof. For example, the multimerization domain is an IgG1 Fc or IgG4 Fc and the IgG1 Fc or IgG4 Fc is also a half-life extending moiety. These constructs contain linkers, including any described herein and any known to those skilled in the art. Exemplary of these constructs are L1, L2 and/or L3 linkers that are (GGGGS) _n (where n = 1-5), and all or part of or at least 95% of the stem region of TNF (SEQ ID NO: 812); independently selected from among variants thereof having 96%, 97%, 98%, 99% sequence identity. These constructs are such that the linker between the TNFR2 agonist and the half-life extending moiety is (GlySer) _n (where n=1-10); (GlySer ₂ ); (Gly ₄ Ser) _n (where n=1-10); (Gly ₃ Ser) _n (where n= 1-5); (SerGly ₄ ) _n (where n=1-5); (GlySerSerGly) _n (where n= 1-5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS; or a linker selected from all or part of the hinge sequence of Trastuzumab and all or part of the hinge sequence of Nivolumab; or a GS linker selected from any combination thereof. The half-life extending moiety can be selected from: the IgG1 Fc, which is the Fc of human IgG1 as shown in SEQ ID NO: 10, or the Fc of Trastuzumab as shown in SEQ ID NO: 27; IgG4 Fc, the Fc of SEQ ID NO: 16 as shown in human IgG4, or the Fc of nivolumab as shown in SEQ ID NO: 30; PEG molecules of at least about 30 kDa in size; Human serum albumin (HSA), and variants of a polypeptide portion having at least 95%, 96%, 97%, 98%, 99% sequence identity therewith.

Constructs that are or include TNFR2 agonist constructs are provided. These constructs include those comprising the formula:

MD-L1-TNFmut-L2-TNFmut-L3-TNFmut (formula II); or

TNFmut-L1-TNFmut-L2-TNFmut-L3-MD (Equation III), where

a) MD is the multimerization domain; TNFmut is a TNFR2 selective TNF mutein; and L1, L2 and L3 are the same or different linkers, wherein

i) MD is EHD2 (SEQ ID NO: 808), MHD2 (SEQ ID NO: 811), the trimerization domain of chicken tenascin C (TNC) (residues 110-139 of SEQ ID NO: 804; SEQ ID NO: 805), or is selected from the trimerization domain of human TNC (residues 110-139 of SEQ ID NO: 806, SEQ ID NO: 807);

ii) L1, L2 and L3 are each (GGGGS) _n (where n = 1-5), or all or part of the stem region of TNF (SEQ ID NO: 812), or a mixture thereof; and

iii) the TNF mutein comprises the TNFR2 selective mutations D143N/A145R;

b) a half-life extending moiety selected from:

an IgG1 Fc that is the Fc of human IgG1 shown in SEQ ID NO: 10 or the Fc of trastuzumab described in SEQ ID NO: 27;

an IgG4 Fc that is the Fc of human IgG4 as shown in SEQ ID NO: 16 or the Fc of nivolumab as shown in SEQ ID NO: 30;

PEG molecules of at least or at least about 30 kDa in size; and

human serum albumin (HSA); and

c) a linker between the TNFR2 selective agonist and the half-life extending moiety comprising:

(GlySer) _n (where n=1-10); (GlySer ₂ ); (Gly ₄ Ser) _n (where n=1-10); (Gly ₃ Ser) _n (where n= 1-5); (SerGly ₄ ) _n (where n=1-5); (GlySerSerGly) _n (where n= 1-5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and a GS linker selected from GSSSGS; or

a linker selected from all or part of the hinge sequence of Trastuzumab and all or part of the hinge sequence of Nivolumab; or

or a combination thereof.

The construct includes a TNFR2 agonist construct comprising the formula:

MD-L1-TNFmut-L2-TNFmut-L3-TNFmut (formula II); or

TNFmut-L1-TNFmut-L2-TNFmut-L3-MD (formula III);

MD is the multimerization domain; TNFmut is a TNFR2 selective TNF mutein; and L1, L2 and L3 are the same or different linkers, wherein

i) MD is selected from IgG1 Fc or IgG4 Fc;

ii) L2 and L3 in formula II, and L1 and L2 in formula III are each independently (GGGGS) _n (where n = 1-5), or all or part of the stem region of TNF (SEQ ID NO: 812), or a combination thereof;

iii) L1 in Formula II and L3 in Formula III are each independently selected from:

(GlySer) _n (where n=1-10); (GlySer ₂ ); (Gly ₄ Ser) _n (where n=1-10); (Gly ₃ Ser) _n (where n= 1-5); (SerGly ₄ ) _n (where n=1-5); (GlySerSerGly) _n (where n= 1-5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and a GS linker selected from GSSSGS; or

a linker selected from all or part of the hinge sequence of Trastuzumab and all or part of the hinge sequence of Nivolumab; or

or a combination thereof.

iv) the TNF mutein comprises the TNFR2 selective mutations D143N/A145R. The MD in these constructs can be selected from:

an IgG1 Fc that is the Fc of human IgG1 shown in SEQ ID NO: 10 or the Fc of trastuzumab described in SEQ ID NO: 27; or

an IgG4 Fc that is the Fc of human IgG4 as shown in SEQ ID NO: 16 or the Fc of nivolumab as shown in SEQ ID NO: 30; or

Combinations or variants thereof having at least 95%, 96%, 97%, 98%, 99% sequence identity.

An exemplary construct is an MD that is an IgG1 Fc of Trastuzumab, wherein the linker between the MD and the adjacent TNF mutein is all or part of the hinge sequence of Trastuzumab corresponding to residues 219-233 of SEQ ID NO: 26, or As MD, the IgG1 Fc of Trastuzumab, the linker between the MD and the adjacent TNF mutein is one comprising the MD comprising the sequence SCDKTH (residues 217-222 of SEQ ID NO:31). An exemplary construct is one comprising MD, an IgG1 Fc of Trastuzumab, wherein the linker between the MD and the adjacent TNF mutein is (Gly ₄ Ser) ₃ and trastuzumab corresponding to residues 219-233 of SEQ ID NO: 26. It includes the hinge sequence of In some embodiments, MD is an IgG1 Fc of Trastuzumab, the linker between MD and adjacent TNF mutein comprises (Gly ₄ Ser) ₃ and SCDKTH (residues 222-227 of SEQ ID NO:31), and MD is an IgG4 Fc of nivolumab, wherein the linker between the MD and the adjacent TNF mutein comprises all or part of the hinge sequence of nivolumab corresponding to residues 212-223 of SEQ ID NO: 29;

MD is the IgG4 Fc of nivolumab, and the linker between the MD and the adjacent TNF mutein contains (Gly ₄ Ser) ₃ and all or part of the hinge sequence of Trastuzumab corresponding to residues 212-223 of SEQ ID NO: 29 do.

Constructs herein, including agonist constructs, may be modified to remove immunogenic sequences, such as sequences immunogenic to humans. Provided herein are TNFR2 agonist constructs that have been modified to remove immunogenic epitopes or immunogenic sequences in which the TNFR2 agonist is immunogenic in a subject, such as a human subject.

In constructs provided herein that are TNFR2 agonist constructs and include modified IgG Fc, the IgG Fc can include one or more of the following modifications:

a) deformation(s) to introduce a knob-into-hole; here

the knob mutation is selected from one or more of S354C, T366Y, T366W and T394W by EU numbering; and

Hall mutations are variants selected from one or more of Y349C, T366S, L368A, F405A, Y407T, Y407A and Y407V by EU numbering;

b) modification(s) to increase or enhance neonatal Fc receptor (FcRn) recycling, selected from one or more of the following:

T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M2 52Y/T256Q, M252F/T256D, M252Y/ S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F, V259I/V308F/M428L, E294del/T307P/N4 34Y, and T256N/A378V/S383N/N434Y ; and

c) modification(s) to reduce or eliminate immune effector function, wherein

the immune effector function(s) is selected from one or more of CDC, ADCC and ADCP; and

The modification(s) to reduce or eliminate immune effector function is selected from one or more of the following:

In IgG1: L235E, L234A/L235A, L234E/L235F/P331S, L234F/L235E/P331S, L234A/L235A/P329G, L234A/L235A/G237A/P238S/H268A/A330S/P331 by EU numbering S, G236R/L328R, G237A , E318A, D265A, E233P, N297A, N297Q, N297D, N297G, N297G/D265A, A330L, D270A, P329A, P331A, K322A, V264A, and F241A; and

In IgG4: L235E, F234A/L235A, S228P/L235E, and S228P/F234A/L235A by EU numbering.

Any of the aforementioned constructs are provided that are TNFR2 agonist constructs comprising a modified IgG Fc, wherein the IgG Fc comprises one or more of the following modifications:

a) deformation(s) to introduce a knob-into-hole; here

the knob mutation is selected from one or more of S354C, T366Y, T366W and T394W by EU numbering; and

Hall mutations are variants selected from one or more of Y349C, T366S, L368A, F405A, Y407T, Y407A and Y407V by EU numbering;

b) modification(s) to increase or enhance neonatal Fc receptor (FcRn) recycling, selected from one or more of the following:

T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M2 52Y/T256Q, M252F/T256D, M252Y/ S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F, V259I/V308F/M428L, E294del/T307P/N4 34Y, and T256N/A378V/S383N/N434Y ; and

c) as modification(s) to increase or enhance immune effector function;

the immune effector function(s) is selected from one or more of CDC, ADCC and ADCP; and

The modification(s) to increase or enhance immune effector function are selected from one or more of the following:

In IgG1: S239D, I332E, S239D/I332E, S239D/A330L/I332E, S298A/E333A/K334A in the first heavy chain; F243L/R292P/Y300L/V305I/P396L; L235V/F243L/R292P/Y300L/P396L; F243L/R292P/Y300L; L234Y/G236W/S298A and S239D/A330L/I332E in the second heavy chain; L234Y/L235Q/G236W/S239M/H268D/D270E/S298A in the first heavy chain and D270E/K326D/A330M/K334E in the second heavy chain; A327Q/P329A; D265A/S267A/H268A/D270A/K326A/S337A; T256A/K290A/S298A/E333A/K334A; G236A; G236A/I332E; G236A/S239D/I332E; G236A/S239D/A330L/I332E; introduction of a biantennary glycan at residue N297; residue N297; K326W; K326A; E333A; K326A/E333A; K326W/E333S; K326M/E333S; K222W/T223W; K222W/T223W/H224W; D221W/K222W; C220D/D221C; C220D/D221C/K222W/T223W; H268F/S324T; S267E; H268F; S324T; S267E/H268F/S324T; G236A/I332E/S267E/H268F/S324T; E345R; and introduction of afucosylated glycans at E345R/E430G/S440Y; by EU numbering.

Provided are any of the TNFR2 agonist constructs of any of the claims comprising an IgG1 Fc modified to increase binding to the inhibitory Fcγ receptor (FcγR) FcγRIIb. An example of this is that the modification that increases binding to FcγRIIb is selected from one or more of S267E, N297A, L328F, L351S, T366R, L368H, P395K, S267E/L328F and L351S/T366R/L368H/P395K by EU numbering.

Constructs provided herein may be multispecific in that they interact with two or more targets. An example of such a multispecific construct is a multispecific TNFR1 inhibitor/TNFR2 agonist construct and is either of the following formulas:

(TNFR1 inhibitor) _n - linker (L) _p - (TNFR2 agonist) _q (Formula I), or

(TNFR1 inhibitor) _n - Linker (L) _p - (TNFR2 agonist) _q , or

(TNFR1 inhibitor) _n - (TNFR2 agonist) _q - linker (L) _p , or

(TNFR2 agonist) _q - (TNFR1 inhibitor) _n - Linker (L) _p , or

Any of the above, including a selective active modifier, wherein: n = 1 or 2, p = 1, 2 or 3, and q = 1 or 2; TNFR1 inhibitors interact with TNFR1 to inhibit its activity; An active modifier is a moiety that modulates or alters the activity or pharmacological properties of a construct compared to a construct without the active modifier; For example, the linker increases the solubility of the construct, increases flexibility, or alters the steric effect of the construct. These constructs include those that are multispecific TNFR1 inhibitor/TNFR2 agonist constructs, wherein: the TNFR1 inhibitor selectively inhibits or antagonizes TNFR1 signaling without inhibiting or antagonizing TNFR2 signaling; TNFR1 inhibitors do not interfere with the activation or action of TNFR2; TNFR2 agonists selectively activate or agonize TNFR2 signaling without activating or agonizing TNFR1 signaling; TNFR2 agonists do not interfere with the inhibition or antagonism of TNFR1. An example of such a construct is the construct of a)-c) as follows:

a) the TNFR1 inhibitor is selected from:

i) an antigen-binding fragment of a human anti-TNFR1 antagonist monoclonal antibody selected from H398 or ATROSAB or a polypeptide having a sequence having at least 95% sequence identity thereto; or

ii) a domain antibody (dAb) of any one of SEQ ID NOs: 52-672, or a scFv of any one of SEQ ID NOs: 673-678 or a Fab of any one of SEQ ID NOs: 679-682, or SEQ ID NOs: 683 or 684 or a TNF mutein of any one of SEQ ID NOs: 701-703, or a polypeptide having a sequence that has at least 95% sequence identity to any of the foregoing polypeptides and is a TNFR1 inhibitor; or

iii) a predominantly-negative tumor necrosis factor (DN-TNF) or TNF mutein comprising a soluble TNF molecule conferring selective inhibition of TNFR1 and having one or more amino acid substitutions selected from:

V1M, L29S, L29G, L29Y, R31C, R31E, R31N, R32Y, R32W, C69V, A84S, V85T, S86T, Y87H, Q88N, T89Q, I97T, C101A, A145R with respect to the sequence of soluble TNF set forth in SEQ ID NO: 2 , E146R, L29S/R32W, L29S/S86T, R32W/S86T, L29S/R32W/S86T, R31N/R32T, R31E/S86T, R31N/R32T/S86T, I97T/A145R, V1M/R31C/C69V/Y87H/C101 A/A145R , and A84S/V85T/S86T/Y87H/Q88N/T89Q;

b) the linker is selected from:

i) (GlySer) _n (where n = 1-10); (GlySer ₂ ); (Gly ₄ Ser) _n (where n=1-10); (Gly ₃ Ser) _n (where n= 1-5); (SerGly ₄ ) _n (where n=1-5); (GlySerSerGly) _n (where n= 1-5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and a GS linker selected from GSSSGS; and/or

ii) all or part of the hinge sequence of Trastuzumab corresponding to residues 219-233 of SEQ ID NO: 26, or all or part of the hinge sequence of Nivolumab corresponding to residues 212-223 of SEQ ID NO: 29; and

iii) IgG1 or IgG4 Fc, wherein:

the IgG1 Fc is selected from the IgG1 Fc of human IgG1 set forth in SEQ ID NO: 10, or the IgG1 Fc of Trastuzumab set forth in SEQ ID NO: 27;

the IgG4 Fc is selected from the IgG4 Fc of human IgG4 set forth in SEQ ID NO: 16 or the IgG4 Fc of nivolumab set forth in SEQ ID NO: 30; and

Optionally, the Fc comprises one or more modifications to introduce a knob-into-hole, increase or enhance neonatal Fc receptor (FcRn) recycling, and/or reduce or eliminate immune effector function; and

c) the TNFR2 agonist is selected from:

i) an antigen-binding fragment that binds to one or more epitopes in human TNFR2 selected from among the epitopes set forth in SEQ ID NOs: 839-865, 1202 and 1204; or

ii) an antigen-binding fragment of a potent human anti-TNFR2 antibody selected from MR2-1 or MAB2261; or

iii) K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145 with respect to SEQ ID NO: 2 R/S147T, Q88N /T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145 T/E146S/S147D, A145Q/E146D /S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/S147D, D143V/F 144L/A145S, S95C/G148C, and a TNFR2 selective TNF mutein, which is a soluble TNF variant comprising one or more TNFR2 selective mutations selected from D143V/A145S; or

iv) a single chain TNFR2 selective TNF mutein trimer comprising the mutations D143N/A145R, wherein the TNF mutein is (GGGGS) _n (where n = 1-5) or all or all of the stem region of TNF (SEQ ID NO: 812) single-chain TNFR2 selective TNF mutein trimers, linked by moieties; or

v) a TNFR2 selective agonist comprising the formula:

MD-L1-TNFmut-L2-TNFmut-L3-TNFmut (formula II); or

TNFmut-L1-TNFmut-L2-TNFmut-L3-MD (formula III);

MD is the multimerization domain; TNFmut is a TNFR2 selective TNF mutein; and L1, L2 and L3 are the same or different linkers, wherein

MD is EHD2 (SEQ ID NO: 808), MHD2 (SEQ ID NO: 811), the trimerization domain of chicken tenascin C (TNC) (residues 110-139 of SEQ ID NO: 804; SEQ ID NO: 805), or human TNC is selected from the trimerization domain of (residues 110-139 of SEQ ID NO: 806, SEQ ID NO: 807);

L1, L2 and L3 are each (GGGGS) _n (where n = 1-5), or all or part of the stem region of TNF (SEQ ID NO: 812), or a mixture thereof; and

TNF muteins include the TNFR2 selective mutations D143N/A145R.

Other such constructs include those that are multispecific TNFR1 antagonist/TNFR2 agonist constructs, wherein:

a) the TNFR1 inhibitor is a domain antibody (dAb) of any one of SEQ ID NOs: 52-672, or a scFv of any one of SEQ ID NOs: 673-678 or a Fab of any one of SEQ ID NOs: 679-682, or SEQ ID NO: a Nanobody of 683 or 684, or a TNF mutein of any one of SEQ ID NOs: 701-703, or a sequence having at least or at least about 95% sequence identity therewith;

b) the linker comprises (GGGGS) ₃ , a polypeptide comprising the sequence SCDKTH (residues 222-227 of SEQ ID NO: 26), and the Fc of Trastuzumab; and

c) the TNFR2 agonist is K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R with respect to SEQ ID NO: 2 , A145R /S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D /S147D, A145T/E146S/S147D A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D S147D, D143V/F144L/A145S, S95C /G148C, and a TNFR2 selective TNF mutein, which is a soluble TNF variant comprising one or more TNFR2 selective mutations selected from D143V/A145S.

Other multispecific constructs are:

a) the TNFR1 inhibitor is a domain antibody (dAb) of any one of SEQ ID NOs: 52-672, or a scFv of any one of SEQ ID NOs: 673-678 or a Fab of any one of SEQ ID NOs: 679-682, or SEQ ID NO: a Nanobody of 683 or 684, or a TNF mutein of any one of SEQ ID NOs: 701-703, or a sequence having at least or at least about 95% sequence identity therewith;

b) the linker comprises (GGGGS) ₃ , all or part of the hinge sequence of nivolumab, and the Fc of nivolumab; and

c) the TNFR2 agonist is K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R with respect to SEQ ID NO: 2 , A145R /S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D /S147D, A145T/E146S/S147D A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D S147D, D143V/F144L/A145S, S95C /G148C, and a TNFR2 selective TNF mutein, which is a soluble TNF variant comprising one or more TNFR2 selective mutations selected from D143V/A145S.

Other multispecific constructs are:

a) the TNFR1 inhibitor is a domain antibody (dAb) of any one of SEQ ID NOs: 52-672, or a scFv of any one of SEQ ID NOs: 673-678 or a Fab of any one of SEQ ID NOs: 679-682, or SEQ ID NO: a Nanobody of 683 or 684, or a TNF mutein of any one of SEQ ID NOs: 701-703, or a sequence having at least or at least about 95% sequence identity therewith;

b) the linker comprises (GGGGS) ₃ and the Fc of Trastuzumab; and

c) the TNFR2 agonist is K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R with respect to SEQ ID NO: 2 , A145R /S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D /S147D, A145T/E146S/S147D A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D S147D, D143V/F144L/A145S, S95C /G148C, and a TNFR2 selective TNF mutein, which is a soluble TNF variant comprising one or more TNFR2 selective mutations selected from D143V/A145S.

Other multispecific constructs are:

a) the TNFR1 inhibitor is a domain antibody (dAb) of any one of SEQ ID NOs: 52-672, or a scFv of any one of SEQ ID NOs: 673-678 or a Fab of any one of SEQ ID NOs: 679-682, or SEQ ID NO: a Nanobody of 683 or 684, or a TNF mutein of any one of SEQ ID NOs: 701-703, or a sequence having at least or at least about 95% sequence identity therewith;

b) the linker comprises (GGGGS) ₃ and the Fc of nivolumab; and

c) the TNFR2 agonist is K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R with respect to SEQ ID NO: 2 , A145R /S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D /S147D, A145T/E146S/S147D A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D S147D, D143V/F144L/A145S, S95C /G148C, and D143V/A145S, and a TNFR2 selective TNF mutein, which is a soluble TNF variant comprising one or more TNFR2 selective mutations selected from any combination of the foregoing mutations.

These multispecific constructs may include a modified Fc, wherein the IgG Fc comprises one or more of the following modifications:

a) deformation(s) to introduce a knob-into-hole;

b) modification(s) to increase or enhance neonatal Fc receptor (FcRn) recycling; and

c) modification(s) to reduce or eliminate immune effector function. An example of an Fc containing a knob-into-hole variant is as follows:

the knob mutation is selected from one or more of S354C, T366Y, T366W and T394W by EU numbering; and

The hole mutation is selected from one or more of Y349C, T366S, L368A, F405A, Y407T, Y407A and Y407V by EU numbering.

Another example is that Fc is T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N4 by EU numbering. 34Y, Y436H, M252Y /T256Q, M252F/T256D, M252Y/S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F, V259I/V 308F/M428L, E294del/T307P/N434Y , and T256N/A378V/S383N/N434Y, wherein the Fc comprises a modification to increase or enhance FcRn recycling. The Fc comprises modifications to an immune effector function selected from one or more of complement dependent cytotoxicity (CDC), antibody dependent cell mediated cytotoxicity (ADCC) and antibody dependent cell mediated phagocytosis (ADCP). An Fc may contain modification(s) to reduce or eliminate immune effector functions in IgG1 and/or IgG4:

In IgG1: L235E, L234A/L235A, L234E/L235F/P331S, L234F/L235E/P331S, L234A/L235A/P329G, L234A/L235A/G237A/P238S/H268A/A330S/P331 by EU numbering S, G236R/L328R, G237A , E318A, D265A, E233P, N297A, N297Q, N297D, N297G, N297G/D265A, A330L, D270A, P329A, P331A, K322A, V264A, and F241A; and/or

In IgG4: L235E, F234A/L235A, S228P/L235E, and S228P/F234A/L235A by EU numbering.

An IgG Fc can include one or more of the following modifications:

a) deformation(s) to introduce a knob-into-hole; here

the knob mutation is selected from one or more of S354C, T366Y, T366W and T394W by EU numbering; and

Hall mutations are variants selected from one or more of Y349C, T366S, L368A, F405A, Y407T, Y407A and Y407V by EU numbering;

b) modification(s) to increase or enhance neonatal Fc receptor (FcRn) recycling, selected from one or more of the following:

T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M2 52Y/T256Q, M252F/T256D, M252Y/ S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F, V259I/V308F/M428L, E294del/T307P/N4 34Y, and T256N/A378V/S383N/N434Y ; and

c) as modification(s) to increase or enhance immune effector function;

the immune effector function is selected from one or more of CDC, ADCC and ADCP; and

The modification(s) to increase or enhance immune effector function are selected from one or more of the following:

In IgG1: S239D, I332E, S239D/I332E, S239D/A330L/I332E, S298A/E333A/K334A in the first heavy chain; F243L/R292P/Y300L/V305I/P396L; L235V/F243L/R292P/Y300L/P396L; F243L/R292P/Y300L; L234Y/G236W/S298A and S239D/A330L/I332E in the second heavy chain; L234Y/L235Q/G236W/S239M/H268D/D270E/S298A in the first heavy chain and D270E/K326D/A330M/K334E in the second heavy chain; A327Q/P329A; D265A/S267A/H268A/D270A/K326A/S337A; T256A/K290A/S298A/E333A/K334A; G236A; G236A/I332E; G236A/S239D/I332E; G236A/S239D/A330L/I332E; introduction of a biantennary glycan at residue N297; residue N297; K326W; K326A; E333A; K326A/E333A; K326W/E333S; K326M/E333S; K222W/T223W; K222W/T223W/H224W; D221W/K222W; C220D/D221C; C220D/D221C/K222W/T223W; H268F/S324T; S267E; H268F; S324T; S267E/H268F/S324T; G236A/I332E/S267E/H268F/S324T; E345R; and introduction of afucosylated glycans at E345R/E430G/S440Y; by EU numbering.

Other such multispecific constructs are: Constructs comprising an IgG1 Fc that has been modified to increase binding to the inhibitory Fcγ receptor (FcγR) FcγRIIb. An example of this is that the modification that increases binding to FcγRIIb is selected from one or more of S267E, N297A, L328F, L351S, T366R, L368H, P395K, S267E/L328F and L351S/T366R/L368H/P395K by EU numbering.

Also provided are constructs that are multispecific TNFR1 antagonists/TNFR2 agonists, wherein the TNFR1 antagonists are monovalent; TNFR2 agonists are monovalent. Also provided are multispecific constructs that are multispecific TNFR1 antagonist/TNFR2 agonist constructs, wherein the TNFR1 antagonist is monovalent; TNFR2 agonists are bivalent.

In some embodiments, the multispecific construct is a multispecific TNFR1 antagonist/TNFR2 agonist construct, wherein:

a) the TNFR1 antagonist is selected from:

i) an antigen-binding fragment of a human anti-TNFR1 antagonist monoclonal antibody selected from H398 or ATROSAB; or

ii) a domain antibody (dAb) of any one of SEQ ID NOs: 52-672, or a scFv of any one of SEQ ID NOs: 673-678 or a Fab of any one of SEQ ID NOs: 679-682, or SEQ ID NOs: 683 or 684 or a TNF mutein of any one of SEQ ID NOs: 701-703, or a sequence having at least or at least about 95% sequence identity therewith; or

iii) a predominantly-negative tumor necrosis factor (DN-TNF) or TNF mutein comprising a soluble TNF molecule conferring selective inhibition of TNFR1 and having one or more amino acid substitutions selected from:

V1M, L29S, L29G, L29Y, R31C, R31E, R31N, R32Y, R32W, C69V, A84S, V85T, S86T, Y87H, Q88N, T89Q, I97T, C101A, A145R with respect to the sequence of soluble TNF set forth in SEQ ID NO: 2 , E146R, L29S/R32W, L29S/S86T, R32W/S86T, L29S/R32W/S86T, R31N/R32T, R31E/S86T, R31N/R32T/S86T, I97T/A145R, V1M/R31C/C69V/Y87H/C101 A/A145R , and A84S/V85T/S86T/Y87H/Q88N/T89Q;

b) the linker is a branched chain PEG molecule of at least or at least about 30 kDa in size; and

c) the TNFR2 agonist is selected from:

i) an antigen-binding fragment that binds to one or more epitopes in human TNFR2 selected from among the epitopes set forth in SEQ ID NOs: 839-865, 1202 and 1204; or

ii) an antigen-binding fragment of a potent human anti-TNFR2 antibody selected from MR2-1 or MAB2261; or

iii) K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145 with respect to SEQ ID NO: 2 R/S147T, Q88N /T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145 T/E146S/S147D, A145Q/E146D /S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/S147D, D143V/F 144L/A145S, S95C/G148C, and a TNFR2 selective TNF mutein, which is a soluble TNF variant comprising one or more TNFR2 selective mutations selected from D143V/A145S; or

iv) a single chain TNFR2 selective TNF mutein trimer comprising the mutations D143N/A145R, wherein the TNF mutein is (GGGGS) _n (where n = 1-5) or all or all of the stem region of TNF (SEQ ID NO: 812) single-chain TNFR2 selective TNF mutein trimers, linked by moieties; or

v) a TNFR2 selective agonist comprising the formula:

MD-L1-TNFmut-L2-TNFmut-L3-TNFmut (formula II); or

TNFmut-L1-TNFmut-L2-TNFmut-L3-MD (formula III);

MD is the multimerization domain; TNFmut is a TNFR2 selective TNF mutein; and L1, L2 and L3 are the same or different linkers, wherein

MD is EHD2 (SEQ ID NO: 808), MHD2 (SEQ ID NO: 811), the trimerization domain of chicken tenascin C (TNC) (residues 110-139 of SEQ ID NO: 804; SEQ ID NO: 805), or human TNC is selected from the trimerization domain of (residues 110-139 of SEQ ID NO: 806, SEQ ID NO: 807);

L1, L2 and L3 are each (GGGGS) _n (where n = 1-5), or all or part of the stem region of TNF (SEQ ID NO: 812), or a mixture thereof; and

TNF muteins include the TNFR2 selective mutations D143N/A145R.

Also provided are multispecific constructs wherein each of the TNFR1 antagonist and the TNFR2 agonist is monovalent. Also provided are constructs wherein the TNFR1 antagonist is monovalent and the TNFR2 agonist is divalent.

Constructs provided herein may be used for the treatment and use of a variety of diseases, disorders and conditions. Multispecific TNFR1 for use in the treatment of chronic inflammatory, autoimmune, neurodegenerative, demyelinating or respiratory diseases or disorders, and diseases, conditions or disorders characterized by overexpression of TNF or deregulated TNFR1 signaling in its pathogenesis. A multispecific construct that is an antagonist/TNFR2 agonist is provided. Multispecific TNFR1 antagonists/ for the treatment of chronic inflammatory, autoimmune, neurodegenerative, demyelinating or respiratory diseases or disorders, or diseases, conditions or disorders characterized by overexpression of TNF or deregulated TNFR1 signaling in its pathogenesis. Uses of TNFR2 agonist constructs are provided.

Also provided are compositions comprising a construct of any of the constructs provided herein in a pharmaceutically acceptable carrier or vehicle. These compositions can be used to treat chronic inflammatory, autoimmune, neurodegenerative, demyelinating or respiratory diseases or disorders, and diseases, conditions or disorders characterized by overexpression of TNF or deregulated TNFR1 signaling in its etiology, diseases, conditions or disorders thereof. , disorders, and conditions, or in a method of treatment thereof. Exemplary chronic inflammatory, autoimmune, neurodegenerative, demyelinating or respiratory diseases or disorders, or diseases, conditions or disorders are diseases, disorders, and conditions characterized by overexpression of TNF or deregulated TNFR1 signaling in its etiology. . These include diseases, disorders, and conditions selected from: rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, juvenile idiopathic arthritis (JIA), spondyloarthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis, inflammatory bowel disease (IBD), uveitis, fibrotic disease, endometriosis, lupus, multiple sclerosis (MS), congestive heart failure, cardiovascular disease, myocardial infarction (MI), atherosclerosis, metabolic disease, cytokine release syndrome, septic shock, Sepsis, acute respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), SARS-CoV-2, influenza, acute and chronic neurodegenerative diseases, demyelinating diseases and disorders, stroke, Alzheimer's disease, Parkinson's disease, Behçet's disease, Dupuytren's disease, tumor necrosis factor receptor-associated periodic syndrome (TRAPS), pancreatitis, type I diabetes, chronic obstructive pulmonary disease (COPD), chronic bronchitis, emphysema, graft rejection, graft-versus-host disease (GvHD), lung inflammation, lung disease and conditions, asthma, cystic fibrosis, idiopathic pulmonary fibrosis, acute fulminant viral or bacterial infections, pneumonia, hereditary genetic diseases with TNF/TNFR1 as a causative pathological vector, periodic fever syndrome, or cancer. In particular, constructs provided herein, such as, but not limited to, TNFR1 antagonist constructs, may be used in uses, methods and compositions for the treatment of rheumatoid arthritis.

Also provided herein are constructs that are TNFR2 antagonists and, optionally, TNFR2 antagonist constructs comprising a linker and optionally an active modifier. For example such a construct has formula 5:

(TNFR2 antagonist) _n - linker _p - (active modifier) _q , or

linker _p - (active modifier) _q - (TNFR2 antagonist) _n , where:

n and q are each an integer and are each independently 1, 2 or 3;

p is 0, 1, 2 or 3;

A TNFR2 antagonist is a molecule capable of inhibiting (antagonizing) the activity of TNFR2 by interacting with TNFR2, thereby inhibiting the proliferation of Treg and/or inducing its death, and also inhibiting the proliferation and inducing the death of TNFR2-expressing tumor cells. ;

An active modifier is a moiety that modulates or alters the activity or pharmacological properties of a construct compared to a construct without the active modifier; and

The linker increases the flexibility of the construct, modulates or reduces the steric effect or interaction of the construct with a receptor, and/or increases the solubility of the construct in aqueous media.

In these constructs, each active modifier and linker is as defined and described for the constructs above and below. They can be used in therapeutic methods and uses and pharmaceutical compositions.

TNFR2 antagonists reduce and/or inhibit proliferation of bone marrow derived suppressor cells (MDSCs); and/or inducing apoptosis in MDSCs by binding to TNFR2 expressed on the surface of MDSCs present in the tumor microenvironment; and/or to induce expansion of T effector cells, including cytotoxic CD8 ⁺ T cells, through suppression of Treg expansion and activity. The TNFR2 antagonist in the construct is an epitope in human TNFR2 containing one or more of the residues KCRPG (corresponding to residues 142-146 of SEQ ID NO: 4), or for example residues 130-149, 137-144 or 142-149, or binds to a larger epitope containing at least 5 contiguous or discontinuous residues within these epitopes, and does not bind to an epitope containing residues KCSPG (corresponding to residues 56-60 of SEQ ID NO: 4); or the TNFR2 epitopes PECLSCGS (corresponding to residues 91-98 of SEQ ID NO: 4), RICTCRPG corresponding to residues 116-123 of SEQ ID NO: 4), CAPLRKCR (corresponding to residues 137-144 of SEQ ID NO: 4), LRKCRPGFGVA (corresponding to residues 140-150 of SEQ ID NO: 4), and/or VVCKPCAPGTFSN (corresponding to residues 159-171 of SEQ ID NO: 4), and/or residues 75-128, 86- of SEQ ID NO: 4 antibodies, antigen-binding fragments thereof, or single chain antibodies that bind to an epitope containing at least 5 contiguous or discontinuous residues within 103, 111-128, or 150-190. For example, an antibody, fragment thereof, or single-chain form thereof may comprise a KCRPG sequence with an affinity that is at least 10-fold greater than the affinity of the same antibody or antigen-binding fragment for a peptide containing the KCSPG sequence of human TNFR2 (SEQ ID NO: 839). (SEQ ID NO: 840). In some embodiments of the TNFR2 antagonist construct, the TNFR2 antagonist is an antibody or fragment or antibody in single chain form selected from:

TNFRAB1 (see SEQ ID NOs: 1212 and 1213 for the heavy and light chain sequences of TNFRAB1, respectively), TNFRAB2 and TNFR2A3 (see, eg, US Patent Publication No. 2019/0144556 for a description of these antibodies);

TNFRAB1 (QRVDGYSSYWYFDV; corresponding to residues 99-112 of SEQ ID NO: 1212), TNFRAB2 (ARDDGSYSPFDYWG; SEQ ID NO: 1217) or TNFR2A3 (ARDDGSYSPFDYFG; SEQ ID NO: 1223), or a CDR with at least about 85% sequence identity there - a CDR-H3 sequence of TNFRAB1 that specifically binds to residues 130-149 containing residues KCRPG of TNFR2 with 40-fold higher affinity than H3 sequence, e.g., residues 48-67 containing residues KCSPG of TNFR2. Antibodies and antibody fragments and single chain forms containing In some embodiments, the TNFR2 antagonist binds to one or more epitopes within TNFR2 selected from:

an epitope containing residues 137-144 (CAPLRKCR; SEQ ID NO: 851);

an epitope comprising one or more residues within positions 80-86 (DSTYTQL; SEQ ID NO: 1247), 91-98 (PECLSCGS; SEQ ID NO: 1248), and/or 116-123 (RICTCRPG; SEQ ID NO: 1249) of human TNFR2 ; and

TNFR2A3 selected from the first epitope is an epitope comprising residues 140-150 of human TNFR2 (LRKCRPGFGVA; SEQ ID NO: 1463) and containing a KCRPG motif, and/or residues 159-171 of human TNFR2 (VVCKPCAPGTFSN; SEQ ID NO: 1464) A second epitope containing

In some embodiments, the TNFR2 antagonist in the construct is an antibody, fragment thereof, or single chain form thereof containing one or more of SEQ ID NOs: 1214, 1215, and 1231-1233 to produce a humanized antagonistic TNFR2 antibody. A CDR-H1 amino acid having the sequence set forth in any, a CDR-H2 sequence set forth in any of 1216, 1224, and 1230, a CDR-H3 sequence set forth in any of SEQ ID NOs: 1217, 1223, and 1225-1229, and /or CDR-H3 of TNFRAB1 corresponding to residues 99-112 of SEQ ID NO: 1212; the CDR-L1 sequence set forth in any of SEQ ID NOs: 1218 and 1234-1236, and/or the CDR-L1 sequence of TNFRAB1 corresponding to residues 24-33 of SEQ ID NO: 1213; the CDR-L2 sequence set forth in any of SEQ ID NOs: 1219, 1220, 1237 and 1238, or the CDR-L2 sequence of TNFRAB1 corresponding to residues 49-55 of SEQ ID NO: 1213; and/or a CDR-L3 sequence set forth in any of SEQ ID NOs: 1221, 1222, and 1241-1244, or a CDR-L3 sequence of TNFRAB1 corresponding to residues 88-96 of SEQ ID NO: 1213; and/or the CDR-H1 and CDR-H2 sequences of the consensus sequence of the human antibody heavy chain variable domain of SEQ ID NO: 1245 replaced by the corresponding CDR sequences of a phenotype neutral TNFR2 specific antibody, and/or of a phenotype neutral TNFR2 specific antibody. CDR-L1, CDR-L2 and CDR-L3 sequences of the sequence of the human antibody light chain variable domain of SEQ ID NO: 1246 replaced by the corresponding CDR sequences. For example, the construct includes a TNFR2 antagonist that specifically binds to an epitope in TNFR2 set forth in any one of SEQ ID NOs: 1247-1464. In some embodiments, the TNFR2 antagonist specifically binds to an epitope(s) selected from:

(a) residues KCRPG corresponding to residues 142-146 of SEQ ID NO: 4, or residues 130-149, 137-144 or 142-149, or a larger epitope containing at least 5 contiguous or discontinuous residues within these epitopes; at least one epitope in human TNFR2 that contains at least one that does not bind to an epitope containing residues KCSPG corresponding to residues 56-60 of SEQ ID NO:4; and/or

(b) one or more TNFR2 epitopes comprising an amino acid sequence comprising:

PECLSCGS corresponding to residues 91-98 of SEQ ID NO: 4, and/or RICTCRPG corresponding to residues 116-123 of SEQ ID NO: 4, and/or

CAPLRKCR (corresponding to residues 137-144 of SEQ ID NO: 4), and/or LRKCRPGFGVA (corresponding to residues 140-150 of SEQ ID NO: 4), and/or VVCKPCAPGTFSN (corresponding to residues 159-171 of SEQ ID NO: 4) equivalent), and/or

An epitope containing at least 5 contiguous or discontinuous residues within residues 75-128, 86-103, 111-128, or 150-190 of SEQ ID NO:4.

In some embodiments, a TNFR2 antagonist construct comprises a TNFR2 antagonist that is a small molecule. For example, a TNFR2 antagonist is thalidomide or its analogues, such as lenalidomide and pomalidomide.

In some embodiments, the TNFR2 antagonist construct comprises a TNFR2 antagonist that reduces FoxP3 expression and inhibits the suppressive activity of Tregs. An example of such an antagonist is a histone deacetylase inhibitor that reduces FoxP3 expression and inhibits the suppressive activity of Tregs. Examples of such inhibitors are panobinostat or cyclophosphamide or triptolide.

TNFR2 constructs can be used in methods and uses for the treatment of infectious diseases, and in the treatment of cancers that express TNFR2. Examples of such cancers are cancers selected from: T cell lymphomas such as Hodgkin's lymphoma and cutaneous non-Hodgkin's lymphoma, ovarian cancer, colon cancer, multiple myeloma, renal cell carcinoma, breast cancer, cervical cancer, endometrial cancer, neurological glioma, head and neck cancer, liver cancer, and lung cancer.

The claims set forth below and in the priority applications and their summary are hereby incorporated by reference into this summary.

Figure 1 shows a plasmid map of the pCBL-1 expression plasmid containing the CMV promoter, a fragment into which TE19080L has been inserted.
Figure 2 shows an exemplary dual-specific construct with a linker (part of the hinge region) and an active modifier connecting two ligands, such as a TNFR1 inhibitor (TNFR1 antagonist) and a TNFR2 agonist.
3A-3D depict exemplary PEG-centric multispecific constructs for presenting/providing one or more targets or two or more moieties that interact with one target at multiple sites. 3A depicts an exemplary bivalent construct. For example, one in the circle is a polypeptide agonist, antagonist or binding protein, such as an antibody or antigen-binding fragment thereof, or an aptamer (nucleic acid or peptide). Other circles represent polysaccharides or receptor ligands or other moieties that interact with the target of interest. The bivalent nature provides clustering of targets for receptor activation. In embodiments provided herein, targets include TNFR1 and TNFR2; As described throughout this specification, moieties include TNFR1 inhibitors, such as moieties that inhibit TNFR1 signaling, and other moieties that are TNFR2 agonists or Treg expanders. 3B shows that monovalent single ligands such as CD3+ prevent cytokine release syndrome linked via PEG moieties to agonists, antagonists or binding proteins that are bivalent for receptor clustering. Again exemplary targets include TNFR1 and/or TNFR2. 3C depicts a heterobifunctional PEG for crosslinking two different cellular targeting agents, or two agents that bind to different sites on the same receptor, such as Trastuzumab and Pertuzumab or portions thereof. This construct can be used, for example, to cluster checkpoint control receptors for stimulation or inhibition of an immune response, or to bridge two different receptors to achieve inhibition of receptor activity (i.e., CD3 versus CD450), or to stimulate and to deliver two different ligands, such as co-stimulatory ligands, to two different receptors on the same cell. Figure 3D depicts homobifunctional PEG for capturing circulatory disease targets such as ligands or soluble receptors such as TNF or clustering the same receptors on the same or different cells depending on the chain length. Additionally in all of these embodiments another reactive group or functional group, such as a serum half-life extending moiety such as HSA, or an additional PEG side chain optionally linked to the FcRn polypeptide may be included in these constructs. The PEG moiety can be modified or replaced by moieties with similar properties to present a binding moiety.
4 depicts additional exemplary configurations and structures of PEG-centric constructs for displaying or providing binding moieties or reactive moieties such as TNFR1 inhibitors and/or TNFR2 agonists as described herein.
5 depicts additional exemplary configurations and structures of PEG-centric constructs for displaying or providing binding moieties or reactive moieties, such as TNFR1 inhibitors and/or TNFR2 agonists. X and Y can be ligands and reactive moieties.

details

outline

A. Definition

B. Overview of Constructs and Methods

C Tumor necrosis factor (TNF) and chronic inflammatory and autoimmune diseases and disorders

1. Tumor necrosis factor ( TNF )

2. Tumor necrosis factor receptor ( TNFR )

a. TNFR1

b. TNFR2

3. Regulatory T cells ( Treg ) and its role in the autoimmune microenvironment

4. Autoimmune/inflammatory diseases mediated by or accompanying TNF

a. arthritis

i. rheumatic arthritis and other types of arthritis

b. inflammatory bowel disease ( IBD ) and uveitis

c. fibrotic disease

d. Tumor Necrosis Factor Receptor Associated Cyclic Syndrome (TRAPS)

e. to TNF due to mediated other diseases that accompany him

i. neurodegenerative disease

a) Alzheimer's disease

b) Parkinson's disease

c) multiple sclerosis (MS)

ii. endometriosis

iii. cardiovascular disease

iv. acute respiratory distress syndrome ( ARDS )

v. severe acute respiratory syndrome (SARS) and COVID -19

D. rheumatic Therapy for arthritis and other chronic inflammatory autoimmune diseases and disorders

1. Conventional synthetic disease modulating anti- rheumatism drug( csDMARD )

2. Anti- TNF therapy/ TNF blocker

E. TNFR1/TNFR2 Targeted Therapies

One. TNFR1 selective antagonist

a. TNFR1 antagonistic antibody

b. 1st TNFR1 Antagonistic antibody/antibody fragment

i. Fab and scFv base TNFR1 antagonist

ii. domain antibody ( dAb ) base TNFR1 antagonist

a) Anti- TNFR1 dAb -anti-albumin dAb fusion construct

b) GSK1995057 and with GSK2862277 designated domain antibody fragments

iii. nanobody ( Nb )

iv. port- TNFR1 nanobody -anti-albumin nanobody fusion construct

c. TNF ( DN - TNF )/ TNF mutein dominant-negative inhibitor

2. TNFR2 optional agonist

a. TNFR2 effective antibody

b. TNFR2 optional TNF mutein and fusions thereof

3. Anti- TNFR2 antagonistic antibodies and small molecule inhibitor

F. Selective targeting of the TNFR1 and/or TNFR2 axis

One. TNFR1 by antagonists TNFR1 selective blocking

2. TNFR2 to agonist by TNFR2 Selective activation

3. TNFR1 antagonist construct , TNFR2 agonist construct ; double be specific multispecific, including TNFR1 antagonist and TNFR2 agonist construct

4. TNFR1 antagonist construct , TNFR2 agonist construct ; and double be specific multispecific, including TNFR1 antagonist/ TNFR2 agonist of the construct ingredient

a. TNFR1 inhibitor moiety ( TNFR1 antagonist)

b. TNFR2 agonist construct and TNFR2 antagonist construct

c. linker

i. peptide linker

a) flexibility linker

b) rigid linkers

ii. chemical linker

d. activation modifier

i. Fc transformation of the part

a) Knob-in-hole

b) newborn Fc receptor ( FcRn ) strains that enhance recirculation

c) Fc immune effector Enhancement or reduction/elimination of function

ii. Fc different variations of parts

iii. human serum albumin

e. multispecific TNFR1 antagonist/ TNFR2 agonist construct

dual specific, TNFR1 antagonist/ TNFR2 agonist construct and Same multispecific construct, PEG centered multispecific of the construct to connect the components PEGization

f. more active modifier - fusion proteins comprising partial or whole polypeptides that increase serum half-life

5. Prediction and elimination of immunogenicity of protein therapeutics

a. B-cells and T-cells epitope

b. in silico epitope Prediction method

i. B-cell epitope in silico prediction

ii. T-cell epitope in silico prediction

iii. peptide- MHC Class II binding prediction

c. in vitro epitope Prediction method

i. B-cells in vitro epitope Prediction method

ii. T-cells in vitro epitope Prediction method

MHC / HLA combination test

iii. In vitro T-cell assay

d. in vivo epitope Prediction method

e. Predicted B-cells and T-cells epitope eliminate( deimmunization )

G. pan-growth factor trap polypeptide

1. Receptor tyrosine Kinase ( RTK )

a. human epidermal growth factor receptor (HER) family

b. human epidermal growth factor receptor (HER) family and diseases associated with its ligands

2. Pan-growth factor inhibition

a. RB242 Ligand Trap

b. RB200 and RB242 for the treatment of autoimmune diseases

c. RB242 Ligand Trap

3. Optimized multispecific, such as bispecific , growth factor trap construct

a. extracellular domain( ECD ) polypeptide

b. extracellular Variations on Domains

c. multimerization domain

d. Fc Variations on Domains

i. Introduction of knob-in-hole

ii. newborn baby Fc receptor ( FcRn ) strains that enhance recirculation

iii. effector function

4. Compositions, therapeutic uses and methods of treatment

a. pharmaceutical composition

b. Therapeutic uses and methods of treatment

5. Combination therapy

H. TNFR1 antagonist and TNFR1 antagonist/ TNFR2 agonist construct Evaluation of activity and efficacy

1. Disease activity score ( DAS28 )

2. analyte for quantification SOMAscan ^® prote analysis and other prote equipment

3. Predict responsiveness to therapy and are likely to benefit from therapy object to choose transcriptome analyze

4. L929 Cytotoxicity Assay

5. HeLa IL-8 assay

6. HUVEC black

7. Treg Quantification and evaluation of cell activity

8. TNFR1 antagonist/ TNFR2 agonist of the construct Bond characterization

9. Antibody-dependent cytotoxicity ( ADCC ) and complement dependent cytotoxicity ( CDC ) black

10. Disease Model

a. Collagen Induced Arthritis (CIA)

b. rheumatic arthritis synovial mononuclear cell culture

c. arthritic Tg197 mouse model

d. arthritis/ of IBD Δ ARE mouse model

e. humanization TNF / TNFR2 mouse

I. TNFR1 antagonist construct and TNFR1 antagonist/ TNFR2 agonist construct Methods of producing nucleic acids that encode

One. TNFR1 antagonist and TNRF2 agonist Isolation or Preparation of Nucleic Acids Encoding Polypeptides

2. Generation of Mutant or Modified Nucleic Acids and Encoding Polypeptides

3. Vectors and Cells

4. Expression

a. prokaryotic cell

b. yeast cells

c. insects and insect cells

d. mammalian expression cells

e. plant

5. Refining

6. Additional variations

a. PEGization

b. albuminization

c. tablet tag

7. Nucleic Acid Molecules and Gene Therapy

J. Composition, Formulation and Dosage

1. Formulation

2. TFNR1 antagonist construct , TNFR2 agonist construct , multispecific, for example bispecific , construct and administration of nucleic acids

3. Administration of Nucleic Acids Encoding Polypeptides (Gene Therapy)

K. Therapeutic Uses and Methods of Treatment

1. Treatment of chronic inflammatory/autoimmune diseases and disorders

2. Neurodegeneration and dehydration Treatment of Diseases and Disorders

3. Cancer and other immunosuppressive diseases, disorders and pathological therapy

4. Combination therapy

L. Examples

A. Definition

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention(s) belong. All patents, patent applications, published applications and publications, GenBank sequences, databases, websites, and other published material referenced throughout the entire disclosure of this specification are incorporated by reference in their entirety unless otherwise indicated. In the event that there are multiple definitions for terms in this specification, the definitions in this section shall prevail. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers may change and certain information may come and go on the Internet, but equivalent information may be found by searching the Internet. Reference thereto demonstrates the availability and public dissemination of such information.

As used herein, a construct is a product containing one or more components, usually at least two. A component can be a polypeptide, small molecule, aptamer, nucleic acid, and/or other such component as described herein or known to those skilled in the art. A variety of constructs are described and exemplified herein; Their components and variations are apparent from the description herein. In view of the description, one skilled in the art may envision other constructs that are within the scope of the present disclosure and claims. The term construct is used because a product can contain a variety of different types of components.

As used herein, a construct that is a TNFR1 construct or a TNFR2 antagonist construct is a construct comprising a TNFR1 inhibitor moiety, which is a moiety that inhibits or reduces TNFR1 activity, such as signaling.

As used herein, a construct that is a TNFR2 construct or a TNFR2 agonist construct is a construct comprising a TNFR2 agonist moiety, which activates the activity of TNFR2, such as signaling or activity that increases Treg cells. It is a moiety that induces or induces.

As used herein, a construct that is a TNFR2 antagonist construct is a construct comprising a TNFR2 antagonist.

As used herein, a construct that is a multispecific construct is a construct containing more than one antagonist or agonist or both moieties, such as a TNFR1 inhibitor and a TNFR2 agonist, or two TNRF1 antagonists, antagonists, each interacting with a different epitope on TNFR1 or each having a different TNFR1 antagonist activity, or two TNFR2 agonists, such as agonists each interacting with a different TNFR2 epitope or each having different TNFR2 agonist activity. It is a construct comprising a construct containing.

As used herein, “tumor necrosis factor”, “tumor necrosis factor alpha”, “TNF”, “TNF-alpha”, “TNF-α” and “TNFα” are members of the TNF superfamily and are tumorigenic / are used interchangeably to refer to pleiotropic pro-inflammatory cytokines that are associated with inflammatory and immune-regulatory activities including regulation of cancer, host defense against pathogenic infection, apoptosis, autoimmunity and septic shock . When other members of the TNF superfamily are intended, they will be identified by name. TNF is involved in the coordination of innate and adaptive immune responses as well as in organogenesis, particularly the formation of lymphoid organs. TNF is produced as a homotrimeric membrane-bound protein containing 233 amino acids that can be cleaved by the protease TACE (TNF alpha converting enzyme; also known as ADAM17) to release soluble TNF (solTNF), which has 157 amino acids contains; Membrane-bound and soluble forms of TNF are biologically active. The homotrimer of TNF binds to and signals through two high-affinity, specific receptors, TNFR1 and TNFR2; Membrane-bound TNF primarily activates TNFR2, whereas soluble TNF primarily activates TNFR1. Dysregulated or dysregulated production of TNF is associated with several conditions including but not limited to, for example, septic shock, rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis and inflammatory bowel disease (IBD). It is associated with chronic inflammatory and autoimmune diseases and conditions, as well as neurodegenerative and demyelinating diseases and conditions including, but not limited to, eg, Alzheimer's disease, Parkinson's disease, stroke and multiple sclerosis.

As used herein, "TNF mutein" or "TNF-α mutein" or "modified TNF polypeptide" refers to the amino acid sequence of the corresponding wild-type TNF (TNFα) and one or more amino acids to TNF from a particular species. Refers to polypeptides with these different amino acid sequences. Generally, such modified TNF polypeptides retain the ability to activate or inhibit TNFR1 and/or TNFR2. Specific mutations within TNF can cause the resulting TNF muteins to act selectively in binding to TNFR1 or TNFR2, resulting in TNF muteins with antagonistic or agonistic properties. For example, as described herein, there are TNFR1-selective antagonistic TNF muteins and TNFR2-selective agonistic TNF muteins.

As used herein, a “dominant-negative inhibitor of TNF” or “DN-TNF” is a TNF mutein that has one or more mutations that abrogate binding to and signaling through TNFR1 and/or TNFR2. DN-TNF selectively inhibits soluble TNF (sTNF or solTNF) by rapidly exchanging subunits with native TNF homotrimers, and forms inactive mixed TNF heterotrimers in which the receptor binding surface is disrupted to interact with TNF receptors. prevent action. DN-TNF does not affect transmembrane TNF (tmTNF) and retains the protective role of TNF signaling through TNFR2. Examples of DN-TNF include substitutions L133Y, S162Q, Y163H, I173T, Y191Q and A221R with reference to the sequence of amino acids set forth in SEQ ID NO: 1 that impairs binding to TNFR (as shown in SEQ ID NO: 2, the sequence of solTNF Corresponding to residues L57Y, S86Q, Y87H, I97T, Y115Q and A145R, see ), there are TNF mutants containing one or more.

As used herein, “modification” refers to a modification of a sequence of amino acids within a polypeptide or a sequence of nucleotides within a nucleic acid molecule, and includes deletions, insertions, translocations, substitutions, and combinations thereof of amino acids or nucleotides, respectively. Methods of modifying polypeptides or nucleic acids are routine to those skilled in the art, such as by using recombinant DNA methodologies.

As used herein, "deletion" when referring to a nucleic acid or polypeptide sequence refers to a deletion of one or more nucleotides or amino acids compared to a sequence, such as a target polynucleotide or polypeptide, or a native or wild-type sequence.

As used herein, "insertion" when referring to a nucleic acid or amino acid sequence describes the inclusion of one or more additional nucleotides or amino acids within a target, native, wild-type or other related sequence. Thus, compared to the wild-type sequence, a nucleic acid molecule containing one or more insertions contains one or more additional nucleotides within the linear length of the sequence.

As used herein, "addition" when referring to a nucleic acid or amino acid sequence describes the addition of one or more nucleotides or amino acids to either end compared to the other sequence.

As used herein, "substitution" or "replacement" refers to the substitution of one or more nucleotides or amino acids in a natural, target, wild-type or other nucleic acid or polypeptide sequence without altering the length of the molecule (as described in number of residues). Refers to replacing with an alternative nucleotide or amino acid. Thus, one or more substitutions in a molecule do not change the number of amino acid residues or nucleotides in the molecule. Amino acid substitutions relative to a particular polypeptide can be expressed as the number of amino acid residues along the length of the polypeptide sequence. For example, a modified polypeptide having a modification at position 100 of an amino acid sequence in which tyrosine (Tyr; Y) is substituted/substituted for glutamic acid (Glu; E) can be expressed as Y100E, Tyr100Glu or 100E. Y100 may be used to indicate that the modified amino acid at position 100 is tyrosine. For purposes of this specification, modifications may also be denoted by reference to HC- or LC- to refer to the chain of a polypeptide, since the modifications are in either the heavy (HC) or light (LC) chain of the antibody.

As used herein, a reference to “at a position corresponding to” or “corresponding to” a nucleotide or amino acid position within a disclosed sequence such that the nucleotide or amino acid position is set forth in a sequence listing is a reference to a standard alignment algorithm, such as the GAP algorithm. Refers to a nucleotide or amino acid position identified when aligned with a referenced sequence to maximize identity using. By aligning the sequences, one skilled in the art can identify corresponding residues, for example using conserved and identical amino acid residues as guides. Generally, sequences of amino acids are aligned such that the highest rank match is obtained to identify corresponding positions (see, e.g., Computational Molecular Biology , Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects , Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I , Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994 Sequence Analysis in Molecular Biology , von Heinje, G., Academic Press, 1987; Sequence Analysis Primer , Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carrillo et al. (1988) SIAM J. Applied Math 48:1073).

As used herein, alignment of sequences refers to the use of homology to align two or more sequences of nucleotides or amino acids. Typically, two or more sequences related by at least 50% identity are aligned. An aligned set of sequences refers to two or more sequences that align in corresponding positions, and may include aligned sequences derived from RNAs such as ESTs and other cDNAs aligned with genomic DNA sequences. Related or variant polypeptides or nucleic acid molecules can be aligned by any method known to those skilled in the art. Such methods typically maximize agreement and include, for example, using manual alignment and using the numerous alignment programs available (eg, BLASTP) and other programs known to those skilled in the art. By aligning the sequences of polypeptides or nucleic acids, one skilled in the art can identify similar portions or positions using conserved and identical amino acid residues as guides. In addition, one skilled in the art may use conserved amino acid or nucleotide residues as a guide to locate corresponding amino acid or nucleotide residues between and among human and non-human sequences. Corresponding positions can also be based on structural alignments, for example by using computer simulation alignments of protein structures. In other instances, corresponding regions can be identified. One skilled in the art can also use conserved amino acid residues as a guide to locate corresponding amino acid residues between and among human and non-human sequences.

As used herein, a reference to proteins being “compared under identical conditions” refers to any one or more conditions under which different proteins may be treated identically or substantially identically to affect the activity or properties of the protein or agent. It means unchanged or substantially unchanged between these test formulations. For example, when the activity of an antibody is compared to another antibody, any one or more conditions, such as the amount or concentration of the polypeptide; the presence (including amounts) of excipients, carriers, or other components in the formulation other than the active agent (eg, antibody); temperature; pH; storage time; storage containers; storage characteristics (eg, shaking); and/or other conditions associated with exposure or use are the same or substantially the same between and among the compared polypeptides/antibodies.

As used herein, “adverse effect” or “side effect” or “adverse event” or “adverse side effect” refers to a detrimental, toxic and/or undesirable effect associated with administration of a therapeutic agent. For example, side effects associated with administration of an anti-TNF antibody such as adalimumab (eg, sold under the trademark Humira ^® ) are known to those skilled in the art, and some are described herein. Such adverse side effects include serious infections, such as, for example, tuberculosis, and other infections caused by viruses, fungi, and bacteria, including upper respiratory tract infections, as well as dermatological and skin toxicities such as rashes, headaches, and nausea. Thus, an “adverse effect” or “side effect” refers to a detrimental, toxic, and/or undesirable effect of administration of a therapeutic agent. Side effects or adverse effects are graded according to toxicity, and there are various toxicity scales that provide definitions for each grade. Examples of such scales include the National Cancer Institute Common Toxicity Criteria version 2.0 toxicity scale, and the World Health Organization or Common Terminology Criteria for Adverse Events (CTCAE) scale. Assigning grading of severity is within the skill of a skilled physician or other healthcare professional. The severity of symptoms can be quantified using the NCI Common Terminology Criteria for Adverse Events (CTCAE) grading system. CTCAE is a descriptive term used for reporting adverse events (AEs). A grading (severity) scale is provided for each AE term. The CTCAE ranks 1 to 5 with a clinical description of the severity of each adverse event based on the following general guidelines: Grade 1 (minor AE); Grade 2 (Intermediate AE); Grade 3 (severe AE); Grade 4 (life-threatening or disabling AE); and Grade 5 (AE-related death/fatal).

As used herein, “characteristics” of a polypeptide, such as an antibody, include, but are not limited to, binding specificity, structural arrangement or conformation, protein stability, resistance to proteolysis, conformational stability, heat resistance, and resistance to pH conditions. Refers to any property exhibited by a non-limiting polypeptide. Changes in properties can alter the “activity” of a polypeptide. For example, a change in the binding specificity of an antibody polypeptide can alter various binding activities such as the ability and/or affinity or avidity to bind antigen or activity of the polypeptide in vivo .

As used herein, “activity” or “functional activity” of a polypeptide, such as an antibody, refers to any activity exhibited by the polypeptide. Such activity can be determined empirically. Exemplary activities include, for example, the ability to interact with biomolecules through antigen binding, DNA binding, ligand binding or dimerization; and enzymatic activity, such as kinase activity or proteolytic activity. In the case of antibodies (including antibody fragments), activities include the ability to specifically bind to a particular antigen, the affinity of antigen binding (e.g., high affinity or low affinity), the avidity of antigen binding (e.g., high or low avidity), on-rate, off-rate, effector functions such as ability to neutralize antigens or promote clearance, neutralize viruses and in vivo activities such as prevent infection or invasion of pathogens or promote clearance or the ability to penetrate certain tissues or fluids or cells in the body, but is not limited thereto. Activity is recognized in assays such as ELISA, flow cytometry, surface plasmon resonance or equivalent assays to measure on- or off-rates, immunohistochemistry and immunofluorescence histology and microscopy, cell-based assays, flow cytometry and binding It can be assessed in vitro or in vivo using an assay (eg, a panning assay). For example, in the case of antibody polypeptides, activity measures binding affinity, avidity and/or binding coefficient (eg, on-/off-rate) and other activities in vitro or immune effects, such as antigen scavenging capacity. ; penetration or localization of antibodies into tissues; protection from diseases such as infections; serum or other fluid antibody titer; or by measuring various effects in vivo , such as other assays well known in the art. Results of such assays showing that a polypeptide exhibits activity can be correlated with the activity of the polypeptide in vivo, where activity in vivo can be referred to as therapeutic activity or biological activity. The activity of the modified polypeptide is 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% of the activity compared to the unmodified polypeptide. , 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%, 500% or more It can be any level of percent activity of the unmodified polypeptide, including but not limited to. Assays for determining the functionality or activity of modified (or variant) antibodies are well known in the art.

As used herein, “bind”, “coupled” and grammatical variations thereof refer to the participation of a molecule in any attractive interaction with another molecule, resulting in two molecules being very close to each other. A stable association in close proximity takes place. Binding interactions include, but are not limited to, non-covalent bonds, covalent bonds (eg, reversible and irreversible covalent bonds), and between molecules such as proteins, nucleic acids, carbohydrates, lipids, and small molecules, such as compounds including drugs. Including but not limited to interactions. Exemplary bonds are antibody-antigen interactions and receptor-ligand interactions. When an antibody “binds” a particular antigen, “binding” refers to the specific recognition of the antigen by the antibody through a cognate antibody-antigen interaction at the antibody binding site. Linkage can also include the association of multiple chains of a polypeptide, such as antibody chains, that interact through disulfide bonds.

As used herein, "binding activity" refers to the characteristic of a molecule, for example, whether and how a polypeptide binds to one or more binding partners. Binding activity may include the ability to bind the binding partner(s), the affinity to bind the binding partner (e.g., high affinity), the ability to bind to the binding partner, the strength of binding to the binding partner, and/or the affinity to bind to the binding partner. Specificity for binding is included.

As used herein, "affinity" or "binding affinity" describes the strength of an interaction between two or more molecules, such as binding partners, and typically the strength of a non-covalent interaction between two binding partners. do. The affinity of an antibody or antigen-binding fragment thereof for an antigenic epitope is a measure of the strength of the total non-covalent interactions between a single antibody binding site and the epitope. Low affinity antibody-antigen interactions are weak and the molecules tend to dissociate rapidly, whereas high affinity antibody-antigen bonds are strong and the molecules remain bound for longer periods of time. Binding affinities may be determined by, for example, association ( ka _or k _on ) and/or dissociation ( k _d or k _off ) rates, half maximal effect concentration (EC ₅₀ ) values, and/or thermodynamic data (eg, Gibbs free energy ( Gibbs free energy (ΔG), enthalpy (ΔH), entropy (-TΔS)) and/or calculating association (K _a ) or dissociation (K _d ) constants. The EC ₅₀ , also referred to as the apparent K _d , is the concentration of antibody (eg, ng/mL) at which 50% of maximal binding is observed for a fixed amount of antigen. EC ₅₀ values are typically determined from a sigmoidal dose-response curve, where EC ₅₀ is the concentration at the inflection point. High antibody affinity for its substrate correlates with low EC ₅₀ values, and low affinity corresponds to high EC ₅₀ values. Affinity constants can be determined by standard kinetic methodologies for antibody responses, eg, immunoassays such as ELISA followed by curve-fitting analysis.

As used herein, “affinity constant” refers to the association constant (K _a ) used to measure the affinity of an antibody for an antigen. The higher the affinity constant, the greater the affinity of the antibody for its antigen. Affinity constants are expressed in units of inverse molarity (ie, M ⁻¹ ) and follow standard kinetic methodologies for antibody responses (eg, immunoassays, surface plasmon resonance, or other kinetic interaction assays known in the art). can be calculated from the rate constants for association-dissociation reactions as measured by The binding affinity of an antibody can also be expressed as the dissociation constant or K _d . The dissociation constant is the reciprocal of the association constant, i.e. K _d = 1/K _a . Thus, the affinity constant can also be given as K _d . Affinity constants can be determined using standard kinetic methodologies for antibody responses, such as immunoassays, surface plasmon resonance (SPR) (see, eg, Rich and Myszka (2000)). Curr . Opin . Biotechnol 11:54; Englebienne (1998) Analyst. 123:1599), isothermal titration calorimetry (ITC) or other kinetic interaction assays known in the art (see, for example, Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989); see also U.S. Patent No. 7,229,619 for a description of exemplary SPR and ITC methods for calculating the binding affinity of antibodies). Instruments and methods for real-time detection and monitoring of binding rates are known and commercially available (e.g., BIAcore 2000, BIAcore AB, Upsala, Sweden and GE Healthcare Life Sciences; Malmqvist (2000) Biochem. Soc. Trans 27 :335).

Methods for calculating affinity, such as determining EC ₅₀ values or determining association/dissociation constants, are well known. For example, a high binding affinity in terms of EC ₅₀ indicates that the antibody is less than about 10 ng/mL, 9 ng/mL, 8 ng/mL, 7 ng/mL, 6 ng/mL, 5 ng/mL, 3 ng/mL. /mL, 2 ng/mL, 1 ng/mL or less EC ₅₀ means that it specifically binds to the target protein. ^A high binding ^{affinity also has an} equilibrium dissociation ^constant ₍ ^{K d} ) can be characterized. In terms of the equilibrium association constant (K _a ), a high binding affinity is generally about 10 ⁶ M ^-1 or greater, about 10 ⁷ M ^{-1 or} greater, about 10 ⁸ M ^-1 or greater, or about 10 ⁹ M ^-1 , It is associated with K _a values greater than 10 ¹⁰ M ^-1 , 10 ¹¹ M ^-1 or 10 ¹² M ^-1 . Affinity can be estimated empirically, or affinity can be determined relatively, eg by comparing the affinity of two or more antibodies for a particular antigen, eg by calculating the pairwise ratio of the affinity of the tested antibodies. can For example, such affinity can be determined using conventional techniques, such as by ELISA; equilibrium dialysis; surface plasmon resonance; by radioimmunoassay using radiolabeled target antigens; or can be readily determined by another method known to those skilled in the art. Affinity data can be found, for example, in Scatchard et al., (1949) Ann NY . Acad . Sci ., 51:660], or for example by the equation: y = ((AD)/(1+((x/C)^B))) + D, where A is the least asymptotes and , where B is the slope factor, C is the inflection point (EC ₅₀ ), and D is the maximum asymptote) by curve fitting analysis using a four parameter logistic nonlinear regression model.

As used herein, "antibody-binding capacity" refers to the strength of multiple interactions between a multivalent antibody and its cognate antigen, such as an antibody containing multiple binding sites associated with an antigen with a repeat epitope or epitope array. High avidity antibodies have a higher strength of such interactions compared to low avidity antibodies.

As used herein, “specificity to a target” such as TNFR1 refers to a preference for binding to a target, higher binding affinity, compared to non-targets. Selective binding is typically at least about 10 ⁷ -10 ⁸ It refers to binding to a target with an affinity of M ^-1 . It can also refer to the relative activity of a moiety or molecule when its affinity for one target molecule is compared to its affinity for another molecule, and the difference is a certain order of magnitude, such as about 10-fold, then the moiety or molecule is It is said to have greater specificity for the first target compared to the second target.

As used herein, "specifically binds" or "immunospecifically binds" with respect to an antibody or antigen-binding fragment thereof are used interchangeably herein and refer to the antibody binding site(s) of an antibody. Refers to the ability of an antibody or antigen-binding fragment to form one or more non-covalent bonds with a cognate antigen by non-covalent interaction between the antigen and the antigen. Typically, for example, an antibody ^that immunospecifically binds ⁽ or specifically binds ⁾ TNFR1 has an ^affinity constant (K _a ) (or 1× It is an antibody that binds to TNFR1 with a dissociation constant (K _d ) of 10 ⁻⁷ M or 1×10 ⁻⁸ M or less. Antibodies or antigen-binding fragments that immunospecifically bind to a particular antigen are, for example, immunoassays such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), or known to those skilled in the art. It can be confirmed by other techniques.

As used herein, "steric effect" refers to the effect of the size of an atom or group on a molecule. Steric effects include, but are not limited to, steric hindrance and van der Waals repulsion. The steric effect is an effect resulting from the fact that atoms occupy space; When atoms are placed close to each other, this consumes energy as the electrons near the atoms repel each other.

As used herein, “exhibits at least one activity” or “retains at least one activity” refers to an antibody polypeptide, such as a variant antibody or other therapeutic, compared to a target or unmodified polypeptide that does not contain a modification. Refers to the activity exhibited by an enemy polypeptide. Modified or variant polypeptides that retain the activity of the target polypeptide may exhibit improved activity, reduced activity, or retain the activity of the unmodified polypeptide. In some cases, a modified or variant polypeptide may retain increased activity compared to a target or unmodified polypeptide. In some cases, a modified or variant polypeptide may retain a reduced activity compared to an unmodified or target polypeptide. The activity of the modified or variant polypeptide is 1% activity compared to the unmodified or target polypeptide, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60% %, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%, It can be any level of the percent activity of the unmodified or target polypeptide, including but not limited to an activity greater than or equal to 500%. In other embodiments, the change in activity is at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold greater than the unmodified or target polypeptide. , 40x, 50x, 60x, 70x, 80x, 90x, 100x, 200x, 300x, 400x, 500x, 600x, 700x, 800x, 900x, 1000x and more big. Assays for retention of activity depend on the activity to be retained. Such assays can be performed in vitro or in vivo . Activity can be measured using assays known in the art for activity and described below, such as, but not limited to, ELISA and panning assays. The activity of a modified or variant polypeptide compared to an unmodified or target polypeptide can also be evaluated in terms of therapeutic or biological activity in vivo or outcome following administration of the polypeptide.

As used herein, “surface plasmon resonance” refers to an optical phenomenon that enables analysis of real-time interactions by detecting changes in protein concentration within a biosensor matrix. Commercial systems are available. For example, the BIAcore system (GE Healthcare Life Sciences) is an exemplary commercial system.

As used herein, “antibody” refers to immunoglobulins and immunoglobulin fragments, whether produced naturally or partially or completely synthetically, such as recombinantly, and which are sufficient and assembled to form an antigen-binding site. and any fragment thereof that contains at least a portion of the variable heavy and/or variable light chain regions of an immunoglobulin molecule sufficient to specifically bind an antigen when rendered. Thus, an antibody includes any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen binding domain (antibody binding site). For example, an antibody refers to an antibody that contains two heavy chains (which may be denoted H and H') and two light chains (which may be denoted L and L'), wherein each heavy chain binds an antigen. may be a full-length immunoglobulin heavy chain or a portion thereof sufficient to form a site (e.g., the heavy chain may be V _H chain, including but not limited to, V _H -C _H 1 chain and V _{H -CH} 1 -C _H 2 -C _H 3 chain), each light chain comprising a full-length light chain sufficient to form an antigen-binding site or (eg, a light chain includes, but is not limited to, a V _L chain and a V _L -C _L chain). Each heavy chain (H and H') is paired with one light chain (L and L', respectively). Typically, an antibody minimally comprises all or at least a portion of a variable heavy chain (V _H ) and/or a variable light chain (V _L ). An antibody may also include other regions, such as, for example, all or part of a constant region, and/or all or part of a hinge region (sufficient to provide flexibility).

For purposes of this specification, the term “antibody” includes full-length antibodies and portions thereof, including antibody fragments, such as, for example, anti-TNFR1 antibody fragments, unless otherwise specified. Antibody fragments include, for example, Fab fragments, Fab' fragments, F(ab') ₂ fragments, Fv fragments, disulfide bonded Fvs (dsFv), Fd fragments, Fd' fragments, single-chain Fvs (scFv), single-chain Fabs (scFabs) , hsFv (helix-stabilized Fv), single domain antibodies (dAb or sdAb), minibodies, diabodies, anti-idiotype (anti-Id) antibodies, nanobodies and camelid antibodies, free light chains, V _HH antibodies (or nanobodies), or antigen-binding fragments of any of the above. Antibody fragments also include, for example, tandem scFv, Fab-scFv (HC C-terminus or LC C-terminus), Fab-(scFv) ₂ (C-terminus), scFv-Fab-scFv, Fab- _CH 2- scFv, scFv fusion (C-terminus or N-terminus), Fab-fusion (HC C-terminus or LC C-terminus), scFv-scFv-dAb, scFv-dAb-scFv, dAb-scFv-scFv and tribodies. A combination of any of the fragments may be included. The term “antibody” includes synthetic antibodies, recombinantly produced antibodies, multispecific and heteroconjugate antibodies (eg, bi-, tri- and tetraspecific antibodies, diabodies, triabodies and tetrabodies), human antibodies, Includes non-human antibodies, humanized antibodies, chimeric antibodies and intrabodies. Antibodies provided herein may be of any immunoglobulin class (eg, IgG, IgM, IgD, IgE, IgA and IgY), any subclass (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or Includes members of sub-classes (eg, IgG2a and IgG2b).

As used herein, “form of antibody” refers to the specific structure of an antibody. Antibodies herein include full-length antibodies and portions thereof, such as, for example, Fab fragments or other antibody fragments. Thus, a Fab is a specific form of an antibody.

As used herein, reference to "corresponding forms" of antibodies means that when comparing the properties or activities of two antibodies, the same forms of the antibodies are used to compare the properties. For example, if an antibody is stated to have a lower activity compared to the activity of the corresponding form of the first antibody, this indicates that the specific form, such as Fab, of the antibody has lower activity compared to the Fab form of the first antibody. it means.

As used herein, a full-length antibody has two full-length heavy chains (e.g., V _H -C _H 1-C _H 2-C _H 3 or V _H -C _H 1-C _H 2-C _H 3- C _H 4), two full-length light chains (V _L -C _L ) and a hinge region, human antibodies produced by antibodies secreting B cells and antibodies with identical domains produced synthetically.

As used herein, a “multispecific construct” refers to a construct, such as a construct comprising an antibody or portion of an antibody that exhibits affinity for more than one target antigen such that it is capable of specifically interacting with a target. refers to The multispecific construct herein may have a structure similar to a whole immunoglobulin molecule and may include an Fc region, eg an IgG Fc region, and an antigen binding region such as a region that specifically binds to TNFR1 or TNFR2. can

As used herein, "bispecific construct" refers to a multispecific construct that has binding specificities for two different antigens. Bispecific constructs include, for example, monoclonal antibodies or antigen-binding fragments thereof linked to polypeptide regions such as Fc or modified Fc that modify the activity of the construct. In the case of human therapeutics, the construct is derived from, is derived from, or humanized from human sources, and the construct has binding specificities for at least two different antigens. The bispecific constructs/molecules provided herein may have binding specificities directed towards TNFR1 and TNFR2. For example, a bispecific construct includes a TNFR1 antagonist and a TNFR2 agonist. Bispecific antibodies or constructs include antibodies and antigen-binding fragments thereof comprising two separate antigen-binding domains (e.g., two scFvs, or two dAbs, or two Fabs joined by a linker). do. Antigen binding domains may bind the same antigen or different antigens.

As used herein, “antibody fragment” or “antibody portion” refers to the variable region(s) (e.g., one or more complementarity-determining regions (CDRs)) of an antibody that are less than full length but sufficient to form an antigen binding site. ), and thus retains the binding specificity and/or activity of the full length antibody; Antibody fragments include antibody derivatives produced by enzymatic treatment of full length antibodies as well as derivatives produced synthetically, eg recombinantly. Examples of antibody fragments include Fab, Fab', F(ab) ₂ , single chain Fv (scFv), Fv, dsFv, diabodies, triabodies, affibodies, nanobodies, aptamers, dAbs, Fd and Fd fragments, but These are not limited (see, eg, Methods in Molecular Biology , Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; pp. 3-25, Kipriyanov). Fragments may include multiple chains linked together, such as by disulfide bridges and/or peptide linkers. Antibody fragments generally contain at least about 50 amino acids, such as at least about or at least 100 amino acids, and typically contain at least about or at least 110, 120, 150, 170, 180 or 200 amino acids.

As used herein, an “Fv antibody fragment” is composed of one variable heavy (V _H ) chain domain and one variable light (V _L ) domain linked by non-covalent interactions.

As used herein, dsFv (disulfide-bonded Fv) refers to an Fv with an engineered intermolecular disulfide bond that stabilizes the V _H -V _L pair.

As used herein, “scFv fragment” refers to an antibody fragment containing a variable light (V _L ) chain and a variable heavy (V _H ) chain covalently linked by a polypeptide linker in any order. A linker is the length by which the two variable domains are bridged without substantial interference. An exemplary linker is a (Gly-Ser) _n residue with some Glu or Lys residues dispersed throughout to increase solubility.

As used herein, a “diabody” is a dimeric scFv; Diabodies typically have shorter peptide linkers than scFvs and preferentially dimerize.

As used herein, a “triabody” is a trimeric scFv; It contains three peptide chains, each of which has one V _H linked by a short linker (e.g., a linker consisting of 1 to 2 amino acids). domain and one V _L domain to allow intramolecular association of the V _H and V _L domains within the same peptide chain; Triabodies are typically trimerized.

As used herein, "Fab fragment" refers to an antibody fragment produced by digesting a full-length immunoglobulin with papain, or, for example, A fragment having the same structure that is produced synthetically by recombinant methods. Fab fragments contain a light chain (containing V _L and C _L ) and another chain containing the variable domain of the heavy chain (V _H ) and one constant region domain of the heavy chain (C _H 1 ).

As used herein, "F(ab') ₂ fragment" refers to an antibody fragment produced by pepsin digestion of an immunoglobulin at pH 4.0 to 4.5, or, for example, A fragment having the same structure that is produced synthetically by recombinant methods. An F(ab') ₂ fragment essentially contains two Fab fragments, wherein each heavy chain portion has flexibility in the hinge region, which includes, for example, a cysteine residue that forms a disulfide bond connecting the two fragments. contains several additional amino acids, such as all or some of which are sufficient to provide

As used herein, a Fab' fragment is a fragment that contains half of an F(ab') ₂ fragment (ie, one heavy chain and one light chain).

As used herein, an Fd fragment is a fragment of an antibody that contains the variable domain (V _H ) and one constant region domain (C _H 1 ) of an antibody heavy chain.

As used herein, an Fd' fragment is a fragment of an antibody that contains one heavy chain portion of an F(ab') ₂ fragment.

As used herein, an Fv' fragment is a fragment that contains only the V _H and V _L domains of an antibody molecule.

As used herein, hsFv (helix-stabilized Fv) refers to an antibody fragment in which the constant domains normally present in Fab fragments have been replaced by heterodimeric coiled-coil domains (see, e.g., Arndt et al. (2001) J. Mol . Biol . 7 :312:221-228).

As used herein, "domain antibody", "single domain antibody", "sdAb" or "dAb", used interchangeably, refers to the variable domain of the heavy (V _H ) or light (V _L ) chain of an antibody. refers to small monomeric antibody fragments that contain A dAb is the smallest antigen-binding fragment of an antibody; It is about 11 to 15 kDa (about 100 to 150 amino acids) in size, which is about 1/10 the size of all monoclonal antibodies (mAbs). Each V _H and each V _L have three complementarity determining regions (CDRs). Each dAb contains three of the six CDRs, highly variable loop regions that bind the target antigen from the V _H -V _L pair of antibodies.

As used herein, camelid antibodies, also referred to as nanobodies or VHHs, lack a light chain and are composed of two identical heavy chains. It occurs naturally in camelids, such as camels and alpacas.

As used herein, a polypeptide "domain" is a structurally and/or functionally distinguishable or definable portion of a polypeptide (a sequence of 3 or more, usually 5, 10 or more amino acids). Exemplary polypeptide domains can form independently folded structures within a polypeptide composed of one or more structural motifs (eg, a combination of alpha helices and/or beta strands connected by loop regions) and/or possess specific functional activities, such as It is the part of a polypeptide that is recognized by enzymatic activity, dimerization or antigen binding. A polypeptide can have one or more, typically more than one distinct domain. For example, a polypeptide can have one or more structural domains and one or more functional domains. Single polypeptide domains can be distinguished based on structure and function. A domain can include a contiguous linear sequence of amino acids. Alternatively, a domain may comprise a plurality of non-contiguous amino acid portions that are non-contiguous along a linear sequence of amino acids of a polypeptide. Typically, a polypeptide contains multiple domains. For example, each heavy chain and each light chain of an antibody molecule contains multiple immunoglobulin (Ig) domains, each about 110 amino acids in length. One skilled in the art is familiar with polypeptide domains and can identify them due to structural and/or functional homology with other such domains. While definitions are provided herein for purposes of illustration, it is understood that recognizing a particular domain by name is within the skill of the art. If necessary, the domain can be verified using appropriate software.

As used herein, a “functional region” of a polypeptide refers to at least one functional domain (e.g., through antigen binding, DNA binding, ligand binding or dimerization, or through enzymatic activity, e.g., kinase activity or a region of a polypeptide that contains certain functions, such as the ability to interact with biomolecules by means of proteolytic activity; Exemplary functional regions of a polypeptide are antibody domains such as V _H , V _L , C _H , C _L and portions thereof such as CDRs including CDR1 , CDR2 and CDR3, or antigen binding portions such as antibody binding sites.

As used herein, a "structural region" of a polypeptide is a region of a polypeptide that contains at least one structural domain.

As used herein, an "Ig domain" is distinguished by a structure called an immunoglobulin (Ig) fold, which contains two beta-pleated sheets, each sheet containing an antiparallel beta chain of looped amino acids. It is a domain recognized per se by those skilled in the art as containing strands. The two beta sheets in the Ig fold are sandwiched together by hydrophobic interactions and conserved intrachain disulfide bonds. Individual immunoglobulin domains within an antibody chain may further be distinguished based on function. For example, a light chain contains one variable region domain (V _L ) and one constant region domain ( _CL ), whereas a heavy chain contains one variable region domain (V _H ) and three or four constant region domains. (C _H ). Each of the V _L , C _L , V _H and C _H domains is an example of an immunoglobulin domain.

As used herein, a “variable domain” in the context of an antibody is a specific immunoglobulin (Ig) domain of an antibody heavy or light chain that contains a sequence of amino acids that varies between different antibodies. Each light chain and each heavy chain has one variable region domain (V _L and V _H , respectively). The variable domain provides antigen specificity and is therefore responsible for antigen recognition. Each variable region contains a complementarity-determining region (CDR) that is part of an antigen binding site domain and a framework region (FR).

As used herein, “hypervariable regions”, “HVs”, “complementarity-determining regions”, “CDRs” and “antibody CDRs” refer to a plurality of regions within each variable region that together form the antigen binding site of an antibody. Used interchangeably to refer to one of the parts. Each variable region domain contains three CDRs designated CDR1, CDR2 and CDR3. The three CDRs are non-adjacent along the linear amino acid sequence, but contiguous in the folded polypeptide. The CDRs are located within loops that join the parallel strands of the beta sheets of the variable domains.

As used herein, "antigen-binding domain", "antigen-binding site", "antigen-binding fragment", "antigen combining site" and "antibody-binding site" are cognate Used synonymously to refer to the domain within an antibody that recognizes and physically interacts with an antigen. An intact, conventional full-length antibody molecule has two conventional antigen binding sites, each containing a portion of a heavy chain variable region and a portion of a light chain variable region. A typical antigen binding site contains loops connecting anti-parallel beta strands within the variable region domain. The antigen binding site may contain other portions of the variable region domains. Each conventional antigen binding site contains three hypervariable regions from the heavy chain and three hypervariable regions from the light chain. Hypervariable regions are also referred to as complementarity-determining regions (CDRs).

As used herein, "part thereof" in reference to an antibody heavy or light chain, or variable heavy or light chain, when assembled into an antibody containing heavy and light chains, is the equivalent of a full-length antibody containing all six CDRs. The antigen is such that it contains at least one or two, typically three, four, five or all six CDRs of the variable heavy (V _H ) and variable light (V _L ) chains sufficient to retain at least some of the binding specificity. refers to an adjacent portion thereof sufficient to form a binding site. Generally, sufficient antigen binding sites require heavy chain CDR3 (CDRH3). This typically requires an additional CDR3 of the light chain (CDRL3). As described herein, one skilled in the art can know and identify CDRs based on Kabat or Chothia numbering (see, e.g., Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest , Fifth Edition, US Department of Health and Human Services, NIH Publication No 91-3242; and Chothia, C. et al. (1987) J. Mol. Biol . 196:901-917).

As used herein, “framework regions” or “FR” are domains within antibody variable region domains located within the beta sheet; FR regions are relatively more conserved than hypervariable regions in terms of their amino acid sequence. Each variable region contains 4 framework regions separating 3 hypervariable regions.

As used herein, a “constant region” domain is a domain within an antibody heavy or light chain that contains a sequence of amino acids that is relatively more conserved among antibodies than variable region domains. Each light chain has a single light-chain constant region (C _L ) domain, and _each heavy chain contains one or more heavy-chain constant region (CH ) domains, including C _H 1 , C _H 2 , C _H 3 and C _H 4 do. Full-length IgA, IgD and IgG isotypes have C _H 1, C _H 2 and C _H 3 domains and hinge regions, whereas IgE and IgM contain the CH ₁ , _CH 2, _CH 3 and _CH 4 domains. The C _H 1 and C _L domains extend the Fab arm of the antibody molecule and contribute to interaction with antigen and rotation of the antibody arm. Antibody constant regions may provide, but are not limited to, effector functions to which the antibody specifically binds, such as clearance of antigens, pathogens, and toxins, for example, through interactions with various cells, biomolecules, and tissues. don't

As used herein, an “antibody hinge region” or “hinge region” occurs between the C _H 1 and C _H 2 domains, connects the Fab and Fc regions, and has no homology to other antibody domains: gamma, delta and the polypeptide region in the heavy chain of the alpha antibody isotype. This region is rich in proline residues, provides flexibility for IgG, IgD and IgA antibodies, and allows the two "arms" of the Fab portion (each containing one antibody binding site) to be resistant to each other when binding antigen. It assumes various angles so that it can be moved. This flexibility allows the Fab arms to move to align the antibody binding site to interact with an epitope on the cell surface or other antigen. A disulfide bond between the two chains in the hinge region stabilizes the interaction between the two heavy chains. In some embodiments provided herein, synthetically produced antibody fragments contain one or more hinge regions to facilitate stability, eg through interactions between two antibody chains. The hinge region is an exemplary part of a dimerization domain and, for purposes of this specification, is part of a linker.

As used herein, "fragment crystallizable region" or "Fc" or "Fc region" or "Fc domain" refers to a polypeptide containing the constant region of an antibody heavy chain, excluding the first constant region immunoglobulin domain. . Fc is the last two constant region immunoglobulin domains of IgA, IgD and IgG (also known as _CH2 and _CH3 , Cγ2 and Cγ3) or the last three constant region immunoglobulin domains of IgE and IgM ( _CH2 , CH3). _H 3 and C _H 4). Optionally, the Fc domains can include all or part of the flexible hinge region that is N-terminal to these domains. For IgA and IgM, Fc may include a J chain. In the case of an exemplary Fc domain of an IgG, the Fc contains all or part of the immunoglobulin domains C _H 2 and C _H 3 and optionally the hinge between C _H 1 and C _H 2 (also referred to as Cγ1 and Cγ2). The boundaries of the Fc region can vary, but typically include at least a portion of the hinge region. For purposes herein, Fc is also any allelic or species variant, or any variant or modified form, such as any variant of Fc that alters binding to an Fc receptor (FcR) or alters an Fc-mediated effector function. or a modified form. Mutations in the Fc region and their effects are well documented in the art.

As used herein, “Fc chimera” refers to chimeric polypeptides in which one or more polypeptides are directly or indirectly linked to an Fc region or derivative thereof. Typically, an Fc chimera combines the Fc region of an immunoglobulin with another polypeptide. Derivatives of Fc polypeptides or modified Fc polypeptides are known to those skilled in the art.

As used herein, “Kabat numbering” refers to the index numbering of IgG1 Kabat antibodies (see, e.g., Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest , Fifth Edition, US Department of Health and Human Services, NIH Publication No 91-3242); This allows easy comparison between antibodies similar to the way chymotrypsin numbering allows comparison between proteases. One skilled in the art can use Kabat numbering to identify regions of the constant region.

As used herein, “EU numbering” or “EU indicator” refers to Edelman et al. (1969) Proc . Natl . Acad . Sci . USA 63:78-85]. "EU indicator as in Kabat" is human IgG1 Kabat as set forth in Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest , Fifth Edition, US Department of Health and Human Services, NIH Publication No 91-3242 Refers to the EU indicator numbering of antibodies. EU numbering, or EU numbering as in Kabat, is frequently used by those skilled in the art to number amino acid residues in the Fc regions of antibody light and heavy chains. For example, one skilled in the art can use EU numbering to identify regions of constant regions. For example, the _CL domain of an Ig kappa light chain corresponds to residues R108-C214 according to Kabat and EU numbering (see eg Table 2 below). The C _H 1 domain of IgG1 corresponds to residues 118-215 (EU numbering) or 114-223 (Kabat numbering); C _H 2 corresponds to residues 231-340 (EU numbering) or 244-360 (Kabat numbering); C _H 3 corresponds to residues 341-447 (EU numbering) or 361-478 (Kabat numbering).

The following table defines the numbering for the IgG1 and IgG4 heavy chain constant domains, and the Ig kappa light chain constant domains by EU, Kabat and sequential numbering. Table 1 shows the IgG1 heavy chain constant domains by EU, Kabat and sequential numbering, wherein the sequential numbering relates to the sequence of amino acids set forth in SEQ ID NO: 9, within the C _H 1 , C _H 2 and C _H 3 domains. Residues as well as hinge regions are identified. Table 2 shows the immunoglobulin (Ig) kappa light chain constant domains by EU, Kabat and sequential numbering, wherein sequential numbering relates to the sequence of amino acids set forth in SEQ ID NO: 17. In Table 2, the top row ( bold ) presents amino acid residue numbers in sequential numbering (see SEQ ID NO: 17); the second row ( bold ) gives the one-letter code for the amino acid residue at the numbered position in the top row; the third row ( italics ) indicates the corresponding Kabat number according to Kabat numbering; and the fourth row shows the corresponding EU indicator number according to EU numbering. Table 3 shows the IgG4 heavy chain constant domains by EU, Kabat and sequential numbering, where the sequential numbering relates to the sequence of amino acids set forth in SEQ ID NO: 15, within the C _H 1 , C _H 2 and C _H 3 domains. Residues as well as hinge regions are identified.

As used herein, when referring to an antibody fragment derived from another antibody, such as a monoclonal antibody, the phrase “derived from” refers to an antibody fragment that retains the binding specificity of the original antibody (e.g., Fab , F(ab'), F(ab') ₂ , single-chain Fv (scFv), Fv, dsFv, dAb, diabodies, Fd and Fd' fragments). Such fragments may be derived by a variety of methods known in the art including, but not limited to, enzymatic cleavage, chemical cross-linking, recombinant means, or combinations thereof. Generally, antibody fragments derived such that the antibody fragment and the parent antibody bind to the same epitope share the same or substantially identical heavy chain (V _H ) and light chain variable regions (V _L ) of the parent antibody.

As used herein, "parent antibody" or "source antibody" refers to an antibody fragment (eg, Fab, F(ab'), F(ab) ₂ , single chain Fv (scFv), Fv, dsFv, dAb , diabodies, Fd and Fd' fragments) are derived.

As used herein, the term "epitope" refers to any antigenic determinant on an antigen or protein to which a paratope of an antibody can bind. Epitopic determinants typically include chemically active surface groupings of molecules such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics as well as specific charge characteristics.

As used herein, "humanized antibody" and human therapeutics refer to antibodies and other protein therapeutics that have been modified to include a "human" sequence of amino acids such that administration to humans does not elicit an immune response. For example, humanized antibodies typically contain complementarity determining regions (CDRs or hypervariable loops) derived from non-human species immunoglobulins and the remainder of the antibody molecule derived primarily from human immunoglobulins. Methods for humanizing proteins, including antibodies, and producing them are well known and readily available to those skilled in the art. For example, DNA encoding a monoclonal antibody can be altered by recombinant DNA techniques to encode an antibody in which the amino acid composition of the non-variable region is based on a human antibody. Methods for identifying variable and non-variable regions of immunoglobulins are known, including computer programs designed to identify such regions. Thus, generally a humanized antibody will contain substantially all of at least one, and typically two, variable domains, wherein the hypervariable loops (e.g., All or substantially all of the CDRs correspond to hypervariable loops of a non-human immunoglobulin, and all or substantially all of the framework regions (FRs) are framework regions of human immunoglobulin sequences. The humanized antibody also optionally contains at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

As used herein, a "multimerization domain" promotes stable interactions of a polypeptide molecule with one or more additional polypeptide molecules, each containing a complementary multimerization domain, which is a stable multimerization domain with the first domain. Refers to a sequence of amino acids that can be the same or different multimerization domains to form a body. Typically the polypeptide is directly or indirectly linked to the multimerization domain. Exemplary multimerization domains include immunoglobulin sequences or portions thereof, leucine zippers, hydrophobic regions, hydrophilic regions and compatible protein-protein interaction domains. For example, the multimerization domain may be an immunoglobulin, such as an Fc domain or portion thereof from IgG, IgA, IgE, IgD and IgM, including, for example, the IgG1, IgG2, IgG3 or IgG4 subtypes, and modified forms thereof. It can be a constant region or domain.

As used herein, a "dimerization domain" is a multimerization domain that facilitates interaction between two polypeptide sequences (eg, but not limited to, antibody chains). A dimerization domain is one or more dimerization sequences that are sequences of amino acids known to promote interactions between two polypeptide sequences, such as all or part of a full-length antibody hinge region, or polypeptides (eg, leucine zippers, GCN4 zippers) amino acid sequences containing cysteine residues that facilitate the formation of disulfide bonds between the liver.

As used herein, “chimeric polypeptide” refers to a polypeptide that contains portions from at least two different polypeptides or two non-contiguous portions of a single polypeptide. Thus, a chimeric polypeptide generally comprises a sequence of amino acid residues from all or part of one polypeptide and a sequence of amino acids from all or part of another, different polypeptide. The two moieties may be linked directly or indirectly, peptide bonds, other covalent bonds, or other ratios of sufficient strength to maintain the integrity of a substantial portion of the chimeric polypeptide under physiological conditions and equilibrium conditions such as isotonic pH 7 buffered saline. - Can be connected through shared interactions.

As used herein, a "fusion protein" refers to a fusion protein from a vector containing two nucleic acids encoding two polypeptides together by expressing the fusion protein in close proximity to each other, e.g., adjacent to each other along the length of the vector. A polypeptide engineered to contain a sequence of amino acids corresponding to two separate polypeptides joined. Thus, a fusion protein refers to a chimeric protein containing parts from two, or two or more proteins or peptides linked directly or indirectly through peptide bonds. The two molecules may be adjacent in the construct or may be separated by a linker or spacer polypeptide.

As used herein, "linker", "linker unit" or "link" is a chain of atoms that covalently attaches an antibody or antigen-binding fragment thereof to another therapeutic moiety or another antibody or fragment thereof. Refers to a peptide or chemical moiety containing For example, linkers are included to increase flexibility, alter steric effects including steric hindrance, and increase solubility in aqueous media.

As used herein, a "linker peptide" or "spacer peptide" refers to a short sequence of amino acids that joins two polypeptide sequences (or encoding nucleic acids, such as amino acid sequences). A "peptide linker" refers to a short sequence of amino acids that joins two polypeptide sequences. Examples of polypeptide linkers are linkers that join a peptide transduction domain to an antibody, or linkers that join two antibody chains in a synthetic antibody fragment, such as a scFv fragment. Linkers are well known, and any known linker can be used in the provided methods. Exemplary polypeptide linkers include the (Gly-Ser) _n amino acid sequence, with some Glu or Lys residues dispersed throughout to increase solubility. Other exemplary linkers are described herein; Any of these and other known linkers can be used with the polypeptides, antibodies, and other products and methods provided herein.

As used herein, a “tag” or “epitope tag” refers to a sequence of amino acids typically added to the N- or C-terminus of a polypeptide provided herein, such as antibodies and antibody fragments/constructs. Inclusion of a tag fused to the polypeptide can facilitate purification and/or detection of the polypeptide. Typically, a tag or tag polypeptide refers to a polypeptide having sufficient residues to provide an epitope recognized by an antibody, or short enough to provide for detection or purification, but not interfering with the activity of the linked polypeptide. The tag polypeptide is typically sufficiently unique that an antibody specifically binding thereto does not substantially cross-react with the epitope of the linked polypeptide. Suitable tag polypeptides generally have at least 5 or 6 amino acid residues, usually about 8 to 50 amino acid residues, typically 9 to 30 residues. A tag may be linked to one or more chimeric polypeptides in a multimer and allow for detection of the multimer, or recovery thereof from a sample or mixture. Such tags are well known and can be easily synthesized and designed. Exemplary tag polypeptides include those used for affinity purification, such as the FLAG tag; his tag; Influenza hemagglutinin (HA) tag polypeptide and its antibody 12CA5 (see, eg, Field et al. (1988) Mol . Cell. Biol. 8 :2159-2165); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (see, eg, Evan et al. (1985) Molecular and Cellular Biology 5 :3610-3616); and the herpes simplex virus glycoprotein D (gD) tag and antibodies thereof (see, eg, Paborsky et al. (1990) Protein Engineering 3 :547-553). Antibodies used to detect epitope-tagged antibodies are typically referred to herein as secondary antibodies.

As used herein, a “label” or “detectable moiety” refers to a molecule (e.g., an antibody or antigen-binding fragment thereof provided herein, such as an anti-TNFR1 antibody or antigen-binding fragment thereof), which may be directly or indirectly attached to or linked to, or which may be associated with a molecule, and which may be detected in vivo and/or in vitro (e.g., a fluorescent molecule, a chemical luminescent molecules, bioluminescent molecules, contrast agents (eg, metals), radionuclides, chromophores, detectable peptides, or enzymes that catalyze the formation of detectable products). The detection method may be a method of known in vivo and/or in vitro detection (e.g., visual inspection, magnetic resonance (MR) spectroscopy, ultrasound signal, X-ray, gamma ray spectroscopy (e.g., positron emission tomography (PET)) scanning, single-photon emission computed tomography (SPECT)), fluorescence spectroscopy or imaging by absorption) may be any method known in the art. Indirect detection refers to a physical phenomenon that directly or indirectly binds to a detectable moiety, such as the measurement of energy or particle emission or absorption of an atom, molecule, or composition (e.g., a labeled secondary antibody that binds to a primary antibody or its antigen). detection of a binding fragment (eg, an anti-TNFR antibody or antigen-binding fragment thereof provided herein).

As used herein, "nucleic acid" refers to at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), typically linked together by phosphodiester linkages. . Also included within the term "nucleic acid" are analogs of nucleic acids, such as peptide nucleic acids (PNAs), phosphorothioate DNA, and other such analogs and derivatives or combinations thereof. Nucleic acids may also include nucleotide analogs other than, for example, phosphodiester linkages, e.g., phosphotriester linkages, phosphoramidate linkages, phosphorothioate linkages, thioester linkages, or peptide linkages (i.e., peptide nucleic acids) or “peptide nucleic acids”. Includes DNA and RNA derivatives containing "backbone" linkages. The term also includes nucleotide analogues, equivalents, derivatives, variants and analogues of RNA or DNA made from single (sense or antisense) and double-stranded nucleic acids. Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine and deoxythymidine. For RNA, the uracil base is uridine.

As used herein, an "isolated nucleic acid molecule" is one that has been separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. An "isolated" nucleic acid molecule, such as a cDNA molecule, may be substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding the provided antibodies or antigen-binding fragments.

As used herein, “operably linked” with respect to nucleic acid sequences, regions, elements or domains means that the nucleic acid regions are in functional relationship with each other. For example, a nucleic acid encoding a leader peptide can be operably linked to a nucleic acid encoding a polypeptide, whereby the nucleic acid can be transcribed and translated to express a functional fusion protein, wherein the leader peptide is responsible for secretion of the fusion polypeptide. It affects. In some cases, a nucleic acid encoding a first polypeptide (e.g., a leader peptide) is operably linked to a nucleic acid encoding a second polypeptide, and the nucleic acid is transcribed as a single mRNA transcript, but translation of the mRNA transcript is It can result in either of the two polypeptides being expressed. For example, an amber stop codon can be placed between a nucleic acid encoding a first polypeptide and a nucleic acid encoding a second polypeptide so that when introduced into a partial amber suppressor cell, the resulting single mRNA transcript is translated to produce the first and a second polypeptide, or can be translated to produce only the first polypeptide. In another example, a promoter can be operably linked to a nucleic acid encoding a polypeptide, whereby the promoter regulates or mediates transcription of the nucleic acid.

As used herein, “synthetic”, e.g., in reference to a synthetic nucleic acid molecule or synthetic gene or synthetic peptide, refers to a nucleic acid molecule or gene or polypeptide molecule produced by recombinant and/or chemical synthetic methods.

As used herein, residues of naturally occurring α-amino acids are those of the 20 α-amino acids found in nature that are incorporated into proteins by specific recognition of charged tRNA molecules with their cognate mRNA codons in humans. .

As used herein, "polypeptide" refers to two or more amino acids covalently linked. The terms "polypeptide" and "protein" are used interchangeably herein.

As used herein, “peptide” refers to a polypeptide that is between 2 and about 40 amino acids in length.

As used herein, “amino acid” is an organic compound containing an amino group and a carboxylic acid group. Polypeptides contain two or more amino acids. For purposes herein, amino acids in polypeptides such as antibodies provided include the 20 naturally occurring amino acids (Table 4), non-natural amino acids and amino acid analogs (eg, amino acids having a side chain at the α-carbon). As used herein, amino acids occurring in various amino acid sequences of polypeptides appearing herein are identified according to their well-known three-letter or one-letter abbreviations (see Table 4). Nucleotides occurring in various nucleic acid molecules and fragments are designated by standard single-letter designations routinely used in the art.

As used herein, “amino acid residue” refers to an amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages. Amino acid residues described herein are generally in the "L" isomeric form. Residues in the "D" isomeric form may be substituted with any L-amino acid residue as long as the desired functional properties are retained by the polypeptide. NH ₂ refers to a free amino group present at the amino terminus of a polypeptide. COOH refers to a free carboxy group present at the carboxyl terminus of a polypeptide. See J. Biol . Chem ., 243:3557-59 (1968) and adopted in 37 CFR §§ 1.821 - 1.822, abbreviations for amino acid residues are shown in Table 4:

All sequences of amino acid residues presented herein by formulas are oriented from left to right in the conventional orientation of amino-terminus to carboxyl-terminus. The phrase "amino acid residue" is also defined to include the amino acids listed in the correspondence table (Table 4), modified non-natural and uncommon amino acids. Moreover, a dash at the beginning or end of an amino acid residue sequence indicates an additional sequence of one or more amino acid residues, or a peptide bond to an amino-terminal group such as NH ₂ , or a carboxyl-terminal group such as COOH. Suitable conservative substitutions of amino acids in a peptide or protein are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. One skilled in the art generally recognizes that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, for example, Watson et al., Molecular Biology of the Gene , 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224).

Such substitutions can be made according to the exemplary substitutions set forth in Table 5 as follows:

Other substitutions are also permissible and may be determined empirically or according to other known conservative or non-conservative substitutions.

As used herein, “naturally occurring amino acids” refers to the 20 L-amino acids that occur in polypeptides.

As used herein, the term “non-natural amino acid” refers to an organic compound that has a structure similar to that of a natural amino acid but has been structurally modified to mimic the structure and reactivity of a natural amino acid. Non-naturally occurring amino acids thus include, for example, amino acids or analogs of amino acids other than the 20 naturally occurring amino acids, and include, but are not limited to, D-stereoisomers of amino acids. Exemplary non-natural amino acids are known to those skilled in the art and include 2-aminoadipic acid (Aad), 3-aminoadipic acid (bAad), β-alanine/β-amino-propionic acid (Bala), 2-aminobutyric acid ( Abu), 4-aminobutyric acid/piperidic acid (4Abu), 6-aminocaproic acid (Acp), 2-aminoheptanoic acid (Ahe), 2-aminoisobutyric acid (Aib), 3-aminoisobutyric acid (Baib) , 2-aminopimelic acid (Apm), 2,4-diaminobutyric acid (Dbu), desmosine (Des), 2,2'-diaminopimelic acid (Dpm), 2,3-diaminopropionic acid (Dpr) , N-ethylglycine (EtGly), N-ethylasparagine (EtAsn), hydroxylysine (Hyl), allo-hydroxylysine (Ahyl), 3-hydroxyproline (3Hyp), 4-hydroxyproline (4Hyp) , Isodesmosine (Ide), allo-isoleucine (Aile), N-methylglycine, sarcosine (MeGly), N-methylisoleucine (MeIle), 6-N-methyllysine (MeLys), N-methylvaline (MeVal) ), norvaline (Nva), norleucine (Nle), and ornithine (Orn).

As used herein, a “DNA construct” is a single- or double-stranded linear or circular DNA molecule that contains segments of DNA combined and juxtaposed in a manner not found in nature. DNA constructs exist as a result of human manipulation, and include clones and other copies of engineered molecules.

As used herein, a "DNA segment" is a portion of a longer DNA molecule having specified properties. For example, a DNA segment encoding a specified polypeptide is part of a longer DNA molecule, such as a plasmid or plasmid fragment, that encodes the amino acid sequence of a specified polypeptide when read in 5' to 3' orientation.

As used herein, the term "polynucleotide" refers to a single- or double-stranded polymer of deoxyribonucleotides or ribonucleotide bases read from the 5' to the 3' end. Polynucleotides include RNA and DNA, and can be isolated from natural sources, synthesized in vitro , or prepared from a combination of natural and synthetic molecules. The length of a polynucleotide molecule is given herein in terms of nucleotides (abbreviated “nt”) or base pairs (abbreviated “bp”). The term nucleotide is used for single- and double-stranded molecules where the context permits. When the term is applied to double-stranded molecules it is used to denote total length and is understood to be equivalent to the term base pair. One skilled in the art will recognize that the two strands of a double-stranded polynucleotide may be slightly different in length and their ends may be staggered; Thus, all nucleotides in a double-stranded polynucleotide molecule are unpairable. Such unpaired ends will generally not exceed 20 nucleotides in length.

As used herein, production by recombinant means using recombinant DNA methods refers to the use of well known molecular biology methods to express proteins encoded by cloned DNA.

As used herein, "expression" refers to the process by which a polypeptide is produced by transcription and translation of a polynucleotide. The level of expression of a polypeptide can be assessed using any method known in the art, including, for example, determining the amount of polypeptide produced from a host cell. Such methods may include quantification of polypeptides in cell lysates by ELISA, Coomassie blue staining after gel electrophoresis, Lowry protein assay and Bradford protein assay. but not limited to these

As used herein, a “host cell” is a cell used to receive, maintain, reproduce, and/or amplify a vector. A host cell can also be used to express the polypeptide encoded by the vector. The nucleic acid in the vector is replicated when the host cell divides to amplify the nucleic acid.

As used herein, a "vector" is a replicable nucleic acid capable of expressing one or more heterologous proteins when the vector is transformed into an appropriate host cell. Reference to a vector includes vectors into which a nucleic acid encoding a polypeptide or fragment thereof may be introduced, typically by restriction digestion and ligation. Reference to a vector also includes vectors containing nucleic acids encoding polypeptides such as modified anti-TNFR1 antibodies. Vectors are used to introduce a nucleic acid encoding a polypeptide into a host cell for amplification of the nucleic acid or for expression/display of the polypeptide encoded by the nucleic acid. Vectors typically remain episomal, but can be designed to effect integration of a gene or portion thereof into a chromosome of the genome. Also contemplated are vectors that are artificial chromosomes, such as yeast artificial chromosomes and mammalian artificial chromosomes. The selection and use of such vehicles is well known to those skilled in the art. A vector also includes a "virus vector" or "viral vector". A viral vector is an engineered virus that is operatively linked to an exogenous gene to transfer (as a vehicle or shuttle) the exogenous gene into a cell.

As used herein, "expression vector" includes vectors capable of expressing DNA in operative link with regulatory sequences capable of affecting the expression of such DNA fragments, such as promoter regions. Such additional segments may include promoter and terminator sequences, and optionally may include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct such as a plasmid, phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in the expression of the cloned DNA. Suitable expression vectors are well known to those skilled in the art, and include those replicable in eukaryotic and/or prokaryotic cells, those that remain episomal, or those that integrate into the host cell genome.

As used herein, “primary sequence” refers to the sequence of amino acid residues of a polypeptide or nucleotide sequence of a nucleic acid molecule.

As used herein, "sequence identity" refers to the number of identical or similar amino acid or nucleotide bases in a comparison between a test and a reference polypeptide or polynucleotide. Sequence identity can be determined by sequence alignment of nucleic acid or protein sequences to identify regions of similarity or identity. For purposes herein, sequence identity is generally determined by alignment to identify identical residues. Alignment can be local or global. Matches, mismatches and gaps can be identified between the compared sequences. A gap is a null amino acid or nucleotide inserted between residues of an aligned sequence such that identical or similar features align. In general, there may be internal and terminal gaps. When using gap penalties, without penalty for end gaps (e.g. end gaps are not penalized) sequence identity can be determined. Alternatively, sequence identity can be determined as the number of identical positions/length Х 100 of the total aligned sequences without accounting for gaps.

As used herein, a “global alignment” is an alignment that aligns two sequences from start to end, aligning each letter of each sequence only once. An alignment is produced regardless of whether there is similarity or identity between the sequences. For example, 50% sequence identity based on "global alignment" means that 50% of the residues, each 100 nucleotides in length, are identical in the full sequence alignment of the two compared sequences. It is understood that global alignments may be used to determine sequence identity even when the lengths of the aligned sequences are not the same. Differences at the ends of sequences are taken into account when determining sequence identity, unless "no penalty for end gaps" is selected. Global alignments are generally used on sequences that share significant similarity over most of their length. Exemplary algorithms for performing global alignment include the Needleman-Wunsch algorithm (Needleman et al. (1970) J. Mol . Biol . 48:443). Exemplary programs for performing global alignments include the Global Sequence Alignment Tool, which is publicly available and available on the National Center for Biotechnology Information (NCBI) website (ncbi.nlm.nih.gov/). Includes programs available at deepc2.psi.iastate.edu/aat/align/align.html.

As used herein, a "local alignment" is an alignment that aligns two sequences, but only portions of the sequences that share similarity or identity. Thus, local alignment determines whether a sub-segment of one sequence is present in another sequence. If there is no similarity, no sort will be returned. Local alignment algorithms include BLAST or the Smith-Waterman algorithm ( Adv . Appl . Math . 2:482 (1981)). For example, 50% sequence identity based on a "local alignment" means that a region of similarity or identity of 100 nucleotides in length in an alignment of the entire sequence of two compared sequences of any length is the same residue in the region of similarity or identity. means that it has 50% of

For purposes herein, sequence identity may be determined by standard alignment algorithm programs used with default gap penalties established by each supplier. Default parameters for the GAP program may include: (1) Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure , National Biomedical Research Foundation, pp. 353-358 (1979), a unary comparison matrix (including values of 1 for identity and 0 for non-identity) and Gribskov et al. Nucl . Acids Res . 14:6745 (1986); the weighted comparison matrix; (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each sign of each gap; and (3) no penalty for end gaps. Any two nucleic acid molecules have a nucleotide sequence, or any two polypeptides are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% “identical” amino acid sequences, or Whether or not it has other similar variances that cite percent identity can be determined using known computer algorithms based on local or global alignments (e.g., providing links to dozens of known and publicly available alignment databases and programs). See wikipedia.org/wiki/Sequence_alignment_software). In general, for purposes herein, sequence identity is determined by a computer algorithm based on global alignment, such as the Needleman- Wunsch Global Sequence Alignment Tool; LAlign (William Pearson implementing the Huang and Miller algorithm ( Adv . Appl . Math . (1991) 12:337-357)); and Xiaoqui Huang's program available at deepc2.psi.iastate.edu/aat/align/align.html. Typically, the full length sequences of each compared polypeptide or nucleotide are aligned over the full length of each sequence in a global alignment. Local alignments can also be used when the sequences being compared are of substantially the same length.

As used herein, the term "identity" refers to a comparison or alignment between a test and a reference polypeptide or polynucleotide. In one non-limiting example, “at least 90% identical” refers to a percent identity between 90% and 100% relative to a reference polypeptide or polynucleotide. Assuming identity at the level of 90% or greater is for illustrative purposes, less than 10% (i.e., 10 out of 100) in the test polypeptide or polynucleotide when comparing a reference polypeptide or polynucleotide to a test of 100 amino acids or nucleotides in length. ) differs from that of the reference polypeptide or polynucleotide. A similar comparison can be made between a test polynucleotide and a reference polynucleotide. Such differences can be presented as point mutations randomly distributed over the entire length of the amino acid sequence, or they can be represented at one or more positions of variable length up to the maximum allowed, e.g., 10/100 amino acid differences (approximately 90% identity). can be clustered. Differences may also be due to deletion or truncation of amino acid residues. Differences are defined as nucleic acid or amino acid substitutions, insertions or deletions. Results at levels of greater than about 85-90% homology or identity, depending on the length of the compared sequences, can be independent of the program and gap parameter set; Such high-level identity can often be easily evaluated without reliance on software.

As used herein, a “disulfide bond” (also called a S—S bond or disulfide bridge) is a single covalent bond derived from the coupling of thiol groups. Disulfide bonds in proteins are formed between thiol groups of cysteine residues and stabilize interactions between polypeptide domains, such as antibody domains.

As used herein, “coupled” or “conjugated” means attached through covalent or non-covalent interactions.

As used herein, the phrases “conjugated to an antibody” or “linked to an antibody” or grammatical variations thereof may refer to the attachment of a moiety, such as a diagnostic or therapeutic moiety, to an antibody or antigen-binding fragment thereof. when the moiety is attached to the antibody or antigen-binding fragment thereof by any known means for linking peptides, such as post-translational by chemical means or by production of a fusion protein by recombinant means, for example. do. Conjugation can be effected using any of a variety of binding agents, including but not limited to peptide or compound linkers, or chemical crosslinkers.

As used herein, “antibody-dependent cell-mediated cytotoxicity”, “antibody-dependent cellular cytotoxicity” and “ADCC” refer to non-specific cytotoxic cells that express Fc receptors (FcRs) (e.g., natural killer ( NK) cells, neutrophils, and macrophages) interchangeably refer to a cell-mediated response that recognizes antibodies bound on target cells and subsequently causes lysis of the target cells. NK cells, the primary cells mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is described by Ravetch et al. (1991) Annu . Rev. Immunol , 9:457-492], summarized in Table 3 on page 464. An in vitro ADCC assay can be performed to assess the ADCC activity of a molecule of interest (see, eg, US Pat. Nos. 5,500,362 and 5,821,337). Exemplary effector cells for such an assay include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of a molecule of interest can be determined, for example, by Clynes et al. (1998) Proc . Natl . Acad . Sci . USA 95: 652-656 ].

As used herein, complement dependent cytotoxicity (CDC) is an effector function of IgG and IgM antibodies. When such antibodies bind to surface antigens on target cells, such as bacterial cells or virus-infected cells, the classical complement pathway is triggered by binding the protein C1q to these antibodies, resulting in the formation of membrane attack complexes (MACs) and subsequent cell lysis. do.

As used herein, antibody dependent cellular phagocytosis (ADCP) is a cellular process by which effector cells with phagocytic potential, such as monocytes and macrophages, internalize target cells. Once phagocytosed, target cells reside in phagosomes that fuse with lysosomes for degradation of the target cell through oxygen-dependent or independent mechanisms.

As used herein, "therapeutic activity" refers to the in vivo activity of a therapeutic polypeptide. In general, therapeutic activity is activity associated with treatment of a disease or condition. The therapeutic activity of the modified polypeptide is 1% activity, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50% of the therapeutic activity compared to the unmodified polypeptide. , 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400 %, 500% or more, but is not limited to, any level of the percentage of therapeutic activity of the unmodified polypeptide.

As used herein, the term “assess” refers to obtaining an absolute value for the activity of a protein, such as an antibody or antigen-binding fragment thereof, present in a sample, and also an indicator, ratio, percentage, visual or other indication of the level of activity. It is intended to include quantitative and qualitative determinations in the sense of obtaining a value. Evaluation can be direct or indirect.

As used herein, “disease or disorder” refers to a pathological state of an organism that results from a cause or condition, including but not limited to infections, acquired conditions, and genetic conditions, and is characterized by identifiable symptoms. do.

As used herein, “treating” a subject with a disease or condition means that the subject's symptoms are partially or completely relieved, or remain static, after treatment. Thus, treatment includes prophylaxis, therapy and/or cure. Prevention refers to preventing an underlying disease and/or preventing worsening of symptoms or progression of a disease. Treatment also includes any pharmaceutical use of any antibody or antigen-binding fragment or composition thereof provided herein.

As used herein, treatment refers to amelioration of symptoms or signs of a disease, disorder or condition.

As used herein, “prevention or prophylaxis” refers to a method of reducing the risk of developing a disease or condition. Preventing a disease means reducing the risk of developing the disease.

As used herein, "pharmaceutically effective agent" refers to any anesthetic, vasoconstrictor, dispersing agent, and conventional therapeutic drug including, but not limited to, small molecule drugs and therapeutic proteins, for example. A therapeutic agent or bioactive agent of

As used herein, “therapeutic effect” means an effect resulting from treatment of a subject that alters, typically ameliorates, alleviates the symptoms of a disease or condition, or cures the disease or condition.

As used herein, “therapeutically effective amount” or “therapeutically effective dose” refers to an amount of an agent, compound, material or composition containing at least sufficient compound to produce a therapeutic effect after administration to a subject. Thus, it is the amount necessary to prevent, cure, ameliorate, arrest, or partially arrest the symptoms of a disease or disorder.

As used herein, “therapeutic efficacy” refers to the ability of an agent, compound, material, or composition containing a compound to produce a therapeutic effect in a subject to which the agent, compound, material, or composition containing the compound is administered. do.

As used herein, "prophylactically effective amount" or "prophylactically effective dose" when administered to a subject, for example, prevents or delays the onset or recurrence of a disease or condition, or prevents or delays the onset or recurrence of a disease or condition Refers to the amount of an agent, compound, substance or composition containing the compound that will have the intended prophylactic effect of reducing the likelihood of recurrence or reducing the incidence of viral infection. A full prophylactic effect does not necessarily occur with administration of a single dose, but may only occur after administration of a series of doses. Thus, a prophylactically effective amount can be administered in one or more administrations.

As used herein, the amelioration of the symptoms of a particular disease or disorder by treatment, such as administration of a pharmaceutical composition or other therapeutic agent, refers to the long-term, permanent, temporary, or permanent improvement of symptoms that may be attributed to or associated with administration of the composition or therapeutic agent. refers to any decrease, whether temporary or instantaneous.

As used herein, "prodrug" is a precursor of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and can be enzymatically activated or converted to a more active parent form. or in derivative form (see, e.g., Wilman, 1986, Biochemical Society Transactions, 615th Meeting Belfast, 14:375-382; and Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery," Directed Drug Delivery , Borchardt et al., (ed.), pp. 247-267, Humana Press, 1985).

As used herein, “anti-cancer agent” refers to any agent that is destructive or toxic to malignant cells and tissues. For example, anti-cancer agents include agents that kill cancer cells or otherwise inhibit or impair the growth of tumors or cancer cells. An exemplary anti-cancer agent is a chemotherapeutic agent.

As used herein, an “anti-angiogenic agent” or “angiogenesis inhibitor” is a compound that blocks or interferes with the development of blood vessels.

As used herein, a TNF-related or TNF-mediated disease refers to a disease, condition or disorder in which TNFR1 or TNFR1 signaling plays a role in pathogenesis; Diseases, disorders and conditions in which inhibition of TNFR1 signaling can ameliorate the symptoms of the disease, condition or disorder are included.

As used herein, a “TNFR2 agonist” or “anti-TNFR2 agonist” refers to an agent that initiates, promotes, or increases activation of TNFR2 and/or potentiates one or more signaling pathways mediated by TNFR2. Small molecules and compounds including TNFR2 antibodies or antigen-binding fragments thereof, and other polypeptides. For example, TNFR2 agonists can promote or increase the proliferation of Treg cell populations. A TNFR2 agonist can promote or increase TNFR2 activation, for example by binding to TNFR2 to induce a conformational change that renders the receptor biologically active. For example, a TNFR2 agonist can induce TNFR2-mediated signaling by nucleating trimerization of TNFR2 in a manner that mimics or mimics the interaction between TNFR2 and its cognate ligand, TNF (TNFα). TNFR2 agonists can also induce proliferation of CD4 ⁺ , CD25 ⁺ , FOXP3 ⁺ Treg cells. TNFR2 agonists may also inhibit cytotoxic T lymphocytes (eg, CD8 ⁺ ) by directly binding to TNFR2 on the surface of autoreactive cytotoxic T-cells and inducing apoptosis, eg, through activation of immunoregulatory Treg cells. T-cells) can be inhibited. A TNFR2 agonist antibody or fragment thereof for use in the methods herein is capable of specifically binding to TNFR2, and generally specifically binds to another receptor of the tumor necrosis factor receptor (TNFR) superfamily member, such as TNFR1. It's specific enough not to.

As used herein, a TNFR2 selective agonist is a TNFR2 agonist that does not elicit or substantially elicit no TNFR1 signaling activity.

As used herein, Treg expanders are molecules, including small molecules and polypeptides, that increase regulatory T cells (Treg cells or Tregs), an immunosuppressive subpopulation of T cells that have immunosuppressive properties, through the production of cytokines. .

As used herein, the terms "pan-growth factor trap construct", "pan-EGFR ligand trap construct", "growth factor trap", "multispecific growth factor trap construct", "bispecific "Growth Factor Trap Construct", "EGFR Ligand Trap Construct", "Pan-HER Ligand Trap Construct", "Pan-HER Therapeutic", "EGFR Ligand Trap Construct", "HER Ligand Trap Construct" and "Growth "Factor trap construct" is used interchangeably to refer to a pan-cell surface receptor molecule comprising a peptide-based compound that modulates the activity of two or more human epidermal growth factor receptors (EGFRs), also called HER or ErbB receptors. do. Generally, pan-growth factor traps target at least two different HER receptors, such as through ligand binding and/or interaction with the receptors.

As used herein, an “extracellular domain” or “ECD” is the part of a cell surface receptor that occurs on the surface of the receptor and includes the ligand-binding site(s). For purposes of this specification, reference to an "ECD polypeptide" is a reference unless the ECD polypeptide contains any contiguous sequence associated with another domain (e.g., transmembrane domain, protein kinase domain, or the like) of a cognate receptor. , any ECD-containing molecule or portion thereof.

As used herein, “knob into hole” or “knob in hole” (KIH) refers to a multimerization domain, such as an immunoglobulin Fc domain, and a steric interaction between and/or between such domains. It is engineered to promote this stable interaction and promote the formation of heterodimers (or heteromultimers) compared to homodimers (or homomultimers) from mixtures of monomers. This can be achieved, for example, by constructing knobs or protrusions and holes or cavities in the complementary multimerization domains. A "knob" can be constructed by replacing a small amino acid side chain from the interface of a first multimerization domain polypeptide (eg, a first Fc monomer) with a larger side chain (eg, tyrosine or tryptophan). Compensatory "holes" of the same or similar size as the knob replace large amino acid side chains with smaller ones (e.g., alanine or threonine) to form a second complementary multimerizing polypeptide (e.g., a second Fc monomer). It is selectively created on the interface.

As used herein, "tethering" refers to an interaction between two domains of a receptor monomer, thereby resulting in a form that makes the monomer less available for interaction. For example, subdomain II of HER1, HER3 and HER4 can interact with subdomain IV to form a tethered inactive structure. When in a tethered state, the receptor or isoform thereof is less or less available for dimerization and/or ligand binding. ECDs of the monomeric forms of HER1, HER3 and HER4 are issued in the tethered form showing lower ligand affinity than the untethered form. HER2 lacking certain residues in subdomain IV occurs in an untethered form and is available for dimerization with HER1, HER3 and HER4. Upon ligand binding to the tethered (monomer) conformation, the tethering interaction is disengaged and the ECD (or receptor) becomes available for dimerization involving interactions between domains II of the two ECDs.

As used herein, a HER (ErbB) associated disease, HER associated disease or HER mediated disease is one in which epidermal growth factor receptor (HER) and/or ligands are implicated in some aspect of the pathogenesis, development of its pathology or symptoms thereof. any disease, condition or disorder. Involvement includes, for example, expression, overexpression or activity of a HER family member or ligand. Diseases include but are not limited to cancers such as gliomas and pancreatic, gastric, head and neck, cervical, lung, colorectal, endometrial, prostate, esophageal, ovarian, uterine, bladder, or breast cancers. diseases, including but not limited to. Other conditions include pathological inflammation and/or autoimmune reactions such as rheumatoid arthritis (RA), non-malignant hyperproliferative diseases, ocular conditions, skin conditions (eg psoriasis), smooth muscle cell proliferation and/or migration, conditions associated with cell proliferation and/or migration, including those involving conditions due to strictures including, for example, restenosis, atherosclerosis, muscle thickening of the bladder, heart or other muscles, or endometriosis.

As used herein, the term “subject” refers to animals, including mammals, such as humans.

As used herein, “patient” refers to a human subject.

As used herein, "animal" includes any animal, such as primates, including humans, gorillas, and monkeys; rodents such as mice and rats; poultry, such as chicken; ruminants such as goats, cows, deer and sheep; pig; and other animals, but are not limited thereto. Non-human animals exclude humans as contemplated animals. Polypeptides provided herein are derived from any source of animals, plants, prokaryotes and fungi. Most polypeptides are of animal origin, including mammalian origin, and are usually human or humanized for therapeutic use.

As used herein, “composition” refers to any mixture. It may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous or any combination thereof.

As used herein, “stabilizer” refers to a compound added to a formulation to protect the antibody or conjugate under conditions (eg, temperature), such as when the formulation of the present disclosure is stored or used. Thus, agents that prevent protein degradation from other components of the composition are included. Examples of such agents are amino acids, amino acid derivatives, amines, sugars, polyols, salts and buffers, surfactants, inhibitors or substrates and other agents as described herein.

As used herein, “combination” refers to any association between two or more items or between items. A combination can be two or more separate items, such as two compositions or two collections, mixtures thereof, such as a single mixture of two or more items, or any variation thereof. The elements of the combination are generally functionally associated or related, such as the elements used in the method.

As used herein, “combination therapy” refers to two or more different therapeutic agents and one or more therapeutic agents, such as anti-TNFR constructs or antibodies or antigen-binding fragments thereof provided herein, or other treatments such as radiation and surgery (surgery). ) refers to the administration of Multiple therapeutic agents may be provided and administered separately, sequentially, intermittently, simultaneously or in a single composition.

As used herein, "kit" refers to other elements, such as additional reagents and combinations or instructions for use of the elements, for purposes including, but not limited to, activation, administration, diagnosis, and evaluation of biological activity or properties. It is a packaged combination that optionally includes.

As used herein, “unit dosage form” refers to individually packaged physically discrete units suitable for human and animal subjects, as is known in the art.

As used herein, “single dosage form” refers to a form for direct administration.

As used herein, “multi-dose formulation” refers to a formulation that contains multiple doses of a therapeutic agent and can be administered directly to provide several single doses of a therapeutic agent. A dose can be administered over the course of minutes, hours, weeks, days or months. Multi-dose formulations may allow for dose adjustment, dose-pooling and/or dose-division. Because multi-dose formulations are used over time, they generally contain one or more preservatives to prevent microbial growth.

As used herein, an “article of manufacture” is a product that is manufactured and sold. As used throughout this application, the term is intended to encompass any of the packaging articles or compositions provided herein contained in packaging articles.

As used herein, “fluid” refers to any composition that can flow. Fluids thus include compositions in the form of semi-solids, pastes, solutions, aqueous mixtures, gels, lotions, creams and other such compositions.

As used herein, an isolated or purified polypeptide or protein (e.g., an isolated antibody or antigen-binding fragment thereof), or a biologically active portion thereof (e.g., An isolated antigen-binding fragment) is substantially free of cellular material or other contaminating proteins from the cell or tissue from which the protein was derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The preparation appears to be free of readily detectable impurities as determined by standard methods of analysis used by those skilled in the art to assess such purity, such as thin layer chromatography (TLC), gel electrophoresis and high performance liquid chromatography (HPLC). Alternatively, it can be determined to be substantially free if it is sufficiently pure that further purification does not detectably alter the physical and chemical properties of the material, such as enzymatic and biological activity. Methods for purifying compounds to produce substantially chemically pure compounds are known to those skilled in the art. However, substantially chemically pure compounds may be mixtures of stereoisomers. In such cases, further purification may increase the specific activity of the compound.

As used herein, "cell extract" or "lysate" refers to a preparation or fraction prepared from lysed or disrupted cells.

As used herein, "control" refers to a sample that is substantially identical to the test sample, except that it has not been treated with a test parameter, or, in the case of a plasma sample, taken from a normal volunteer unaffected by the condition of interest. can A control can also be an internal control.

As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a polypeptide containing an “immunoglobulin domain” includes polypeptides having one or a plurality of immunoglobulin domains.

As used herein, the term “or” is used to mean “and/or” unless explicitly indicated to refer only to alternatives or the alternatives are mutually exclusive.

As used herein, ranges and amounts can be expressed as “about” a specific value or range. “About” also includes precise amounts. Thus "about 5 amino acids" means "about 5 amino acids" and also means "5 amino acids". For a particular parameter, about is a range within experimental error or acceptable to one skilled in the art for that particular parameter.

As used herein, “optional” or “optionally” means that the subsequently described event or circumstance does or does not occur, and the description includes instances where said event or circumstance occurs and instances where it does not. means to be For example, optionally a variant portion means that the portion is a variant or non-mutant.

As used herein, the abbreviations for any protecting groups, amino acids, and other compounds are in accordance with their common usage, recognized abbreviations, or IUPAC-IUB Committee on Biochemical Nomenclature unless otherwise indicated ( Biochem . (1972)). 11(9):1726-1732).

For clarity of disclosure and without limitation, the detailed description is divided into the following subsections.

B. Overview of Constructs and Methods

Autoimmune diseases occur when the body's immune system attacks itself. The resulting inflammation and tissue destruction is initiated by an inflammatory hormone called tumor necrosis factor (TNF). There are over 100 autoimmune diseases; Overall, about 75% of humans with autoimmune diseases are female. Previous drugs for autoimmune diseases have harmful side effects including infections, heart problems and other diseases and disorders.

TNF interacts with immune cells through two receptors: TNFR1, which is overactivated in autoimmune diseases, and TNFR2, which suppresses autoimmune diseases but is inhibited when TNRF1 is overactivated. TNF blockers such as infliximab (marketed by Remicade ^® ), adalimumab (marketed by Humira ^® ) and etanercept (marketed by Enbrel ^® ) block TNFR1 and TNFR2, causing harmful side effects. The constructs provided herein address this issue. The constructs provided herein not only treat autoimmune disease symptoms by blocking only TNFR1 that increases TNFR2 activity, but also provide improved treatment and reduced or no adverse side effects because TNFR2 activity is not blocked. Various constructs are provided herein that address the problems of prior art TNF blockers. The types of constructs identified by activity and detailed and provided herein are summarized in the following table.

Constructs for the treatment of TNF-mediated diseases, disorders and conditions, or diseases, disorders and conditions in which TNF plays a role in pathogenesis or in which interference with TNFR1 signaling has an ameliorative effect are provided. For example, TNFR1 antagonists are useful in a variety of disorders, including autoimmune disorders, and also in diseases and conditions, such as endometriosis, brain fog, such as from chemotherapy and COVID, Alzheimer's disease, acute inflammation, such as influenza It can be used in the treatment of SARS-CoV2, which results from infection with the virus, which causes long-lasting or permanent damage to the lungs, kidneys and other tissues. Due to the adverse effects of previous TNF blockers and the resulting safety concerns, they cannot be used for most indications. TNFR1 antagonist constructs provided herein may be used. These constructs as described herein are monovalent in that they inhibit only TNFR1 and do not cause receptor clustering, are specific and non-immunogenic, and have a half-life of at least about 3-4 weeks, so that approximately once a month dosing is sufficient. allowed

Thus, multispecificities such as TNFR1 antagonist constructs, TNFR2 agonist constructs, and bispecific constructs comprising TNFR1 antagonist and TNFR2 agonistic activity are provided. The construct may include at least one moiety that specifically interacts with TNFR1 or TNFR2, and typically additional moieties that directly or indirectly modulate the interaction or provide pharmacological (pharmacodynamic or pharmacokinetic or both) properties to the construct. contains a moiety Thus, constructs provided herein include at least two moieties: a binding moiety that interacts with TNFR1 or TNFR2, and a second moiety that modulates or alters a pharmacological property or activity of the construct or binding moiety. tea.

Some of the constructs provided herein are antagonists of TNFR1 activity. A TNFR1 antagonist construct imparts a moiety that binds to or interacts with TNFR1 and inhibits TNFR1-mediated signaling, and additional properties such as extended serum half-life, ablation of ADCC and/or CDC activity, and modulation of interaction with specific receptors. It contains a second part that TNFR1 antagonists and constructs may include modification(s) such that they are not immunogenic or reduced, particularly in humans, and may also contain modifications to eliminate or reduce binding to preexisting antibodies.

The TNFR1 antagonist construct is selected to bind specifically to TNFR1 and to have minimal or no binding to TNFR2 or no TNFR2 antagonist activity. Thus, the construct only modulates TNFR1. In some embodiments, the TNFR1 antagonist construct is also selected to have or be linked to a second domain or moiety that has TNFR2 agonistic activity. TNFR1 constructs have an affinity, such as K _d < 50 nM or < 10 nM or < 5 nM, and particularly higher affinity (K _d < 1 nM or < 0.1 nM or higher affinity) and/or TNFR1 signal those designed or selected to interact with potent inhibition of delivery (eg, IC ₅₀ 50 nM or < 10 nM or < 5 nM or < 3 nM, or 1 nM or < 0.5 nM).

Also provided are multispecific constructs, such as bispecific, containing a TNFR1 antagonist moiety linked directly or via a linker to a TNFR2 agonist moiety. Linkers provide molecules with advantageous properties such as, for example, increased serum half-life, increased stability, appropriate three-dimensional structure and flexibility, and improved pharmacological properties. Such constructs solve problems associated with the administration of other therapies, such as anti-TNF therapies (“TNF blockers”, examples of which include etanercept, adalimumab (Humira ^® ), infliximab), This suggests that these constructs increase the specificity of TNFR1 inflammation blockade and result in preservation or amplification of the natural immunosuppressive TNFR2 function, at least in part by upregulation and protection of immunosuppressive Tregs and induction of anti-inflammatory signaling pathways. In addition, TNF blockade resulting in suppression of TNFR2 function also reduces T cell-induced monocyte activation, increasing the likelihood of opportunistic infections (see, e.g., Rossel et al. (2007) J. Immunol . 179: 4239-48).

There are many differences between the activity of the exemplary TNFR1 antagonist constructs provided herein and existing approved TNF blockers: TNF blockers such as etanercept, adalimumab, infliximab are not specific for TNFR1 . Other blockers such as IL6, IL17, and IL23 block only parts of the cytokine cascade, but not the whole. Existing TNF blockers have the same mechanism of action for TNFR1 and TNFR2, thus blocking the activity of both. JAK inhibitors pose similar problems; They have inflammatory and anti-inflammatory action. For example, the inflammatory cytokine IL1 is not blocked by JAK inhibitors, the inflammatory cytokine IL6 is blocked by JAK inhibitors (secondary use in rheumatoid arthritis treatment), and the anti-inflammatory drug IL10 is blocked by JAK inhibitors. It doesn't work. In contrast, constructs provided herein combine the effects of TNFR1 and TNF inhibitor therapy with the benefits of TNFR2 agonists that eliminate or reduce the side effects of anti-TNFR1/anti-TNF therapy, and also upregulate immunosuppressive Tregs , contributing to additional therapeutic modality benefits including induction of protective and anti-inflammatory signaling pathways.

A TNFR1 antagonist construct contains one or more TNFR1 inhibitors, one or more linkers, and one or more active modifiers. For example, the structure of a TNFR1 antagonist construct provided herein can be represented by Formula 1 below:

(TNFR1 inhibitor) _n - linker _p - ring activity modifier) _q , Formula 1a, or

(active modifier) _q - linker _p - ring TNFR1 inhibitor) _n , Formula 1b, where:

(n and q are each an integer, each independently 1, 2 or 3; p is 0, 1, 2 or 3); activity modifiers are polypeptides such as albumin that increase the serum half-life of the TNFR1 inhibitor, or moieties such as Fc modified to have reduced or no ADCC activity; A TNFR1 inhibitor is a molecule, such as a polypeptide or small drug molecule, that binds to TNFR1 and inhibits its activity, such as its signaling activity. The active modifier is not a human serum albumin antibody or an unmodified single Fc. Active modifiers include modified Fc regions such as Fc, Fc dimers and other antibody domains that have been modified to eliminate ADCC and/or CDC activity. Linkers include chemical linkers, polypeptides such as GS linkers, hinge regions such as antibodies, such that constructs include chemical conjugates, fusion proteins, and combinations thereof.

Multispecific constructs are also provided. The structure of a multispecific, e.g., bispecific, construct provided herein is represented by the following formula (Equation 2):

(TNFR1 inhibitor) _n - (active modifier) _r1 - (linker (L) _p - (active modifier) _r2 - (TNFR2 agonist) _q ,

wherein n is 1, 2 or 3, p is 1, 2 or 3, q is 0, 1 or 2, and r1 and r2 are each independently 0, 1 or 2. As with the constructs of Formula 1, the order of components may vary and additional linkers may be present as desired. The construct may include additional linkers necessary to impart properties such as flexibility. Each linker may contain multiple components. Formula 2 may also include active modifiers in place of or in addition to linkers. Active modifiers and linkers include Fc or Fc with a hinge region, or Fc with a GS linker, or other combinations of elements. The Fc of these constructs includes an unmodified Fc region; Linkers are as described above and are detailed below.

Also provided is a TNFR2 agonist construct having Formula 3:

(TNFR2 agonist) _n - linker _p - (active modifier) _q , Formula 3a, or

(active modifier) _q - linker _p - (TNFR2 agonist) _n , formula 3b,

wherein n, p and q are as described in Formula 1, and the linker and active modifier are as described in Formula 1.

Components of formulas 1-3, discussed in detail in the next section, may be other molecules or polypeptides such as small drugs that specifically bind or interact with the targeting receptor. Each component of the construct/molecule provided herein is described in turn in the sections below.

The properties of each component of the constructs provided herein are discussed in detail in the sections below. Components of the construct thus include, but are not limited to, the following components detailed in the following sections.

1. TNFR1 antagonists

2. TNFR2 agonists

3. Linker

a. glycine-serine linker

b. hinge area

c. chemical linker

4. Active modifiers

a. Modified Fcs

b. Polypeptides and other moieties that confer improved or altered pharmacological properties such as increased serum half-life, resistance to degradation by endogenous proteases, and other such properties.

Other constructs are provided, which are detailed in the following sections.

The construct is characterized in that TNF is a pathological modifier of a disease, condition or disorder such that inhibition of TNFR1 signaling is reduced or inhibited and/or inhibition of TNF or TNFR1 signaling can inhibit or cause regression of a disease, disorder or condition; / or inhibition is used in methods of treating diseases, disorders, and conditions, wherein the disease, disorder, and/or condition ameliorates symptoms of the disease, disorder, and/or condition. These diseases, conditions and disorders including inflammatory diseases including autoimmune diseases are discussed in the following sections.

Also provided are pharmaceutical compositions for use in the methods and uses, and nucleic acids and vectors for generating constructs, including those that are polypeptides and fusion proteins. The following sections cover diseases, disorders, and conditions, TNFR1/TNFR2 activity and its role in diseases, disorders, and conditions, existing treatments for diseases, disorders, and conditions, constructs provided herein and their components, and constructs. Methods of producing, pharmaceutical compositions containing the constructs and/or encoding nucleic acids, and methods of treatment are described.

C Tumor necrosis factor (TNF) and chronic inflammatory and autoimmune diseases and disorders

This section describes the role that tumor necrosis factor (TNF) and/or its receptors play in inflammatory and autoimmune diseases, characterization of exemplary diseases, and problems with existing therapies, and constructs provided herein are useful in these problems. show how to solve

1. Tumor Necrosis Factor (TNF)

Tumor necrosis factor (TNF; see eg SEQ ID NO: 1; also referred to as TNF alpha, TNF-α or TNFα) is involved in the regulation of tumorigenesis/cancer, host defense against pathogenic infection, apoptosis, autoimmunity and sepsis. It is a pleiotropic, pro-inflammatory cytokine that is associated with inflammatory and immunomodulatory activities, including shock, and plays an important role in organogenesis, particularly of lymphoid organs, as well as coordination of innate and adaptive immune responses. In humans, TNF is produced primarily by macrophages and may also be produced by monocytes, dendritic cells (DCs), B-cells, T cells, fibroblasts and other cell types. It is a homotrimeric membrane-bound protein containing 233 amino acids (26 kDa) that can be cleaved by the protease TACE (TNF alpha convertase; also known as ADA17) to release soluble TNF containing 157 amino acids (17 kDa). produced as a protein; Both membrane bound and soluble forms of TNF are biologically active. Transmembrane human TNF contains 233 amino acids and, with respect to SEQ ID NO: 1, a cytoplasmic domain corresponding to residues 1-35, a transmembrane domain corresponding to residues 36-56, and an extracellular domain corresponding to residues 57-233 contains a domain. The soluble form of TNF corresponds to amino acid residues 77-233 as set forth in SEQ ID NO: 1 (see SEQ ID NO: 2 for the amino acid residue sequence of soluble TNF).

Uncontrolled production of TNF is associated with several inflammatory and autoimmune diseases including, for example, septic shock, rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis, and inflammatory bowel disease (IBD); and related to the condition Overexpression of TNF is also associated with neurodegenerative diseases and conditions such as, for example, Alzheimer's disease, Parkinson's disease, stroke and multiple sclerosis. TNF also promotes osteoclastogenesis, and overproduction of TNF is associated with bone loss. In rheumatoid arthritis (RA), TNF is overexpressed in the synovial fluid and synovial membrane, whereas the expression of the TNF receptor (TNFR) is upregulated in the synovial membrane. For example, overexpression of human TNF in mice results in the development of spontaneous RA-like lesions in joints with formation of hyperplastic synovium and destruction of cartilage and bone (see, e.g., Bluml et al. (2010) Arthritis & Rheumatism 62(6):1608-1619;Keffer et al. (1991) EMBO J. 10(13):4025-4031; Esperito Santo et al. (2015) Biochem . Biophys . Res. Commun. 464:1145-1150; Bluml et al. (2012) International Immunology 24(5):275-281; Dong et al. (2016) Proc. Natl. Acad. Sci. USA 113(43):12304-12309).

As discussed further below, TNF signaling signals through two high-affinity specific receptors, TNFR1 and TNFR2; TNFR1 is associated with detrimental inflammatory processes, whereas TNFR2 is associated with beneficial immune regulatory processes. Membrane-bound TNF has been shown to primarily activate TNFR2, whereas soluble TNF has been shown to primarily activate TNFR1 (Bluml et al. (2010) Arthritis & Rheumatism 62(6):1608-1619]). Soluble TNF (solTNF; corresponding to residues 77-233 of SEQ ID NO: 1; see sequence given in SEQ ID NO: 2) involved in paracrine signaling (mainly via TNFR1) is associated with chronic inflammation, whereas cell Transmembrane TNF (tmTNF), which acts through liver contact to induce juxtacrine signaling (mainly through TNFR2), relieves inflammation and kills pathogens such as Listeria monocytogenes and Mycobacterium tuberculosis Rosis ( Mycobacterium tuberculosis ) It is associated with the induction of immunity against. Thus, TNF signaling through TNFR1 and TNFR2 leads to different results depending on the receptor type.

Because of the association between TNF overexpression and the development of inflammatory and autoimmune diseases and conditions, blockade of TNF is effective in rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis (JIA), and inflammatory bowel disease ( It has been used in the treatment of a variety of such diseases and conditions, including but not limited to IBD; The use of TNF blockers, which block TNF and prevent signaling through TNFR1 and TNFR2, is associated with an increased risk of serious infections such as tuberculosis and listeriosis due to immunosuppression. TNF blockers not only block detrimental inflammatory signaling through TNFR1, but also block beneficial immune regulatory signaling through TNFR2. As a result, the use of TNF blockers may be limited, especially in chronic diseases/conditions requiring long-term administration, such as arthritis or IBD. About one-third of RA patients are unresponsive or do not receive sustained therapeutic benefit with anti-TNF therapy. Thus, there is a need for therapies with improved therapeutic efficacy and safety, particularly those that block the inflammatory effects of TNFR1 signaling while maintaining or enhancing the beneficial anti-inflammatory effects of TNFR2 signaling. Such therapies are provided herein.

2. Tumor Necrosis Factor Receptor (TNFR)

Homotrimers of TNF form two specific, high affinity homotrimeric receptors, TNFR1 (TNF receptor type 1; also known as TNFRI, p55, p60, CD120a, TNF receptor superfamily member 1A and TNFRSF1A) and TNFR2 (TNF receptor type 1). 2; TNFRII, p75, p80, CD120b, also known as TNF receptor superfamily member 1B and TNFRSF1B). TNFR1 is expressed by all nucleated cell types; TNFR2 expression affects immune cells (e.g., monocytes, macrophages, activated T cells, regulatory T cells (Treg), B cells, and natural killer (NK) cells), endothelial cells, certain central nervous system (CNS) cells, and limited to certain heart cells. TNFR2 expression on Tregs is induced upon T-cell receptor activation.

In vivo, TNFR1 and TNFR2 exist as membrane-bound receptors and soluble "decoy" (ie, non-signaling) receptors after shedding from the cell surface. Soluble TNF binds preferentially/selectively to TNFR1; Binding of membrane-bound and soluble forms of TNF activates TNFR1. The primary ligand of TNFR2 is membrane-bound TNF. Although soluble TNF does not fully activate TNFR2, the soluble form of TNFR2 (after TNFR2 shedding) has a high binding affinity for TNF, thus allowing TNF to bind to membrane-bound signaling receptors contributing to the anti-inflammatory effects of TNFR2. eliminate and suppress Membrane-bound TNFR2 binds to TNF with rapid on/off kinetics, whereby TNFR2 concentrates TNF on the cell surface and transfers its ligand to TNFR1 to mediate TNFR1 signaling. TNFR1 and TNFR2 each contain extracellular, transmembrane and cytoplasmic domains. The extracellular domains of TNFR1 and TNFR2 contain four cysteine-rich domains (CRDs) required for ligand binding. The intracellular domains of TNFR1 and TNFR2 initiate different signaling cascades and mediate different effector functions in response to TNF ligand binding.

Abnormalities in TNFR signaling are associated with several autoimmune diseases, and administration of TNF can be used as a therapeutic strategy for these diseases. For example, low-dose TNF selectively destroys autoreactive T cells in blood samples from patients with type I diabetes and scleroderma and in animal models of Sjögren's syndrome. Administration of TNF can cause systemic toxicity, for example in cancer patients with high TNF levels. As described herein, toxicity results from ubiquitous cellular expression of TNFR1; As described herein, functional TNFR2 is a safer treatment option than administration of TNF due to more restricted cellular expression. Promotion of TNF signaling through TNFR2 can be accomplished by administering a TNFR1 antagonist (see, eg, Faustman et al. (2013) Front. Immunol. 4:478])

a. TNFR1

Human TNFR1 (see SEQ ID NO: 3) is a major inflammatory receptor and accounts for most of the inflammatory, cytotoxic and apoptotic effects attributed to TNF. Human TNFR1 is a homotrimeric receptor, and its binding by TNF induces pro-inflammatory responses (see, e.g., Morton et al. (2019) for a description of TNFR1 signaling). Sci Signal. 12(592):eaaw2418). TNFR1 contains 455 amino acid residues; Residues 1-29 correspond to the signal peptide, residues 30-211 correspond to the extracellular domain, residues 212-232 correspond to the transmembrane domain and residues 233-455 correspond to the cytoplasmic domain. Within the extracellular domain, TNFR1 contains cysteine rich domains (CRDs) 1-4 corresponding to amino acid residues 43-82, 83-125, 126-166 and 167-196 of SEQ ID NO:3, respectively. CRDs 2 and 3 contact bound TNF, and amino acid residues 30-82 with respect to CRD1, particularly SEQ ID NO: 3, form the pre-ligand binding assembly domain (PLAD), a hemophilia interaction motif required for ligand binding and receptor function. form The cytoplasmic domain contains a death domain (corresponding to residues 356-441 of SEQ ID NO: 3) that binds to TNFR1-associated death domain (TRADD) and Fas-associated death domain (FADD) after TNF binds to TNFR1, and binds caspases to activates and creates signaling pathways that induce apoptosis. Binding of TNF to TNFR1 is also via the MAPK (mitogen activated protein kinases; e.g., p38, JNK, ERK) and NF-κB (nuclear factor kappa-light chain-enhancer of activated B cells) signaling pathways. initiates an inflammatory cascade. TNFR1 plays a role in lymphoid organogenesis and the immune response to pathogens and is a key receptor involved in host anti-viral defense mechanisms. It has been shown that mycobacterial blockade is dependent on TNF-derived signaling and patients treated with TNF blockers may suffer from endogenous reactivation of latent tuberculosis.

TNFR1, which is mainly involved in proinflammatory signaling, is a driving force behind the development of arthritis. For example, knockout of TNFR1 in mice and silencing of TNFR1 expression by RNA interference result in attenuation of collagen-induced arthritis (CIA), an animal model of arthritis. TNFR1-deficient mice overexpressing TNF are protected from developing arthritis, and reintroduction of TNFR1 to mesenchymal cells causes TNF-dependent arthritis. In addition, TNFR1 causes local bone destruction by enhancing the production of osteoclasts, and TNFR1 deficiency in hematopoietic cells has been shown to attenuate bone destruction in an erosive arthritis model. TNFR1 has also been associated with cardiotoxic effects in TNF-induced heart failure and myocardial infarction models and has been shown to promote neurodegeneration in animal models of retinal ischemia (see, e.g., Schmidt et al. (2013) Arthritis & Rheumatism 65(9):2262-2273; Goodall et al. (2015) PLoS ONE 10(9):e0137065; McCann et al. (2014) Arthritis & Rheumatology 66(10):2728-2738; Ruspi, etc. (2014) Cellular Signaling 26:683-690; Faustman and Davis (2013) Front. Immunol . 4:478; Bluml et al. 2012) International Immunology 24(5):275-281; Dong et al. (2016) Proc. Natl. Acad. Sci. USA 113(43):12304-12309).

b. TNFR2

Human TNFR2 (reference, SEQ ID NO: 4) contains 461 amino acid residues; Residues 1-22 correspond to the signal peptide, residues 23-257 correspond to the extracellular domain, residues 258-287 correspond to the transmembrane domain and residues 288-461 correspond to the cytoplasmic domain. Unlike TNFR1, TNFR2 without a death domain has a TNF receptor-associated factor 2 (TRAF2) binding site. TNFR2 signaling through TRAF2 promotes cell survival and proliferation through NF-κB and activator protein 1 (AP1) activation and is associated with PI3K-PKB/Akt-mediated repair and migration. As discussed elsewhere herein, TNF signaling through TNFR2 also promotes the expansion and activation of regulatory T cells (Tregs), which play an important role in the suppression of inflammatory and autoimmune diseases and disorders. TNFR2 signaling has been implicated in wound healing and repair and regeneration in myocardial infarction models, whereas knockout of TNFR2 in erosive arthritis mouse models results in joint inflammation and bone destruction.

TNFR2, which is primarily involved in anti-inflammatory signaling, is associated with neurological, cardiac, intestinal and bone protective effects. TNFR2 exhibits anti-inflammatory and protective effects; This effect has been demonstrated, for example, in experimental autoimmune encephalomyelitis (EAE), experimental colitis, heart failure/heart disease, myocardial infarction, inflammatory arthritis, demyelination and neurodegenerative disorders, and infectious diseases. For example, activation of TNFR2 by TNF inhibits seizures, attenuates cognitive dysfunction after brain injury, promotes survival after myocardial infarction in mice, protects against myocardial ischemia/reperfusion injury, remodels and Reduces hypertrophy. TNFR2 agonism is also associated with pancreatic regeneration, remyelination, survival of neuronal subtypes and stem cell proliferation. TNFR2 agonism selectively destroys autoreactive T cells rather than healthy cells in blood samples from patients with type I diabetes, multiple sclerosis, Graves' disease and Sjogren's syndrome. Depletion of autoreactive T cells using low-dose TNF in animal models of type I diabetes regenerates pancreatic tissue. TNF signaling through TNFR2 has been shown to induce the regeneration of oligodendrocyte precursors in the myelin and can thus be used in the treatment of demyelinating disorders such as multiple sclerosis (MS). TNFR2 has also been shown to promote neuroprotection in animal models of retinal ischemia.

TNFR2 also regulates osteoclastogenesis. Osteoclast is a type of bone cell that decomposes bone tissue; Regulation of osteoclastogenesis is important for maintaining bone mass and protecting against joint inflammation and erosive destruction. In TNFR2-deficient mice, osteoclastogenesis is enhanced, leading to exacerbation of TNF-induced arthritis and local bone destruction. Deficiency of TNFR2 in animal models of erosive arthritis leads to disease progression, and TNFR2-deficient mice overexpressing TNF develop worse arthritis and joint destruction compared to control mice. Expression of TNFR2 in hematopoietic cells attenuates TNF-induced arthritis, whereas loss of TNFR2 in hematopoietic cells increases recruitment of inflammatory cells to the synovial membrane. In experimental colitis, lack of TNFR2 expression on CD4 ⁺ T cells accelerates the onset of disease and increases the severity of inflammation, whereas in experimental autoimmune encephalitis (EAE) symptoms are exacerbated in TNFR2 deficient mice (see, e.g., [Schmidt et al. (2013) Arthritis & Rheumatism 65(9):2262-2273; Goodall et al. (2015) PLoS ONE 10(9):e0137065; McCann et al. (2014) Arthritis & Rheumatology 66(10):2728-2738; Ruspi, etc. (2014) Cellular Signaling 26:683-690; Faustman and Davis (2013) Front. Immunol . 4:478; Bluml et al. (2012) International Immunology 24(5):275-281; Dong et al. (2016) Proc . Natl . Acad . Sci . USA 113(43):12304-12309). Polymorphisms in the TNFR2 gene are correlated with a variety of autoimmune diseases including, for example, RA, Crohn's disease, systemic lupus erythematosus, ankylosing spondylitis, inflammatory bowel disease, ulcerative colitis and scleroderma; The polymorphism prevents the binding of TNF to TNFR2, limiting the activation of NF-κB and interfering with the TNFR2 signaling pathway in Tregs (see, e.g., Yang et al. (2018) Front. Immunol. 9:784]).

TNFR1 contains an intracellular death domain and can activate apoptotic and/or inflammatory pathways, whereas TNFR2 can bind TRAF and activate canonical and non-canonical NF-κB pathways to control cell survival and proliferation. In general, cells expressing TNFR2 also express TNFR1 in varying proportions depending on the cell type and function. Since TNFR1 signaling generally induces apoptosis, while TNFR2 signaling generally induces cell survival, their co-expression ratio to cells shifts the balance towards either apoptosis or cell survival. As discussed above and elsewhere herein, TNFR1 is the primary TNF receptor involved in the pathogenesis of RA, whereas TNFR2 has been shown to play an immunomodulatory role. However, both receptors are involved in mediating the antiviral activity of TNF. For example, animal disease models show that TNFR1 is associated with inflammatory neurodegeneration, while TNFR2 is associated with neuroprotection.

Selective inhibition of TNFR1 or selective activation of TNFR2 was achieved by ATROSAB (antagonistic TNF receptor monospecific antibody), TNFR1-selective antagonistic IgG1 antibody, or EHD2-scTNF _R2 , an agonistic TNFR2-selective antibody, in a mouse model of NMDA-induced acute neurodegeneration. demonstrated by TNF muteins (i.e., mutant proteins). EHD2-scTNF _R2 is the mutation D143N/A145R fused to the dimerization domain EHD2 (residue numbering for soluble TNF shown in SEQ ID NO: 2 and corresponding to D219N and A221R with respect to SEQ ID NO: 1, respectively; these mutations are ), which produces a disulfide-linked dimer derived from the heavy chain C _H 2 domain of IgE and containing a hexameric TNF domain. .

Simultaneous injection of NMDA and ATROSAB, or NMDA and EHD2-scTNF _R2 into nucleus basal cerebral cells showed a significant but incomplete neuroprotective effect compared to the control group in an in vivo mouse model. The incomplete nature of this response was due to the agonistic activity of ATROSAB, a by-product of the bivalent antibody, which induces aberrant receptor clustering and activation (Richter et al. (2013) PLoS One 8(8):e72156]). Similarly, EHD2-scTNFR2 is immunogenic in humans because of its multiple fusion partners and an immune response to the IgE fragment results in an autoimmune response in toxicity studies (see, e.g., Weeratna et al. (2016) Immun . Inflamm. Dis . 4(2) :135-147). Thus, there is a need for improved TNFR1 antagonists and improved TNFR2 agonists that overcome these limitations.

3. Regulatory T cells (Tregs) and their role in the autoimmune microenvironment

Regulatory T cells (Treg cells or Tregs) are an immunosuppressive subpopulation of T cells that possess immunosuppressive properties through cytokine production. These are transforming growth factor beta, interleukins 35, and interleukin 10. Induction of Treg function can suppress several pathologies. Induction can enhance transplant success rates, suppress allergies, and modulate responses to autoimmunity and infectious diseases such as severe acute respiratory syndrome. Tregs can inhibit and/or downregulate the induction and proliferation of effector T cells (Teffs), modulate the immune system, maintain immune homeostasis and tolerance to self antigens, and prevent the development of autoimmune diseases and tissue destruction. . Tregs express CD4, CTLA-4, CD25 (also known as IL-2 receptor alpha chain or IL2RA), and FOXP3 (transcription factor forkhead box P3), among other markers, and express TNFR1 10-fold higher Density expresses TNFR2. TNFR2 is expressed only by a subpopulation of Tregs, the maximally suppressive subset; This subset contains TNFR2 expressing CD4 ⁺ FoxP3 ⁺ Tregs. TNF promotes Treg cell proliferation, upregulation of FoxP3 expression, and Treg cell suppressive activity/function through TNFR2 signaling. The autoimmune microenvironment contains more autoreactive CD8 ⁺ effector T cells than immunosuppressive CD4 ⁺ Tregs, resulting in tissue destruction. Consequently, preservation or enhanced TNFR2 function to expand Tregs and eliminate autoreactive T cells restores the immune balance (Sharma et al. (2018) Front Immunol . 9: 883]). For these reasons and others described below, pharmacological maintenance of Treg function by selective inhibition of TNFR1 in combination with TNFR2 stimulation (agonism) results in many acute and chronic inflammatory conditions (severe acute respiratory syndrome, autoimmune diseases). will improve

In addition to upregulating the expression of TNFR2 on Tregs, TNF upregulates Treg surface expression of other costimulatory members of the TNF receptor superfamily (TNFRSF), such as 4-1BB and OX40, resulting in optimal activation and proliferation of Tregs. Attenuates the excessive inflammatory response. Neutralization of TNF (blocking TNFR2) blocks expansion of Tregs in vivo (see, for example, Hamano et al. (2011) Eur. J. Immunol. 41:2010-2020]).

Compared to CD4 ⁺ FoxP3 ⁻ conventional T cells, CD4 ⁺ FoxP3 ⁺ Tregs constitutively express TNFR2 to promote Treg cell activation, expansion and survival. TNF signaling through TNFR2 (i.e., TNFR2 agonism) promotes activation and expansion of Tregs, whereas TNFR2 antagonism results in Treg contraction. For example, TNFR2 agonism selectively kills autoreactive T cells and expands suppressive Tregs in humans with autoimmune disease and in animal models of autoimmunity. TNFR2 signaling promotes Treg cell expansion and suppressive activity in experimental autoimmune encephalomyelitis (EAE; an animal model of inflammatory CNS demyelinating diseases such as multiple sclerosis) and murine diabetes models and induces human antigen-specific Treg cells by tolerogenic dendritic cells. induce TNFR2-deficient Tregs have a reduced ability to protect against experimental colitis in vivo, and TNFR2 is required for sustained FoxP3 expression in Tregs, and consequently, TNFR2 is required for the immunosuppressive function of Tregs in vivo to maintain their phenotypic and functional stability. (See, for example, McCann et al. (2014) Arthritis & Rheumatology 66(10):2728-2738; Faustman and Davis (2013) Front. Immunol . 4:478; Schmidt et al. (2013) Arthritis & Rheumatism 65(9):2262-2273; Vanamee et al. (2017) Trends in Molecular Medicine 23(11):P1037-P1046; Chen et al. (2013) J. Immunol . 190(3):1076-1084). In one study, antigen-specific Tregs produced in vitro were shown to suppress disease and reduce joint inflammation and bone destruction in a well-established model of antigen-induced arthritis (AIA), where they were shown to induce T cell-mediated tissue damage. To immunize mice with methylated bovine serum albumin (mBSA) (see, e.g., Wright et al. (2009) Proc . Natl . Acad . Sci . USA 106(45):19078-19083). Although the use of Tregs in cell therapy is promising, manufacturing and other complications require traditional biotherapeutic agents that deliver the benefits of Tregs without complications.

As described and provided herein, TNFR2 and its expression by Tregs are required for the suppression of inflammatory and autoimmune diseases and conditions. For example, Mycobacterium bovis Bacillus calmetr-Guerin (BCG) induces transient expansion of Tregs. In a clinical trial, BCG induced Treg production in patients with type I diabetes, suppressed the disease and temporarily restored islet cell function, indicating the use of Treg and/or modulators to enhance Treg function in the treatment of type I diabetes. (See, e.g., Spence et al. (2016) Curr Diab Rep 16(11):110. doi : 10.1007/s11892-016-0807-6).

It is described and established herein that modulation of Treg function represents a therapeutic approach for the prevention or treatment of inflammatory and autoimmune diseases and conditions. However, Tregs constitute only ~1-5% of total CD4+ T cells in the blood. Their low numbers prevent their clinical use. Stimulation of ex vivo and/or in vivo production of Tregs is a limiting factor for their therapeutic use. For example, in vivo stimulation with IL-2, anti-CD3 or anti-CD28 is too toxic, whereas ex vivo stimulation with these agents can release pro-inflammatory cytokines and may have antagonistic properties. Generate a heterogeneous CD4 ⁺ population. An alternative approach has been to expand Tregs in vivo and ex vivo using naturally occurring TNF receptor superfamily agonists, TL1A-Ig or TNFR2 monoclonal antibody agonists, respectively. TNFR2 agonist constructs and multispecific constructs provided herein can preserve and/or expand Treg populations in vivo without interfering with the therapeutic activity of anti-TNFR1 activity. As described and provided herein, selective inhibition of inflammatory TNFR1 activity is beneficial in the treatment of inflammatory and autoimmune diseases and conditions while maintaining or increasing TNFR2-associated Treg suppressive activity. Such diseases and conditions include, but are not limited to, RA, type I diabetes, heart failure, and multiple sclerosis (see, e.g., Goodall et al. (2015) PLoS ONE 10(9):e0137065).

In the tumor microenvironment (TME), unlike the autoimmune microenvironment where expansion of TNFR2 ⁺ Tregs prevents tissue destruction, tumors are infiltrated by large numbers of immunosuppressive TNFR2 ⁺ Tregs, resulting in tumor-killing CD8 ⁺ cytotoxic T lymphocytes ( CTLs) (also called effector T cells (Teffs)) to allow tumor growth. In the TME, TNFR2's antagonism on lymphocytes restores the balance between the two types of T cells by suppressing or eliminating Tregs and allowing the activation and expansion of effector T cells, a condition in which tumor growth can be controlled or reversed. To be useful as therapeutics, TNFR2 inhibitors must not have the ability to aggregate immune cells via ADCC for two reasons: 1) aggregation transiently causes 'super induction' of TNFR2-mediated immunosuppression; 2) eventually leading to systemic depletion of Tregs, which would be detrimental to patients as maintaining basal levels of Treg activity is essential to maintain immune homeostasis. Tumor cells and myeloid-derived suppressor cells (MDSCs) also express TNFR2, and inhibition of TNFR2 in MDSCs controls metastasis as seen in murine liver cancer models. Thus, blockade of TNFR2, such as through the use of non-aggregation antagonistic antibodies or other therapeutic agents as provided herein, through suppression of immunosuppressive Tregs, represents a useful treatment for certain types of cancer. However, TNFR2 antagonists should only be administered to patients with overexpression of TNFR2 in the tumor compared to adjacent normal tissue, as judged by immunohistochemistry. Thus, such treatment should be accompanied by a diagnosis to confirm overexpression (see, e.g., Zhang et al. for an exemplary assay). (2019) Thorac Cancer 10(3): 437-444. doi:10.1111/1759-7714.12948; Yang et al. (2017) Oncol Lett.14 (2) :2393-2398. doi:10.3892/ol.2017.6410; and Yang et al. (2018) Oncol Lett. 16(3) :2971-2978. doi:10.3892/ol.2018.8998).

4. Autoimmune/inflammatory diseases mediated by or accompanying TNF

Elevated levels or uncontrolled expression of TNF and deregulation of TNF signaling can lead to chronic inflammation, which can lead to the development of autoimmune diseases and tissue damage. TNF-α is involved in numerous diseases, disorders and conditions. Constructs provided herein can be used for the treatment of these diseases, disorders and conditions. The discussion below describes some exemplary diseases, disorders and conditions for which TNF blockade may have a therapeutic effect. TNF blockers such as etanercept, infliximab, adalimumab, certolizumab and golimumab have side effects that may limit their use in the treatment of these diseases, disorders and conditions. Constructs provided herein that avoid some or all of these adverse effects can be used to treat such diseases, disorders and conditions (e.g., a review of the role of TNF in disease and the use of TNF blockers for its treatment). For, Lis et al. (2014) Arch Med Sci.10(6) :1175-1185).

Inflammatory diseases include a number of disorders and conditions characterized by inflammation and include autoimmune diseases. The immune system protects the body by producing antibodies or activating lymphocytes in response to invasion by microorganisms such as viruses and bacteria. In a healthy individual, the immune system does not trigger a response against the body's own (ie, “self”) cells; Autoimmune diseases occur when the immune system attacks healthy non-invasive self, cells and tissues. Autoimmune/inflammatory diseases and disorders associated with elevated TNF levels include, for example, arthritis (eg, rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis, spondyloarthritis), inflammatory bowel disease (eg, among others) eg, Crohn's disease and ulcerative colitis), uveitis, fibrotic disease, endometriosis, lupus, ankylosing spondylitis, psoriasis, multiple sclerosis (MS), Parkinson's disease, and Alzheimer's disease. Exemplary autoimmune and inflammatory diseases and disorders that can be treated with the constructs provided herein are discussed below.

a. arthritis

rheumatoid arthritis and other types of arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease. Inflammation associated with rheumatoid arthritis affects the lining of joints (i.e., the synovial lining) and may also inflame the membranes lining blood vessels and the heart. RA is characterized by infiltration of immune cells (eg, activated B-cells) into the synovial membrane and proliferation of synovial cells, which causes the synovial lining to thicken. A proliferative mass known as a pannus invades and destroys cartilage and bone, irreversibly destroying joint structure and function. This is mediated by the induction of pro-inflammatory cytokines such as TNF, IL-1 and IL-6. Tumor necrosis factor α (TNFα) is a key regulator of the induction and persistence of pro-inflammatory activity associated with RA. TNF is overexpressed in synovial fluid and synovial membrane, and expression of TNFR is upregulated in synovial membrane (see, e.g., Bluml et al. (2012) International Immunology 24(5):275-281; Schmidt et al. (2013) Arthritis & Rheumatism 65(9):2262-2273; Keffer et al. (1991) EMBO J. 10(13):4025-4031). Other types of arthritis that can be treated with the constructs herein include, for example, psoriatic arthritis, juvenile idiopathic arthritis and spondyloarthritis.

b. Inflammatory bowel disease (IBD) and uveitis

Inflammatory bowel disease (IBD) includes Crohn's disease and ulcerative colitis, which are inflammatory diseases of the intestine and colon. Mice overexpressing TNF develop intestinal inflammation similar to Crohn's disease, whereas TNFR1 deficiency protects against Crohn's disease (see, e.g., Fischer et al. (2015) Antibodies 4:48-70).

Uveitis is a form of eye inflammation that affects the eye wall (uvea), the middle layer between the retina and the sclera (the white of the eye), and can lead to vision loss. TNF-alpha has been implicated in its pathophysiology and TNF blockers have been used for treatment.

c. fibrotic disease

The constructs herein may be used for the treatment of fibrotic diseases. Dupuytren's disease is an example of such a disease. Dupuytren's disease (DD) is a common fibrotic disease of the hand characterized by irreversible flexion contractures of the fingers; This condition is limited to the palm and irreversibly bends the fingers, severely impairing hand function. There is no approved therapy for the disease in its early stages, and the nodules become dormant for a time and then become active, progressing to spinal cord and flexion deformities of the fingers, resulting in loss of hand function. Treatment includes surgical removal of the diseased tissue or spinal cord (myofasnotomy) or destruction of the spinal cord using collagenase or needle fasciotomy. Surgical and non-surgical treatments have high rates of recurrence and complications. In the early stages of the disease, therapeutic intervention to prevent progression to spinal cord development and consequent flexion contracture of the fingers is advantageous (see, e.g., Nanchahal et al. (2018) EBioMedicine 33:282-288).

Myofibroblasts that express the contractile protein α-smooth muscle actin (α-SMA) and aggregate to nodules accumulate excess collagenous extracellular matrix and are responsible for remodeling and contraction in all fibrotic conditions, including DD. TNF converts palmar fibroblasts of DD patients to myofibroblasts through the Wnt signaling pathway, and DD myofibroblasts show a dose-dependent decrease in contractility and decreased expression of α-SMA and pro-collagen after treatment with anti-TNF therapy. Treatment with the fully humanized IgG mAbs adalimumab and golimumab was most effective. However, use of anti-TNF therapies such as adalimumab is associated with an increased risk of infection, and 1 patient (of 21 patients receiving adalimumab) in a phase 2a trial evaluating the therapeutic efficacy of adalimumab in DD developed wound infections requiring hospitalization (see, e.g., Nanchahal et al. (2018) EBioMedicine 33:282-288]). Therefore, other therapies are needed.

d. Tumor Necrosis Factor Receptor Associated Cyclic Syndrome (TRAPS)

Tumor necrosis factor receptor-associated periodic syndrome (TRAPS) is the second most common congenital autosomal dominant autoinflammatory disease and is caused by mutations in the TNFRSF1A gene, which encodes TNFR1. TRAPS is characterized by periodic, long-lasting fever, systemic inflammation, abdominal pain, skin lesions, conjunctivitis, myalgia, and pericarditis, inflammatory attacks lasting up to several weeks. A complication associated with the more severe clinical phenotype of TRAPS is type AA serum amyloidosis, which can lead to renal damage and renal failure. Disease onset usually occurs in early childhood, but TRAPS can also appear in adults. Most of the TRAPS-associated mutations occur in the extracellular domain of TNFR1 involved in ligand binding. High penetrance mutations associated with the most severe clinical phenotypes occur in the extracellular cysteine rich domain (CRD). Mutations can affect the folding and secondary structure of TNFR1 resulting in defective TNFR1 trafficking, altered ligand binding affinity, reduced activation-induced shedding and impaired cell signaling. For example, ligand-independent gain of function of TNFR1 leads to TRAPS pathophysiology and certain mutations result in constitutive activity of TNFR1, NF-κB and caspase 1. Traditional anti-TNF therapies, including etanercept, infliximab, etc., are only partially effective in treating TRAPS (see, for example, Greco et al. (2015) Arthritis Research & Therapy 17:93), so other therapies are needed.

e. Other diseases mediated by or involving TNF

i. neurodegenerative disease

Aging and several neurodegenerative diseases are associated with elevated levels of TNF in the central nervous system (CNS). TNF is involved in initiating and maintaining neuroinflammation and regulating other neurological processes such as synaptic function and plasticity. The level of TNFR1 in the hippocampus of aged rats is approximately 3-fold higher than that of TNFR2. In animal models of the disease, TNF has been implicated in chronic glial activation and impaired neuronal viability through its action on TNFR1. Neurological changes in aged animals include synaptic dysfunction and Ca ²⁺ dysregulation, which can be replicated in healthy young animals and in neuronal ^cultures using artificial elevations of TNF. TNF also enhances ^the activity of L-type voltage-sensitive Ca ²⁺ channels (L-VSCC); A similar effect is observed in hippocampal neurons of aging rats with memory impairment. A study in rats showed that TNF blockade in the cerebellum accelerated learning on a delayed eye-blink task. Selective blockade of TNFR1 signaling using XPro1595, a soluble dominant-negative TNF (DN-TNF) that preferentially inhibits TNFR1 signaling, resulted in improved behavioral performance in the Morris swim task, reduced microglia activation, and hippocampal long-term Depression (LTD) was prevented and the activity of L-VSCC in CA1 neurons was reduced. These results indicate that TNF signaling through TNFR1 is involved in modifying the neurological phenotype of aged animals and can lead to pathological changes associated with neurological diseases. (See, e.g., Sama et al. (2012) PLoS ONE 7(5):e38170).

a) Alzheimer's disease

TNF plays a central role in the inflammatory response; TNF protein levels are low in the healthy brain but are chronically elevated in many neuroinflammatory diseases, including Alzheimer's disease (AD). In animal models of AD, TNF promotes microglial activation, synaptic dysfunction, neuronal apoptosis, accumulation of plaques and tangles, and cognitive decline. For example, in a triple transgenic AD mouse model (3xTg-Ad) harboring mutations in presenilin 1, amyloid precursor protein (APP) and tau, TNF levels were increased in the entorhinal cortex consistent with the early appearance of the pathology. (See, e.g., McCoy et al. (2006) J. Neurosci . 26(37):9365-9375). TNF-driven processes are implicated in AD pathology and contribute to cognitive dysfunction and accelerated AD progression. The bacterial endotoxin lipopolysaccharide (LPS), which induces inflammation and TNF production, accelerates the appearance and severity of AD pathology in several animal models of AD. Overproduction of proinflammatory mediators, including TNF, occurs in the brain when microglia, which are closely physically associated with amyloid plaques in the AD brain, are chronically activated. High levels of TNF inhibit phagocytosis of amyloid beta (Aβ) in the brains of AD patients, preventing efficient plaque clearance by microglia. Chronic inhibition of solTNF by administration of DN-TNF, such as XENP345, or a lentivirus encoding DN-TNF, prevents acceleration of AD-like pathology induced by chronic systemic inflammation in an AD animal model (3xTgAD mouse) and in the hippocampus , reduced LPS-derived intraneuronal accumulation of 6E10-immunoreactive proteins, particularly the C-terminal amyloid precursor protein (APP) fragment (β-CTF), in the cortex and amygdala. Genetic deletion of TNFR1 in 3xTgAD mice also prevents LPS-induced accumulation of neurotoxic β-CTF. Neuronal cells with familial AD (FAD) mutations accumulate β-CTF intracellularly, suggesting involvement in the pathogenesis of AD. These results suggest that soluble TNF is a mediator of neuroinflammatory effects on early pre-plaque pathology in 3xTgAD mice and that targeted inhibition of solTNF in the central nervous system (CNS) can slow the emergence of amyloid-associated pathology, cognitive deficits and progressive loss of neurons in AD. (See, e.g., McAlpine et al. (2009) Neurobiol. Dis. 34(1):163-177).

b) Parkinson's disease

Parkinson's disease (PD) is the second most common neurodegenerative disease in the United States, with an incidence of 5% in individuals over the age of 65. Clinical symptoms of Parkinson's disease result from selective loss of dopaminergic neurons in the ventral midbrain substantia nigra compacta (SNpc), resulting in a decrease in striatal dopamine. Cerebrospinal fluid (CSF) and postmortem brains of PD patients and animal models of PD show high levels of TNF. A cohort of patients with early-onset PD in Japan showed an increased frequency of a polymorphic allele (-1031C) in the TNF gene promoter, resulting in higher transcriptional activity and higher TNF levels. TNFR1 is highly expressed in nigrostriatal dopaminergic neurons, increasing susceptibility to TNF-induced neuroinflammation and dopaminergic neuron toxicity. Neutralization of soluble TNF (solTNF) in vivo by a dominant-negative TNF mutein (XENP345) is neuroprotective and prevents retrograde substantia nigra degeneration induced by striatal injection of the oxidative neurotoxin 6-hydroxydopamine (6-OHDA) 50 %, and amphetamine-induced turning behavior in rats was attenuated, indicating preservation of striatal dopamine levels. In embryonic murine midbrain neuron/glia cultures exposed to LPS (lipopolysaccharide), delayed administration of XENP345 prevented dopaminergic neuron degeneration despite sustained microglia activation and solTNF secretion. XENP345 also attenuated 6-OHDA-induced dopaminergic neuronal toxicity in vitro. Thus, TNF is implicated in the pathogenesis of Parkinson's disease, and it may be possible to delay the progressive degeneration of the nigrostriatal pathway in humans using TNF-blocking therapeutics, particularly in the early stages of Parkinson's disease (see, e.g., McCoy et al. (2006) J. Neurosci . 26(37):9365-9375).

c) multiple sclerosis (MS)

CNS-specific overexpression of TNF in transgenic mice results in spontaneous demyelination, indicating a role for TNF in multiple sclerosis (MS). Polymorphisms in the gene encoding TNFR1 are associated with increased susceptibility to developing MS. TNFR1 is required for disease induction in experimental autoimmune encephalomyelitis (EAE), an animal model of MS, and TNFR2 deficiency exacerbates the disease. Mice expressing non-cleavable membrane-bound TNF are protected against EAE, indicating that the interaction of soluble TNF with TNFR1 is associated with disease pathology (see, e.g., Fischer et al. (2015) Antibodies 4:48-70).

ii. endometriosis

TNF-α has been implicated in the pathophysiology of endometriosis. TNF-α levels are increased in the peritoneal fluid of women with endometriosis, and levels correlate with disease severity (see, eg, Koninckx (2008) Hum Reprod . 23 : 2017-2023). Peritoneal fluid TNF-α is produced locally by activated peritoneal macrophages, and TNF-α induces IL-8 secretion by peritoneal mesothelial cells. Peritoneal fluid concentrations of TNF-α and IL-8 correlate with the size and number of active peritoneal lesions (Bullimore, (2003) Med hypotheses. 60 :84-88]). Serum TNF-α levels are increased and monocytes from endometriosis patients release more TNF-α in vitro compared to monocytes from controls. Peritoneal fluid levels of MCP-1 are increased in endometriosis patients. TNF-α, IL-8 and MCP-1 induce an inflammatory Th-1 type response in the peritoneal fluid of endometriosis patients. TNF-α-mediated inflammation may be a causal factor in the pain associated with endometriosis. TNF-α blockade appears to inhibit the development of the disease in animal models and may be effective in humans as well. Treatment of endometriosis with these blockers is not recommended because of the harmful side effects of existing TNF blockers (Koninckx (2008) Hum Reprod. 23 : 2017-2023). However, the constructs provided herein are designed to avoid deleterious effects and can be considered for treating TNF-a mediated inflammation in endometriosis.

iii. cardiovascular disease

TNFα was the first cytokine identified in human atherosclerotic plaque; TNFα is involved in the activation of endothelium and the upregulation of adhesion molecules that occur in the early stages of atherosclerotic disease. TNF has also been implicated in the pathogenesis of atherosclerosis by influencing lipid metabolism and inducing vascular inflammation. Blockade of TNFα by TNF binding protein or IL-1 blockade by IL-1 receptor antagonists partially protects apoE knockout mice from atherosclerosis. Atherogenesis is primarily a result of TNFα production by bone marrow cells. The plaque area of apoE ^-/- and TNF ^-/- mice fed a high-fat diet was half the size of the plaque area of apoE ^-/- mice. Transplantation of bone marrow from apoE ^-/- and TNF ^-/- mice into apoE ^-/- mice reduced the size of atherosclerotic lesions by 83%. Atherosclerotic lesion size was also reduced after treatment of apoE ^-/- mice with recombinant soluble p55 (TNFR1) TNF blocker, indicating a role that TNF plays in atherosclerosis. NF-κB signaling is involved in the production of TNF-α in human atherosclerotic plaque. Peripheral blood TNFα levels in patients with cardiovascular disease are also associated with the development of myocardial infarction. Cardiotoxicity is primarily due to TNF-induced cardiomyocyte apoptosis. The use of anti-TNF therapies such as infliximab and etanercept in clinical trials for the treatment of heart failure has failed and increased mortality; Accordingly, anti-TNF therapies have not been tested for the treatment of cardiovascular disease (see, e.g., Udalova et al. (2016) Microbial Spectrum 4(4):MCHD-0022-2015; Kalliolias and Ivashkiv (2016) Nat. Rev. Rheumatol. 12(1):49-62]).

Consequently, alternative therapies are needed.

iv. Acute Respiratory Distress Syndrome (ARDS)

Acute respiratory distress syndrome (ARDS) affects approximately 190,000 patients per year in the United States and has a mortality rate of up to 40%. There are no effective treatments targeting the underlying pathophysiological mechanisms of ARDS. ARDS is characterized by immune cell-mediated lung injury associated with the release of inflammatory cytokines and proteases. The uncontrolled local inflammatory response in ARDS results in damage to the alveolar-capillary barrier and non-cardiogenic pulmonary edema. The recruitment of pulmonary neutrophils, which is central to the pathogenesis of ARDS, is mediated by the interaction of primed and activated neutrophils with the pulmonary microvascular endothelium and is augmented by damage to the alveolar-capillary barrier due to the action of pro-inflammatory mediators. TNF-α has been identified as a key effector molecule not only in ARDS but also in sepsis, a common cause of ARDS. For example, TNF-α contributes to increased endothelial permeability. Clinical trials involving administration of non-selective anti-TNF antibodies for the treatment of sepsis have not demonstrated any survival benefit, and one trial indicated that higher doses were detrimental.

TNFR1-deficient mice are protected from lung injury, sepsis and other acute organ damage, whereas TNFR2-deficient mice are more susceptible to injury in this model, indicating that selective antagonism of TNFR1 may be therapeutically effective. GSK1995057, a short-acting, fully human domain antibody (dAb) fragment that selectively antagonizes TNFR1 but not TNFR2, attenuated disease severity in a murine model of acute respiratory distress syndrome and attenuated signs of inflammation and lung injury in non-human primates. In a randomized, placebo-controlled trial of nebulized GSK1995057 in 37 healthy humans challenged with low doses of inhaled endotoxin, treatment with GSK1995057 resulted in pulmonary neutrophilia, inflammatory cytokine release, and signs of endothelial damage in bronchoalveolar lavage fluid and serum samples. has been weakened These results indicate the potential for pulmonary delivery of selective TNFR1 antagonists for the prevention and treatment of ARDS (see, e.g., Proudfoot et al. (2018) Thorax 73:723-730).

v. Severe Acute Respiratory Syndrome (SARS) and COVID-19

Subjects infected with severe acute respiratory syndrome coronavirus (SARS-CoV) develop lower respiratory tract symptoms including fever and respiratory illness, general malaise, cough and shortness of breath, with an overall mortality rate of -10%. TNF signaling promotes the pathogenesis of SARS by triggering excessive inflammation that causes severe tissue damage. The development of cytokine release syndrome (CRS) plays a role in severe COVID-19, a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Sustained viral stimulation increases the levels of circulating cytokines such as IL-6 and TNFα in some subjects, reducing the number of lymphocytes and causing inflammatory organ damage, particularly to the lungs and blood vessels. SARS-CoV-2 shares some similarities with SARS-CoV, the coronavirus strain responsible for the 2002 SARS pandemic. SARS-CoV and SARS-CoV-2 use the spike (S) protein to bind to the cellular receptor ACE2 (angiotensin converting enzyme 2) to invade cells. Expression of the ACE2 receptor is upregulated by SARS-CoV-2 infection and inflammatory cytokine stimulation. In SARS-CoV infection, the S-protein induces TNF-α-converting enzyme (TACE)-dependent shedding of the ACE2 ectodomain, a process tightly coupled with TNFα production. Loss of ACE2 activity due to shedding is associated with lung damage due to increased activity of the renin-angiotensin system. ACE2 knockout mice are prone to severe respiratory failure after chemical challenge and ACE2 has been shown to ameliorate ACE-induced intracellular inflammation. ACE2 downregulation is associated with severe respiratory distress associated with SARS-CoV infection. Therefore, increased TNFα production can promote viral infection and lead to organ damage such as lung damage.

As discussed, regulatory T cells (Tregs) are a class of immunosuppressive cells that have diverse clinical applications in transplantation, allergy, infectious disease, GVHD, autoimmunity and cancer. Tregs co-express the interleukin-2 receptor alpha chain CD25 ^hi with CD4+ and are characterized by inducible levels of the intracellular transcription factor forkhead box P3 (FOXP3). Naturally occurring Tregs express TNFR2 at higher densities than TNFR1. TNF signaling through TNFR2 promotes Treg activity: TNF-mediated TNFR2 activates and induces the proliferation of Tregs (100), and TNFR2 expression represents maximally suppressive Tregs, thus preventing infection (influenza, SARS type virus, endotoxemia). ), when TNF is overproduced in response to inflammatory stimuli, Tregs can prevent overreaction to inflammatory stimuli.

Anti-TNF could be a treatment for SARS and COVID-19. Adalimumab is being used to treat COVID-19 (China Clinical Trial, February 2020 (ChiCTR2000030089); see, e.g., Lucchino et al. (2020) Rheumatology (Oxford) 59(6) :1200-1203; Haga et al. (2008) Proc. Natl . Acad . Sci . USA . 105 :7809-7814). Knockout of TNFR2 in mice infected with SARS-CoV did not provide any protective effect; Double knockout of TNFR1 and TNFR2 protected infected mice from infection-related weight loss (see, e.g., McDermott et al. (2016) BMC Systems Biology 10:93]). These results indicate that TNF signaling through TNFR1 mainly contributes to the pathogenesis of SARS-CoV infection by increasing the pro-inflammatory process, and that selective inhibition of TNFR1 rather than inhibition of TNF is a better therapeutic approach. Constructs provided herein may be used to treat the acute inflammatory aspects of SARS and COVID-19. The constructs are used with anti-infective agents; The constructs are used to inhibit or ameliorate the acute effects of a cytokine storm.

TNFa inhibition reduces the severity of lung disease caused by viruses, such as those caused by respiratory syncytial virus (RSV) or influenza virus, in mice. In this mouse model, depletion of TNF using an anti-TNF antibody reduces the recruitment of inflammatory cells to the lungs and decreases the production of pro-inflammatory cytokines (e.g., IFNγ, IL-4, IL-5, TNF) by T-cells. production is reduced and reduces disease severity without interfering with viral clearance (see, for example, Hussell et al. (2001) Eur . J. Immunol . 31:2566-2573]). These results indicate that TNF inhibitors and TNF receptor antagonists may be beneficial in the treatment of human viral lung diseases, such as those caused by SARS-CoV and SARS-CoV2, by preventing or reducing TNF-induced immune activation and lung damage.

Allogeneic hematopoietic stem cell transplantation is complicated by the occurrence of non-infectious idiopathic pneumonia syndrome (IPS), an acute lung dysfunction similar to SARS pneumonia. High levels of TNFα have been found in the serum of patients with lung injury after allogeneic stem cell transplantation (SCT), and donor-derived alloreactive T-cells have been shown to be involved in this process. In humans, anti-TNF therapy with etanercept is beneficial for IPS treatment after allogeneic stem cell transplantation. Recipients of allogeneic stem cell transplants may be subject to the immunosuppressive effects of SCT control regimens, the requirement for long-term use of immunosuppressive drugs to prevent or treat graft-versus-host disease (GvHD), and other factors that may impair host defenses. There is a high risk of developing bacterial and fungal infections with SCT complications (including acute GvHD) (see, e.g., Yanik et al. (2002) Biol . Blood Marrow Transplant. 8:395-400]). Other indications that can be treated by the constructs provided herein include women undergoing treatment for chemobrain, particularly breast cancer, a condition experienced during and after chemotherapy. In addition, therapies and constructs herein may be used to treat long-term COVID.

As a result, the use of selective TNFR1 antagonists that maintain protective TNF signaling through TNFR2 and, unlike anti-TNF therapies, do not increase the risk of serious infection, can treat, prevent or ameliorate viral- and non-viral induced lung injury. offer safer and more effective treatment options for Thus, the constructs provided herein are ideal therapeutics for these indications.

D. rheumatic Therapy for arthritis and other chronic inflammatory autoimmune diseases and disorders

Although there is no cure for rheumatoid arthritis (RA), treatment can improve symptoms and slow disease progression, for example by minimizing pain and swelling, preventing bone deformities, and maintaining daily function. The first-line treatment for RA is disease-modifying antirheumatic drugs (DMARDs), which are effective against other chronic inflammatory and autoimmune diseases and disorders, such as psoriasis, plaque psoriasis, psoriatic arthritis, juvenile idiopathic arthritis, ankylosing spondylitis, and Behçet's disease. , inflammatory bowel disease (IBD; eg, Crohn's disease and ulcerative colitis), multiple sclerosis, and lupus, as well as treatment of some cancers.

DMARDs are immunosuppressive and immunomodulatory agents that are classified as conventional synthetic DMARDs (csDMARDs), or biological DMARDs (bDMARDs; eg, antibodies and fusion proteins). Conventional synthetic DMARDs include, for example, methotrexate (MTX), chemotherapeutic agents and immunosuppressive agents; hydroxychloroquine (HCQ; Plaquenil ^® ), an anti-malarial agent; sulfasalazine (Azulfidine ^® ), an anti-inflammatory drug; and leflunomide ( ^Arava® ), an immunosuppressive agent that inhibits dihydroorotate dehydrogenase. Biological DMARDs include, for example, abatacept (Orencia ^® ), a fusion protein that prevents T cell activation and contains the Fc region of IgG1 fused to the extracellular domain of CTLA-4; anakinra, a recombinant human IL-1 receptor antagonist (sold eg under the trade name ^Kineret® ); Rituximab, a chimeric monoclonal antibody to CD20 that induces apoptosis in CD20 ⁺ cells such as B cells (sold under trademarks including Rituxan ^® , Truxima ^® , MabThera ^® ); tocilizumab (atlizumab, ^Actemra® , ^RoActemra® ), a humanized monoclonal antibody to the IL-6 receptor (IL-6R); corticosteroids; tofacitinib (Xeljanz ^® ), a small molecule inhibitor of Janus kinase (JAK), a protein tyrosine kinase involved in mediating cytokine signaling; and TNF-inhibitor/anti-TNF agents such as certolizumab pegol ( ^Cimzia® ), infliximab ( ^Remicade® ), adalimumab ( ^Humira® ), golimumab ( ^Simponi® ), and etaner including sept (Enbrel ^® ). In particular, combination therapy of methotrexate and biologic DMARDs is more effective than monotherapy. Combination therapy may also include multiple csDMARDs together with multiple csDMARDs and one biologic DMARD. Due to the risk of serious side effects, including serious infections, multibiological DMARDs, particularly anti-TNF DMARDs, are not generally used in combination therapy regimens.

The following sections describe existing therapies and the problems associated with each to highlight how the therapies provided herein address the problems.

1. Conventional Synthetic Disease Modifying Anti-Rheumatic Drugs (csDMARDs)

Conventional synthetic disease-modifying antirheumatic drugs (csDMARDs) are generally first-line treatments for RA and other autoimmune and chronic inflammatory diseases and disorders. csDMARDS includes drugs such as methotrexate, leflunomide, hydroxychloroquine, and sulfasalazine, and methotrexate is the most commonly used agent for initial treatment, and its mechanism of action is to stimulate the release of adenosine from fibroblasts, reduce neutrophil adhesion , inhibition of leukotriene B4 synthesis by neutrophils, inhibition of local IL-1 production, reduction of IL-6 and IL-8 levels, suppression of cell-mediated immunity, and inhibition of synovial collagenase gene expression. Other conventional synthetic DMARDs act by inhibiting the proliferation of lymphocytes or by causing lymphocyte dysfunction. For example, leflunomide inhibits dihydroorotate dehydrogenase, thereby inhibiting pyrimidine synthesis and blocking lymphocyte proliferation. Sulfasalazine mediates anti-inflammatory effects by preventing oxidative, nitrifying and nitric damage, and hydroxychloroquine is a weak immune modulator that inhibits intracellular Toll-like receptor 9 (TLR9).

Hydroxychloroquine, which has the best safety profile of conventional DMARDs, does not increase the risk of infection and does not cause hepatotoxicity or renal dysfunction; Common side effects of hydroxychloroquine include rash and diarrhea. Retinopathy/maculopathy is a rare but serious side effect of hydroxychloroquine therapy and is associated with doses greater than 5 mg/kg/day, long-term use (>5 years of therapy), old age and chronic kidney disease. Other rare side effects of hydroxychloroquine include anemia, leukopenia, myopathy and cardiomyopathy. Therapy with methotrexate, leflunomide, and sulfasalazine is associated with increased rates of nausea, abdominal pain, diarrhea, rash/allergic reactions, bone marrow suppression, hepatotoxicity, and common and sometimes serious infections. Methotrexate and leflunomide also cause alopecia. Other side effects associated with methotrexate therapy include interstitial lung disease, folic acid deficiency and cirrhosis of the liver. Leflunomide has also been associated with hypertension, peripheral neuropathy and weight loss. Sulfasalazine carries a very high risk of gastrointestinal upset and may rarely cause DRESS syndrome (a drug reaction with eosinophilia and systemic symptoms) (see, e.g., Benjamin et al. Disease Modifying Anti-Rheumatic Drugs (DMARD) [February 2020 Updated 27th]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2020 Jan. Source: URL: ncbi.nlm.nih.gov/books/NBK507863/]). These drugs are effective because they are immunosuppressive. Constructs provided herein that are selective anti-TNFR1 antagonists that advantageously preserve TNFR2 immunosuppressive activity may avoid the need for these immunosuppressive drugs.

2. Anti-TNF Therapy/TNF Blockers

Anti-TNF therapy/TNF-blockers (a type of biological DMARD) are typically prescribed after failure of conventional DMARDs and include monoclonal antibodies (mAbs) such as the chimeric mAb infliximab ( ^Remicade® ); contains a murine variable region and a human IgG1 constant region, and fully humanized mAbs (IgG1s) adalimumab ( ^Humira® ) and golimumab ( ^Simponi® ); PEGylated humanized Fab' fragment of a mAb targeting TNF, certolizumab pegol ( ^Cimzia® ); and TNFR2 fusion proteins such as etanercept ( ^Enbrel® ), a TNFR2-Fc fusion protein containing an extracellular receptor region containing the binding site of human TNFR2 fused to the Fc of human IgG1. Remsima ^® and Inflectra ^® are biosimilars of infliximab approved for use in the European Union for the treatment of a variety of autoimmune and chronic inflammatory diseases and disorders. Such TNF inhibitors that sequester TNF can treat, for example, RA, psoriasis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis (JIA), and/or inflammatory bowel disease (IBD; eg, Crohn's disease and ulcerative colitis). It is used for the treatment of a variety of diseases and conditions including

Because of the immunosuppressive effect of therapies targeting TNF, these therapies are, for example, sepsis and serious infections such as listeriosis, reactivation of tuberculosis, reactivation of hepatitis B/C, shingles, and reactivation of invasive fungi. It is associated with severe side effects including increased risk of activation and other opportunistic infections. TNF is a key cytokine in the inflammatory and immune response to infection, and the use of drugs that remove TNF impairs host immunity to microorganisms, increasing the risk of infection. For example, TNF blockers have been implicated in reactivation of M. tuberculosis infection. TNF plays an important role in resistance to Mycobacterium tuberculosis , and adalimumab therapy in RA patients significantly reduces responsiveness to M. tuberculosis . As described herein, reduced immune reactivity can be associated with activation of Tregs and induction of apoptosis in effector lymphocytes. Anti-TNF therapy has been shown to induce macrophage apoptosis in rheumatoid synovial membranes. Infliximab is associated with increased apoptosis of inflammatory cells infiltrating the digestive tract of patients with Crohn's disease. Other anti-rheumatic drugs such as methotrexate and glucocorticoids can also induce apoptosis in immune cells (see, eg, Vigna-Pιrez et al. (2005) Clin . Exp . Immunol . 141(2):372-380). The TNFR2-Fc fusion proteins adalimumab and infliximab, but not etanercept, induce caspase-dependent apoptosis in cultured monocytes and downregulate IL-10 and IL-12 production by monocytes (e.g. For example, see Shen et al. (2005) Ailment Pharmacol . Ther . 21:251-258]). The most common fungal infections associated with TNF blockers are histoplasmosis, candidiasis and aspergillosis. Anti-TNF agents can also cause aggravation of severe congestive heart failure, drug-induced lupus, demyelinated central nervous system (CNS) disease, as well as lymphomas and non-melanoma skin cancers (see, e.g., Benjamin et al. Disease Modifying Anti -Rheumatic Drugs (DMARD) [updated 27 Feb 2020].In: StatPearls [Internet].Treasure Island (FL): StatPearls Publishing;Jan 2020. Source: ncbi.nlm.nih.gov/books/NBK507863/] reference).

Infliximab has also been associated with the development of leukopenia, neutropenia, thrombocytopenia and pancytopenia (some fatal). Etanercept is associated with an increased incidence of opportunistic bacterial and viral infections in patients with RA. Etanercept is also used to treat severe refractory graft-versus-host disease (GvHD). Severe GvHD subjects treated with etanercept have Aspergillus fumigatus fumigatus ) is very high (100% in one study, see Zoran et al. (2019) Sci. Rep. 9:17231), a life-threatening fungus (i.e., fungus). ). Treatment with etanercept upregulates CXCL10, a chemokine from immune cells, such as CXCL10, as well as downregulation of genes involved in immune response and TNF signaling, including genes involved in NF-κB signaling, antimicrobial humoral response, and apoptotic processes. results in decreased secretion of (see, eg, Zoran et al. (2019) Sci. Rep. 9:17231).

Other side effects associated with the use of TNF blockade therapy are congestive heart failure, liver damage, demyelinating disease/CNS disorders, increased susceptibility to the development of additional autoimmune diseases as well as cancer, including lupus, psoriasis, sarcoidosis, lymphoma and solid malignancies. (see, e.g., Dong et al. (2016) Proc . Natl . Acad . Sci . USA. 113(43):12304-12309; Zalevsky et al. (2007) J. Immunol . 179:1872-1883; Zoran et al. (2019) Sci . Rep. 9:17231]). Therefore, abrogation of all TNF-mediated signaling by sequestering TNF is not an ideal therapeutic strategy as it results in severe immunosuppression that can lead to serious, sometimes fatal infections and other dangerous side effects.

Anti-TNF therapy improves RA but is not curative and requires years of continuous and costly therapy. Inhibition/blockade of TNF in RA reduces inflammation and joint destruction, but, as discussed above, is associated with an increased risk of serious infections such as tuberculosis and listeriosis due to immunosuppression. As a result, the use of TNF blockers is limited, particularly in chronic diseases/conditions requiring long-term administration, such as arthritis and IBD. About 30% of RA patients are unresponsive or do not receive sustained therapeutic benefit with anti-TNF therapy (see, e.g., McCann et al. (2014) Arthritis & Rheumatology 66(10):2728-2738). Failure to respond also occurs in non-RA patients receiving anti-TNF therapy. Depending on the anti-TNF agent, 13-33% of treated patients do not respond to treatment, and up to 46% stop responding, leading to discontinuation of treatment or increase in dose (see, for example, Richter et al. (2019) MABS 11(4):653-665). Therefore, there is a need for therapies with improved therapeutic efficacy and safety.

Anti-TNF therapeutics block/sequester TNF and inhibit soluble TNF (solTNF) and transmembrane TNF (tmTNF) signaling through TNFR1 and TNFR2, respectively; solTNF signaling is associated with chronic inflammation, whereas tmTNF signaling is associated with resolution of inflammation and pathogens such as Listeria monocytogenes. monocytogenes ) and Mycobacterium tuberculosis ( Mycobacterium tuberculosis ) are associated with the induction of immunity. The major anti-inflammatory effect of anti-TNF therapy is achieved by blocking TNFR1, whereas blocking TNFR2 inhibits Treg cell activity. As discussed elsewhere herein, TNFR1 signaling is primarily inflammatory and is involved in the pathogenesis of inflammatory and autoimmune diseases and conditions such as RA, psoriasis, IBD, and neurodegenerative disorders such as MS; On the other hand, TNFR2 signaling has anti-inflammatory and protective effects in various cell and organ types, including nerve, heart, digestive tract and bone tissue, and is also involved in host defense mechanisms against pathogen infection. Thus, as described herein, selective blockade of TNFR1 can be used with anti-TNF therapy by preserving the beneficial effects of TNFR2 signaling while eliminating undesirable pro-inflammatory signaling associated with RA and other autoimmune and inflammatory diseases and conditions. to improve therapeutic efficacy (see, eg, McCann et al. (2014) Arthritis & Rheumatology 66(10):2728-2738; Schmidt et al. (2013) Arthritis & Rheumatism 65(9):2262-2273; Bluml et al. (2012) International Immunology 24(5):275-281; Zalevsky et al. (2007) J. Immunol. 179:1872-1883]).

Anti-TNF therapy has failed to treat neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, stroke and multiple sclerosis (MS) associated with overexpression of TNF. For example, in a phase 2 trial for the treatment of relapsing-remitting MS, the TNFR1 receptor-Fc IgG1 fusion protein anti-TNF treatment Renecept (Ro 45-2081) failed, and symptoms increased compared to patients receiving a placebo. / has worsened, and the neurological deficit is more severe in patients treated with Renercept. These results indicate that anti-TNF therapy can exacerbate demyelinating disease. TNFR1 has been shown to mediate inflammatory neurodegeneration, but TNFR2 induces neuroprotection, so blockade of signaling through both receptors by anti-TNF therapy eliminates the neuroprotective effects of TNFR2 signaling. Blocking TNFR1 with ATROSAB, a humanized TNFR1-blocking monoclonal antibody, or activating TNFR2 with EHD2-scTNF _R2 , an potent TNFR2-selective TNF mutein, protects cholinergic neurons against apoptosis and in a mouse model of NMDA-induced acute neurodegeneration. Reverse neurodegenerative-associated memory impairment. It is likely to be immunogenic. ATROSAB is a partial TNFR1 agonist; One skilled in the art would not administer a TNFR1 agonist. However, blockade of TNFR1 and TNFR2 negates the therapeutic effect, indicating that TNFR2 plays an essential role in neuroprotection and that selective blockade of TNFR1 can be used in the treatment of neurodegenerative diseases in which anti-TNF therapy has failed (see, for example, Dong etc. (2016) Proc . Natl . Acad . Sci. USA 113(43):12304-12309).

Due to side effects associated with the use of anti-TNF agents, non-response in some patients, continued lack of response in patients who had an initial response, treatment failure and/or worsening of a neurodegenerative disease such as MS, other therapies are needed. Such therapies are provided herein.

E. TNFR1/TNFR2 Targeted Therapies

The following section discusses exemplary therapeutics targeting TNFR1/TNFR2 and describes some of the problems and limitations of these therapeutics. Existing therapeutics may be modified as described in Section F and the Examples, used in whole or in part, or modified to improve properties for use in the constructs provided herein.

1. TNFR1 selective antagonists

As discussed and provided herein, therapy with TNF blockers such as etanercept, infliximab, adalimumab, and the like, abrogates TNF signaling through TNFR1 and TNFR2. TNFR1 signaling induces inflammation, cytotoxicity and apoptosis, whereas TNFR2 signaling protects in part due to expansion and activation of immunosuppressive Tregs that destroy effector T cells in an autoimmune environment, preventing tissue destruction and disease progression. and anti-inflammatory. Treatment with TNF blockers may result in an inflammatory microenvironment through inhibition of TNFR2 signaling and consequent depletion of immunosuppressive Tregs, failing to treat or exacerbating autoimmune and inflammatory diseases and disorders. Double blockade of TNFR1 and TNFR2 can also lead to opportunistic infections and cancer.

As provided herein, specific inhibition of TNFR1 signaling maintains normal TNFR2 function, which maintains an equilibrium between pro-inflammatory and anti-inflammatory activities through the generation of subsets of both regulatory and cytotoxic T cells. needed to Selective TNFR1 inhibition retains the potent anti-inflammatory activity of TNFR2 signaling, reduces opportunistic infections and cancer, and preserves TNF-induced Treg function.

a. TNFR1 antagonistic antibody

Among the TNFR1 antagonist antibodies is ATROSAB (antagonistic TNF receptor monospecific antibody). ATROSAB, the first TNFR1 blocking antibody, is a full-length IgG1 humanized version of the neutralizing mouse anti-human TNFR1 monoclonal antibody H398. It was abandoned as a therapeutic because it has partial agonistic activity to mimic TNF activity, a toxic pathway by activating TNFR1. ATROSAB maintains the TNFR1 conformation in an inactive state and blocks TNF binding. The Fc region of ATROSAB has been mutated to eliminate FCγR receptor binding and complement fixation, thereby avoiding unwanted immune system activation (see, e.g., Kalliolias and Ivashkiv (2016) Nat. Rev. Rheumatol. 12(1):49- 62]).

Full-length antibodies have the advantage of increased half-life in vivo, but are not suitable for development of TNFR1 antagonists due to receptor crosslinking, which tends to agonize instead of antagonize TNFR1, as discussed elsewhere herein. This is not a result of Fc cross-linking as the Fc-interacting portion of the antibody has been removed by mutation. For example, IgG ATROSAB exhibited some TNFR1 agonistic activity in the absence of TNF observed to a limited extent over a narrow concentration range due to its bivalent molecular structure. Cross-linking of TNFR1 can occur due to secondary events such as interaction with FcγRs or anti-drug antibodies (ADAs), which should be avoided to maintain the antagonistic properties of TNFR1 inhibitors. ADA was observed at rates of 50% and 31%, respectively, in patients treated with infliximab or adalimumab (see, eg, Richter et al. (2019) mAbs 11(4):653-665; See Richter et al. , (2019) mAbs 11(1):166-177).

b. Monovalent TNFR1 antagonistic antibodies/antibody fragments

Small antibody fragments such as domain antibodies and derivatives and modified forms thereof have been developed, and exemplary antibody fragments and modified forms are discussed in the following sections. However, small antibody fragments have not been successfully developed as pharmaceuticals. They have limited use as therapeutics; They have a short serum half-life and rapid peripheral clearance, which is a result of their small size. For example, molecules less than 50-60 kDa in size are subject to renal filtration; dAbs and other antibody fragments less than 50-60 kDa in size are rapidly cleared by the kidney. For example, dAb designated DMS5541 and similar molecules exhibit selectivity for TNFR1 and can potentially inhibit the detrimental effects of TNFR1 signaling. DMS5541, composed of two dAbs (anti-TNFR1 and anti-human serum albumin), is only about 25 kDa in size and is too small to have desirable pharmacokinetics for therapeutic purposes. Its binding to HSA, implying half-life stabilization, is only 34 nM, meaning that it is often in a dissociated state with respect to HSA. The single domain antibodies (sdAbs) tested so far are expressed in E. coli and are prone to aggregation (unfolding) during manufacturing. In addition, cytoplasmically produced sdAbs (directly expressed in E. coli ) often lack the conserved disulfide bonds found in the variable heavy chain domain, which can reduce melting point and reduce refolding ability. The rapid clearance and short elimination half-lives of small antibody fragments, which may be less than a few hours, may reduce efficacy in vivo and require frequent dosing and/or continuous infusion, reducing patient compliance. Such molecules (see, for example, Holland et al. (2013) J Clin Immunol 33(7) :1192-203]) was produced in E coli and was often incorrectly folded resulting in poor solubility and immunogenicity, resulting in clinical failure (see, for example, adisinsight.springer.com/ drugs/800037882).

Constructs, such as the TNFR1 antagonist constructs provided herein, address these issues as well as other issues such as immunogenicity and reaction with pre-existing antibodies. Provided herein are constructs containing small antibody fragments, such as dAbs, having specificity for TNFR1 and/or TNFR2, which exhibit prolonged serum half-life, increased stability, reduced/slowed peripheral clearance and immunogenicity compared to prior art dAbs. It exhibits improved pharmacological properties, such as pharmacokinetic properties, including reduction.

Therapeutic antibodies of various structures can be potent and well-tolerated therapeutics. Antibodies may be used for, for example, rheumatoid arthritis (eg, adalimumab, sold under the trademark ^Humira® ); cancers such as non-Hodgkin's lymphoma (eg, rituximab and ibritumomab tiuxetan, sold under the tradenames ^Rituxan® and ^Zevalin® , respectively) and breast and gastric cancer (eg, trastuzumab, tradename Herceptin sold as ^® ); and respiratory syncytial virus infections (eg, palivazumab, sold under the tradename ^Synagis® ). Preparation of intact antibodies has several limitations, such as reliance on mammalian cell expression. As a result, antigen-binding fragments of antibodies such as Fab (~57 kDa) and single chain Fv fragments (scFv, ~27 kDa) and phage display (escape animal immunization) can be selected in vitro and using bacterial or yeast cell cultures. Other structures have been developed that can be manufactured in large quantities. Fab fragments contain a V _H -C _H 1 polypeptide linked to a V _L -C _L polypeptide via a disulfide bond; A scFv is a fusion protein containing a V _H domain and a V _L domain linked by a short polypeptide linker. Another class of therapeutics, which are small fragments of antibodies, are domain antibodies (dAbs; also known as single domain antibodies or sdAbs), which are monomeric and contain the variable domain of either the heavy (V _H ) or light (V _L ) chain of an antibody. dAb is the smallest antigen-binding fragment of an antibody; They are about 11-15 kDa in size, which is about 1/10 the size of all monoclonal antibodies (mAbs) (see, e.g., Holt et al. (2003) Trends in Biotechnology 21(11):484-490). Similar to dAbs, nanobodies (Nb) are small antigen-binding fragments derived from camel heavy chain antibodies that lack light chains. Nanobodies are small (~15 kDa), low immunogenic, high affinity, soluble, stable, encoded by a single gene/exon (VHH), and modular, allowing high-yield production in bacteria and yeast. (See, e.g., Steeland et al. (2015) J. Biol . Chem . 290(7):4022-4037; Steeland et al. (2017) Sci . Reports 7:13646]).

i. Fab- and scFv-based TNFR1 antagonists

As discussed above, ATROSAB, a humanized semi-agonistic/antagonistic TNFR1 specific antibody, inhibits TNFR1 mediated cellular responses. ATROSAB exhibits some TNFR1 agonistic activity due to its bivalent molecular structure or due to binding to TNFR1 in the absence of TNF. The parental mouse antibody, H398, possesses stronger inhibitory potential due to the faster dissociation of ATROSAB (ie higher k _off value) compared to H398. This was determined using quartz crystal microbalance (QCM) measurements where the antigen density of the chip was reduced to favor monovalent interactions; The slower dissociation of monovalently bound H398 from TNFR1 and consequently longer receptor occupancy contributes to improved blockade of TNFR1. Therefore, in order to eliminate the TNFR1 agonistic activity of ATROSAB and improve the TNFR1 antagonistic activity of ATROSAB, monovalent derivatives of ATROSAB have been developed.

To increase the affinity and antagonistic activity of ATROSAB, single-chain variable fragments (scFvs) of ATROSAB are prepared by site-directed mutagenesis of exposed residues within individual CDRs or combinations of CDRs and selection by phage display against human TNFR1-Fc. It has undergone first-order affinity maturation. The scFv of ATROSAB contains a V _H domain corresponding to residues 1-115 of the ATROSAB heavy chain (see SEQ ID NO: 31) linked to a VL domain by a short peptide linker corresponding to residues 1-113 of the ATROSAB light chain (SEQ ID NO: 31). 32). A clone with six mutations in CDR-H2 of the ATROSAB heavy chain, Y52V, Y54T, S55Q, H57E, Y59K and E62D, scFv IG11 (see SEQ ID NO: 674) exhibits slower receptor dissociation and human TNFR1 with respect to SEQ ID NO: 31 - Improved equilibrium binding to Fc, and improved inhibition of TNF-induced TNFR1 activation. This clone was further subjected to random mutagenesis to obtain mutations Q1H, Y52V, Y54S, S55Q, H57E, Y59K, and E62D (see SEQ ID NO:31) in the V _H domain, and S96G (see SEQ ID NO:32) in the V _L domain. ) to create clone scFv T12B (reference, SEQ ID NO: 675) containing. scFv T12B reduced dissociation from immobilized TNFR1-Fc and increased TNFR1 inhibitory activity compared to the scFv of ATROSAB and scFv IG11 (see, e.g., Richter et al. (2019) mAbs 11(1):166-177; See also Richter, F. Thesis, entitled "Evolution of the Antagonistic Tumor Necrosis Factor Receptor One-Specific Antibody ATROSAB," Universitδt Stuttgart, 2015; available at pdfs.semanticscholar.org/d8e7/8b87d76dce36225c1d497939ef37445cfa8a.pdf).

The humanization of H398 was reengineered by exchanging the VH and VL framework regions of H398 with alternative germline genes to optimize the CDR arrangement. scFv 13.7 containing the VH domain of scFV T12B linked to the newly humanized VL domain of H398 by a short peptide linker, binding similar to human TNFR1-Fc in ELISA and QCM, improved inhibition of TNF-induced TNFR1 activity, improved thermal stability, scFv It showed 10 ℃ higher melting point than T12B. Based on scFv 13.7, IgGs and Fabs with the same constant region as ATROSAB were generated (IgG 13.7 and Fab 13.7, respectively), compared to ATROSAB (1.4-fold) and the Fab of ATROSAB (Fab ATR; 8.7-fold), respectively, to TNFR1. binding to increased. Fab 13.7 also has reduced dissociation from immobilized TNFR1-Fc compared to Fab ATR and has an 18.8-fold improved monovalent affinity. Thus, affinity maturation and framework replacement resulted in improved binding to TNFR1 for Fab 13.7. Fab 13.7 and IgG 13.7 showed selectivity for TNFR1-Fc and did not bind the TNFR2-Fc fusion protein; Fab 13.7 bound human and rhesus TNFR1-Fc, but not mouse and rat TNFR1-Fc, showing a similar binding pattern to ATROSAB. In vitro, monovalent Fab ATR and Fab 13.7 did not activate TNFR1, whereas ATROSAB showed minor activation of TNFR1 activity and IgG 13.7 strongly activated TNFR1. The potent activity of IgG 13.7 may be due to improved affinity and slow dissociation from TNFR1, resulting in the formation of stable signaling competent receptor-antibody complexes. Fab 13.7 showed improved inhibition of TNFR1 activity compared to Fab ATR and ATROSAB and lacked any agonistic activity. Incubation of Fab 13.7 or ATROSAB with cross-linked anti-human Fab serum has shown that Fab 13.7 does not activate TNFR-1 whereas ATROSAB does (see, for example, Richter et al. (2019) mAbs 11(1):166-177).

Compared to ATROSAB with an initial half-life of 0.44 hours, a terminal half-life of 32.1 hours, and an area under the curve (AUC) of 181 μg/ml xh, Fab 13.7 (molecular mass ~47 kDa). Fab 13.7 exhibited an initial half-life of 0.08 hours, a terminal half-life of 1.4 hours and an AUC of 4.2 μg/ml xh, similar to the values obtained for Fab ATR. To prolong the half-life, a Fab' fragment, Fab' 13.7, was created by introducing a free cysteine residue at the C-terminus of the CH1 domain, which is a branched PEG ₄₀ Chemically coupled to _{the kDa} moiety yielded Fab 13.7PEG. Fab 13.7 was also fused to Fd via a short flexible linker to the N-terminus of mouse serum albumin (MSA) to create Fab13.7-MSA. A monovalent Fab-Fc fusion protein is generated by fusing Fab 13.7 to a modified Fc that lacks the cysteine residues in the hinge region and the ability to dimerize through the CH3 domain, resulting in a one-armed half-IgG molecule (IgG1 _half 13.7) did A monovalent Fv-Fc molecule was generated by fusing the VH and VL domains to a heterodimerizing knob-into-hole (kih) Fc chain lacking a cysteine residue in the hinge region (Fv13.7-Fc _kih ). None of the derivatives showed any potent TNFR1 activity and compared to Fab 13.7, slightly reduced binding to human TNFR1-Fc was observed for Fab13.7PEG, Fab13.7-MSA and IgG1 _half 13.7; The binding of Fv13.7-Fc _kih was not affected. Inhibition of TNF-mediated TNFR1 activity was reduced 1.5-3.3 fold compared to Fab 13.7; Fab13.7PEG showed the strongest impairment in function, and Fv13.7-Fc _kih showed the lowest change in bioactivity. IgG _half 13.7 showed a half-life similar to Fab 13.7, and the AUC value increased 7.1 times. The terminal half-lives of Fab13.7PEG, Fab13.7-MSA and Fv13.7-Fc _kih were extended to 14.4 hours, 9.7 hours and 10.5 hours, respectively, and the AUC values were increased. Therefore, the fusion protein Fv13.7-Fc _kih engineered for heterodimeric assembly of two peptide chains using the knobs-into-holes technology has improved pharmacokinetic properties and TNFR1 antagonistic activity. The best combination is indicated (see, eg, Richter et al. (2019) mAbs 11(1):166-177; See also Richter, F. Thesis, entitled "Evolution of the Antagonistic Tumor Necrosis Factor Receptor One-Specific Antibody ATROSAB," Universitδt Stuttgart, 2015; available from pdfs.semanticscholar.org/d8e7/8b87d76dce36225c1d497939ef37445cfa8a.pdf).

In another study, to improve pharmacokinetic properties such as serum half-life of Fab 13.7, an IgG-like Fc was incorporated into the molecule while maintaining the Fab-like heterodimerization of the polypeptide chain. To achieve this, the variable domains of the heavy and light chains of Fab 13.7 were fused to the N-terminus of a newly generated heterodimerizing Fc chain known as Fc-on/kappa (Fc1κ). The Fc heterodimerization approach is based on interspersed Ig domains derived from heterodimerizing IgG1 constant heavy chain domains (CH1) and kappa light chain constant domains (CLκ), mediating FcRn binding and FcRn-mediated drug recycling in vivo. contains a section of the IgG1 CH3 sequence that enables Interspersed Ig domains include “CH31”, which contains amino acid sequence fragments of CH1 and CH3, and “CH3kappa” (CH3), which contains amino acid sequence fragments of CLκ and CH3. The IgG1 CH2 domain was also fused to the N-terminus of the CH31 and CH3κ domains to encompass the entire FcRn binding region of the IgG molecule. Addition of an IgG1 hinge region to the N-terminus of the CH2 domain creates a covalently linked heterodimerizing Fc moiety known as Fc1κ. Unlike other Fc heterodimerization techniques, such as knob-into-hole, which involve one or more amino acid replacements at the CH3-CH3 interface, Fc heterodimerization was achieved by exchanging larger stretches of amino acid sequences obtained from human antibody sequences. . Asymmetric scFv-Fc1κ fusion proteins were prepared and compared to scFv fusions with Fcs containing knob-into-holes, and heterodimer formation was similar or improved compared to fusions containing knob-into-hole technology (e.g. See, eg, Richter et al. (2019) mAbs 11(4):653-665).

The variable domains of the TNFR1-specific Fab 13.7 molecule fused VH to the CH2-CH3κ chain and VL to the CH2-CH31 chain (VL13.7-CH2-CH31/VH13.7-CH2-CH3κ; VL1C/VHκC) , generating the monovalent TNFR1-specific antagonistic antibody-derived molecule (Fv-Fc1κ fusion protein) known as atroximab (size 72 kDa), using a short peptide linker as a CH31- or CH2 domain N- of a CH3κ-containing Fc chain. fused at the end. Atroximab lacks the ability to mediate Fc effector function due to mutations introduced into Fc1κ; Lack of binding to effector molecules of the immune system prevents activation of TNFR1 due to secondary cross-linking of atroximab bound to cells expressing FcγRs. Atroximab, which binds TNFR1 with high affinity (K _D 2.7 nM), inhibited TNF-induced activation of TNFR1 with IC ₅₀ values of 16-55 nM in various in vitro assays and was compared with anti-human IgG antibodies (i.e., cross-linked antibodies). ) showed improved pharmacokinetic properties in the presence of Compared to the parental Fab 13.7 molecule, TNFR1 binding and inhibition were slightly reduced, which could be attributed to alterations in VH and VL pairing after fusion to the CH2 domain. The early and terminal half-lives of atrosimab were determined to be 2.2 +/- 1.2 h and 41.7 +/- 18.1 h, respectively, with an AUC of 5856 +/- 1369.9 μg/ml xh. The terminal half-life of Atroximab was extended almost 40-fold compared to Fab 13.7 and extended 1.3-fold compared to ATROSAB; These values may be imprecise, but since the infusion doses of Fab 13.7 and ATROSAB were lower, this may affect the pharmacokinetic properties (see, e.g., Richter et al. (2019) mAbs 11(4):653- 665]).

ii. TNFR1 antagonists based on domain antibodies (dAbs)

Another class of therapeutic agent, which is a small fragment of an antibody, is a domain antibody (dAb; also known as a single domain antibody or sdAb), which is a monomer and contains the variable domain of either the heavy (V _H ) or light (V _L ) chain of an antibody. dAb is the smallest antigen-binding fragment of an antibody; They are about 11-15 kDa in size, which is about 1/10 the size of all monoclonal antibodies (mAbs). Similar to dAbs, nanobodies originating in Camelidae produce antibodies containing only a heavy chain in which the antigen binding site is a single unpaired variable domain known as _VHH . In the dAb, there are three complementarity determining regions (CDRs) in each V _H and each V _L ; Thus, each dAb contains three of the six CDRs from a V _H -V _L pair of antibodies, which are highly divergent loop regions that bind the target antigen.

Because of their smaller size, dAbs are produced in higher yields in bacterial culture and are better suited for phage display because only a single polypeptide chain is produced. Specific dAbs with high affinity and potency can be rapidly produced by protein engineering. The small size of dAbs also allows for increased tissue penetration, stability and choice of delivery formulation. Because of their small size, it is possible to generate molecules containing linked dAbs specific for different antigens/targets, which is impossible with conventional antibodies and difficult to achieve with other antibody fragments such as Fabs and scFvs. Due to the monomeric and monovalent binding behavior of dAbs, they are suitable for use where the target cannot be intervened with monoclonal antibodies. TNFR1 is one such target and TNFR1 is activated/agonized by antibody-induced receptor cross-linking (see, e.g., Holt et al. (2003) Trends in Biotechnology 21(11):484-490; Schmidt et al. (2013) Arthritis & Rheumatism 65(9):2262-2273; Goodall et al. (2015) PLoS ONE 10(9):e0137065).

Small size antibody fragments such as dAbs, scFvs, Fvs, disulfide-linked Fvs and Fabs are easier to produce and handle and rapidly distribute throughout the body compared to larger molecules; The short in vivo half-life limits therapeutic efficacy. As with other antibody fragments, increasing the serum half-life of a dAb increases therapeutic efficacy and reduces dosing frequency, particularly in applications where binding antigen is required in the bloodstream, such as rheumatoid arthritis or cancer treatment. This can be achieved by PEGylation, conjugation to serum albumin, fusion to a second dAb that specifically binds serum albumin, or fusion to an Fc fragment or complete antibody constant region. Fusion to an Fc region also allows recruitment of Fc effector functions, including complement activation, antibody-dependent cytotoxicity or Fc-mediated clearance of immune complexes (see, e.g., Holt et al. (2003) Trends in Biotechnology 21(11):484-490; Goodall et al. (2015) PLoS ONE 10(9):e0137065).

a) anti-TNFR1 dAb-anti-albumin dAb fusion constructs

DMS5540 is a 25 kDa mouse TNFR1 antagonist, a bispecific single variable domain antibody containing a non-competitive (does not interfere with TNF binding) anti-TNFR1 dAb fused to an albumin binding dAb (AlbudAb; to extend serum half-life). DMS5540, which does not bind human TNFR1, was found to inhibit TNFα-mediated cytotoxicity in the mouse fibroblast cell line L929, which is highly sensitive to TNFα-mediated cytotoxicity. Serum IL-6 levels were evaluated by intravenous bolus injection of TNFα 4 hours after intravenous injection of DMS5540 into mice. DMS5540 lacks specific antigen binding but has a capacity for TNFα-mediated signaling effects in vivo as determined by reduced IL-6 responses when compared to mice dosed with a control dAb fused to AlbudAb (DMS5538) or a control dAb without dAb. Dependent inhibition was demonstrated. (See, e.g., Goodall et al. (2015) PLoS ONE 10(9):e0137065).

In another study, mice with collagen-induced arthritis (CIA) were treated with DMS5540, an isotype (negative) control dAb (DMS5538), or a drug that blocks both receptors (TNFR1 and TNFR2) and inhibits mouse TNF for 10 days from the onset of arthritis. Treatment with murine TNFR2 genetically fused to mouse IgG1 Fc and disease progression monitored. Concentrations of systemic cytokines were measured, numbers of T cell sub-sets in lymph nodes and spleen were evaluated, and intrinsic Treg cell function was assessed. Disease progression was similarly inhibited by TNFR1 blockade with DMS5540 and TNFR1/2 blockade with mTNFRII-Fc compared to negative controls, indicating that TNFR1 or TNF blockade protects joints from inflammatory mediators that lead to joint damage in arthritis . Effector T cell activity, as measured by expression levels of pro-inflammatory cytokines (e.g., IFNγ, IL-10, and RANTES), increased after TNFR1/2 blockade with mTNFRII-Fc, but not after selective TNFR1 blockade with DMS5540. did not increase, indicating an immune modulatory role for TNFR2 signaling (eg suppression of T cell effector function). In addition, blocking TNFR1 but not TNFR1/2 resulted in expansion and activation of Treg cells, whereas increased expression of FoxP3 and TNFR2 expressed by Treg was observed in joints that went through remission, indicating a role in relieving inflammation. These results indicate that inhibition of signaling of TNFR1, but not TNFR2, inhibits inflammation and promotes Treg cell suppressor activity, resulting in enhanced therapeutic efficacy compared to conventional TNF inhibition methods (see, for example, McCann et al. (2014) Arthritis & Rheumatology 66(10):2728-2738).

DMS5540 also prevents inflammation-induced osteoclastogenesis and bone loss more effectively than mTNFR2.Fc (anti-TNF) in an in vivo mouse model of lipopolysaccharide (LPS) induced osteolysis. TNFR2-deficient mice showed increased LPS-induced bone destruction. In vitro, DMS5540, the human equivalent of DMS5541, containing the anti-human TNFR1 dAb reduced human osteoclastogenesis more effectively than etanercept in the presence and absence of low-dose TNF. These results indicate a bone protective role for TNFR2 signaling. Consequently, selective inhibition of TNFR1 may be used for therapeutic intervention in inflammatory bone loss disorders such as osteomyelitis and periarticular osteolysis and aseptic relaxation (see, for example, Esperito Santo et al. Biochem . Biophys . Res. Commun . 464:1145-1150).

DMS5541 (also known as TNFRI-AlbudAb), which contains an uncompetitive human TNFR1-specific dAb fused to AlbudAb, is an ex vivo cultured human rheumatoid cell that expresses TNFR1 and TNFR2 and produces inflammatory cytokines and chemokines spontaneously in the absence of exogenous stimuli. Selective blockade of TNF signaling through TNFR1 in rheumatoid arthritis (RA) synovial mononuclear cells (MNC) was evaluated. DMS5541 inhibited the pro-inflammatory cytokines GM-CSF, IL-10, IL-1 and IL-6, chemokines IL-8, RANTES (CCL5) and MCP-1 (CCL2) together with etanercept at levels similar to TNF ligand blockade. inhibited the formation of This inhibition was not due to cytotoxicity, as DMS5541 inhibited TNFα-induced cytotoxicity in human rhabdomyosarcoma KYM-1D4 cells in a dose-dependent manner, similar to TNF blockade by etanercept. In addition, DMS5541 inhibited the production of soluble TNFR1 but not soluble TNFR2, demonstrating selectivity for TNFR1. These results indicate that the TNFR1 pathway is the predominant inflammatory pathway responsible for the TNF response observed in the ex vivo cultured RA synovial MNC disease model (see, e.g., Schmidt et al. (2013) Arthritis & Rheumatism 65(9):2262-2273).

b) domain antibody fragments designated GSK1995057 and GSK2862277

The domain antibody fragment designated GSK1995057 (see SEQ ID NO: 55) is a short acting fully human domain antibody (dAb) fragment (containing V _H chain) that selectively antagonizes TNF signaling through TNFR1 but not TNFR2. Because of its small size, GSK1995057 can be nebulized directly into the lungs and has been investigated for the treatment of animal and human models of acute respiratory distress syndrome (ARDS) via inhalation. GSK1995057 reduces lung inflammation in non-human primates (cynomolgus monkeys) and human ARDS models. Pulmonary neutrophil infiltration is central to the etiology of ARDS and is increased due to damage to the alveolar-capillary barrier due to the action of proinflammatory mediators. TNF-α contributes to an increase in endothelial permeability and GSK1995057 prevents this increase, indicating that TNFR1 signaling mediates TNF-induced endothelial permeability (see, e.g., Proudfoot et al. (2018) Thorax 73:723-730). The experiment failed due to its inherently short half-life and neutralization by autoantibodies. The immunogenicity of GSK1995057 may be due to improper folding of the protein made in E. coli rather than to the failure to properly humanize the dAb; It was derived from a human antibody fragment, only the hypervariable sequence was altered to adapt specificity to TNFR1 (see, eg, International PCT Publication No. WO2008/149148A2).

In monkeys exposed to a single inhaled lipopolysaccharide (LPS) challenge, a well-established model that induces a clinically relevant inflammatory response modeling subclinical tissue damage, pretreatment with GSK1995057 resulted in pulmonary neutrophil infiltration, proinflammatory chemokine levels, and endothelial damage. Indicator and alveolar-capillary leakage are reduced in a dose-dependent manner. The results indicate that inhaled GSK1995057 can produce the same results as higher doses of parenterally administered antibody. In a clinical trial in which healthy human subjects were pretreated with a single nebulized dose of GSK1995057 and then exposed to low doses of inhaled LPS, pretreated subjects showed systemic inflammation and neutrophilic lung inflammation and endothelial damage in response to LPS challenge compared to subjects receiving placebo. There were few signs. Despite these findings, clinical translation is unlikely. In trials, GSK1995057 was administered prior to LPS challenge, but patients with ARDS usually require treatment after an initial injury (e.g., Proudfoot et al. (2018) Thorax 73:723-730]), but not before.

Another difficulty is the detrimental effect of anti-drug antibodies (ADAs) to anti-TNFR1 agents observed in the phase 1 clinical study of GSK1995057, and the high levels of pre-existing naturally occurring drugs present in approximately 50% of naïve and healthy subjects. A cytokine release infusion response was observed at doses of 2-10 μg/kg due to anti-immunoglobulin autoantibodies (i.e., ADA). Specifically, ADA was a human anti-V _H (HAVH) autoantibody, and the complex of the HAVH autoantibody with the framework sequence of GSK1995057 activated TNFR1 signaling and resulted in mild to moderate infusion in subjects with high HAVH autoantibody titers. reaction (see, e.g., Cordy et al. (2015) Clin. Exp. Immunol. 182:139-148]).

Binding of the HAVH autoantibody to the framework regions of dAb GSK1995057 induces cytokine release in vitro. The epitope of GSK1995057 for the autoantibody was characterized. Existing anti-drug antibodies (ADA) bind epitopes close to the C-terminal region of V _H dAbs, including dAb GSK1995057. To combat this, a modified dAb designated GSK2862277 (see SEQ ID NO: 56) was created by adding a single alanine residue to the C-terminus of the modified dAb. This modification reduced binding to the HAVH autoantibody. In serum samples from healthy subjects screened positive for HAVH autoantibodies that bind GSK1995057, the frequency of preexisting autoantibodies decreased from 51% for GSK1995057 specific HAVH autoantibodies to 7% for GSK2862277 specific autoantibodies. In human in vitro systems and animal in vivo experiments, GSK2862277 does not induce TNFR1 activation even in the presence of GSK2862277-specific autoantibodies, and pharmacology and biology of GSK2862277, including target affinity, in vitro potency, and in vivo pharmacokinetics and pharmacodynamics Physical properties are similar to those of the parent dAb (GSK1995057).

A phase 1 clinical trial to investigate the safety, tolerability, pharmacokinetics and pharmacodynamics of single and repeated doses of inhaled (ih) and intravenous (iv) GSK2862277 found that GSK2862277 was generally well tolerated when administered by inhalation or intravenously. . However, one subject had a mild infusion reaction with cytokine release after repeated IV dosing; This subject had high serum levels of a pre-existing antibody to GSK2862277, and this subject's serum antibody was shown to activate TNFR1 signaling in an in vitro assay. Interaction between GSK2862277 and autoantibodies results in antibody-mediated, GSK2862277-dependent cross-linking of cellular TNFR1 agonizing the receptor and leads to cytokine release. Thus, despite the reduced binding of GSK2862277 to the pre-existing HAVH autoantibody, side effects were still associated with the presence of a new pre-existing antibody response specific to the modified dAb framework. These results highlight the difficulty of developing biological antagonists for TNFR1 (see, e.g., Cordy et al. (2015) Clin . Exp . Immunol . 182:139-148]). Thus, there is still a need for improved TNFR1 antagonists.

iii. Nanobody (Nb)

Similar to dAbs, nanobodies (Nb) are small antigen-binding fragments derived from camel heavy chain antibodies that lack light chains. They are small (15 kDa), low immunogenic, high affinity, soluble, stable, encoded by a single gene/exon (VHH) and modular, enabling high-yield production in bacteria or yeast.

iv. Anti-TNFR1 nanobody-anti-albumin nanobody fusion construct

TROS (TNF Receptor One-Silencer; also known as Nb Alb-70-96) is a trivalent high-affinity nanobody-based selective inhibitor of human TNFR1 that competes with TNF for binding to TNFR1. To generate TROS, two anti-human TNFR1 nanobodies (Nb 70 and Nb 96; see SEQ ID NOS: 683 and 684, respectively) generated from a VHH library constructed by immunization of alpaca with recombinant human soluble TNFR1 are linked , via a (Gly ₄ Ser) ₃ linker, to an anti-albumin nanobody (Nb Alb) to increase serum half-life to generate trivalent TROS. The serum half-life of TROS produced is -24 hours; The serum half-life of monovalent Nb is only -1.5 hours. Treatment with TROS delays disease onset and prevents established disease in mouse experimental autoimmune encephalomyelitis (EAE; a model of MS); The therapeutic effect is due to the conversion of TNF to signaling through TNFR2 and the effect of such signaling. TROS also inhibits inflammation in ex vivo cultured colon biopsies of patients with Crohn's disease and antagonizes inflammation in an acute TNF-induced liver inflammation model in liver chimeric humanized mice (see, e.g., Steeland et al. (2015) J. Biol . Chem . 290(7):4022-4037; Steeland et al. (2017) Sci . Reports 7:13646]).

c. Dominant-negative inhibitors of TNF (DN-TNF)/TNF muteins

Another class of TNF inhibitors are dominant-negative inhibitors that lack the signaling ability of TNF (DN-TNF), also known as TNF muteins. DN-TNF is an engineered variant of TNF with mutations that abrogate binding and signaling to TNFR1 and TNFR2. DN-TNF interacts with TNF receptors by selectively inhibiting soluble TNF (sTNF or solTNF) by rapidly exchanging subunits with native TNF homotrimers and forming inactive mixed TNF heterotrimers with disrupted receptor-binding surfaces to prevent DN-TNF retains the protective role of TNF signaling through TNFR2 without affecting transmembrane TNF (tmTNF). DN-TNF inhibits TNF-induced NF-κB activity and caspase-mediated apoptosis and reduces disease severity in animal models of arthritis and Parkinson's disease. Because of their structure, these molecules are likely to be immunogenic.

As a selective inhibitor of soluble TNF, DN-TNF does not inhibit tmTNF signaling and, unlike anti-TNF therapies that bind to solTNF and tmTNF, does not inhibit tmTNF signaling and is resistant to infection by L. monocytogenes in mice. do not suppress An example of DN-TNF is the amino acid sequence set forth in SEQ ID NO: 1 that impairs binding to TNFR (corresponding to residues L57Y, S86Q, Y87H, I97T, Y115Q and A145R relative to the sequence of solTNF set forth in SEQ ID NO: 2) is a TNF mutant containing one or more of the substitutions L133Y, S162Q, Y163H, I173T, Y191Q and A221R with respect to. Additional modifications to improve expression may also be included, such as site-specific PEGylation (see, for example, Zalevsky et al. (2007) J. Immunol. 179:1872-1883]).

For example, the TNF mutations R32W and S86T with respect to SEQ ID NO: 2 result in a multifold loss in affinity for TNFR2 but do not affect binding to TNFR1. The R32W/S86T double mutant eliminates all binding to TNFR2 without loss of binding to TNFR1. Mutation L29S, L29G, L29Y, R31E, R31N, R32Y, R32W, S86T, L29S/R32W, L29S/S86T, R32W/S86T, L29S/R32W/S86T, R31N/R32T, R31E/S86T, R31E/S86T, R31N/R32T , And E146R, in relation to SEQ ID NO: 2, also confers selectivity for TNFR1. The mutations D143N, D143Y, A145R and D143N/A145R with respect to SEQ ID NO: 2 provide TNF variants that are selective for TNFR2 (eg, Loetscher et al. (1993) J. Biol. Chem. 268(35):26350-26357; See U.S. Patent No. 5,422,104).

The modified TNF designated as XPro1595 (see INmuneBio; SEQ ID NO: 701) is a PEGylated soluble DN-TNF mutein that preferentially inhibits TNFR1 signaling and has mutations V1M, R31C, C69V, Y87H, C101A with respect to SEQ ID NO: 2 and A1456R (see, eg, US Publication No. 2015/0239951). XPro1595 reduces neuroinflammation and is being studied for treatment of Alzheimer's disease (see, eg, clinical trial accession number NCT03943264). XPro1595 Blocks Amyloid Pathology Development in Alzheimer's Disease (3xTgAD) Mouse Model, Prevents Loss of Neuronal Communication and Cognitive Impairment in Different (tgCRND8) Mouse Model of Alzheimer's Disease, and Functions in Neurocommunication and Cognitive Deficits in Normal Aged Rats attenuates disability and prevents juvenile mice from developing amyloid pathology, cognitive impairment and dysfunction of neuronal communication in a third model of Alzheimer's disease (5xFAD). In aged mice with pathologies such as Alzheimer's disease, XPro1595 reduced amyloid, improved cognition, rescued neural communication, and normalized innate and adaptive immune responses.

Levels of TNFR1 are higher in the hippocampus compared to TNFR2, but not in young adult (6 months) Fischer 344 rats. When treated with ^XPro1595 , aged rats showed improved Morris Water Maze performance, reduced microglia activation, reduced susceptibility to hippocampal long-term depression, increased levels of GluR1 type glutamate receptors, and L-type voltage sensitive Ca ²⁺ channels ( L-VSCC), indicating that functional changes associated with brain aging may arise from selective alteration of TNF signaling. In animal models of Parkinson's disease and aging, XPro1595 inhibits neuroinflammation and microglia activation. In EAE (MS model), XPro1595 ameliorates disease, improves remyelination, and reduces CNS lesions and neuroinflammation. XPro1595 also improves inflammatory arthritis and reduces the susceptibility of treated animals to infection. Compared to etanercept, which has no therapeutic effect, treatment with XPro1595 delays the onset of EAE and improves symptoms more efficiently. XPro1595 administration increases TNR2 expression levels in the lesional area of EAE, indicating that tmTNF signaling through TNFR2 is implicated in nerve regeneration (see, e.g., Yang et al. (2018) Front. Immunol . 9:784; Sama et al. (2012) PLoS ONE 7(5):e38170). XPro1595 does not inhibit the activity of transmembrane TNF (which activates TNFR1 and TNFR2) and therefore cannot block the inflammatory effects of TNFR1. This also applies to other predominantly negative TNF reagents described below.

XENP345 (see SEQ ID NO: 702) is a PEGylated DN-TNF mutein containing the mutations I97T/A145R with respect to SEQ ID NO:2. In vivo neutralization of soluble TNF (solTNF) by XENP345 in animal models of Parkinson's disease and Alzheimer's disease is neuroprotective, reduces neurodegeneration and cognitive dysfunction, and slows neurodegenerative disease progression (see, e.g., McCoy et al. (2006) J. Neurosci . 26(37):9365-9375; McAlpine et al. (2009) Neurobiol. Dis. 34(1):163-177).

R1antTNF (see SEQ ID NO: 703) is a TNFR1 selective antagonistic mutant TNF identified in a phage library displaying structural human TNF variants, wherein each of the six amino acid residues of the receptor binding site corresponding to residues 84-89 of SEQ ID NO: 2 has been mutated. R1antTNF containing the mutations A84S, V85T, S86T, Y87H, Q88N and T89Q, like wild-type human TNF, has a similar affinity to TNFR1 and does not interfere with TNFR2 activity. R1antTNF ameliorated liver damage as evidenced by reduced serum levels of alanine aminotransferase and pro-inflammatory cytokines IL-2 and IL-6 in two models of acute hepatitis. However, the plasma half-life of R1antTNF is very short (12 minutes), similar to that of wild-type TNF. To increase the half-life of R1antTNF in vivo, PEG-R1antTNF, a PEGylated version in which PEG is linked to the N-terminal region of R1antTNF, was created. PEG-R1antTNF reduces morbidity, ameliorates disease symptoms, ameliorates demyelination in an EAE mouse model, and inhibits Th1 and Th17 cell activation and inflammatory T-cell infiltration in the spinal cord. PEG-R1antTNF also induced femoral artery injury in an external vascular cuff model by inhibiting NF-κB, inhibiting smooth muscle cell proliferation, and reducing chemokine and adhesion molecule expression, followed by intimal hyperplasia and arterial hyperplasia in IL-1 receptor antagonist-deficient mice. Reduces inflammation. As a result of comparing the effects of PEG-R1antTNF and etanercept on antiviral immunity using recombinant adenoviral vectors, PEG-R1antTMF did not reactivate the viral infection and did not affect the clearance of the injected adenovirus. Viral load increased after nercept treatment. PEG-R1antTNF treatment also delayed and ameliorated CIA symptoms in both prophylactic and therapeutic settings and was more effective than etanercept when used in the treatment of established CIA (see, e.g., Yang et al (2018) Front. Immunol.9:784; Shibata et al. (2008) J. Biol. Chem. ) Antibodies 4:48-70; Horiuchi et al (2010) Rheumatology (Oxford) 49:1215-1228).

Soluble TNFR1 is also associated with an increased risk of developing MS; Therefore, neutralization of soluble TNFR1, which cannot be achieved with DN-TNFs/TNF muteins, may be beneficial. Unlike inhibitors of solTNF, such as DN-TNF, TNFR1 antagonists can block the binding of another member of the TNF superfamily, lymphotoxin-α (LT-α), to TNFR1. LT-α may play pro-inflammatory roles in RA and animal disease models such as CIA and EAE; Thus, simultaneous blockade of TNF and LT-α binding to TNFR1 by a TNFR1 antagonist may have additional advantages compared to solTNF inhibition in acute and chronic inflammatory diseases and disorders (see, e.g., Fischer et al. (2015) Antibodies 4:48-70).

2. TNFR2 Selective Agonists

CD4 ⁺ FoxP3 ⁺ regulatory T cells (Tregs) maintain immunological homeostasis and suppress autoimmune responses. Tregs also modulate the antitumor immune response, allowing tumor immune evasion. Thus, Tregs are therapeutic targets, for example, in the treatment of autoimmune and chronic inflammatory diseases and conditions, graft versus host disease (GvHD), transplant rejection and cancer. TNF signaling through TNFR2 regulates Treg function and activity. TNFR2 agonists upregulate Treg activity, whereas TNFR2 antagonists downregulate Treg activity. The Treg-stimulating effect of the TNF-TNFR2 signaling pathway can be exploited for the treatment of several human diseases and disorders, including autoimmune and chronic inflammatory diseases through agonism, and cancer through antagonism (see, e.g., Zou et al. (2018) Front. Immunol. 9:594]).

TNFR2 agonists include antibodies and antigen binding fragments thereof, such as monoclonal TNFR2 agonist antibodies, peptides and proteins, such as TNFR2 selective TNF muteins, fusion proteins, and small molecules. As provided herein, specific agonism of TNFR2 leads to expansion and activation of Tregs, which modulate the immune system, reduce the activity of autoreactive CD8 ⁺ T cells that damage tissue, and provide neuroprotective, cardioprotective , induces signaling pathways with cell survival, regeneration and protective effects, including intestinal and osteoprotective effects, as well as anti-inflammatory. Thus, enhancement of TNFR2 signaling by TNFR2 selective agonists may have a therapeutic effect of TNFR1 specific antagonism, particularly in the treatment of autoimmune and chronic inflammatory diseases and disorders, including neurodegenerative diseases where anti-TNF therapy/TNF-blockers have failed. can be used to reinforce

a. TNFR2 agonistic antibody

Human TNFR2 selective agonist antibodies include the commercially available MR2-1 (monoclonal mouse IgG1 binding to human, cynomolgus monkey and rhesus monkey TNFR2; Hycult Biotech) and clone MAB2261 (monoclonal mouse IgG2A binding to human TNFR2). ; R&D Systems). TNFR2 agonists such as antibodies can potently stimulate the expansion of a homogeneous population of FoxP3 ⁺ Tregs in CD4 cell culture and upregulate the expression of TNF, TRAF2, TRAF3, BIRC3 (cIAP2) and FoxP3 mRNA. Magnetic Activated Cell Sorting (MACS)-purified CD4 ⁺ CD25 ⁺ cells cultured using standard in vitro human Treg expansion protocols (i.e., containing anti-CD3 antibody, anti-CD28 antibody, IL-2, and rapamycin), TNFR2 Generates expanded Tregs with higher levels of FoxP3 (and other characteristic Treg markers) and stronger suppressive capacity when expanded in the presence of the TNFR2 agonist antibody compared to in the absence of the agonist. Tregs isolated from type 1 diabetic patients exhibiting a resting phase phenotype are activated and expanded upon in vitro treatment with a TNFR2 agonist antibody; These Tregs are more potent at suppressing autologous CD8 ⁺ T cells (see, e.g., Zou et al. (2018) Front. Immunol . 9:594]).

Treatment of the expanded isolated Tregs using standard in vitro protocols with MR2-1, a commercially available potent human TNFR2 monoclonal antibody (mAb) containing mouse IgG1, resulted in a homogeneous population of FoxP3 ⁺ Helios ⁺ CD127 ^low Tregs. create; These Tregs retain their phenotype and high suppressive activity in humanized mouse models. Thus, TNFR2 agonists can enhance ex vivo expansion of Treg cells in impure cell populations for use in Treg-based immunotherapy (see, e.g., Zou et al. (2018) Front. Immunol. 9:594]).

b. TNFR2 selective TNF muteins and fusions thereof

As described herein, TNF can be engineered to selectively bind to TNFR1 or TNFR2; For example, a TNFR2 selective TNF mutein is a variant of TNF that contains one or more mutations that increase binding to TNFR2 and/or reduce or eliminate binding to TNFR1. A TNFR2 selective mutation is a non-conservative substitution of the Asp residue at position 143 of soluble TNF (see SEQ ID NO: 2), for example D143Y, D143F or D143N, or a non-conservative substitution of the Ala residue at position 145 of soluble TNF; eg A145R (see eg US Pat. No. 9,081,017). Other mutations in TNF conferring selectivity for TNFR2 include, for example, K65W, D143E, D143W, D143V, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, see SEQ ID NO: 2 , A145R/S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145 N/E146D/S147D, A145T/E146S /S147D, A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E 146D/S147D, D143V/F144L/A145S , and D143V/A145S (see, eg, [US Patent Publication No. 2020/0102362]).

TNF ligand trimerization is essential for signaling through TNFR. At low concentrations, such as in serum, the trimer dissociates and degrades. Generation of functionally active receptor-specific TNF muteins requires the creation of stable trimers. TNF07 is a soluble TNF (sTNF or solTNF) mutein containing the mutation S95C/G148C (with respect to the sequence of residues set forth in SEQ ID NO: 2) that forms stable TNF trimers and functions as a TNFR2 agonist.

The S95C/G148C mutation forms an intermolecular Cys-Cys covalent bond; Stable trimers are thus formed as a result of covalent internal disulfide bridges of sTNF at strategic locations between TNF monomers. TNF07 acts as a TNFR2 agonist despite the absence of TNFR2 selective mutations. TNF07 induces strong TNFR2 signaling, expands FoxP3 ⁺ Treg cells, and selectively induces the death of autoreactive CD8 ⁺ T cells isolated from patients with type 1 diabetes (see, e.g., Ban et al. (2015) Molecular and Cellular Therapies 3:7; Zou et al. (2018) Front. Immunol. 9:594]).

Several TNFR2 agonists have been created that contain a fusion of a single chain TNFR2 selective TNF mutein trimer with a multimerization domain. As described herein, the primary ligand for TNFR2 is membrane bound TNF (memTNF; also referred to herein as transmembrane TNF or tmTNF). Addition of a multimerization domain, such as a dimerization or trimerization domain, creates a hexameric or 9meric molecule for the TNF subunit, respectively; These hexamers and 9mers of TNF can effectively activate TNFR2 signaling as they mimic membrane-bound TNF trimers. Commonly used dimerization domains include EHD2 derived from the heavy chain C _H 2 domain of IgE and MHD2 derived from the heavy chain C _H 2 domain of IgM. Dimerization domains may also include Fc domains, such as those derived from IgG1 and IgG4, optionally including modifications that alter immune effector function. Commonly used trimerization domains include chicken tenascin C (TNC) and human TNC. Dimerization and trimerization enhance TNFR2 signaling and improve the half-life of the fusion protein, eg by increasing the molecular weight of the molecule and/or by introducing FcRn recycling, eg when the dimerization domain is Fc.

STAR2 (also known as TNC-sc-mTNF(221N/223R)) is a 9-mer potent TNFR2 specific mouse TNF variant that does not bind TNFR1 and is a single chain mouse TNF timer, wherein each TNF subunit has SEQ ID NO: 804 are residues 91-235 of SEQ ID NO: 5 fused to the trimerization domain of chicken tenascin C (cTNC), corresponding to residues 110-139 of (see also SEQ ID NO: 805). The three single-chain mouse TNF subunits are linked by two (GGGS) _4- peptide linkers (see for example residues 116-120 of SEQ ID NO: 707), and the TNC trimerization domain is the first TNF subunit in the single-chain trimer. is connected to the N-terminus of The specificity of STAR2 for TNFR2 results from mutations D221N and A223R (see the sequence of mouse TNF set forth in SEQ ID NO: 5) in individual TNF subunits that create steric clashes between STAR2 and mouse TNFR1. Fusion to the TNC trimerization domain causes spontaneous oligomerization to produce three covalently linked TNF trimers and mimics membrane-bound TNF. STAR2 stimulates the proliferation of Tregs in vitro and in vivo in a TNFR2 dependent, IL-2-independent mechanism. Pretreatment of allogeneic hematopoietic stem cells with STAR2 prior to transplantation into mice prolongs survival and reduces the severity of GvHD in a TNFR-2 and Treg dependent manner. The human equivalent of the TNFR2-specific STAR2 agonist, TNC-scTNF (143N/145R) made from residues 9-157 of soluble TNF containing mutations D143N/A145R with reference to SEQ ID NO: 2 (solTNF) (SEQ ID NO: 2 ) potently stimulated CD4 ⁺ FoxP3 ⁺ Treg expansion in vitro from CD4 ⁺ T cells isolated from healthy donors (see, eg, Chopra et al. (2016) J. Exp . Med . 213(9):1881-1900; Zou et al. (2018) Front. Immunol . 9:594]).

TNC-scTNF _R2 is human tenascin C (hTNC) containing residues 110-139 of SEQ ID NO: 806 (see also SEQ ID NO: 807) at the N-terminus of a TNFR2 selective short-chain TNF variant (scTNF _R2 ; SEQ ID NO: 803). ) and is a soluble human TNFR2 agonist containing three TNF domains connected by two short peptide linkers (GGGGS). The TNF _R2 selective TNF molecule, scTNF _R2 , is similar to soluble trimeric TNF and each TNF subunit contains the amino acids of full-length TNF set forth in SEQ ID NO: 1 together with mutations D143N/A145R referenced to SEQ ID NO: 2 that abolish binding to TNFR1. 80-233 (corresponding to residues 4-157 of SEQ ID NO: 2). Since TNFR2 is fully activated only by membrane-bound TNF and not by soluble TNF trimer, the trimerization domain of TNC is fused to the N-terminus of scTNF _R2 , resulting in TNC-scTNF _R2 . TNC-scTNF _R2 is present in a trimeric assembly of single-stranded fusion proteins and resembles nonameric TNF molecules; This oligomeric TNF mutein mimics the activity of membrane-bound TNF (memTNF) due to its increased avidity and efficiently activates TNFR2 by inducing clustering of TNFR2 and formation of the TNFR2 signaling complex. TNC-scTNF _R2 exhibits neuroprotective properties; It protects neurons from superoxide-induced cell death and rescues neurons from catecholamine-induced apoptosis. In an in vitro model of Parkinson's disease, TNC-scTNF _R2 rescued neurons after induction of apoptosis by 6-OHDA. These results indicate that TNC-scTNF _R2 can ameliorate the neurodegenerative process (see, eg, Fischer et al. 2011 PLoS ONE 6(11):e27621).

EHD2-scTNF _R2 (see SEQ ID NO: 810) is a potent TNFR2 selective TNF mutein fusion protein, which is derived from the heavy chain C _H 2 domain of IgE and is a dimer producing a disulfide-linked dimer containing a hexameric TNF domain. A covalently stabilized human TNFR2 selective single-chain TNF trimer with mutations D143N/A145R (residue numbering for soluble TNF shown in SEQ ID NO: 2) with no binding to TNFR1 fused to the embodied domain EHD2 (SEQ ID NO: 808) scTNF _R2 ; SEQ ID NO: 803). Each TNF subunit within scTNF _R2 contains residues 4-157 of SEQ ID NO:2. EHD2 is fused to the N-terminus of trivalent human short chain scTNF _R2 via a peptide linker (GGGSGGGSGGGSGGGSGGGSGGSEFLA; SEQ ID NO: 809), and the three TNF domains of scTNF _R2 are linked via two GGGGS peptide linkers. EHD2-scTNF _R2 exhibits neuroprotective properties in a mouse model of NMDA-induced acute neurodegeneration (see, e.g., Dong et al. (2016) Proc . Natl . Acad . Sci . USA . 113(43):12304-12309; and US Patent Publication No. 2020/0102362).

The TNFR2 agonist fusion protein also includes a single chain TNFR2 agonist (scTNF _R2 ) containing three TNF muteins with mutations D143N/A145R (see SEQ ID NO: 2), which is TNFR1 fused with a dimerization domain that is Fc. and as a result a protein that is a hexamer for the TNF domain (scTNF _R2 -Fc) is obtained. The Fc may be an IgG4 or IgG1 Fc, optionally containing mutations that abolish Fc effector functions such as ADCC and CDC. The three TNF muteins containing residues 12-157 of SEQ ID NO: 2 are linked together by two short peptide linkers and the dimerization domain is linked to the N of a single-chain trimeric TNF molecule (scTNF _R2 ) by a third short peptide linker. It is linked to the -terminus or C-terminus. All three linkers can be the same or different, including a GS linker (such as (GGGGS) _n residues 116-121 of SEQ ID NO:707, and/or other combinations of Gly and Ser), where n = 1-5 or may contain all or part of the stem region of TNF-α (GPQREEFPRDLSLISLISPLAQAVRSSSRTPSDK (SEQ ID NO: 812) corresponding to residues 57-87 of SEQ ID NO: 1), at least 10, 15 or 20 contiguous residues. Dimerization enhances signaling by TNFR2 agonists and improves the half-life of the fusion protein. Alternative dimerization domains that can be used in the fusion protein are Fc fusions derived from other dimerization molecules, such as IgE heavy chain domain 2 (EHD2; see SEQ ID NO: 808) and IgM heavy chain domain 2 (MHD2; see SEQ ID NO: 811). protein (MHD2; see SEQ ID NO: 811) (see, eg, International Application Publication No. WO 2019/226750).

3. Anti-TNFR2 Antagonistic Antibodies and Small Molecule Inhibitors

TNFR2 antagonists can inhibit Treg proliferation and induce Treg apoptosis, and also inhibit proliferation and induce apoptosis of TNFR2-expressing tumor cells. TNFR2 antagonists can reduce or inhibit the proliferation of myeloid-derived suppressor cells (MDSCs) and/or induce apoptosis within MDSCs by binding to TNFR2 expressed on the surface of MDSCs present in the tumor microenvironment. TNFR2 antagonists induce expansion of T effector cells, including cytotoxic CD8 ⁺ T cells, through suppression of Treg expansion and activity. As a result, TNFR2 antagonists are effective against infectious diseases and certain cancers that express TNFR2, such as T-cell lymphoma (eg, Hodgkin's lymphoma and cutaneous non-Hodgkin's lymphoma), ovarian cancer, colon cancer, multiple myeloma, renal cell may be useful in the treatment of carcinoma, breast, cervical, endometrial, glioma, head and neck, liver, and lung cancers (see, e.g., U.S. Patent Publication No. 2019/0144556; Torrey et al. (2017) Sci. Signal. 10:eaaf8608]).

As discussed herein, expression of TNFR2 is restricted to certain immune cells, including Tregs and MDSCs, endothelial cells, and certain neurons and cardiac cells. Restricted expression of TNFR2 makes it an ideal drug target because systemic toxicity from anti-TNFR2 therapeutics is less likely to occur.

TNFR2 antagonist antibodies and antigen-binding fragments thereof, for example, residues KCRPG (corresponding to residues 142-146 of SEQ ID NO: 4), or residues 130-149, 137-144 or 142-149, or at least five residues within these epitopes. Binds to at least one epitope in human TNFR2 that contains at least one of the larger epitopes containing contiguous or discontinuous residues and does not bind to an epitope containing residues KCSPG (corresponding to residues 56-60 of SEQ ID NO: 4) . TNFR2 antagonists also include the TNFR2 epitopes PECLSCGS (corresponding to residues 91-98 of SEQ ID NO: 4), RICTCRPG corresponding to residues 116-123 of SEQ ID NO: 4), CAPLRKCR (corresponding to residues 137-144 of SEQ ID NO: 4) ), LRKCRPGFGVA (corresponding to residues 140-150 of SEQ ID NO: 4), and/or VVCKPCAPGTFSN (corresponding to residues 159-171 of SEQ ID NO: 4), and residues 75-128, 86 of SEQ ID NO: 4 -103, 111-128, or 150-190 include antibodies, antigen-binding fragments thereof, or single chain antibodies that bind to an epitope containing at least 5 contiguous or discontinuous residues (see, e.g., U.S. Patent Publication No. 2019/ See 0144556.

Generally, an antagonistic TNFR2 antibody or antigen-binding fragment thereof exhibits, for example, at least 10-fold greater affinity than the affinity of the same antibody or antigen-binding fragment for a peptide containing the KCSPG sequence of human TNFR2 (SEQ ID NO: 839). It also binds to an epitope containing one or more residues of the KCRPG sequence (SEQ ID NO: 840). An antibody or antibody fragment that binds an epitope containing one or more residues of a KCRPG sequence and an epitope containing a KCSPG motif with similar affinity (eg, less than a 10-fold difference in affinity) is not a antagonistic TNFR2 antibody. Antagonistic TNFR2 antibodies include TNFRAB1 (see SEQ ID NOs: 1213 and 1213 for the heavy and light chain sequences of TNFRAB1, respectively), TNFRAB2 and TNFR2A3 (see, e.g., U.S. Patent Publication No. 2019/0144556 for descriptions of these antibodies). reference). The TNFR2 antagonist may also be a CDR-H3 sequence of TNFRAB1 (QRVDGYSSYWYFDV; corresponding to residues 99-112 of SEQ ID NO: 1212), TNFRAB2 (ARDDGSYSPFDYWG; SEQ ID NO: 1217) or TNFR2A3 (ARDDGSYSPFDYFG; SEQ ID NO: 1223), or antibodies and antibody fragments containing CDR-H3 sequences having at least about 85% sequence identity. For example, TNFRAB1 specifically binds residues 130-149 containing residue KCRPG of TNFR2 with 40-fold higher affinity than residues 48-67 containing residue KCSPG of TNFR2 (see, e.g., [U.S. Patent Publication No. 2019/0144556).

NFRAB1 (see SEQ ID NOs: 1212 and 1213 for heavy and light chains, respectively) is a murine antibody that antagonizes the TNF-TNFR2 interaction and, in addition to binding to the KCRPG sequence of TNFR2, binds to residues 161- of TNFR2 (SEQ ID NO: 4). 169 (CKPCAPGTF; SEQ ID NO: 1258). Another TNFR2 antagonistic antibody, TNFRAB2, has an epitope containing residues 137-144 (CAPLRKCR; SEQ ID NO: 851), as well as positions 80-86 of human TNFR2 (DSTYTQL; SEQ ID NO: 1247), 91-98 (PECLSCGS; SEQ ID NO: 1248), and one or more residues within 116-123 (RICTCRPG; SEQ ID NO: 1249). TNFR2A3 is a murine antagonistic human TNFR2 antibody discovered by CDR mutagenesis after immunization of mice with human TNFR2, wherein the CDR-H3 of the resulting precursor antibody was replaced with the CDR-H3 sequence ARDDGSYSPFDYFG (SEQ ID NO: 1223) . TNFR2A3 binds to two distinct epitopes in human TNFR2; The first epitope comprises residues 140-150 of human TNFR2 (LRKCRPGFGVA; SEQ ID NO: 1463) and contains the KCRPG motif, and the second epitope contains residues 159-171 of human TNFR2 (VVCKPCAPGTFSN; SEQ ID NO: 1464) is the downstream sequence. These data suggest that the CDR-H3 sequence of an antagonistic TNFR2 antibody primarily directs its antigen-binding properties, and that the CDR-H3 motif is an anti-TNFR2 that does not exhibit antagonistic activity to confer TNFR2-dominant antagonistic characteristics to such antibody or antigen-binding fragment thereof. Indicates a modular domain that can be substituted into an antibody. For example, replacement of the CDR-H3 sequence of a neutral anti-TNFR2 antibody (i.e., an antibody that is neither antagonistic nor agonistic) with a CDR-H3 from an antagonistic TNFR2 antibody, such as the CDR-H3 sequence of TNFRAB1, TNFRAB2 or TNFR2A3, may alter the phenotype. -conversion of the neutral antibody to an antagonistic TNFR2 antibody, such as a predominantly antagonistic TNFR2 antibody, which is an antagonist that inhibits TNFR2 activation even in the presence of a TNFR2 agonist such as TNF or IL-2 (see, e.g., [U.S. Patent Publication No. 2019 /0144556]).

TNFR2 antagonist antibodies or antigen-binding fragments thereof include SEQ ID NOs: 1214, 1215, and 1231-1233; CDR-H2 sequences set forth in any of 1216, 1224, and 1230; the CDR-H3 sequence set forth in any of SEQ ID NOs: 1217, 1223, and 1225-1229, or the CDR-H3 of TNFRAB1 corresponding to residues 99-112 of SEQ ID NO: 1212; the CDR-L1 sequence set forth in any of SEQ ID NOs: 1218 and 1234-1236, or the CDR-L1 sequence of TNFRAB1 corresponding to residues 24-33 of SEQ ID NO: 1213; the CDR-L2 sequence set forth in any of SEQ ID NOs: 1219, 1220, 1237 and 1238, or the CDR-L2 sequence of TNFRAB1 corresponding to residues 49-55 of SEQ ID NO: 1213; or a CDR-L3 sequence as presented in any of SEQ ID NOs: 1221, 1222, and 1241-1244, or a CDR-L3 sequence as presented in any of the CDR-L3 sequences of TNFRAB1 corresponding to residues 88-96 of SEQ ID NO: 1213 may contain. Exemplary framework regions that can be used in the development of humanized anti-TNFR2 antibodies containing one or more of the above CDRs include, without limitation, those described in U.S. Patent Nos. 7,732,578 and 8,093,068, and International Application Publication No. WO 2003/105782. Another approach to engineering humanized anti-TNFR2 antagonistic antibodies is to align the sequences of the heavy chain variable region and light chain variable region of an antagonistic TNFR2 antibody, such as TNFRAB1, TNFRAB2 or TNFR2A3, with the heavy chain variable region and light chain of a common human antibody. Common human antibody heavy and light chain sequences are known in the art (see, eg, the "VBASE" human germline sequence database; see also Kabat, et al., Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No 91-3242, (1991) Tomlinson et al., (1992) J. Mol . Biol . 227:776-798 and Cox et al., (1994) Eur. J. Immunol . reference). In this way, variable domain framework residues and CDRs can be identified by sequence alignment. For example, a humanized TNFR2 antagonist antibody can be produced by replacing the CDR-H3 of a consensus human antibody with the CDR-H3 of an antagonistic TNFR2 antibody, such as the CDR-H3 of TNFRAB1, TNFRAB2 or TNFR2A3. Exemplary variable domains of common human antibodies include the heavy chain variable domain set forth in SEQ ID NO: 1245 and the light chain variable domain set forth in SEQ ID NO: 1246 identified in U.S. Patent No. 6,054,297 (see, e.g., [U.S. Patent Publication No. ] reference). The CDR-H1 and CDR-H2 sequences of the exemplary consensus sequence of the human antibody heavy chain variable domain of SEQ ID NO: 1245 can be replaced with the corresponding CDR sequences of, for example, a phenotype-neutral, TNFR2 specific antibody, SEQ ID NO: The CDR-L1, CDR-L2 and CDR-L3 sequences of the exemplary consensus sequence of the human antibody light chain variable domain of 1246 can be replaced with the corresponding CDR sequences of a phenotype-neutral, TNFR2 specific antibody to produce a humanized antagonistic TNFR2 antibody. there is.

Other TNFR2 antagonists include techniques known in the art, such as phage display, bacterial display, yeast display, mammalian display, ribosome display, mRNA display and cDNA display, or sugars such as those described in US Patent Publication No. 2019/0144556. It can be identified by screening for peptides that bind to an epitope in TNFR2 such as set forth in any one of SEQ ID NOs: 1247-1464 using any other method known in the art.

Human TNFR2 antagonist mAb inhibits expansion of Tregs and reduces suppressive activity when added to standard Treg expansion culture conditions (see, e.g., Zou et al. (2018) Front. Immunol. 9:594]). Two potent, predominantly anti-human TNFR2 antagonistic antibodies that surpass TNF (a natural agonist of TNFR2) and inhibit the TNF-induced in vitro expansion of human Tregs derived by TNF are capable of inducing apoptosis of Treg in vitro. can The TNFR2 antagonist specifically binds to TNFR2 through the F(ab) region independently of cross-linking of the Fc region or antibody, binds to the antiparallel dimer of TNFR2, and blocks TNF from binding to TNFR2. As a result, TNF-induced activation of the NF-κB pathway in Tregs is inhibited and the conversion of transmembrane TNFR2 (tmTNFR2) to soluble TNFR2 (sTNFR2) is inhibited. Tregs isolated from ovarian cancer tissue were shown to be more susceptible to TNFR2 antagonist mAb-induced apoptosis due to higher levels of TNFR2 expression in tumor-infiltrating Tregs. TNFR2 antagonists also induced the death of TNFR2 ⁺ OVCAR3 (ovarian cancer) tumor cells expressing TNFR2. These results indicate the therapeutic potential of TNFR2 antagonists in tumor treatment by targeting tumor cells as well as tumor-infiltrating Tregs (see, e.g., Zou et al. (2018) Front. Immunol . 9:594; Torrey et al. (2017) Sci. Signal. 10:eaaf8608]).

In addition to anti-TNFR2 antagonist mAbs, small molecules can inhibit TNFR2. For example, thalidomide is a small molecule synthetic glutamic acid derivative with immune modulatory and anti-inflammatory properties; Thalidomide and its structural analogues, lenalidomide and pomalidomide are classified as immunomodulatory drugs. Thalidomide and its analogs down-regulate NF-κB, destroy TNF mRNA, inhibit TNF synthesis by targeting reactive oxygen species and α1-acid glycoprotein, and also inhibit intracellular TNFR2 transport to the cell surface. Inhibits the surface expression of TNFR2 on T cells. Thalidomide reduces the number and function of Tregs in patients with chronic lymphocytic leukemia, and the combination of lenalidomide and azacitidine in patients with acute myeloid leukemia downregulates TNFR2 expression on CD4 ⁺ T cells and decreases the number of TNFR2 ⁺ Tregs. has been shown to enhance effector immune function by reducing However, in patients with multiple myeloma, treatment with thalidomide and its analogues increased the number of Tregs, most likely due to elevated serum levels of TNF after treatment, suggesting that the effect of thalidomide on TNFR2 ⁺ Tregs is disease-specific. indicates (see, e.g., Zou et al. (2018) Front. Immunol. 9:594]).

Another small molecule inhibitor of TNFR2 is panobinostat, a histone deacetylase inhibitor that can reduce FoxP3 expression and inhibit the suppressive activity of Tregs. Combination therapy of panobinostat and azacytidine reduces the number of TNFR2 ⁺ Tregs in the blood and bone marrow of patients with acute myeloid leukemia, and consequently increases the production of IFNγ and IL-2 by effector T cells, resulting in a therapeutic effect in these patients. indicates (see, e.g., Zou et al. (2018) Front. Immunol . 9:594]). Cyclophosphamide, a DNA alkylating agent commonly used as a cytotoxic chemotherapeutic agent in cancer treatment, is capable of suppressing the immunosuppressive function of Tregs at low doses and depletes maximally suppressive Tregs in mice with PROb colon cancer after a single dose administration, resulting in an anti-inflammatory effect. resulting in activation of the tumor immune response. In a mouse model of mesothelioma, cyclophosphamide treatment depleted TNFR2 ^hi Tregs. Combination of cyclophosphamide with etanercept inhibited the growth of established CT26 tumors in mice by blocking the TNF-TNFR2 interaction and abolishing TNFR2-expressing Treg activity (see, e.g., Zou et al. (2018) Front. Immunol . 9:594]). Chinese herbal medicine Tripterygium Wilfordi triptolide, an immunosuppressive molecule isolated from wilfordii ) suppresses TNF and TNFR2 expression in the colon of a mouse colitis model and also reduces the number of Tregs and inhibits tumor growth in mice with melanoma (see, e.g., [ Zou et al. (2018) Front. Immunol. 9:594]).

F. Selective targeting of the TNFR1 and/or TNFR2 axis

As described herein, existing anti-TNF therapies that block TNF and inhibit signaling through TNFR1 and TNFR2 have limited therapeutic efficacy, tolerability and safety. Anti-TNF therapy ameliorates RA and other autoimmune and inflammatory diseases and conditions by preventing TNF signaling through TNFR1 and blocking apoptotic and inflammatory pathways. However, these anti-TNF therapies block the beneficial effects of TNFR2 signaling, including protective, pro-survival, regenerative and anti-inflammatory signaling pathways, as well as TNFR2-associated expansion of immunosuppressive Tregs, resulting in severe, including severe infections. Sometimes fatal, side effects result. Other side effects associated with the use of TNF blockade therapy are congestive heart failure, liver damage, demyelinating disease/CNS disorders, increased susceptibility to the development of additional autoimmune diseases as well as cancer, including lupus, psoriasis, sarcoidosis, lymphoma and solid malignancies. includes Anti-TNF therapy has failed to treat demyelination and neurodegenerative diseases and may exacerbate disease symptoms.

TNFR1 antagonist constructs, TNFR2 agonist constructs, constructs including multispecific, such as bispecific, TNFR1 antagonist/TNFR2 agonist constructs, and nucleic acids and methods for selective inhibition of TNF signaling through TNFR1 provided herein (eg, FIG. 2 , which depicts exemplary bispecific constructs). Also provided are constructs and methods for selective inhibition of TNF signaling through TNFR1, including maintaining or enhancing TNFR2 signaling. These constructs and methods provide improved therapeutic approaches for the treatment of diseases and disorders of the TNF/TNFR1 axis. Such therapeutic approaches include, but are not limited to, treatment of autoimmune, chronic inflammatory, neurodegenerative, and demyelinating, diseases, disorders and conditions, and cancers with inflammatory factors. As described herein, simultaneous or sequential selective agonism of TNFR2 together with TNFR1 antagonism activates desirable signaling pathways such as the anti-inflammatory pathway and the NF-κB pathway that control cell survival and proliferation, and the autoimmune microenvironment. It has a therapeutic effect and can enhance the therapeutic index of selective TNFR1 antagonists by inducing expansion of immunosuppressive Tregs that eliminate excessive autoreactive/effector T cells that lead to tissue destruction in .

Sections 1 and 2 describe methods for targeting TNRF1 and TNRF2, respectively; Section 3 provides an overview of constructs provided herein that address problems with previous approaches, particularly those targeting TNFR1; Section 4 describes the structures and components provided herein.

1. Selective blockade of TNFR1 by TNFR1 antagonists

However, the use of multivalent agents such as antibodies against TNFR1 is not feasible. The TNF trimer binds to the three TNFR1 chains as a pre-ligand assembly complex mediated by the pre-ligand assembly domain (PLADD) of each monomeric TNFR. This differs from most receptor systems, which require ligand binding before clusters form on the cell surface. The TNF receptor is a single transmembrane glycoprotein with about 28% homology in most of its extracellular domain with both receptors containing four tandem repeating cysteine rich motifs. Their intracellular sequences are largely unrelated and show little homology to each other, and early studies provided an explanation for their signaling function (Grell et al. (1994) J. Immol . 153(5):1963- 72]). It contains several motifs of known functional significance. Each TNFR1 and TNFR2 contains an extracellular pre-ligand binding assembly domain (PLAD) domain (distinct from the ligand binding domain) that pre-complexes the receptor. The conformational change results when the trimeric TNF ligand binds to the TNFR trimer at the cell membrane, resulting in signal activation (MacEwan (2002) Br J Pharmacol . 135(4) :855-875; and Lo et al. (2019) Sci Signal. 12(592) :eaav5637]).

As a result, antibodies and other multivalent agents that bind to TNFR1 may not be suitable for use as antagonists because they may cause superclustering leading to activation of TNFR signaling. On the other hand, monovalent antagonists such as single domain antibodies (dAb or sdAb), nanobodies (Nb; camelid single domain antibodies), scFv fragments, and Fab fragments bind to one TNFR1 molecule and inhibit cross-linking or clustering of receptors at the cell surface. block the activation of TNFR1 signaling. Monovalent antagonists can bind domains 1, 2, 3 or 4 of the TNFR1 extracellular domain or epitope spanning multiple domains (see, eg, U.S. Patent Nos. 9,028,817 and 9,028,822). It was a cure without treatment. Among the various problems were short serum half-life, immunogenicity and other problems. Selective blockade of TNFR1 can be achieved with TNFR1 antagonists described herein and having the properties provided.

2. Selective activation of TNFR2 by TNFR2 agonists

As described herein, selective activation of TNFR2 is achieved using TNFR2 specific agonists, which can include, for example, TNFR2 agonistic antibodies and antigen-binding fragments thereof, and TNFR2 selective TNF muteins and fusion proteins thereof. It can be. Antigen-binding fragments of antibodies that bind to the first and/or second epitopes of human TNFR2 may be used. A first epitope of TNFR2 comprises amino acid residues 48-67 of SEQ ID NO:4, and a second epitope comprises, for example, residues 128-147, 130-149, 135-147, or 135-153 of SEQ ID NO:4 and position 135 of SEQ ID NO: 4 (see, eg, International Application Publication No. WO 2014/124134; and US Patent No. 9,821,010). Other epitopes of TNFR2 have been identified and can be used to design antigen-binding fragments with TNFR2 selectivity, as discussed below.

In contrast to the antagonism of TNFR1, to agonize TNFR2, dimeric and trimeric molecules are used that mimic the action of membrane bound TNF, the primary ligand that activates TNFR2. As such, TNFR2 agonists include TNFR2 selective TNF muteins and antibody fragments. Examples are TNF muteins and antibody fragments fused with a multimerization domain, particularly a dimerization or trimerization domain, as discussed below. To extend the half-life of these molecules, they can be associated with or coupled to polyethylene glycol with or without a cleavable linker (see, for example, Santi et al. (2012) Proc . Natl . Acad . Sci . USA 109 :6211-6216)), half-life extending proteins or peptides such as human serum albumin (with or without FcRn optimization, itself PEGylated or not); Can be fused or linked to the ADCC-inactivated/FcRn optimized Fc domain of an antibody with or without PEGylation (see e.g. Strohl (2015) BioDrugs 29(4):215-239). Half-life extenders include, for example, PEGylation, modification of glycosylation, sialylation, PASylation (polymers of PAS amino acids of about 100-200 residues in length), ELPylation (see, for example, Floss et al. (2010) J. Trends Biotechnol.28 (1): 37-45), Hapylation (glycine homopolymer), fusion to human serum albumin, fusion to GLK, fusion to CTP, fusion to GLP , fusions to constant fragment (Fc) domains of human immunoglobulin (IgG), fusions to transferrin, XTEN (also known as rPEG, of incorrectly repeating peptide sequences containing A, E, G, P, S and T). Genetic fusion, e.g., see Schellenberger et al. (2009) Nat Biotechnol . 27(12):1186-90), and to increase size, increase hydrodynamic radius, alter charge or rather than clearance other such modifications and fusions that target receptors for recycling, and combinations of such modifications. Specific examples of half-life extenders are discussed and exemplified in detail below.

3. TNFR1 antagonist construct , TNFR2 agonist construct ; double be specific Multispecific, TNFR1 antagonist and TNFR2 agonist constructs comprising

Accordingly, provided herein are constructs for inhibiting TNRFR1 signaling/activity and/or agonizing TNFR2. Constructs discussed below that are multispecific, such as bispecific, that inhibit TNFR1 signaling and agonize TNFR2, are included in the constructs provided herein. Bispecific antagonists TNFR1 or TNFR2 should be used with care when designing such constructs, as they can prevent signaling by inhibiting the ability of TNF to induce an activation change in the conformation of the resting trimeric TNFR. Other multimeric molecules risk receptor aggregation, allowing TNFR to signal for cell inflammation and apoptosis. Multispecific constructs herein generally target different receptors, such as TNFR1 and TNFR2, respectively. By inhibiting TNFR1 signaling and advantageously agonizing TNFR2 activity, this provides improved treatment of diseases, conditions and disorders in which TNF is involved.

Among the constructs provided herein are TNFR1 antagonist constructs. Included herein are fusion protein constructs such as TNFR1 antagonist-Fc fusion constructs. As described herein and exemplified in the Examples, TNFR1 antagonists can be selected, created, or designed that specifically target TNFR1 or contain or exhibit TNFR2 agonistic activity without antagonizing or substantially antagonizing TNFR2. TNFR1 antagonist constructs improve the therapeutic efficacy and safety of previous TNFR1 antagonists, including monovalent antagonists such as dAbs, scFvs and Fabs.

Also provided are selective TNFR2 agonist constructs such as TNFR2-Fc fusion constructs that improve the therapeutic efficacy of previous TNFR2 agonists. For example, as shown herein, the half-life of an Fc fusion construct increases the half-life of a previous TNFR1 antagonist or TNFR2 agonist, which, for example, reduces dosing frequency, improves patient compliance, and improves therapeutic index improve Also provided are selective TNFR2 agonist constructs such as TNFR2-Fc fusion constructs that improve the therapeutic efficacy of previous TNFR2 agonists. For example, as shown herein, the half-life of an Fc fusion construct increases the half-life of a previous TNFR1 antagonist or TNFR2 agonist, which, for example, reduces dosing frequency, improves patient compliance, and improves therapeutic index improve Alternative candidate half-life extenders, including PEGylation and fusions to peptides, are discussed above, and exemplary extenders are described in detail below (Strohl (2015) BioDrugs 29(4) :215-239). Reviewed, also see Tan et al. (2018) Current Pharmaceutical Design 24 :4932-4946), including PEGylation with linear or branched PEGs (see, e.g., Swierczewska et al. (2015) Expert Opin Emerg Drugs 20(4) :531-536).

A TNFR1 agonist construct includes an optional linker and an optional active modifier. Can be assembled in any order. The structure of the TNFR1 antagonist construct can be shown in Formula 1 below:

(TNFR1 inhibitor) _n - linker _p - ring activity modifier) _q , Formula 1a, or

(active modifier) _q - linker _p - ring TNFR1 inhibitor) _n , Formula 1b, where:

(n and q are each an integer, each independently 1, 2 or 3; p is 0, 1, 2 or 3); activity modifiers are polypeptides such as albumin that increase the serum half-life of TNFR1 inhibitors, or moieties such as Fc that have been modified to have reduced or no ADCC activity; TNFR1 inhibitors are molecules such as polypeptides or small drug molecules that bind to and inhibit the activity of TNFR1. Active modifiers are not human serum albumin antibodies or unmodified Fc. Also provided are TNFR2 agonists of formula 3: (TNFR2 agonist) _n -linker _p -ring activity modifier) _q , where n, p and q, linker, and activity modifier are as shown in Formula 1.

Also provided are bispecific, including multispecific constructs containing a TNFR1 antagonist (TNFR1 inhibitor) and a TNFR2 agonist linked either directly or via a linker. Such constructs may include a TNFR1 antagonist of the formula above or may have a structure as set forth in formula 2 below. Bispecific and multispecific constructs selectively inhibit inflammatory and deleterious TNFR1 signaling and enhance protective and anti-inflammatory TNRFR2 signaling. They contain moieties that provide advantageous pharmacokinetic properties, including increased serum half-life and stability, and reduced peripheral clearance compared to previous TNFR1 antagonists and TNFR2 agonists.

The structure of a multispecific, e.g., bispecific, molecule/construct provided herein is represented by the following formula (Equation 2):

(TNFR1 inhibitor) _n - (active modifier) _r1 - linker (L) _p - (active modifier) _r2 - (TNFR2 agonist) _q ,

where n = 1, 2, or 3, p = 1, 2, or 3, r1 and r2 each independently = 0, 1, 2, and q = 0, 1 or 2.

As in Equation 1, the order of the components may be different. The linker may contain multiple components, such as a GS linker, a polymeric moiety such as PEG, or other such linkers, or a hinge region, or other combination of components, and the active modifier is an Fc region, or a modified Fc region , or a moiety that modifies the activity of a construct such as a polypeptide that increases half-life or resistance to endogenous inhibitors. Components of Formulas 1 and 2 may be polypeptides or may contain other molecules such as small drugs or chemical linkers that bind specifically or non-peptide activity modifiers. Listed below each component.

Also provided are constructs containing (Equation 5).

(TNFR2 agonist) _n - linker _p - (active modifier) _q , Formula 5a, or

(active modifier) _q - linker _p - (TNFR2 agonist) _n , formula 5b,

wherein each component is as defined in Formula 1 above, and the TNFR2 agonist can be a small molecule, or a polypeptide, such as a TNFR2 single chain antibody agonist or a portion thereof.

4. TNFR1 antagonist construct , TNFR2 Efficacy agent construct , and bispecific , TNFR1 Multispecific Components Containing Antagonist/TNFR2 Agonist Constructs

Descriptions and examples of the constructs provided herein, and each component of the constructs, are described in the sections below. Exemplary forms of each construct are shown and described by Formulas 1 and 2 above, and Formulas 3 and 4 below.

a. TNFR1 inhibitor moiety (TNFR1 antagonist)

A TNFR1 inhibitor moiety in Formula 1 above and in the multispecific molecules/constructs provided herein (Equation 2 above) is any molecule comprising a polypeptide or small molecule that inhibits TNFR1 signaling. Included herein are TNFR1 inhibitors that selectively inhibit TNFR1 signaling without inhibiting TNFR2 signaling.

To avoid receptor clustering that agonizes TNFR1, TNFR1 antagonist constructs are usually monomeric/monovalent. The TNFR1 antagonist inhibitor component of the construct may be one known to have TNFR1 antagonist activity or may be identified, for example, by selection from a library such as a phage library, an antibody library, or an aptamer library. TNFR1 inhibitor moieties are those that have been modified or selected to have increased specificity or affinity for TNFR1 (adverse effects resulting from such activity are less than Grade 2 and are generally Grade 1 or less according to the NCI Common Terminology Criteria for Adverse Events (CTCAE) grading system). ), has little or no agonist activity for TNFR1, and optionally has agonist activity for TNFR2.

In such cases, the TNFR1 inhibitor moiety may be provided as a single chain antibody or in any other form described herein, including, for example, a half-life extender such as any described above and below, a modified Fc region or an Fc dimer. sieve, or other part or parts that increase serum half-life.

For example, as provided herein, the TNFR1 inhibitor component of a TNFR1 antagonist construct may be or include a human domain antibody (dAb) that specifically binds to TNFR1. A dAb may contain a variable region heavy (V _H ) or light (V _L ) chain domain. dAbs for use herein include, for example: the dAb designated DOM1h-574-208 (SEQ ID NO: 54) (from DMS5541; see SEQ ID NO: 38), GSK1995057 (see SEQ ID NO: 55) and GSK2862277 (see SEQ ID NO: 56), as well as the dAb set forth in any of SEQ ID NOs: 57-672; See, for example, US Pat. Nos.: 9,028,817 and 9,028,822; US Publication Nos.: 2006/0083747, 2010/0034831, and 2012/0107330; and International Application Publication Numbers: WO 2004/058820, WO 2004/081026, WO 2005/035572, WO 2006/038027, WO 2007/049017, WO 2008/149144, WO 2008/149148, WO 2010/094720, WO 20 11/051217 , WO 2011/006914, WO 2012/172070, WO 2012/104322, and WO 2015/104322, and other related family member applications and patents; See also Enever et al., (2015) Protein Engineering, Design & Selection 28(3) :59-66, which provides sequences and discussion of various dAbs. Vhh dAbs containing heavy chains are provided. These dAbs can be directly or indirectly linked to moieties such as Fc or HSA, which can increase serum half-life and also confer other properties or activities to the construct.

The anti-TNFR1 inhibitor component may be or may include a Nanobody. Examples of these are (Nb) Nb 70 and/or Nb 96 (see SEQ ID NOs: 683 and 684, respectively). These dAbs and Nbs are investigated for immunogenicity and, if necessary, modified to remove predicted immunogenic sequences using molecular modeling and mutagenesis. Immunogenic sequences can be removed by standard methods known in the art. For example, each amino acid is conservatively replaced to identify potentially antigenic peptides and to identify those that are not antigenic and retain activity. Other methods are known (see, for example, Schubert et al., which describe methods for deimmunizing proteins). (2018) PLoS Comput Biol.14(3) :e1005983)]).

Thus, for example, the TNFR1 antagonist dAb portion is about or at least about 85%, 90%, 95% of the dAb set forth in any of SEQ ID NOs: 54-672, or the dAb set forth in any of SEQ ID NOs: 54-672. , a dAb with 98%, 99%, or greater sequence identity, or a TNFR1 antagonist dAb known to those skilled in the art.

Other TNFR1 antagonists include, for example, antigen-binding antibody fragments. For example, a TNFR1 antagonist may be a Fab fragment, Fab' fragment, single chain Fv (scFv), disulfide linked Fv (dsFv), Fd fragment, Fd' fragment, single chain Fab (scFab), hsFv (helix-stabilized Fv), free light chain, or an antigen-binding fragment of any of the above. It is also possible to include a linker, such as a GS linker, in the construct to increase flexibility, for example.

For example, the TNFR1 inhibitor portion of an antagonist may contain an antigen-binding fragment of an antagonistic TNFR1 antibody designated ATROSAB. The fragment has at least 85%, 90%, 95% or more sequence identity therewith (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) of the heavy or light chain CDRs of ATROSAB, or one or more (or all) of the CDRs. The TNFR1 antagonist is at least 85%, 90% of V _H of ATROSAB (residues 1-115 of SEQ ID NO: 31) and/or V _L (residues 1-113 of SEQ ID NO: 32), or V _H or V _L of ATROSAB , V _H or V _L containing 95%, or greater, sequence identity. For example, it may contain a dAb derived from ATROSAB. TNFR1 antagonists include, for example, Fab or scFv fragments such as ATROSAB Fab (FabATR) light and heavy chains set forth in SEQ ID NOs: 679 and 680, respectively, or ATROSAB including the ATROSAB scFv (scFv IZI06.1) set forth in SEQ ID NO:673. may contain other monovalent antibody fragments of For example, the scFv can be linked to the VL by a short peptide linker corresponding to residues 1-113 of the ATROSAB light chain (eg, GGGGSGGGGSGGSAQ as in SEQ ID NO:673, or the linker set forth in any of SEQ ID NOs: 813-834). It contains a V _H domain (see SEQ ID NO: 31) corresponding to residues 1-115 of the ATROSAB heavy chain linked to the domain (see SEQ ID NO: 32). A TNFR1 antagonist may contain a variant of ATROSAB scFV with increased affinity or selectivity for TNFR1 or both, including scFv IG11, which contains the sequence set forth in SEQ ID NO: 674, the sequence set forth in SEQ ID NO: 675 or scFv 13.7 containing the sequence set forth in SEQ ID NO: 676, or variants containing at least 90% sequence identity to the sequences of scFv IG11, scFv T12B, and scFv 13.7. The TNFR1 antagonist may also include sequences of amino acid residues from Fab 13.7 light and heavy chains (derived from scFV 13.7) set forth in SEQ ID NOs: 681 and 682, respectively.

TNFR1 inhibitors within a TNFR1 antagonist construct also include TNF variants (muteins) that bind to TNFR1 and reduce or inhibit signaling. These are, for example, TNF variants (muteins), such as the mutations L29S, L29G, L29Y, R31E, R31N, R32Y, R32W, S86T, L29S/R32W, L29S with reference to SEQ ID NO: 2, which confer selectivity for TNFR1. /S86T, R32W/S86T, L29S/R32W/S86T, R31N/R32T, R31E/S86T, R31N/R32T/S86T, and TNF variants containing one or more of E146R. A TNFR1 antagonist may contain, for example, the TNFR1 selective antagonist TNF mutein derived from the mutein designated XPro1595 (see SEQ ID NO: 701). XPro1595, with reference to SEQ ID NO: 2, contains the mutations V1M, R31C, C69V, Y87H, C101A and A1456R. Another exemplary TNFR1 selective antagonistic TNF mutein is derived from XENP345 (see SEQ ID NO: 702) containing the mutations I97T/A145R with respect to SEQ ID NO: 2; The TNFR1 selective antagonistic TNF mutein is designated R1antTNF (see SEQ ID NO: 703) containing the mutations A84S, V85T, S86T, Y87H, Q88N and T89Q with reference to SEQ ID NO: 2. TNFR1 inhibitors for use in TNFR1 antagonists also include small molecule inhibitors that can be chemically conjugated to a linker.

As described herein, and referring to the Examples, for example, a TNFR1 inhibitor (antagonist) moiety can be modified to improve its specificity/selectivity for TNFR1, and also optionally to have TNFR2 agonistic activity. can TNF binds to TNFR1 with low pM affinity (K _d 19 pM); In general, the antagonists herein have at least the same affinity as TNF, and unless their activity is due to 'locking' the inactive form of the receptor, it will lock the receptor and is therefore not required. A TNFR1 antagonist construct provided herein can be about 100 nM or less (e.g., 95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55 nM, 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, or a _KD value of 1 nM or less) specifically for TNFR1 including bonding In certain embodiments, the TNFR1 antagonist is 1 nM or less (e.g., 990 pM, 980 pM, 970 pM, 960 pM, 950 pM, 940 pM, 930 pM, 920 pM, 910 pM, 900 pM, 890 pM, 880 pM, 870 pM, 860 pM, 850 pM, 840 pM, 830 pM, 820 pM, 810 pM, 800 pM, 790 pM, 780 pM, 770 pM, 760 pM, 750 pM, 740 pM, 730 pM, 720 pM, 710 pM, 700 pM, 690 pM, 680 pM, 670 pM, 660 pM, 650 pM, 640 pM, 630 pM, 620 pM, 610 pM, 600 pM, 590 pM, 580 pM, 570 pM, 560 pM, 550 pM , 540 pM, 530 pM, 520 pM, 510 pM, 500 pM, 490 pM, 480 pM, 470 pM, 460 pM, 450 pM, 440 pM, 430 pM, 420 pM, 410 pM, 400 pM, 390 pM, 380 pM, 370 pM, 360 pM, 350 pM, 340 pM, 330 pM, 320 pM, 310 pM, 300 pM, 290 pM, 280 pM, 270 pM, 260 pM, 250 pM, 240 pM, 230 pM, 220 pM, 210 pM, 200 pM, 190 pM, 180 pM, 170 pM, 160 pM, 150 pM, 140 pM, 130 pM, 120 pM, 110 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM , 40 pM _, 30 pM, 20 pM, 10 pM, 5 pM, or 1 pM or less) and binds specifically to TNFR1.

TNFR1 antagonist constructs provided herein are also selected or designed to lack or have reduced binding to other TNFR superfamily members. For example, they are evaluated to confirm that they do not specifically bind to other TNFR superfamily members, such as TNFR2, using any suitable in vitro binding assay. Assays include, for example, ELISA-based methods. For example, a TNFR1 antagonist construct may specifically bind to human TNFR1 or a TNFR1 derived peptide with greater affinity than it does to other family members or their corresponding peptides. The increased affinity is, for example, at least or at least about 5-fold greater than the affinity of a TNFR1 antagonist for another TNFR superfamily member, such as TNFR2 (eg, at least or 5-fold or greater, 6-fold or greater, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-times or more, 90-times or more, 100-times or more, 200-times or more, 300-times or more, 400-times or more, 500-times or more, 600-times or more, 700-times or more, 800-times or more, 900-fold, 1,000-fold, 2,000-fold, 3,000-fold, 4,000-fold, 5,000-fold, 6,000-fold, 7,000-fold, 8,000-fold, 9,000-fold, 10,000-fold or more).

Among the TNFR1 antagonist constructs provided herein are those that exhibit high k _on and low k _off values upon interaction with TNFR1 consistent with high affinity receptor binding. For example, a TNFR1 antagonist construct provided herein may have a TNFR1 expression of about 10 ⁴ M ^-1 s ^-1 or higher (eg, 1.0 x 10 4 M -1 s -1 , 1.5 x ^{10 4} M ^-1 s ^-1 , 1.5 x 10 ⁴ M -1 ^{s -1} in the presence of TNFR1). s ^-1 , 2.0 x 10 ⁴ M ^-1 s ^-1 , 2.5 x 10 ⁴ M ^-1 s ^-1 , 3.0 x 10 ⁴ M ^-1 s ^-1 , 3.5 x 10 ⁴ M ^-1 s ^-1 , 4.0 x 10 ⁴ M ^-1 s ^-1 , 4.5 x 10 ⁴ M ^-1 s ^-1 , 5.0 x 10 ⁴ M ^-1 s ^-1 _, 5.5 x 10 ⁴ M ^-1 s ^-1 , 6.0 x 10 ⁴ M ^-1 s ^-1 , 6.5 x 10 ⁴ M ^-1 s ^-1 , 7.0 x 10 ⁴ M ^-1 s ^-1 , 7.5 x 10 ⁴ M ^{-1 s -1} , 8.0 x 10 ⁴ M ^-1 s ^-1 , 8.5 x 10 ⁴ M ^-1 s ^-1 , 9.0 x 10 4 M ^-1 s ^-1 , 9.5 x 10 ⁴ M ^-1 s ^-1 , 1.0 x 10 ⁵ M ^-1 s ^-1 , 1.5 x 10 ⁵ M ^-1 s ^-1 , 2.0 x 10 ⁵ M ^-1 s ^-1 , 2.5 x 10 ⁵ M ^-1 s ^-1 , 3.0 x 10 ⁵ M ^{- 1} s ^-1 , 3.5 x 10 ⁵ M ^-1 s ^-1 , 4.0 x 10 ⁵ M ^-1 s ^-1 , 4.5 x 10 ⁵ M ^-1 s ^-1 , 5.0 x 10 ⁵ M ^-1 s ^-1 , 5.5 x 10 ⁵ M ^-1 s ^-1 , 6.0 x 10 ⁵ M ^-1 s ^-1 , 6.5 x 10 ⁵ M ^-1 s ^-1 , 7.0 x 10 ⁵ M ^-1 s ^-1 , 7.5 x 10 ⁵ M ^-1 s ^-1 , 8.0 x 10 ⁵ M ^-1 s ^-1 , 8.5 x 10 ⁵ M ^-1 s ^-1 , 9.0 x 10 5 M ^-1 s ^-1 , 9.5 x ^{10 5} M ^-1 s ^-1 , 1.0 x 10 ⁶ M ^-1 s ^-1 or more) may indicate a k _on value. For example, a TNFR1 antagonist provided herein, when complexed with TNFR1, has about 10 ⁻³ s ⁻¹ or less (eg, about 1.0 x 10 ⁻³ s ⁻¹ , 9.5 x 10 ⁻⁴ s ⁻¹ , 9.0 x 10 ^-4 s ^-1 , 8.5 x 10 ^-4 s ^-1 , 8.0 x 10 ^-4 s ^-1 , 7.5 x 10 ^-4 s ^-1 , 7.0 x 10 ^-4 s ^-1 , 6.5 x 10 ^-4 s ^-1 , 6.0 x 10 ^-4 s ^-1 , 5.5 x 10 ^-4 s ^-1 , 5.0 x 10 ^-4 s ^-1 , 4.5 x 10 ^-4 s ^-1 , 4.0 x 10 ^-4 s ^-1 , 3.5 x 10 ^-4 s ^-1 , 3.0 x 10 ^-4 s ^-1 , 2.5 x 10 ^-4 s ^-1 , 2.0 x 10 ^-4 s ^-1 , 1.5 x 10 ^-4 s ^-1 , 1.0 x 10 ^-4 s ^-1 , 9.5 x 10 ^-5 s ^-1 , 9.0 x 10 ^-5 s ^-1 , 8.5 x 10 ^-5 s ^-1 , 8.0 x 10 ^-5 s ^-1 , 7.5 x 10 ^-5 s ^-1 , 7.0 x 10 ^-5 s ^-1 , 6.5 x 10 ^-5 s ^-1 , 6.0 x 10 ^-5 s ^-1 , 5.5 x 10 ^-5 s ^-1 , 5.0 x 10 ^-5 s ^-1 , 4.5 x 10 ^-5 s ^{- 1} , 4.0 x 10 ^-5 s ^-1 , 3.5 x 10 ^-5 s ^-1 , 3.0 x 10 ^-5 s ^-1 , 2.5 x 10 ^-5 s ^-1 , 2.0 x 10 ^-5 s ^-1 , 1.5 x 10 ^-5 s ^-1 , or 1.0 x 10 ^-5 s ^-1 or less) may indicate a k _off value.

The C-terminus of a TNFR1 antagonist (the TNFR1 inhibitor portion of a construct of Formula 1 and also of Formula 2), such as any TNFR1 antagonist construct described herein, is directly or more generally via a linker or combination of elements of a linker. or fused to the N-terminus of a TNFR2 agonist (or to a small molecule TNFR2 agonist) via one or more linkers, as discussed below and elsewhere herein. Alternatively, the N-terminus of a TNFR1 inhibitor moiety can be fused to the C-terminus of a TNFR2 agonist, or the C-terminus of a TNFR1 inhibitor moiety (or to a small molecule TNFR2 agonist) is active modified directly or via a linker. It can be fused to an agent or linker.

Linker (L), discussed in more detail below, is any that improves pharmacological properties, including increased stability and flexibility and reduced steric hindrance, and optionally imparts additional properties to the construct. A linker may comprise more than one component, where each component confers a particular characteristic, e.g., a TNFR1 antagonist may comprise an Ig Fc region, and/or an antibody hinge region, and/or a short peptide linker such as glycine-serine. Any one or more of the linkers may be included. The Fc region is modified, for example, to eliminate or reduce ADCC activity and/or to alter receptor binding and/or for other such activities and properties. As discussed below, linkers also include chemical linkers. For example, in some embodiments, the linker is a poly(ethylene glycol) (PEG) molecule, or a branched PEG molecule, such as having a molecular weight of about 30 kDa or greater.

b. TNFR2 Agonist Constructs and TNFR2 Antagonist Constructs

TNFR2 agonist (regulatory T cell generator) constructs can be used to treat inflammatory and autoimmune diseases, and also solid tumors, among others diseases, disorders and conditions. Regulatory T cells (Treg) suppress autoimmunity and have immunosuppressive effects such as the tumor microenvironment. Treg proliferation is positively regulated by TNFR2, and the absence of TNFR2 correlates with reduced Treg numbers and exacerbated experimental arthritis. Thus, TNFR2 agonist constructs can be used in the treatment of many autoimmune diseases, other chronic inflammatory, and other acute inflammatory conditions (eg, SARS, COVID-19).

TNFR2 antagonist constructs inhibit regulatory T cells and are used in the treatment of cancer and other hyperproliferative diseases (TNFR2 is a 'checkpoint receptor'). Regulatory T cells accumulate in the tumor microenvironment and play a role in suppressing antitumor immune responses. TNFR2 antagonist constructs are for the treatment of cancer and other hyperproliferative diseases such as Dupuytren's contracture, and idiopathic pulmonary fibrosis.

As discussed above, TNFR2 agonist constructs containing TNFR2 agonists are also provided. They include a TNFR2 agonist linked to an active modifier, either directly or via a linker, and also contain multispecific constructs, such as bispecific constructs containing a TNFR1 antagonist and a TNFR2 agonist in various configurations with a linker having the appropriate structure and properties. including enemy constructs. In some embodiments, the TNFR2 agonist is in a bispecific construct. A TNFR2 agonist does not or does not activate TNFR1, particularly in a multispecific molecule/construct, such as a bispecific, and/or interferes with inhibition of TNFR1 signaling through a multispecific, eg, TNFR1 antagonist portion of a bispecific molecule. activating or agonizing TNFR2 without

The TNFR2 agonist can be anything known to those of skill in the art, including agonist antibodies and antigen binding portions thereof and single chain and other arrangement derivatives of antibodies, and can also be small molecule agonists. TNFR2 agonists can also be generated through in silico design and/or candidate preparation and library screening. For example, TNFR2 agonists can be identified by screening phage libraries or antibody libraries or aptamer libraries. TNFR2 agonist antibodies or antigen-binding fragments thereof can be generated by screening libraries of antibodies and antigen-binding fragments thereof for functional molecules that bind to epitopes within TNFR2 and selectively promote receptor activation. Examples of such methods and molecules are those described in International Application Publication No. WO 2017/040312.

Development of TNFR2 selective agonists may involve elucidation of epitopes within TNFR2 that promote agonist receptor binding. Epitope mapping analyzes using linear, constrained cyclic and bicyclic peptides from various regions of TNFR2 indicate that potent TNFR2 antibodies bind epitopes from distinct regions of the TNFR2 polypeptide in a conformation dependent manner. For example, one identified epitope of TNFR2 includes residues 56-60 of SEQ ID NO:4 (KCSPG). The potent TNFR2 antibody MR2-1 binds to this epitope; It does not bind to the epitope containing residues 142-146 of SEQ ID NO:4 (KCRPG). Human TNFR2 can be selected to bind to an epitope (eg comprising residues 56-60 of SEQ ID NO:4). In general, human TNFR2 agonists can be selected or designed to bind to an epitope in human TNFR2 containing at least 5 discontinuous or contiguous residues within residues 96-154 of SEQ ID NO: 4 (CGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVARPGT; SEQ ID NO: 841); / or may bind to an epitope within residues 111-150 of SEQ ID NO: 4 (TREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVA; SEQ ID NO: 842) to which MR2-1 further binds. The human TNFR2 agonist may also comprise residues 115-142 of SEQ ID NO: 4 (NRICTCRPGWYCALSKQEGCRLCAPLRK; SEQ ID NO: 843), and/or residues 122-136 of SEQ ID NO: 4 (PGWYCALSKQEGCRL; SEQ ID NO: 844), and/or SEQ ID NO: : an epitope within residues 96-122 of SEQ ID NO: 4 (CGSRCSSDQVETQACTR; SEQ ID NO: 845), and/or an epitope within residues 101-107 of SEQ ID NO: 4 (SSDQVET; SEQ ID NO: 846; to which MR2-1 further binds) , and/or an epitope within amino acids 48-67 of SEQ ID NO: 4 (QTAQMCCSKCSPGQHAKVFC; SEQ ID NO: 847), and/or residues 130-149 of SEQ ID NO: 4 (KQEGCRLCAPLRKCRPGFGV; SEQ ID NO: 848), and/or SEQ ID NO: : an epitope containing residues 110-147 of 4 (CTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGF; SEQ ID NO: 849), and/or positions 106-155 of SEQ ID NO: 4 (ETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVARPGTE; SEQ ID NO: 850), and/or SEQ ID NO: 4 residues 137-144 (CAPLRKCR; SEQ ID NO: 851), and/or residues 141-149 of SEQ ID NO: 4 (RKCRPGFGV; SEQ ID NO: 852).

In another aspect, the TNFR2 agonist antibody and antigen-binding fragment thereof specifically binds an epitope within or containing an amino acid residue of any one of SEQ ID NOs: 853-1211, such that the antibody or antigen-binding fragment is human It binds specifically to TNFR2, but not to other TNFR superfamily members, particularly TNFR1. Human TNFR2 agonist antibodies or antigen-binding fragments thereof do not bind or have impaired/reduced binding to other members of the TNFR superfamily, including TNFR1 (see, eg, International Application Publication No. WO 2017/040312).

Epitopes within TNFR2 that can be used to screen for TNFR2 agonists include peptides whose sequences are set forth in any one of SEQ ID NOs: 853-1211. These peptides mimic the structurally fixed framework of TNFR2 and the structural constraints of the peptide fragments within TNFR2 to constrain the peptide fragments into distinct three-dimensional conformations (e.g., cysteine residues in N- and C-terminal positions). or by incorporating at various internal positions in the peptide chain) into cyclic and polycyclic forms. Cyclic and polycyclic peptide fragments can then be immobilized on a solid surface and screened for molecules that bind to, for example, the TNFR2 agonistic antibody MR2-1 using ELISA. Using this assay, peptides containing residues within an epitope of TNFR2 that promote receptor activation can be structurally preconfigured with these amino acids to mimic the conformation of that peptide in native proteins. Cyclic and polycyclic peptides thus obtained (e.g. peptides having the sequence of any one of SEQ ID NOs: 853-1194, in particular those containing a KCSPG motif such as SEQ ID NOs: 905, 921, 927, 970 and 1085) ) can be used to screen libraries of antibodies and antigen-binding fragments thereof to identify TNFR2 agonists for use herein. The constrained peptide acts as a surrogate for an epitope within TNFR2 that promotes receptor activation, and thus antibodies or antigen-binding fragments generated using this screening technique bind to the corresponding epitope of TNFR2 and are agonists of receptor activity ( See, eg, International Application Publication No. WO 2017/040312). To generate TNFR2 agonists, phage display is used. Phage display libraries are contacted under conditions that allow specific binding to occur. The TNFR2 derived peptide(s) (eg, any of SEQ ID NOs: 853-1194) are immobilized on a solid support or within the phage. Phage containing the TNFR2 binding moiety are complexed with the target on a solid support and unbound phage are washed away. The bound phage are then separated from the target by changing the buffer to an extreme pH (pH 2 or 10), changing the ionic strength of the buffer, adding a denaturant, or using other known methods. To isolate bound phage, protein elution can be performed (see, eg, International Application Publication No. WO 2017/040312).

MR2-1 is an exemplary potent TNFR2 antibody that binds to TNFR2 and enhances TNFR2-mediated Treg cell proliferation. Although MR2-1 binds osteoprotegerin, the heavy and/or light chain variable regions of this antibody, or particularly the heavy and/or light chain CDRs of MR2-1, are modified to eliminate the ability of the resulting antibody or fragment to bind to other than TNFR2. to a member of the TNFR superfamily of to generate an agonistic TNFR2 antibody or antigen-binding fragment thereof. This can be achieved using genetic engineering and/or antibody library screening techniques as described, for example, in International Application Publication No. WO 2017/040312.

As provided herein, TNFR2 agonists include antigen-binding fragments of potent human anti-TNFR2 antibodies, such as MR2-1 and MAB2261, such as MR2-1 commercially available from Hycult Biotech; and MAB2261 from R&D Systems. For example, the V _H and V _L domains of MR2-1 or MAB2261, or one or more CDRs contained therein, are used to create TNFR2 agonists. Such agonists may contain human domain antibodies (dAbs) specific for TNFR2; The dAb has at least or at least about 85%, 90% sequence identity to the variable region heavy (V _H ) or light chain (V _L ) domain of MR2-1 or MAB2261, or to V _H or V _L or MR2-1 or MAB2261; contains at least 95% of V _H or V _L , with the proviso that

The resulting TNFR2 retains TNFR2 agonist activity. A TNFR2 agonist may also be MR2-1 or another antigen-binding fragment derived from the MAB2261 antibody, or an amino acid sequence having at least or at least about 85%, 90%, 95%, or greater sequence identity thereto, e.g., Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, disulfide-linked Fv (dsFv), Fd fragment, Fd' fragment, single-chain Fv (scFv), single-chain Fab (scFab), hsFv (helix-stabilized Fv), minibodies, diabodies, anti-individual (anti-Id) antibodies, free light chains, or antigen-binding fragments of any of the foregoing. Antibody fragments can be any of the above fragments, e.g., tandem scFv, Fab-scFv (HC C-terminus, or LC C-terminus), Fab-(scFv) ₂ (C-terminus), scFv-Fab-scFv , Fab- _CH 2 -scFv, scFv fusion (C-terminus, or N-terminus), Fab-fusion (HC C-terminus, or LC C-terminus), scFv-scFv-dAb, scFv-dAb-scFv, dAb- scFv-scFv, and combinations of tribodies. A TNFR2 agonist includes any dAb provided herein or known in the art that has about or at least about 85%, 90%, 95% or more sequence identity thereto and TNFR2 agonist activity.

In some embodiments, the TNFR2 agonist is a scFv of a TNFR2 agonistic monoclonal antibody, including any known in the art, or about or at least about 85%, 90%, 95%, or greater than 95% of such a scFv. scFvs with sequence identity, provided that the resulting construct retains TNFR2 agonist activity. In some embodiments, a TNFR2 agonist can be a Fab fragment of a TNFR2 agonistic monoclonal antibody or a Fab thereof or a Fab having at least about 85%, 90%, 95% or greater sequence identity and TNFR2 agonistic activity.

A TNFR2 agonist can also be or include a TNF mutein that has been modified to bind TNFR2 and have agonist activity (see, eg, SEQ ID NOs: 765-800). Examples of such embodiments are for example the TNFR2 selective mutations K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, with respect to SEQ ID NO: 2; D143N/A145R, A145R/S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/ S147D, A145N/E146D/S147D, A145T/E146S/S147D, A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A1 45R/S147T, E146D/S147D, D143V/ A TNFR2 agonist containing a TNFR2 selective TNF mutein such as a TNF variant having one or more of F144L/A145S, and D143V/A145S, and combinations thereof, such as a combination of D143V/A145S and S95C/G148C.

For example, a TNF variant with mutations D143N/A145R (SEQ ID NO: 781) binds to and agonizes TNFR2 and can be used in the constructs provided herein. The combination of the TNF mutein with the mutation S95C/G148C and any others listed, known or identified with respect to SEQ ID NO: 2 is also a TNFR2 selective agonist that can be included in the constructs provided herein.

A TNFR2 agonist may contain a fusion of a single chain TNFR2 selective TNF mutein trimer with a multimerization domain. As described herein, the primary ligand for TNFR2 is membrane bound TNF (memTNF; also referred to herein as transmembrane TNF or tmTNF). Addition of a multimerization domain, such as a dimerization or trimerization domain, results in a hexameric or 9meric molecule for the TNF subunit, respectively; These hexamers and 9mers of TNF mimic membrane-bound TNF trimers and thus activate TNFR2 signaling. The dimerization domain includes, for example, EHD2 (SEQ ID NO: 808) discussed above. EHD2 is derived from MHD2 (SEQ ID NO: 811) derived from the heavy chain C _H 2 domain of IgE and the heavy chain C _H 2 domain of IgM. The dimerization domain also includes Fc domains derived from IgG1 (see SEQ ID NO: 10) and IgG4 (see SEQ ID NO: 16), optionally altering immune effector function and/or enhancing FcRn recycling. including variations such as The trimerization domain includes, for example, the trimerization domain of chicken tenascin C (TNC) (SEQ ID NO: 805) and the trimerization domain of human TNC (SEQ ID NO: 807). Dimerization and trimerization enhance TNFR2 signaling and improve the pharmacological properties of the construct. For example, the half-life of the fusion protein is increased by increasing the molecular weight of the molecule and/or introducing FcRn recycling, eg when the dimerization domain is Fc.

As provided herein, a TNFR2 agonist may contain a TNF mutein (TNFmut) trimeric chain together with any of the mutations described herein that confer selectivity for TNFR2 and/or reduce or eliminate binding to TNFR1. can An example of such a mutation is the replacement D143N/A145R with respect to SEQ ID NO: 2 fused to a multimerization domain (MD) such as a dimerization or trimerization domain. The multimerization domain may be fused to the N- or C-terminus of the TNF mutein trimeric chain, and a linker is included between each TNF mutein and between the TNF mutein trimeric chain and the multimerization domain. These TNFR2 agonists have formulas 4 and 5:

MD-L1-TNFmut-L2-TNFmut-L3-TNFmut (Equation 4); or

TNFmut-L1-TNFmut-L2-TNFmut-L3-MD (Equation 5);

where MD is the multimerization domain (active modifier); TNFmut is a TNFR2 selective TNF mutein such as the mutein with mutations D143N/A145R; L1, L2 and L3 are linkers described below, such as the Gly-Ser linker, which may be the same or different.

In certain embodiments, the multimerization domain is EHD2 (SEQ ID NO: 808), MHD2 (SEQ ID NO: 811), the trimerization domain of chicken TNC (SEQ ID NO: 805), the trimerization domain of human TNC (SEQ ID NO: 807) , IgG1 Fc or IgG4 Fc. If the dimerization domain is an IgG1 Fc or an IgG4 Fc, it is the same Fc used to link the TNFR1 antagonist to the TNFR2 agonist and not an additional Fc. An IgG1 or IgG4 Fc can be modified to enhance or eliminate immune effector functions such as ADCC, ADCP and/or CDC activity and/or to enhance FcRn binding. The multimerization domain, such as the Fc region, increases the in vivo stability and serum half-life of the construct. For purposes herein, the Fc region in a construct of Formulas 1-5 or variations thereof is generally modified to alter or modulate the pharmacological properties or activity of the construct. Fc modifications are discussed in more detail below. Any multimerization domain known in the art is also contemplated for use in the TNFR2 agonists herein.

A TNF mutein can be a TNF variant having any one or more mutations conferring TNFR2-selectivity. Mutations are for example K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A1 with respect to SEQ ID NO: 2 45R/ S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/ S147D, A145T/E146S/S147D, A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/S1 47D, D143V/F144L/A145S, and D143V Include /A145S. TNF variants with the mutations D143N/A145R are contemplated for use herein. Any other mutation conferring TNFR2-selectivity known in the art is also contemplated for use herein. A TNF mutein may contain the entire sequence of soluble TNF (i.e., residues 1-157 of SEQ ID NO: 2), or a partial sequence of soluble TNF, e.g., of sufficient length to bind and/or agonize TNFR2. , for example residues 4-157, 9-157 or 12-157 of SEQ ID NO:2.

The L1, L2 or L3 linkers may be the same or different. In particular, the linker may contain a short peptide linker such as a GS linker. For example, the linker may contain (GGGGS) _n (where n = 1-5) (SEQ ID NO: 1471). The linker may also include all or part of the stem region of TNF-α containing the sequence of amino acids GPQREEFPRDLSLISLISPLAQAVRSSSSRTPSDK (SEQ ID NO: 812) corresponding to residues 57-87 of the full-length sequence of TNF (transmembrane TNF) set forth in SEQ ID NO: 1 ( at least 10, 15, or 20 contiguous residues). For example, a linker containing all or part of the stem region, including at least 10, 15 or 20 contiguous amino acid residues, may be between the N- or C-terminal TNF mutein and the multimerization domain. All three linkers (GGGGS) _n (n is usually 1-10) (SEQ ID NO: 1472), or other combinations of Gly-Ser (GGGGS) Gly-Ser residues such as _n and TNF may contain a mixture of all or part of the stem region of at least 10, 15 or 20 contiguous amino acid residues. Exemplary linkers are set forth in SEQ ID NOs: 813-834, 1471 and 1472.

A TNFR2 agonist provided herein is less than or equal to about 100 nM (e.g., 95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55 nM, 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM) specifically for _TNFR2 . including bonding In certain instances, the TNFR2 agonist is 1 nM or less (e.g., 990 pM, 980 pM, 970 pM, 960 pM, 950 pM, 940 pM, 930 pM, 920 pM, 910 pM, 900 pM, 890 pM, 880 pM, 870 pM, 860 pM, 850 pM, 840 pM, 830 pM, 820 pM, 810 pM, 800 pM, 790 pM, 780 pM, 770 pM, 760 pM, 750 pM, 740 pM, 730 pM, 720 pM, 710 pM, 700 pM, 690 pM, 680 pM, 670 pM, 660 pM, 650 pM, 640 pM, 630 pM, 620 pM, 610 pM, 600 pM, 590 pM, 580 pM, 570 pM, 560 pM, 550 pM , 540 pM, 530 pM, 520 pM, 510 pM, 500 pM, 490 pM, 480 pM, 470 pM, 460 pM, 450 pM, 440 pM, 430 pM, 420 pM, 410 pM, 400 pM, 390 pM, 380 pM, 370 pM, 360 pM, 350 pM, 340 pM, 330 pM, 320 pM, 310 pM, 300 pM, 290 pM, 280 pM, 270 pM, 260 pM, 250 pM, 240 pM, 230 pM, 220 pM, 210 pM, 200 pM, 190 pM, 180 pM, 170 pM, 160 pM, 150 pM, 140 pM, 130 pM, 120 pM, 110 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM , 40 pM, 30 pM, 20 pM, 10 pM, 5 pM, or 1 pM) and binds specifically to _TNFR2 .

The TNFR2 agonist can be administered to Tregs (e.g., CD4 + , CD25 + FOXP3, CD4 ⁺ , CD25 ⁺ FOXP3) in vivo, for example, in a subject to whom the agonist is administered, or in vitro in a sample containing Tregs that come into contact with the TNFR2 agonist for testing purposes. ⁺ ) can induce proliferation. Proliferation of Tregs is, for example, between 0.00001% and 100.0% (eg, 0.00001%, 0.00002%) as measured by FACS analysis on subjects or samples containing cell populations not treated with a TNFR2 agonist. , 0.00003%, 0.00004%, 0.00005%, 0.00006%, 0.00007%, 0.00008%, 0.00009%, 0.0001%, 0.0002%, 0.0003%, 0.0004%, 0.0005%, 0.00 06%, 0.0007%, 0.0008%, 0.0009%, 0.001 %, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0% , 8.0%, 9.0%, 10.0%, 20.0%, 30.0%, 40.0%, 50.0%, 60.0%, 70.0%, 80.0%, 90.0%, or 100%).

Thus, TNFR2 agonists can be used to promote Treg cell proliferation and can be administered to a mammalian subject, such as a human patient with an autoimmune or chronic inflammatory disease or disorder, to elicit an immune response in the patient (e.g., an autoimmune or non-threatening disease or disorder). Attenuates the size and duration of CD8 ⁺ cytotoxic T lymphocytes produced in vivo in response to foreign antigens. For example, a population of Treg cells expanded ex vivo by administration of a TNFR2 agonist or treatment with a TNFR2 agonist to a human patient is, for example, about 0.00001 mg/mL to 10.0 mg/mL, relative to a subject not treated with a NFR2 agonist. (e.g., 0.00001 mg/mL, 0.0001 mg/mL, 0.001 mg/mL, 0.01 mg/mL, 0.1 mg/mL, 1.0 mg/mL, or 10.0 mg/mL), or 0.001 to 1.0 mg/mL mL (eg, 0.001 mg/mL, 0.005 mg/mL, 0.010 mg/mL, 0.050 mg/mL, 0.10 mg/mL, 0.20 mg/mL, 0.30 mg/mL, 0.40 mg/mL, 0.50 mg/mL , 0.60 mg/mL, 0.70 mg/mL, 0.80 mg/mL, 0.90 mg/mL, or 1.0 mg/mL) of secreted immunoglobulins (e.g., IgG) that cross-react with self or non-threat antigens. quantity can be reduced. Additionally or alternatively, the TNFR2 agonist increases cytotoxic T cell numbers (eg, the level of CD8+ T cells), eg, as measured by FACS analysis on subjects not treated with the TNFR2 agonist. For example, from about 0.00001 to 100.0% (e.g., 0.00001%, 0.00002%, 0.00003%, 0.00004%, 0.00005%, 0.00006%, 0.00007%, 0.00008%, 0.00009%, 0.000 1%, 0.0002%, 0.0003%, 0.0004 %, 0.0005%, 0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0. 01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0% , 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10.0%, 20.0%, 30.0%, 40.0%, 50.0%, 60.0%, 70.0%, 80.0%, 90.0 %, or 100%). For example, a TNFR2 agonist can be administered to a subject (eg, a mammalian subject such as a human) to treat an autoimmune or chronic inflammatory disease or disorder as described herein. Treating a subject in this manner reduces the amount of autoreactive CD8 ⁺ T-cells in the subject.

TNFR2 agonists provided herein can be evaluated to confirm that they lack specific binding to another TNFR superfamily member, particularly TNFR1. This can be accomplished using any of a variety of in vitro binding assays, such as ELISA-based methods known to those skilled in the art. For example, a TNFR2 agonist has an affinity greater than that of the agonist for other TNFR superfamily members, such as TNFR1, i.e., at least or at least about 2-, 3-, 4-, or 5-fold greater. (e.g., 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, 10-fold or more, 20-fold or more, 30-fold or more, 40-fold or more, 50-times or more, 60-times or more, 70-times or more, 80-times or more, 90-times or more, 100-times or more, 200-times or more, 300-times or more, 400-times or more, 500-times or more, 600-fold, 700-fold, 800-fold, 900-fold, 1,000-fold, 2,000-fold, 3,000-fold, 4,000-fold, 5,000-fold, 6,000-fold, at least 7,000-fold, at least 8,000-fold, at least 9,000-fold, at least 10,000-fold) human TNFR2 or a TNFR2-derived peptide, such as residues 48-67 of SEQ ID NO: 4 in human TNFR2 (QTAQMCCSKCSPGQHAKVFC, SEQ ID NO: 847) It includes specifically binding to a peptide fragment containing

TNFR2 agonists provided herein include those that exhibit high k _on and low k _off values upon interaction with TNFR2 consistent with high affinity receptor binding. For example, a TNFR2 agonist provided herein may be about 10 ⁴ M ^-1 s ^-1 or greater (eg, about 1.0 x 10 ^{4 M -1 s -1 , 1.5 x 10 4} M ^-1 s ^-1 , 1.5 x 10 ⁴ M -1 ^{s -1} in the presence of TNFR2). s ^-1 , 2.0 x 10 ⁴ M ^-1 s ^-1 , 2.5 x 10 ⁴ M ^-1 s ^-1 , 3.0 x 10 ⁴ M ^-1 s ^-1 , 3.5 x 10 ⁴ M ^-1 s ^-1 , 4.0 x 10 ⁴ M ^-1 s ^-1 , 4.5 x 10 ⁴ M ^-1 s ^-1 , 5.0 x 10 ⁴ M ^-1 s ^-1 _, 5.5 x 10 ⁴ M ^-1 s ^-1 , 6.0 x 10 ⁴ M ^-1 s ^-1 , 6.5 x 10 ⁴ M ^-1 s ^-1 , 7.0 x 10 ⁴ M ^-1 s ^-1 , 7.5 x 10 ⁴ M ^{-1 s -1} , 8.0 x 10 ⁴ M ^-1 s ^-1 , 8.5 x 10 ⁴ M ^-1 s ^-1 , 9.0 x 10 4 M ^-1 s ^-1 , 9.5 x 10 ⁴ M ^-1 s ^-1 , 1.0 x 10 ⁵ M ^-1 s ^-1 , 1.5 x 10 ⁵ M ^-1 s ^-1 , 2.0 x 10 ⁵ M ^-1 s ^-1 , 2.5 x 10 ⁵ M ^-1 s ^-1 , 3.0 x 10 ⁵ M ^{- 1} s ^-1 , 3.5 x 10 ⁵ M ^-1 s ^-1 , 4.0 x 10 ⁵ M ^-1 s ^-1 , 4.5 x 10 ⁵ M ^-1 s ^-1 , 5.0 x 10 ⁵ M ^-1 s ^-1 , 5.5 x 10 ⁵ M ^-1 s ^-1 , 6.0 x 10 ⁵ M ^-1 s ^-1 , 6.5 x 10 ⁵ M ^-1 s ^-1 , 7.0 x 10 ⁵ M ^-1 s ^-1 , 7.5 x 10 ⁵ M ^-1 s ^-1 , 8.0 x 10 ⁵ M ^-1 s ^-1 , 8.5 x 10 ⁵ M ^-1 s ^-1 , 9.0 x 10 ⁵ M ^-1 s ^-1 , 9.5 x 10 ⁵ M ^-1 s ^-1 , or 1.0 Exceeding x 10 ⁶ M ^-1 s ^-1 ) may indicate a k _on value. For example, a TNFR2 antagonist provided herein, when complexed with TNFR2, exhibits about 10 ⁻³ s ⁻¹ or less (eg, about 1.0 x 10 ⁻³ s ⁻¹ , 9.5 x 10 ⁻⁴ s ⁻¹ , 9.0 x 10 ^-4 s ^-1 , 8.5 x 10 ^-4 s ^-1 , 8.0 x 10 ^-4 s ^-1 , 7.5 x 10 ^-4 s ^-1 , 7.0 x 10 ^-4 s ^-1 , 6.5 x 10 ^-4 s ^-1 , 6.0 x 10 ^-4 s ^-1 , 5.5 x 10 ^-4 s ^-1 , 5.0 x 10 ^-4 s ^-1 , 4.5 x 10 ^-4 s ^-1 , 4.0 x 10 ^-4 s ^-1 , 3.5 x 10 ^-4 s ^-1 , 3.0 x 10 ^-4 s ^-1 , 2.5 x 10 ^-4 s ^-1 , 2.0 x 10 ^-4 s ^-1 , 1.5 x 10 ^-4 s ^-1 , 1.0 x 10 ^-4 s ^-1 , 9.5 x 10 ^-5 s ^-1 , 9.0 x 10 ^-5 s ^-1 , 8.5 x 10 ^-5 s ^-1 , 8.0 x 10 ^-5 s ^-1 , 7.5 x 10 ^-5 s ^-1 , 7.0 x 10 ^-5 s ^-1 , 6.5 x 10 ^-5 s ^-1 , 6.0 x 10 ^-5 s ^-1 , 5.5 x 10 ^-5 s ^-1 , 5.0 x 10 ^-5 s ^-1 , 4.5 x 10 ^-5 s ^{- 1} , 4.0 x 10 ^-5 s ^-1 , 3.5 x 10 ^-5 s ^-1 , 3.0 x 10 ^-5 s ^-1 , 2.5 x 10 ^-5 s ^-1 , 2.0 x 10 ^-5 s ^-1 , 1.5 x 10 ^-5 s ^-1 , or 1.0 x 10 ^-5 s ^-1 or less) may indicate a k _off value.

As provided herein, a TNFR2 agonist is directly or indirectly linked through a linker to a TNFR1 antagonist, such as any of those described above, in any order or suitable arrangement. For example, the N-terminus of a TNFR2 agonist, such as any TNFR2 agonist described herein, is fused to the C-terminus of a TNFR1 antagonist via one or more linkers as discussed below and elsewhere herein. Alternatively, the C-terminus of a TNFR2 agonist may be fused to the N-terminus of a TNFR1 antagonist. When the TNFR2 agonist has the structure shown in Formula 3, the N-terminus of the multimerization domain is linked to the C-terminus of the TNFR1 antagonist, and when the TNFR2 agonist has the structure shown in Formula 4, the C-terminus of the multimerization domain is It is linked to the N-terminus of the anti-TNFR1 antagonist. The linker (L) between the TNFR1 antagonist and the TNFR2 agonist can be any suitable linker and combination thereof, such as one or more of an Ig Fc region, and/or an antibody hinge region, and/or a short peptide linker, such as a glycine-serine linker. can include In some embodiments, the linker is a poly(ethylene glycol) (PEG) molecule or a branched PEG molecule of 30 kDa or greater. As discussed above, when a TNFR2 agonist has the structure shown in Formula 3 or 4, if the multimerization domain is an Fc then it is the same Fc used to link the TNFR1 antagonist to the TNFR2 agonist.

c. linker

The TNFR1 antagonist constructs (eg Formula 1), multispecific TNFR1 antagonist-TNFR2 agonist constructs (eg Formula 2) and TNFR2 agonist constructs (eg Formulas 3-5) optionally comprise a linker, as well as Contains active modifiers. Linkers have a variety of functions, including providing additional or improved biological and pharmacological properties, and are used for structural purposes to link different molecules. An exemplary linker is a Gly-Ser polypeptide, hinge region (eg, see Tables 1-4 above which set forth sequences of various hinge regions and combinations thereof).

Polypeptide linkers and also chemical linkers for chemical conjugation are included. Linker peptides are incorporated into the space between polypeptides and can promote proper protein folding and stability of the polypeptides, improve protein expression, and enhance the bioactivity of components of the construct. Peptide linkers are designed primarily as unstructured flexible peptides. A linker may be included as described in Exemplary Formulas 1-4 above. For example, in a provided bispecific construct, the components are fused via a linker (L) in an N-terminus to C-terminus or C-terminus to N-terminus arrangement. The linker is usually alone or in combination with one or more other linkers, including, for example, a short peptide linker, such as a glycine-serine (GS) linker, and/or the hinge region of an immunoglobulin (Ig) polypeptide, such as an Fc region. It is a peptide linker containing In embodiments herein, for example, the C-terminus of the TNFR1 antagonist is fused to the N-terminus of the peptide linker(s) and the C-terminus of the peptide linker(s) is fused to the N-terminus of the TNFR2 agonist do. In another embodiment, the C-terminus of TNFR2 is fused to the N-terminus of the peptide linker(s) and the C-terminus of the peptide linker(s) is fused to the N-terminus of the TNFR1 antagonist. The linker improves the therapeutic index of the molecule by providing increased molecular weight, increasing stability and serum half-life, enhancing tissue retention, and reducing or reducing peripheral clearance. The linker also increases the flexibility of the molecule, allowing each part of the molecule to interact with target antigens/epitopes such as TNFR1 and TNFR2 as provided herein. As discussed below and elsewhere herein, in embodiments where the linker contains an Fc region of an immunoglobulin, usually a modified Fc region, additional properties including, for example, neonatal Fc receptor (FcRn) recycling are present. may be conferred, which further increases serum stability and half-life and/or increases the enhancement or elimination of immune effector functions.

i. peptide linker

Linkers for fusion proteins are well known to those skilled in the art. See, eg, Chen et al (2013) Adv . Drug. Deliv . Rev. 65 :1357-1369, entitled " Fusion Protein Linkers: Property, Design and Functionality . Linkers may be designed or may be based on or from linkers of naturally occurring multi-domain proteins. Empirical linkers designed by researchers are generally classified into three categories according to their structure: flexible linkers, rigid linkers, and in vivo cleavage used to deliver prodrugs that are activated, for example, by cleavage of the linker in situ. Possible linker.

In addition to their role in linking functional domains together (as in flexible and rigid linkers) or releasing free functional domains in vivo (as in in vivo cleavable linkers), linkers outline the properties of the linked moieties. can do. Included herein are, for example, improving biological activity, increasing expression yield, and achieving a desirable pharmacokinetic profile. Databases and methods for selecting linkers are known to those skilled in the art (see, e.g., George et al. (2002) " An analysis of protein domain linkers: their classification and role in protein folding ," Protein Eng . 15 :871-879]).

a) a flexible linker

Flexible linkers are generally applied when the linked domains require some degree of movement or interaction. Flexible linkers are usually enriched with small or polar amino acids such as Gly and Ser to provide good flexibility and solubility. It is a suitable choice when a specific motion or interaction (eg in scFv) is required for the fusion protein domain. In addition, the flexible linker is not a rigid structure, but can act as a passive linker that maintains the distance between functional domains. The length of the flexible linker can be adjusted to allow for proper folding or to achieve optimal biological activity of the fusion protein.

Flexible linkers are generally described in Argos (1990) J. Mol . Biol . 211(4):943-958] consists of small, non-polar (eg Gly) or polar (eg Ser or Thr) amino acids. The small size of these amino acids provides flexibility and allows mobility of the linking functional domains. Incorporation of Ser or Thr can form hydrogen bonds with water molecules to maintain the stability of the linker in aqueous solution, thereby reducing undesirable interactions between the linker and the protein moiety.

An exemplary flexible linker is a linker containing primarily or only stretches of Gly and Ser residues ("GS" linkers). An example is a flexible linker with the sequence of (Gly-Gly-Gly-Gly-Ser) _n . By adjusting the copy number “n”, one can choose the length of this GS linker to achieve proper separation of the functional domains or to maintain the necessary inter-domain interactions. Flexible linkers are also rich in small or polar amino acids, such as Gly and Ser, and can contain additional amino acids, such as Thr and Ala, to maintain flexibility, and polar amino acids, such as Lys and Glu, to improve solubility. can do.

To confer protease resistance and increase the flexibility of the fusion protein, the SCDKTH hinge sequence and other hinge sequences can be replaced or preceded by a short polypeptide linker. An example of a polypeptide linker is the (Gly-Ser) _n amino acid sequence (GS linker), with some Glu or Lys residues dispersed throughout to increase solubility. For example, a polypeptide linker can be (GlySer) _n (where n=1-10); (GlySer ₂ ); (Gly ₄ Ser) _n (where n=1-10); (Gly ₃ Ser) _n (where n= 1-5); (SerGly ₄ ) _n (where n=1-5); (GlySerSerGly) _n (where n= 1-5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS (see SEQ ID NOs: 816-827 for the GS linker). The linker can be a poly-Gly peptide of at least 2-18 residues in length or longer, or a similar linker of the same length and flexibility. Exemplary polypeptide linkers in molecules provided herein include, but are not limited to, eg, GSGS, GGGGS or GGGGSGGGGSGGGGS (see SEQ ID NOs: 816-827 for Gly-Ser linkers). Another linker that provides similar performance is the (GGGGS) ₄ (SEQ ID NO: 819) linker. Another flexible linker rich in Gly and Ser is GSAGSAAGSGEF (SEQ ID NO: 828). This linker has been shown to maintain good solubility in aqueous solutions. Linkers containing only glycine may be used. For example, (Gly) ₆ (SEQ ID NO: 1473) and (Gly) ₈ (SEQ ID NO: 1474) linkers are known and have been shown to be stable against protease digestion during protein purification from the expressing organism.

Several other types of flexible linkers including KESGSVSSEQLAQFRSLD (SEQ ID NO: 829) and EGKSSGSGSESKST (SEQ ID NO: 830). The linker's Gly and Ser residues provide flexibility and Glu and Lys improve solubility.

b) rigid linkers

Flexible linkers have the advantage of passively connecting functional domains and allowing some movement, but the lack of rigidity of such linkers can be limiting. Rigid linkers are selected when spatial separation of domains is required to preserve the stability or bioactivity of the fusion protein. Rigid linkers exhibit relatively rigid structures by adopting an α-helical structure or by containing several Pro residues. The length of the linker can be easily tuned by changing the copy number to achieve an optimal distance between domains.

(EAAAK) The alpha helix forming linker with the sequence of _n (SEQ ID NO: 831) has been applied to the construction of many recombinant fusion proteins. The α-helical structure is rigid and stable, with intrasegmental hydrogen bonds and a densely packed backbone. Rigid α-helical linkers can serve as rigid spacers between protein domains. An example of a rigid linker is A(EAAAK) _n A (SEQ ID NO: 832), where n = 2-5. This linker exhibits an α-helical conformation stabilized by GluLys ⁺ salt bridges within the segment. Another type of rigid linker has a Pro-rich sequence, (XP) _n , where X represents any amino acid and is usually Ala, Lys or Glu. The presence of Pro in the non-helical linker increases stiffness and effectively separates the protein domains. An example of such a linker is a 33 residue peptide repeating -Glu-Pro- and -Lys-Pro-.

One skilled in the art can select from known linkers or designed linkers. Desirable properties and requirements for them are known. The following discussion summarizes some exemplary linkers (Chen et al. (2013) Adv . Drug. Deliv . Rev. 65 :1357-1369], which provides details of flexible, rigid, cleavable linkers and may be used). Flexible linkers have been used to link functional domains that are enriched in small and/or hydrophilic amino acids such as Gly or Ser, providing structural flexibility and favoring interactions or movement between domains. Rigid linkers can be used if sufficient separation of protein domains is desired. Rigid linkers are designed or selected to adopt an α-helical structure or to contain proline. Rigid linkers can distance protein moieties. Flexible and rigid linkers are stable in vivo and do not allow separation of bound proteins. The cleavable linker allows release of the free functional domain in vivo via reductive or proteolytic cleavage. They are generally used to deliver prodrugs to the target site.

In Formula 2 above, additional linkers such as between a TNFR1 antagonist and/or TNFR2 agonist portion and an active-modifying portion such as an Fc portion may be included; Such a linker may include all or part of the hinge sequence sufficient to provide flexibility of trastuzumab, including at least residues SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26), or flexibility of the hinge region of nivolumab. To provide the sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29) or a sequence having at least 98% or 99% sequence identity thereto, or any other suitable antibody hinge region or sequence known in the art It may contain all or part, including sufficient portions.

In certain embodiments, only the GS linker is included. Other short peptide linkers known in the art are also contemplated for use in the bispecific molecules provided herein. For example, N- or C-terminal extensions of Fc can be used as linkers. Human IgG, ELQLEESSAEAQDGELDG (SEQ ID NO: 833) or a sequence having at least 98% or 99% sequence identity thereto, or a variant containing the sequence ELQLEESSAEAQGG (SEQ ID NO: 834) or a sequence having at least 98% or 99% sequence identity thereto A C-terminal extension from can also be used as a linker.

A second Fc subunit, either a fusion protein or not a fusion protein, may be included (see, eg, FIG. 2 , may be modified to include a knob-in-hole (see discussion below). It may be used within mammalian cell expression systems. assembles to form a knob-in-hole mediated Fc dimer to produce an Fc dimer, which further increases the serum half-life and stability of the molecule In certain embodiments, the second Fc subunit has a second TNFR2 potency fused with an antibody to create a bivalent antibody-like structure, in another embodiment, only one Fc subunit (Fc monomer) is included.

ii. chemical linker

In some embodiments, a linker is a chemical linker. Included herein are polypeptide modifiers such as linkers that are non-cleavable moieties, chemical cross-linking reagents, and polymeric molecules that include PEGylated moieties. Chemical linkers are better suited for the creation of branched constructs and other structures that cannot be achieved with peptide linkers.

Exemplary linkers include non-cleavable linkers. Non-cleavable linkers include, for example, amide linkers and amide and ester linkages with succinate spacers (see, eg, Dosio et al., (2010) Toxins 3:848-883). Exemplary chemical cross-linking linkers include but are not limited to SMCC (succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate) and SIAB (succinimidyl (4-iodoacetyl)aminobenzoate) It doesn't work. SMCC is an amine-to-sulfhydryl crosslinker containing NHS-ester and maleimide reactive groups at opposite ends of a mid-length cyclohexane stabilized spacer arm. SIAB is a short NHS-ester and iodoacetyl crosslinker for amine-to-sulfhydryl conjugation. Other exemplary cross-linking reagents include thioether linkers, chemically labile hydrazone linkers, 4-mercaptovaleric acid, BMPEO, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SMPB, SMPH , sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfonyl)benzoate), and bis -maleimide reagents such as DTME, BMB, BMDB, BMH, BMOE, BM(PEO) ₃ , and BM(PEO) ₄ (which are commercially available from Pierce Biotechnology, Inc.). Bis-maleimide reagents allow for sequential or simultaneous attachment of the free thiol groups of cysteine residues of antibodies to thiol-containing targeting agents or linker intermediates. Other thiol-reactive functional groups besides maleimide include iodoacetamide, bromoacetamide, vinyl pyridine, disulfide, pyridyl disulfide, isocyanates, and isothiocyanates. Other exemplary linkers and methods of use are well known to those skilled in the art and are described in, for example, US Patent Publication No. 2005/0276812 and Ducry et al. (2010) Bioconjug. Chem. 21:5-13].

Linkers may optionally be replaced with groups that modulate properties such as solubility and reactivity. For example, a sulfonate substituent can increase the water solubility of a reagent, facilitate a coupling reaction between a linker reagent and an antibody or drug moiety, and/or facilitate a coupling reaction. Linker reagents may also be obtained from commercial sources such as Molecular Biosciences Inc. (Boulder, CO.) or as described in Toki et al. (2002) J. Org . Chem . 67:1866-1872; U.S. Patent No. 6,214,345; US Publication Nos. 2003/130189, and 2003/096743; and International Application Publication Nos. WO 02/088172, WO 03/026577, WO 03/043583, and WO 04/032828. For example, a linker reagent such as DOTA-maleimide (4-maleimidobutyramidobenzyl-DOTA) is described in Axworthy et al. (2000) Proc. Natl . Acad . Sci . USA . 97(4):1802-1807] by the reaction of aminobenzyl-DOTA with 4-maleimidobutyric acid (Fluka) activated with isopropylchloroformate (Aldrich). The DOTA-maleimide reagent reacts with the free cysteine amino acid of the cysteine engineered antibody and gives the antibody a metal complex ligand (Lewis et al. (1998) Bioconj . Chem . 9:72-86]). Chelating linker labeling reagents such as DOTA-NHS (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono (N-hydroxysuccinimide ester) are commercially available. Possible (Macrocyclics, Dallas, TX.).

The linker can be a dendritic type linker for covalent attachment of more than one moiety to an antibody via a branched multifunctional linker moiety (see, e.g., Sun et al. (2002) Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al. (2003) Bioorganic & Medicinal Chemistry 11:1761-1768; King et al. (2002) Tetrahedron Letters 43:1987-1990). Where an antibody possesses only one reactive cysteine thiol group, multiple other moieties may be attached via the dendritic linker. Exemplary dendritic linker reagents are known (see, eg, US Patent Publication No. 2005/0276812).

Other examples of chemical linkers (which may also be active modifiers for use in the constructs herein) are PEG molecules and branched PEG molecules, particularly molecules having a molecular weight greater than 30 kDa. The PEG linker provides for multispecific and bivalent (in the case of TNFR2 agonists where receptor clustering enhances signaling) incorporation and increases the molecular weight of the molecule, thereby increasing serum half-life in vivo. PEG linkers also ameliorate the difficulty of reengineering antibodies, for example by avoiding the introduction of non-native structures that are rapidly degraded and removed and/or cause immunogenicity.

d. active modifier

Among the components of the construct are portions or regions that modulate or alter the activity and/or pharmacological properties of the construct (see formulas 1 and 2 above). Examples of these include Fc regions, modified Fc regions, other multimerization domains, polymeric moieties comprising dimers of Fc and modified Fc, and other moieties such as polypeptides such as half-life extending polypeptides, albumin such as human serum albumin (HSA), and transferrin, and polymers such as PEG discussed elsewhere herein that can increase serum half-life. Active modifiers may increase plasma half-life by reducing access to proteases, reducing renal filtration, and/or altering intracellular routing through receptor-mediated recycling; providing uptake across the epithelial bilayer by binding to receptors that undergo transexocytosis; targeting sites in vivo that overexpress or natively express specific receptors or antigens; and other properties as exemplified in the discussion below and also known in the art.

As provided herein, the construct is an Fc region of a human immunoglobulin as an active modifier, such as an IgG, e.g., an IgG1 Fc (SEQ ID NO: 10), an IgG2 Fc (SEQ ID NO: 12), an IgG3 Fc ( SEQ ID NO: 14), or IgG4 Fc (SEQ ID NO: 16). In particular Fc is derived from IgG1 or IgG4 antibodies. For example, the linker can include an IgG1 kappa Fc region, such as an IgG1 Fc derived from Trastuzumab that contains the C _H 2 and C _H 3 domains of Trastuzumab heavy chain (eg, SEQ ID NO: 26 See also residues 234-450 of SEQ ID NO: 27). The Fc subunit of the bispecific molecules provided herein may also be an IgG4 Fc, such as, for example, an IgG4 Fc derived from nivolumab ( ^Opdivo® ) that contains the C _H 2 and C _H 3 domains of the nivolumab heavy chain. (See eg residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30).

An Fc region can, for example, inhibit any one or more of antibody-dependent cellular cytotoxicity (ADCC; also known as antibody-dependent cell-mediated cytotoxicity), antibody-dependent cell-mediated phagocytosis (ADCP), and complement dependent cytotoxicity (CDC). It can be mutated or modified as discussed below to eliminate, reduce or enhance immune effector function, including. In some embodiments herein, immune effector function is eliminated or reduced, for example when the construct is a bispecific molecule used to treat inflammatory and autoimmune diseases and conditions. When a therapeutic agent is used in the treatment of a tumor or cancer, the immune effector function can be enhanced to improve the anti-tumor immune response and therapeutic efficacy. Additionally or alternatively, the Fc region can be modified to enhance FcRn recycling to increase serum stability and half-life in vivo of the molecules provided herein.

For purposes herein, the Fc region or domain is specifically modified to reduce or eliminate ADCC. Small molecule therapeutics such as antibody fragments (eg Fab, scFv, dAb) are advantageous. They can be produced in high yield and have other advantageous properties. They exhibit enhanced tissue penetration and target accessibility compared to monoclonal antibodies (mAbs), such as receptor clustering, activation of immune effector functions, poor tissue penetration, and lack of access to targets in poorly vascularized areas. Undesirable effects of mAbs can be prevented. However, small antibody fragments have poor pharmacokinetic properties. For example, because of their small size, dAbs and other antibody fragments are rapidly cleared by the kidneys as molecules less than 50-60 kDa in size undergo renal filtration. The rapid clearance and short elimination half-life of small antibody fragments, which may be less than a few hours, may reduce efficacy in vivo and require frequent dosing and/or continuous infusion.

Several methods can be used to increase the retention and in vivo half-life of small antibody fragments, such as dAbs. For example, as provided herein, the dAb(s) in a TNFR1 antagonist, TNFR2 agonist, and combination/multispecific construct are half-life extenders or linkers comprising them, e.g., IgG, such as IgG1 or It is fused to the Fc region of IgG4. Fc can be monomeric or dimeric. Fusing a small antibody fragment, such as a dAb, to the Fc region of an IgG molecule increases the size of the molecule, preventing clearance/excretion from the body and mediating binding to the neonatal Fc receptor (FcRn) expressed on endothelial cells to protect it from lysosomal degradation. Protects antibodies and prolongs half-life in vivo. However, the addition of Fc may introduce undesirable properties such as complement activation, release of pro-inflammatory cytokines and induction of immune effector functions that can lead to cytotoxicity. Because TNFR1 is almost universally expressed and TNFR2 is expressed in many tissues, it is generally undesirable to use ADCC-enhancing antibodies and rely on the antagonist activity of the antibody for efficacy.

As described herein, the Fc region in TNFR1 antagonists, TNFR2 agonists and multispecific, such as bispecific, constructs can be modified to improve pharmacokinetic and pharmacodynamic (i.e., pharmacological) properties and to eliminate undesirable properties. do. For example, the Fc region is modified to utilize/enhance neonatal FcR recycling to increase half-life in vivo and/or Fc-related immune effector functions such as antibody-dependent cellular cytotoxicity (ADCC; also known as antibody-dependent cell-mediated cytotoxicity). ), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). Additionally, in embodiments where the construct contains a TNFR1 antagonist and a TNFR2 agonist and is multispecific, such as bispecific, such as embodiments containing an Fc dimer, the dimer is mutated to introduce a knob-in-hole Prevent homodimerization. Numerous modifications to the Fc portion (or region) are known to those skilled in the art (see, eg, Li et al., (2014) Expert Opin Ther Targets 18 :335-350).

i. Variations on the Fc part

a) knob-in-hole

Bispecific antibodies (bsAbs) can contain two distinct antigen binding sites, allowing an alternative therapeutic approach to conventional therapeutic monoclonal antibodies (mAbs), avoiding the limitations associated with mAbs such as receptor co-clustering. there is. Small antibody fragments are easy to produce in high yield, inexpensive, and readily penetrate tissues, but are associated with limitations such as poor stability, solubility and pharmacokinetic properties. For example, because of their small size, they have a short serum half-life, reduced tissue retention, and are rapidly eliminated from the blood by the kidneys. Consequently, IgG-like bispecific (bs) Abs without the same limitations are advantageous. For example, a bsAb may include an Fc region to increase serum half-life and also allow effector function if desired. However, production of purified bsAb in high yield can be difficult because homodimerization of the heavy chain must be avoided. The “knob-in-hole” (KiH; also known as “knob-in-hole”) approach provides a solution to this problem. The C _H 3 domains of antibody (IgG) heavy chains can be engineered for heterodimerization to construct Fc-containing bifunctional therapeutic molecules that do not self-associate.

The knob-in-hole approach involves interfacial residues asymmetrically mutating in the C _H 3 domains of the two parental heavy chains in a complementary manner. A "knob" is created by replacing an amino acid with a small side chain at the interface between the _CH3 domains with an amino acid with a larger side chain such as tyrosine or tryptophan, and a "hole" is created by replacing an amino acid with a large side chain. It is produced by replacing an amino acid with a smaller amino acid such as alanine or threonine. Knob and hole variants are heterodimerized by insertion of the knob into a correspondingly designed hole in the partner C _H 3 domain. The steric repulsion prevents knob-knob association and destabilizes the hole-hole homodimer. For example, a knob mutation can be S354C, T366Y, T366W or T394W and a hole mutation can be Y349C, T366S, L368A, F405A, Y407T, Y407A or Y407V (all by EU numbering). Knobs generated towards the center of the dimer interface, such as residue T366, appeared to be more disruptive to homodimer formation than knobs located near the edge of the dimer interface. Residue T366 on the first C _H 3 domain is within hydrogen bonding distance of residue Y407 on the second or partner C _H 3 domain, so T366Y and Y407T represent a common knob-in-hole pair; This pair has been found to produce heterodimers in greater than 90% yield (see, e.g., Ridgway et al. (1996) Protein Eng. 9(7):617-621]).

For example, the IgG Fc region in the bispecific TNFR1 antagonist/TNFR2 agonist construct provided herein can be modified using a knob-in-hole approach to generate heterodimerized molecules in high yield. . Table 6 below shows the corresponding knob and hole mutations by Kabat numbering and sequential numbering with respect to the sequence of the IgG heavy chain constant domain set forth in SEQ ID NO:9. Any mutation known to one skilled in the art that introduces a knob-in-hole can be used in the constructs herein.

Ligand Trap Construct

Fc modified to have a “knob-in-hole” as described above can also be used with other bispecific molecules to create heterodimers. See, eg, US Patent Publication No. 2010/0055093 and Jin et al. (2009) Mol . Med . 15 :11-20] describe bispecific “ligand” trap constructs that target EGF receptor family ligands, including one designated RB200 and the other designated RB242. A problem with these constructs is that they are heterogeneous and contain homodimers and heterodimers, the latter being the intended therapeutic. RB200 and RB242 are examples of ligand traps that can be modified by replacing the Fc region with a modified Fc region with complementary knobs and holes, so the resulting dimers are all heterodimers. RB242 targets HER1 (EGFR), HER2, and HER3 ligands, and some HER4 ligands. HER4 was designed not to capture HER4-specific ligands because it plays a role in neurodevelopment not shared by other members of the EGFR family. RB242 is HER1/ErbB1 (amino acids 1-621 of SEQ ID NO: 41) and HER3/ErbB3 (amino acids 1-621 of SEQ ID NO: 45) fused to the Fc domain of human immunoglobulin G1 (IgG1) (HER1-HER3/Fc). ) and acts as a chimeric bispecific ligand trap. The HER3/Fc component of RB242 contains a 6Υ histidine tag at the COOH terminus (see, e.g., Jin et al. (2009) Mol . Med . 15 :11-20]). RB200 binds with high affinity to HER1/ErbB1 ligands (EGF, TGF-α, HB-EGF, AR, BTC, EPR and EPG) and HER3/ErbB3 ligands (NRG1-α and NRG1-β3). RB242 inhibited EGF-stimulated and NRG1-1-stimulated tyrosine phosphorylation of HER family proteins (HER1, HER2 and HER3) and showed efficacy in various cell proliferation assays. RB200 inhibits tumor growth in an in vivo animal model.

The epidermal growth factor (EGF) ligand/receptor family plays a role in a variety of diseases, disorders and conditions including rheumatoid arthritis (RA). The EGF family of cell surface receptors (ErbB and human epidermal growth factor receptor (HER)) belong to the receptor tyrosine kinase (RTK) superfamily and contain an extracellular domain (ECD) and an intracellular tyrosine kinase signaling domain. The EGF family includes members: EGF, transforming growth factor α (TGF-α), heparin-binding EGF-like growth factor (HB-EGF), amphiregulin (AR), β-cellulin (BTC), epiure. EGF receptor (EGFR)/HER1/ErbB1, HER2/ErbB2, HER3/ErbB3, and HER4/ErbB4, activated by a large family of ligands, including gulin (EPR), epigen (EPG) and neuregulin (NRG). There are 4 ECDs within EGFR; Domains I and III are the ligand binding domains, and domains II and IV mediate binding to each other and to other members of this receptor family. Ligand binding leads to the formation of homodimers or heterodimers between receptors. For example, TGF-α and EGF bind EGFR/HER1/ErbB1 whereas NRG4 binds HER4/ErbB4. Depending on the dimer formed, phosphorylation translocation of intracellular regions occurs leading to activation of numerous downstream signaling pathways resulting in cell proliferation, survival and differentiation (see, e.g., Jin et al. (2009) Mol. Med. 15 :11-20]).

The epidermal growth factor receptor family consists of four closely related receptor tyrosine kinases: EGFR (ErbB-1), HER2 (Erb-B2), HER3 (ErbB-3) and HER 4 (ErbB-4). In many cancer types, mutations or amplifications in one of the family members are associated with poor survival of cancer patients. In autoimmune diseases, TNF signaling induces the synthesis of epiregulin and heparin-binding EGF (HB-EGF) in macrophages to transcriptionally activate the EGFR signaling pathway, and both growth factors activate EGFR.

In a complementary manner, EGFR and HER2 are upregulated in synovial fibroblasts, leading to proliferation. EGFR, HER2 (ErbB2) and EGF-like growth factors are overexpressed, for example, in RA synovial fibroblasts and macrophages. Thus, the TNF and EGFR pathways cooperate in the progression of lupus, rheumatoid arthritis and other autoimmune diseases. Among the constructs provided herein are those designated as “ligand traps”. Ligand trap constructs inhibit the growth of rapidly growing synovial fibroblasts in affected RA joints by blocking most inflammatory growth factors of the EGFR family. These ligand traps are intended for administration in combination therapy protocols with TNF blocker constructs, which are TNFR1- and/or TNFR2-targeting constructs provided herein. This combination therapy, such as rheumatoid arthritis, can be combined synergistically to achieve disease regression.

The EGFR family of growth factors is overexpressed in hyperproliferative/inflammatory diseases such as RA, and is also overexpressed in ovarian cancer and other cancers. Elevated levels of the EGFR family and/or cognate are common components of many types of cancer. When overexpressed (or sometimes mutated), these receptors are causally associated with shorter survival in many types of malignancies. Examples of targeted therapeutics that act through the EGFR family are cetuximab (Erbitux ^® ), panitumumab (Vectibix ^® ), trastuzumab (Herceptin ^® ) and pertuzumab (Perjeta ^® ) (generic and exemplary trademarks listed with the serving source). Small molecule inhibitors also target intracellular tyrosine kinase activity of the EGFR family. Examples of small molecules are lapatinib (Tykerb ^® ), erlotinib (Iressa ^® ) and neratinib (Nerlynx ^® ). Because the drug targets only one member of the EGFR family, other members of the family can upregulate and compensate for tumor growth. Likewise, an antibody versus a single growth factor (eg, TGF-α, EGF, HB-EGF, etc.) inhibits only that growth factor, and tumor cells compensate by upregulating other growth factors. The ligand trap constructs provided herein address this by blocking HER1, HER2 and HER3 together. This results in global inhibition of the EGFR family on cancer cells. Ovarian cancer is one of the cancers that require treatment.

The ligand trap constructs provided herein are improved by optimizing heterodimer production and FcRn recycling using modified Fc regions as described herein below for TNFR1/TNFR2 constructs. A ligand trap construct may be a TNFR1 antagonist construct, and/or a TNFR2 agonist construct, and/or a multispecific TNFR1 antagonist/bispecific construct, and/or a TNF described herein and/or known to those skilled in the art. It is administered in combination therapy protocols with any of the other constructs provided herein for the treatment of diseases, disorders and conditions for which they play a role.

b) Modifications that Enhance Neonatal Fc Receptor (FcRn) Recycling

There are numerous approaches to increasing the short serum half-life of small polypeptide or protein therapeutics. PEGylation, which increases the serum half-life of small protein therapeutics, has its drawbacks. PEGylation can reduce the efficacy or activity of a protein therapeutic, can lead to heterogeneity, and can lead to immunoreactivity of the protein. Another approach involves fusing with albumin, which can improve protein circulation by increasing molecular weight and reducing kidney clearance.

Serum half-life can also be increased by fusion with the Fc portion of an IgG. The long circulating half-life of about 2-3 weeks and the slow clearance of IgG are due at least in part to its interaction with the neonatal Fc receptor (FcRn), which binds IgG with high affinity at acidic pH and releases it at above-neutral pH. FcRn binds in a 2:1 FcRn:IgG configuration (bivalent interaction) to the Fc portion of pinocytosylated IgG (within the _CH 2- _CH 3 domain) in acidic (~pH 6) endosomes and lysosomes. They transport them from the degradation pathway and are recycled back into the circulation after exposure to the extracellular physiological pH (~7.4) at which the Fc-FcRn complex dissociates. Poor binding to FcRn at acidic pH results in migration of the antibody to lysosomes where it is degraded. Recycling receptors, such as FcRn, also provide a route for IgG to move across the epithelium (transexocytosis) into the bloodstream. Utilizing interactions with FcRn can improve protein transport across epithelial barriers such as the gut and lung, allowing for non-invasive administration. Residues within the Fc _CH 2 and _CH 3 domains are involved in FcRn binding, and mutations in mAbs have been shown to affect serum half-lives in vivo. Circulation and delivery of small protein therapeutics can be improved by fusing them to the Fc domain of an IgG, and the resulting fusion protein binds FcRn and utilizes the IgG serum stabilization pathway. Fusion with the Fc domain also increases the molecular weight of the therapeutic and reduces renal clearance, but may be undesirable due to potentially reduced tissue penetration and specific activity of the fusion protein. Alternatively, studies have shown that short FcRn binding peptides (FcRnBPs) allow interaction of FcRn with small proteins so that they do not need to be fused to high molecular weight Fc domains. For example, fusion with FcRnBP increases the molecular weight by about 3 kDa compared to fusion with Fc or albumin, which increases the molecular weight by about 50-70 kDa (see, for example, Datta-Mannan et al. (2019) Biotechnol . J. 14:1800007; Sockolosky et al. (2012) Proc . Natl . Acad . Sci . USA 109(40):16095-16100).

For example, short (16 residues) linear and cyclic FcRnBPs (e.g. SEQ ID NOs: 48-51) are placed at the C-terminus, N-terminus or both of the Fab heavy and light chains (FcRnBP-Fab constructs), or both Fabs. fused to 1-4 FcRnBPs per. Pharmacokinetic studies in cynomolgus monkeys showed that the FcRn binding of the FcRnBP-Fab constructs increased as the number of peptides fused to the Fab increased. This is due to the increased avidity that constructs containing four linear FcRnBPs fused to the N- and C-terminus of the heavy and light chains of the Fab show the greatest improvement in pharmacokinetics in cynomolgus monkeys compared to the parental Fab. For example, the half-life improved from 3.7 hours for the parental Fab to 15-60 hours for the various FcRnBP-Fab constructs (see, e.g., Datta-Mannan et al. (2019) Biotechnol . J. 14:1800007]). These results indicate an improvement in serum half-life, but still much lower than that of IgG, which is about 2-3 weeks. The use of FcRnBP also does not significantly increase the molecular weight of the therapeutic and therefore does not reduce renal clearance.

As discussed above, fusion with an IgG Fc increases the half-life of a small protein therapeutic by taking advantage of FcRn binding and also increasing the molecular weight of the therapeutic so that it is less rapidly cleared from the body by the kidneys. To improve pharmacokinetics and overall pharmacology, residues within the Fc region can be mutated to increase the affinity for FcRn, typically more than 30-fold, further increasing the half-life in vivo. The Fc region spanning the interface of the _CH 2 and _CH 3 domains interacts with FcRn. Human Fc residues identified that play a role in FcRn binding include, for example, L251, M252, I253, S254, L309, H310, Q311, L314, E380, N434, H435 and Y436 (by EU numbering, see Table 1). include Mutations of residues located at the Fc-FcRn interface, including M252, S254, T256, H433, N434 and Y436 (by EU numbering), enhance the stability of the human FcRn-IgG1 complex. For example, the replacements M252Y/S254T/T256E and H433K/N434F/Y436H improve binding to human FcRn by 11-fold and 6.5-fold, respectively, at pH 6.0 compared to wild-type IgG1 with efficient release at pH 7.4. The combination of these replacements increases the binding affinity for FcRn 57-fold. Additional mutations of IgG1 Fc that have shown improved binding to FcRn include, for example, M252W, M252Y, M252Y/T256Q, M252F/T256D, E380A and N434F/Y436H (see for example Dall'Acqua et al. (2002) J. Immunol. 169:5171-5180]).

The triple substitutions M252Y/S254T/T256E, when introduced into the _CH 2 domain of MEDI-524, a humanized antirespiratory syncytial virus (RSV) mAb, compared to unmodified MEDI-524, significantly improved the serum efficiency of the mAb in cynomolgus monkeys. The half-life was increased about 4-fold. When introduced into the Fc portion of MEDI-522, a humanized affinity-optimized mAb for the human α _v 3 integrin complex, the alternative M252Y/S254T/T256E (YTE) reduces ADCC activity and binding to human FcγRIIIA (F158 allotype) made it The ADCC activity of MEDI-522-YTE can be restored and increased by introducing the ADCC-enhanced substitutes S239D/A330L/I332E (by EU numbering) compared to unmodified MEDI-522, which suggests that the substitute YTE can reduce the ADCC of human IgG1. indicate that it provides a reversible mechanism to modulate function (see, e.g., Dall'Acqua et al. (2006) J. Biol. Chem . 281(33):23514-23524).

Residues at positions 250, 314 and 428 (by EU numbering) of the human IgG heavy chain, which are conserved among all four human IgG subtypes, are also located near the Fc-FcRn interface. Mutations T250Q, M428L and T250Q/M428L, when introduced to the Fc of human IgG2 mAb, increased binding to FcRn by ~3-fold, 7-fold and 28-fold respectively at pH 6.0 and no binding was observed at pH 7.5. When the pharmacokinetics of the mutants were evaluated in rhesus monkeys, the average clearance, i.e., the volume of serum antibody cleared per unit time, was found to be ~1.8-fold lower for the M428L mutant and ~2.8-fold lower for the T250Q/M428L mutant. However, the elimination half-life was ~1.8-fold longer for the M428L mutant and ~1.9-fold longer for the T250Q/M428L mutant compared to the unmodified antibody. As these residues are conserved between IgG subtypes, the mutations M428L and T250Q/M428L are expected to have similar effects in human IgG1, IgG3 and IgG4 antibodies (see, for example, Hinton et al. (2004) J. Biol . Chem . 279(8):6213-6216). The variant T250R/M428L has been shown to reduce selective binding to FcRn at pH 6.0 and degradation of serum IgG2 and IgG1 in rhesus monkeys by 2.8-fold (see, e.g., Saxena et al. (2016) Front. Immunol. 7:580]).

Mutation N434A (by EU numbering), when incorporated into human anti-HER2 IgG1 trastuzumab, exhibited approximately 4-fold higher affinity for human FcRn compared to the unmodified antibody at pH 6, but negligible at pH 7.4. it was a bond The N434A variant showed increased exposure, decreased clearance (~2-fold) and increased half-life (~2-fold) compared to the wild-type antibody when tested in vivo in cynomolgus monkeys. In contrast, mutant N434W, which resulted in ∼80-fold increased binding to FcRn at pH 6, showed clearance similar to wild type; This mutant also showed significant binding to FcRn at pH 7.4, indicating that maintaining the pH-dependent binding of the Fc mutant to FcRn is important to improve pharmacokinetics in vivo (Yeung et al. (2009) J. Immunol . 182:7663-7671). The N434A mutation also corresponds to poor FcRn affinity that may arise due to the introduction of mutations that increase binding to FcγR; N434A is typically added to mutations S298A/E333A/K333A to create variants with enhanced FcγR binding and normal or improved FcRn binding. Fc mutations that improve FcRn binding also include N434Y, E294del/T307P/N434Y and T256N/A378V/S383N/N434Y. E294 deletion results in higher sialylation of the N297 glycan in the Fc, increasing antibody half-life in vivo. Sialylation has also been shown to play a role in regulating serum half-life (see, eg, Saunders, KO (2019) Front. Immunol . 10:1296).

Alternate M428L/N434S (by EU numbering), when incorporated into the humanized anti-VEGF IgG1 antibody bevacizumab (Avastin ^® ), showed an 11-fold increase in affinity for FcRn at pH 6.0 and in vivo serum of cynomolgus monkeys. The half-life was extended from 9.7 days to 31.1 days, a 3.2-fold improvement. The M428L/N434S variant resulted in a similar increase in FcRn binding and half-life extension when introduced to the anti-EGFR antibody cetuximab, which is rapidly cleared due to receptor-mediated internalization. The half-life extension of these antitumor antibodies is associated with enhanced in vivo tumor reduction in mouse models, indicating that the in vivo therapeutic efficacy of the antibodies increases when pharmacokinetics, such as clearance, are improved. Other mutations engineered into the bevacizumab Fc include (by EU numbering): N434S with -3-fold improvement in FcRn binding and -2.8-fold increase in serum half-life in mice; V259I/V308F with about -6-fold improvement in FcRn binding and -about 3-fold and -2-fold increases in serum half-life in mice and cynomolgus monkeys, respectively; M252Y/S254T/T256E with -7-fold improvement in FcRn binding and -4-fold and 2.5-fold increase in serum half-life in mice and cynomolgus monkeys, respectively; and V259I/V308F/M428L with ~20-fold improvement in FcRn binding and ~4-5-fold and 2.6-fold increases in serum half-life in mice and cynomolgus monkeys, respectively (Zalevsky et al. (2010) Nat. Biotechnol. 28(2):157-159).

The mutations identified above and other such mutations can be introduced into the IgG Fc region in the constructs provided herein. These include constructs such as those of Formulas 1 and 2 wherein the linker comprises Fc or an Fc dimer, depending on the structure of the construct.

In some embodiments, the bispecific TNFR1 antagonist/TNFR2 agonist constructs provided herein, and the IgG Fc regions in constructs herein, such as the TNFR1 antagonist constructs, enhance neonatal FcR recycling to increase in vivo half-life. transformed to do This can be effected by mutating residues at the interface of the C _H 2 and C _H 3 domains of IgG Fc that are responsible for binding to FcRn. These include residues T250, L251, M252, I253, S254, T256, V259, T307, V308, L309, H310, L314, Q311, A378, E380, S383, M428, H433, N434, H435 and Y436 by EU numbering Not limited to this. Exemplary Fc modifications that increase binding to FcRn are T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434 by EU numbering. W, N434S, N434Y, Y436H, M252Y/T256Q, M252F/T256D, M252Y/S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N4 34S, V259I/V308F, V259I/V308F/ M428L, E294del/T307P/N434Y, T256N/A378V/S383N/N434Y, and combinations thereof. Table 7 below shows the corresponding mutations by Kabat numbering and sequential numbering with respect to the sequence of the IgG heavy chain constant domain set forth in SEQ ID NO:9. Other modifications known in the art that confer enhanced or increased FcRn binding are also contemplated for use herein.

c) enhancement or reduction/elimination of Fc immune effector function

There are four human IgG subclasses that differ in effector function, circulating half-life and stability. IgG1 has Fc effector functions, is the most abundant IgG subclass and is the most commonly used subclass in FDA-approved therapeutic proteins. IgG2 lacks Fc effector function, but dimerizes with other IgG2 molecules and is unstable due to scrambling of disulfide bonds in the hinge region. IgG3 has Fc effector functions and a very long and tight hinge region. IgG4 lacks Fc effector function, has a shorter circulating half-life than other subclasses, and IgG4 dimers are biochemically unstable due to the presence of a single disulfide bond in the hinge region, leading to H-chain exchange between different IgG4 molecules. Thus, the Fc regions of IgG2 and IgG4 do not possess effector functions and can be used where effector functions are not needed or may be detrimental, for example in the context of autoimmune and inflammatory diseases and disorders.

Most approved therapeutic mAbs belong to the human IgG1 subclass and can interact with humoral and cellular components of the immune system. For example, antibodies participate in the humoral immune response through interaction with the complement protein C1q, which initiates the complement cascade, resulting in cytolysis in target cells (i.e., complement dependent cytotoxicity (CDC)) and Fc gamma Membrane attack complexes involved in cellular immune responses are formed by interaction with receptors (FcγR). FcγRs include the FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) families, which differ in cell surface expression and Fc binding affinity. The five activating FcγRs include the high-affinity FcγRI, which can bind monovalent antibodies, and the low-affinity FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb, which require avidity-based interactions. FcγRIIb is the only inhibitory receptor. When Fc binds to an activating receptor, intracellular signaling pathways that are regulated through phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) activate effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC; also called antibody-dependent cellular cytotoxicity). and antibody-dependent cell-mediated phagocytosis (ADCP; also called antibody-dependent cellular phagocytosis), as well as inflammation due to induction of cytokine secretion. Signaling through the inhibitory FcγRIIb, regulated through phosphorylation of the immunoreceptor tyrosine-based inhibitory motif (ITIM), recruits phosphatases that balance activation signaling pathways (see, e.g., Wang et al. (2018) Protein Cell 9(1):63-73).

Hinge and proximal C _H 2 amino acid sequences (lower hinge-upper C _H 2 domain region), and glycosylation of the conserved N297 residue in the C _H 2 domain Asn-X-Ser/Thr glycosylation motif of the Fc region (see EU numbering). by) mediates the interaction of the antibody with FcγR and the complement protein C1q. Antibody/Fc engineering has been used to modify the immune effector functions of antibodies by altering binding to C1q and various Fcγ receptors. Therefore, the CDC, ADCC and ADCP activities of therapeutic mAbs may increase or decrease depending on the field of application. For example, the efficacy of anti-cancer mAbs depends in part on induction of FcγR effector function. Effector functions include activation of natural killer (NK) cells via FcγRIIIa and subsequent ADCC activity and release of inflammatory cytokines, induction of macrophage-mediated ADCP through interaction with multiple FcγRs, and other immune cells such as NK cell-mediated ADCC It involves the recruitment and activation of neutrophils, the main receptors for There are two polymorphic variants of FcγRIIIa: the one with V158 with higher affinity for IgG1; and those having F158 with low affinity for IgG1. Cancer patients with the high-affinity V158 polymorphism may have better outcomes after treatment with cetuximab, trastuzumab, and rituximab compared to patients with the low-affinity F158 polymorphism. These results highlight the role FcγR-mediated immune effector functions play in therapy and indicate that antibodies and related molecules can be engineered to have increased affinity for FcγRs to improve therapeutic efficacy (see, e.g., Wang et al. (2018) Protein Cell 9(1):63-73).

Residues in the lower hinge and proximal CH2 regions of IgG were determined to be important for binding to FcγR. Residues within 5 angstroms of the FcγR:Fc interface for FcγRI, FcγRIIa, FcγRIIb and FcγRIIIb are residues (by EU numbering) P232, E233, L234, L235, G236, G237, P238, S239 (corresponding to residues P115-S122 , see SEQ ID NO: 9), D265, V266, S267, H268, E269, D270 (corresponding to residues D148-D153, see SEQ ID NO: 9), Y296, N297, S298, T299 (corresponding to residues Y179-T182) , see SEQ ID NO: 9), and N325, K326, A327, L328, P329, A330, P331 and I332 (corresponding to residues N208-I215, see SEQ ID NO: 9) (eg, [Wang et al. (2018) Protein Cell 9(1):63-73).

Modifications of Fc that enhance or decrease ADCC activity and/or enhance affinity/binding to a receptor are known to those skilled in the art. For example, Fc modifications that increase IgG1 affinity and binding to FcγRIIIa and/or enhance ADCC function include the replacements (by EU numbering): F243L/R292P/Y300L/V305I/P396L, L235V/F243L /R292P/Y300L/P396L, F243L/R292P/Y300L, S239D, I332E, S239D/I332E, S239D/A330L/I332E, S298A/E333A/K334A, and L234Y/L235Q/G236W/S in one heavy chain 239M/H268D/D270E/ Combinations of S298A and D270E/K326D/A330M/K334E in opposing heavy chains, and L234Y/G236W/S298A in one heavy chain and S239D/A330L/I332E in opposing heavy chains. Also, the mutations A327Q/P329A (interacts with FcγRI), D265A/S267A/H268A/D270A/K326A/S337A (interacts with FcγRIIa), G236A (interacts with FcγRIIa), T256A/K290A/S298A/E333A/K334A (interacts with FcγRIIa) with a Interaction) FcγRIIIa) results in high affinity interactions with FcγR.

Fc modifications that increase binding to FcγRIIa and FcγRIIIa and enhance ADCC and ADCP are (by EU numbering) G236A/I332E, G236A/S239D/I332E (also increased binding to FcγRI), and G236A/S239D/A330L/I332E (See, for example, Wang et al. (2018) Protein Cell 9(1):63-73; Saxena et al (2016) Front. Immunol. 7:580; and Saunders, KO (2019) Front. Immunol . 10:1296]).

Glyco-engineering of IgGs containing a conserved N-linked glycosylation site at residue N297 in the C _H 2 domain can enhance Fc effector function. Glycosylation of N297 is essential for maintaining Fc conformation and mediating interactions with FcγR (and C1q). The glycan at residue N297 is typically two N-acetylglucosamines (GlcNAc), three mannoses and more than two additional GlcNAc linked to the mannose to form a biantennary complex glycan. Additional fucose, galactose, sialic acid and GlcNAc can be added to the core glycan structure. IgG circulating in human serum is usually fucosylated, but recombinant IgG production can be achieved by expressing antibodies in plant cells, by knocking down or knocking out specific glycosidases, or by in vitro enzymatic digestion of glycosylated IgG to reduce the glycan composition. can change; Since both heavy chains are glycosylated, a single IgG molecule can have glycan heterogeneity. Glycans directly affect FcγR binding. For example, the N297 glycan of Fc may collide with the glycan of the FcγRIII protein, resulting in poor binding of effector cells mediating ADCC. The Fc region containing different glycans at N297 adopts a different hinge region conformation that may affect the Fc's ability to interact with FcγRs. Expression of β(1,4)-N-acetylglucosaminyltransferase III when expressing IgG produces antibodies glycosylated with biantennary glycans at position N297; This antibody showed increased binding to FcγRIIIa and enhanced ADCC activity. Fucose deficient (non-fucosylated/non-fucosylated) IgG1 demonstrated up to a 50-fold increase in binding to FcγRIIIa and enhanced ADCC activity. Two glycoengineered (afucosylated) mAbs, obinutuzumab (anti-CD20) and mogamulizumab (anti-CCR4), have been approved for clinical use, demonstrating the potential of glycoengineering for enhanced effector function and Indicates translation into clinically approved therapeutics (see, e.g., Wang et al. (2018) Protein Cell 9(1):63-73; Saxena et al. (2016) Front. Immunol . 7:580; and Saunders, KO (2019) Front. Immunol . 10:1296]).

Fc can also be modified to bind to a wider range of Fc receptors. Fc receptors for isotypes other than gamma (i.e., IgA, IgM, and IgE) are present in certain leukocytes, and by modifying the Fc region to bind multiple Fc receptors, antibodies with expanded ability to bind effector cells are generated. Neutrophils, the most abundant white blood cells in the body, bind to the Fc of IgA antibodies through the FcαRI receptor. For example, to link FcγR and FcαRI, a single domain of IgA2 was added to the end of the IgG1 constant region to create a four domain constant region, CH1g-CH2g-CH3g-CH3a. The C _H 1 domain of IgG1 was replaced with an alpha 1 constant region domain to create a constant region (CH1a-CH2g-CH3g-CH3a) that is structurally closer to the alpha constant region. Similar to native IgA2, these 4-domain, cross-isotype IgGA chimeric antibodies coupled to the J chain exhibited reduced transport by the polymeric Ig receptor, a 3-5-fold reduction in FcγRI affinity, and delayed serum circulation of IgG1 instead of IgA2. Serum half-life was short. However, 4-domain, a 4-domain, cross-isotype IgGA chimeric antibody, was capable of mediating complement-dependent lysis of sheep erythrocytes and was more pH resistant than IgG1. Another cross-isotype Fc was created by fusing the gamma 1 and alpha constant regions together to create a tandem G1-A Fc region in which the hinge, C _H 2 and C _H 3 domains of IgA2 were fused to the C-terminus of IgG1 did This tandem cross-isotype IgG/IgA fusion exhibited similar expression levels, antigen binding and thermostability to IgG1 and bound FcαRI and FcγRI, FcγRII, FcγRIIIa and FcRn in vitro with similar affinity to wild-type IgA and IgG, respectively. Binding to various FcRs resulted in ADCC activity on polymorphonuclear cells and NK cells; However, C1q binding was reduced 3-fold compared to IgG1. The tandem IgG/IgA had an in vivo half-life similar to that of IgG1 in BALB/c mice. An alternative cross-isotype antibody comprises the CH3 domain of the IgG1 constant region and the CH2 α1 loop residues 245-258 (by EU numbering, corresponding to the sequence PKPKDTLMISRTPE; (residues 128-141 of SEQ ID NO: 9)) in the IgA constant region It is created by replacing with a structurally similar region of This chimeric Fc bound FcγRI, FcγRIIa and FcαRI, and antibodies containing chimeric Fc-mediated ADCC with polymorphonuclear cells and ADCP with macrophages, and activated complement, but lacked binding to FcRn, which regulates antibody half-life. lacking; Thus, further optimization is required for effective in vivo use (see, eg, Saunders, KO (2019) Front. Immunol . 10:1296).

Another approach to enhance FcγR binding is the multimerization of IgG with potential for the treatment of autoimmune diseases. IgG multimers are created by adding a heteromultimerization domain, for example an isoleucine zipper, or by adding another hinge region to the N-terminus of the natural hinge or by adding another hinge region to the C-terminus of the _CH 3 domain. . An IgG hexamer is created by adding an IgM tailpiece to the C-terminus of an IgG1 Fc and creating a cysteine bond at position 309; This multimeric IgG strongly binds to FcγRI, FcγRIIa and FcγRIIIa and weakly binds to FcγRIIb and FcγRIIIb. Various multimeric IgGs increased binding to FcγRI, FcγRIIb and FcγRIII compared to monomeric IgGs and showed promise in preclinical models of arthritis, neuropathy and autoimmune myasthenia gravis. This multimeric IgG design is being further optimized to fine-tune the ability of immune receptors, including FcRn, to bind multimers (see, e.g., Saunders, KO (2019) Front. Immunol . 10:1296). .

Residues in the Fc region of IgG involved in interaction and binding to C1q (and therefore CDC) include (by EU numbering) S267, D270, K322, K326, P329, P331 and E333. Fc variants that have been shown to enhance CDC by increasing C1q binding include, for example, K326A, E333A, K326A/E333A, K326W, K326W/E333S, K326M/E333S, C220D/D221C, H268F/S324T, S267E, H268F, S324T, S267E/H268F/S324T, and G236A/I332E/S267E/H268F/S324T (all by EU numbering). Replacing Trp at positions 222, 223, and 224 (i.e., K222W, T223W, and H224W by EU numbering) in various combinations in the upper hinge region of IgG1 Fc resulted in no effect on FcγRIIIa binding and ADCC activity against wild-type IgG1. Increased C1q binding and CDC activity. Specifically, mutations included K222W/T223W, K222W/T223W/H224W and D221W/K222W. Mutations C220D/D221C and C220D/D221C/K222W/T223W also increased C1q binding and CDC activity (see, for example, Wang et al. (2018) Protein Cell 9(1):63-73; Saxen et al. (2016) Front. Immunol . 7:580; Saunders, KO (2019) Front. Immunol . 10:1296; and Dall'Acqua et al. (2006) J. Immunol. 177:1129-1138]).

IgG3 has the best in vitro binding to C1q; The C _H 1 and hinge regions of IgG1 are combined with the C _H 2 and C _H 3 regions of IgG3 (retaining the ADCC activity of IgG1 and the CDC activity of IgG3) to create an IgG1/IgG3 cross-subtype antibody and binding C1q and enhance CDC activity. Other IgG1/IgG3 cross subtype antibodies with increased C1q binding and enhanced CDC activity include C _H 1, hinge and C _H 3 of IgG1, and C _H 2 of IgG3; This modification not only allows increased Cq1 binding since C1q binds to the C _H 2 domain, but also facilitates purification because Protein A binds to the C _H 3 domain. Additionally, variants E345R/E430G/S440Y resulted in the formation of IgG hexamers with K322 oriented in a position that favorably interacted with the hexameric C1q headpiece, enhancing CDC activity. Mutation E345R alone also results in IgG hexamer formation with increased C1q binding and enhanced CDC activity (see, e.g., Wang et al. (2018) Protein Cell 9(1):63-73; Saxena et al. (2016) Front. Immunol . 7:580; Saunders, KO (2019) Front. Immunol . 10:1296]).

Glycoengineering can also be used to improve complement binding; The N297 glycan in the C _H 2 domain of Fc can be modified to improve CDC activity. For example, excessive galactosylation in IgG1 Fc increases C1q binding and CDC activity and also improves thermostability compared to unmodified IgG1 glycoforms. Thus, galactosylation of Fc can be used to generate stable biologics with enhanced CDC activity (see, eg, Saunders (2019) Front. Immunol . 10:1296).

Table 8 below summarizes Fc modifications that enhance binding to FcγR or C1q, thereby enhancing immune effector functions, including ADCC, ADCP and CDC, and Kabat numbering and Provide corresponding variants by sequential numbering. Any one or more of these modifications, alone or in various combinations, can be introduced into the IgG1 Fc portion of the constructs provided herein. Other modifications known in the art that confer enhanced or increased immune effector function are also contemplated for use herein.

Therapeutic antibodies can also be engineered to reduce or eliminate immune effector function. For purposes herein, in some embodiments it is important to reduce or eliminate, for example, ADCC activity. Constructs herein comprising an Fc are generally modified to reduce or eliminate ADCC activity.

For example, it is important to reduce or eliminate immune effector function, where: the therapeutic antibody is antagonistic to prevent receptor-ligand interaction and signaling; Antibodies are receptor agonists that cross-link receptors and induce signaling; Antibodies are drug delivery vehicles that deliver drugs to antigen-expressing target cells; and reduction or elimination of effector function prevents target apoptosis or unwanted cytokine secretion. Reduced effector function also prevents antibody-drug conjugates from interacting with FcγRs, reducing off-target cytotoxicity. The importance of reducing or eliminating effector function became apparent after the side effects associated with administration of muromonab, the first approved mAb designed to prevent T cell activation in transplant patients receiving donor kidney, lung or heart. Patients receiving muromonab experienced a dangerous induction of pro-inflammatory cytokines (ie, cytokine storm); This was in part due to the interaction of muromonap with FcγR (see, for example, Wang et al. (2018) Protein Cell 9(1):63-73; and Saunders, KO (2019) Front. Immunol . 10:1296]).

There are many known mutations that reduce or eliminate receptor function. For example, the replacements L235E and F234A/L235A in human IgG4, and L235E and L234A/L235A in human IgG1 (all by EU numbering) reduce FcγR and C1q binding and reduce effector functions such as inflammatory cytokine release. The release of inflammatory cytokines from therapeutic antibodies can cause side effects. Alternate S228P/L235E also reduces binding to FcγR when introduced into IgG4; The S228P mutation improves the stability of IgG4. Mutations S228P/F234A/L235A of IgG4 Fc reduce binding to FcγRI, IIa and IIIa and reduce ADCC and CDC. Triple mutant L234E/L235F/P331S of IgG1 Fc reduces binding to FcγRI, FcγRII, FcγRIII and C1q and reduces CDC, and mutation L234A/L235A/P329G of IgG1 Fc abolishes FcγRI, FcγRII, FcγRIII and C1q binding and decrease ADCP. Mutations L234F/L235E/P331S also reduce binding to FcγR and C1q and reduce effector functions of IgG1 Fc. Mutations G237A and E318A of IgG1 Fc reduce binding to FcγRII and decrease ADCP, respectively; Mutations D265A and E233P reduce binding to FcγRI, FcγRII and FcγRIII and reduce ADCC and ADCP, mutations G236R/L328R reduce binding to all FcγRs and reduce ADCC. Crystal structure data suggest that conformational changes in residue P329, which packs between two conserved tryptophan residues that occur in all FcγRs to form a “proline sandwich,” may be detrimental to interactions with FcγRs and that modifications of residue D270 may be detrimental to interactions with C1q. action (see, for example, Wang et al. (2018) Protein Cell 9(1):63-73; Saunders, KO (2019) Front. Immunol . 10:1296; International Application Publication No. WO 2019/226750).

Induction of the complement cascade is associated with abnormal antibody injection site reactions and also eliminates C1q binding to Fc, which is an early stage in CDC activation. Modification of the Fc region that removes C1q binding can be used to remove CDC in constructs containing the Fc region. Many mutations that abolish FcγR binding also ablate C1q binding, as shown above. For example, mutation A330L disrupts C1q binding, reduces CDC and also abolishes FcγRIIb binding. Mutations D270A, P329A, K322A and P331A also result in reduced C1q binding and reduced CDC activity (see, eg, Saunders, KO (2019) Front. Immunol . 10:1296).

Glyco-engineering can be used to remove FcγR and C1q binding. As discussed elsewhere herein, the glycan at residue N297 is a complex biantennary glycan. Modification of this glycan to a high mannose glycan (i.e., high mannose glycosylation) reduces the affinity of the IgG1 Fc for C1q and reduces CDC activity. Mutations in Fc that reduce or eliminate C1q and FcγRI binding can also increase galactosylation and sialylation of the N297 glycan; Such mutations include, for example, F241A, V264A and D265A. Mutations N297A, N297Q, N297D and N297G by EU numbering remove the glycosylation site in N297 and reduce effector functions such as CDC and ADCC by removing Fc interactions with C1q and FcγR, respectively. The N297G/D265A combination almost completely abolishes binding to FcγR and C1q. An IgG3 Fc lacking glycosylation (aglycon Fc) has reduced binding to FcγRI and C1q (see, e.g., Wang et al. (2018) Protein Cell 9(1):63-73; Saunders, KO (2019) Front. Immunol . 10:1296]).

In order to reduce or eliminate Fc effector function, large portions of Fc regions from different subclasses lacking the opposite function can be exchanged to create inter-subclass Fc regions. For example, IgG2 has weak FcγR binding but binds C1q, IgG4 lacks C1q binding but reacts with FcγR; Combinations of IgG2 and IgG4 CH domains lacking both C1q and FcγR binding can thus be constructed. In general, in an IgG1/IgG4 chimera, the hinge family C _H 1 domain is derived from IgG2, and the C _H 2 and C _H 3 domains are derived from IgG4. Since IgG1 and IgG3 recruit complement more effectively than IgG2 and IgG4, and IgG2 and IgG4 have a limited ability to induce ADCC, a cross-subclass approach may reduce effector function. For example, the anti-C5 mAb eculizumab is IgG2 residues 118-260 (by EU numbering; corresponding to residues 114-273 by Kabat numbering and residues 1-139 with respect to SEQ ID NO: 11), and IgG4 Contains residues 261-447 (by EU numbering; corresponding to residues 274-478 by Kabat numbering and residues 141-327 with respect to SEQ ID NO: 15) and has limited or undetectable effector function. Similarly, point mutations H268Q/V309L/A330S/P331S (by EU numbering; H281Q/V328L/A349S/P350S by Kabat numbering and H147Q/V188L/A209S/P210S by SEQ ID NO: 11) from IgG4 were all It lacks binding to FcγR and C1q and exhibits reduced effector function. IgG2 to IgG4 cross-subclass mutations V309L/A330S/P331S (by EU numbering; corresponding to V328L/A349S/P350S by Kabat numbering and V188L/A209S/P210S with reference to SEQ ID NO: 11), and non-reproductive variants containing the series mutations V234A/G237A/P238S/H268A (by EU numbering; corresponding to V247A/G250A/P251S/H281A by Kabat numbering and V114A/G116A/P117S/H147A with reference to SEQ ID NO: 11) So-called IgG2σ) abolishes binding to FcγR and C1q and exhibits undetectable CDC, ADCC and ADCP activities. The IgG1/IgG4 cross subclass variant IgG1σ containing the mutations L234A/L235A/G237A/P238S/H268A/A330S/P331S lacks binding to FcγRI and IIIa and has very weak, reduced binding to FcγRIIa and IIb at high concentrations of the antibody. ADCC and CDC activity (see, for example, Wang et al. (2018) Protein Cell 9(1):63-73; Saunders, KO (2019) Front. Immunol . 10:1296]).

Tables 9 and 10 below show reduction or elimination of binding to FcγR and/or C1q to reduce or eliminate immune effector functions including ADCC, ADCP and CDC that can be introduced into the Fc region in the constructs herein. Some IgG1 and IgG4 Fc modifications are summarized.

The table provides the corresponding modifications by Kabat numbering and sequential numbering with respect to the sequence of the IgG1 heavy chain constant domain set forth in SEQ ID NO: 9 or the IgG4 heavy chain constant domain set forth in SEQ ID NO: 15. Any one or more of these modifications, alone or in various combinations, can be introduced into the IgG1 Fc portion of the constructs provided herein. Other modifications known in the art to reduce or eliminate immune effector function are also contemplated for use herein.

ii. Fc different variations of parts

The Fc portion can also be modified to increase binding to inhibitory FcγRs to suppress the immune response. Therapeutic antibodies with immunosuppressive Fc modifications are beneficial for the treatment of inflammatory diseases. These mutations may be incorporated into the Fc portion of the constructs herein for treatment of diseases and conditions in which an inflammatory factor or pathogenesis or involvement is involved. For example, an immunosuppressive version of the anti-CD19 antibody (XmAb5871; Xencor) containing the mutations S267E/L328F (by EU numbering) binds the inhibitory FcγRIIb with approximately 430-fold increased affinity and prevents systemic lupus erythematosus (SLE). ) depletes CD19 ⁺ B-cells in patients with When the same mutation is introduced into a humanized anti-IgE antibody (XmAb7195; Xencor), it prevents binding of IgE to the high-affinity receptor (FcεRI) present on basophils and mast cells and increases the affinity for FcγRIIb ~430-fold. It is used in the treatment of allergies, including allergic asthma. Anti-CD3 antibody TRX4 (Tolerx) containing the aglycosylated Fc mutation N297A (by EU numbering) suppresses pathogenic T-cells and restores normal Treg cell activity in patients with type 1 diabetes (autoimmune) (e.g. , Saxena et al. (2016) Front. Immunol. 7:580]).

A further example is a monomeric IgG1 Fc (mFc) containing the mutations L351S/T366R/L368H/P395K (by EU numbering), which binds FcRn, exhibits a half-life in vivo similar to dimeric Fc, and binds to FcγRI with high affinity. It selectively binds, but does not bind FcγRIIIa, eliminating Fc-mediated cytotoxicity including ADCC and CDC. FcγRI is expressed in inflammation-related cells such as inflammatory macrophages. Targeting this receptor can be used for the treatment of chronic inflammatory diseases such as arthritis, multiple sclerosis and cancer. When the variant mFc is fused to the Pseudomonas exotoxin A fragment (PE38), FcγRI⁺ Kill the macrophage-like U937 cells. Neither the variant mFc nor the fusion protein exhibits any cytotoxicity (ADCC or CDC) in vitro (see, e.g., Ying et al. (2014)mAbs6(5):1201-1210).

Modifications that increase binding to, or confer selective binding to, inhibitory FcγRIIb and/or FcγRI, but not FcγRIIIa, can be engineered into the IgG Fc region in the TNFR1 antagonist and TNFR1 antagonist/TNFR2 agonist constructs provided herein. . Such variations include, but are not limited to, one or more of S267E, N297A, L328F, L351S, T366R, L368H, P395K, S267E/L328F, L351S/T366R/L368H/P395K, and combinations thereof by EU numbering. Table 11 below shows the corresponding replacements by Kabat numbering and sequential numbering with respect to the sequence of the IgG heavy chain constant domain set forth in SEQ ID NO:9.

iii. Human Serum Albumin (HSA)

A problem with previously provided dAbs (see, eg, International PCT Application No. 2008/149144) has been that the serum half-life is insufficient for use as a therapeutic agent. They were linked to anti-HSA antibodies to bind HSA; The half-life was insufficient. wherein the dAb or Vhh antibody is linked to HSA. HSA has 33 cysteines; Cys34 is the only cysteine with a free sulfhydryl group that does not participate in disulfide bonds. HSA can be linked directly to the dAb via its N or C terminus or via a linker such as the Gly-Ser linker to extend the serum half-life of the dAb. It can also be linked through free cysteines. Example 6 illustrates a construct containing a dAb linked to the N-terminus of HSA via a Gly-Ser linker.

e. Multispecific TNFR1 antagonist/TNFR2 agonist constructs

To selectively inhibit TNFR1 signaling, while enhancing the beneficial effects of TNFR2 signaling, multispecific, such as bispecific constructs containing a TNFR1 antagonist and a TNFR2 agonist are provided (e.g., formula 2 above). reference). Such multispecific constructs, as discussed above, may contain linkers and active modifiers to impart advantageous properties as desired.

The TNFR1 inhibitor and TNFR2 agonist portions of the constructs provided herein can be polypeptides or small molecules or combinations thereof; Can be linked directly in any order, directly or indirectly via a linker, such as a Gly-Ser linker, including any described herein, and/or a hinge region, or can be linked via a chemical linker. The constructs may contain active modifiers, such as an Fc region or modified Fc, and/or other active modifiers, such as polypeptides, such as HAS, which extend half-life, and may be polymers, such as PEG or polymeric moieties.

A human TNFR1 antagonist, such as a TNFR1 antagonist set forth in any one of SEQ ID NOs: 54-703, or the C-terminus of a TNFR1 antagonist having about or at least about 95% sequence identity to a TNFR1 antagonist set forth in any of SEQ ID NOs: 54-703. is fused to the N-terminus of a first IgG1 Fc, such as an IgG1 Fc derived from Trastuzumab. The order can be reversed.

The Fc region contains the C _H 2 and C _H 3 domains of Trastuzumab heavy chain (see, eg, residues 234-450 of SEQ ID NO: 26). In some embodiments, the linker between the TNFR1 antagonist and the first Fc subunit contains all or part of the hinge sequence of an antibody such as Trastuzumab (SCDKTH; corresponding to residues 222-227 of SEQ ID NO: 26). To confer protease resistance and increase flexibility of the fusion protein, the SCDKTH hinge sequence or protease cleavage site or both Gly-Ser short peptide linkers such as GSGS, GGGGS or GGGGSGGGGSGGGGS, and/or sugars described herein Others known in the art may be substituted. In other embodiments, only the GS linker is included. In another embodiment, the linker contains a branched PEG or PEG having a molecular weight of 30 kDa or greater.

In some embodiments, Fc subunits (also referred to as regions or domains) can multimerize. The first Fc subunit is attached to the second Fc subunit via a disulfide bond. For the bispecific construct, the C-terminus of the second Fc subunit is a TNFR2 agonist, e.g., a TNFR2 agonist of any of SEQ ID NOs: 765-801, 803, and 810, or SEQ ID NO: 765 -801, 803, and 810, or a TNFR2 agonist having about or at least about 95% sequence identity to the TNFR2 agonist, or a small molecule TNFR2 agonist. The second Fc subunit and the TNFR2 agonist are linked through a linker such as the SCDKTH hinge sequence of Trastuzumab, either alone or in combination with a short GS linker as described above. In other embodiments, only the GS linker is included. In an alternative embodiment, a single chain Fv fragment (scFv), or Fab region, or other antigen binding fragment of a TNFR2 agonistic monoclonal antibody may be used; A scFv or Fab dimerizes by fusion of the C-terminus and the N-terminus of Fc. As provided herein, antigen-binding fragments can be derived from TNFR2 agonizing mAbs MR2-1 and MAB226.

An Fc subunit can be modified to alter its activity. For example, dimers prevent homodimerization and/or inhibit immune effector functions such as antibody dependent cellular cytotoxicity (ADCC), antibody dependent cell mediated phagocytosis (ADCP), and/or complement dependent cytotoxicity (CDC). and/or modified to enhance neonatal FcR (FcRn) recycling to increase the in vivo half-life and stability of the recombinant construct, as described below.

In embodiments where the construct is for treatment of an inflammatory disease, the Fc portion is modified such that effector function is reduced or eliminated. In an embodiment, the Fc dimer is modified to enhance immune effector function such as ADCC, ADCP and/or CDC, eg where the construct is for the treatment of cancer. The specific Fc modification depends on the intended disease target.

In some embodiments, the Fc subunit(s) may contain an IgG4 Fc region, such as an IgG4 Fc derived from nivolumab ( ^Opdivo® ) that contains the C _H 2 and C _H 3 domains of a nivolumab heavy chain (e.g. See, eg, residues 224-440 SEQ ID NO: 29). ESKYGPPCPPCP (see eg residues 212-223 of SEQ ID NO: 29), a short peptide linker containing all or part of the nivolumab hinge sequence sufficient to provide flexibility, can be used to link the nivolumab Fc region with a TNFR1 antagonist and/or TNFR2 agonists. Optionally or alternatively, a GS linker may also be included.

In exemplary embodiments, since TNFR2 may require receptor aggregation/clustering for signaling, bivalent antibody-like structures can be created to achieve superior agonism. In this embodiment, the C-terminus of each of the first and second Fc subunits is fused to the N-terminus of the TNFR2 agonist as described above. Fc dimers, as described elsewhere herein, prevent homodimerization, eliminate antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and enhance neonatal FcR recycling to enhance the bioactivity of recombinant constructs. modified to increase my half-life.

dual specific, TNFR1 antagonist/ TNFR2 agonist construct and same multispecific Composition PEGylation to Link Components of Water, PEG-Centric Multispecific Constructs

PEGylation, which refers to the covalent attachment of the biocompatible and biologically inert polymer poly(ethylene glycol) (PEG) to molecules such as proteins, peptides, drugs and other molecules, is another modifier of the activity of a construct. PEGylation increases the water solubility of a molecule, increases the molecular weight of the molecule, prolongs circulation time in vivo , reduces peripheral clearance rate, minimizes non-specific uptake, and enhances permeability and retention (EPR) effect to target tumors. PEGylation of therapeutics, such as protein therapeutics, can protect the therapeutic from the action of antibodies and antigen processing cells by masking undesirable antigenic surface markers, thereby reducing degradation by proteolytic enzymes and other inactivation processes. let it PEGylation also increases the molecular weight of the protein therapeutic, thereby prolonging the half- life in vivo and reducing peripheral clearance, thus allowing for less frequent dosing.

Chemical conjugation of a therapeutic molecule with a polymer, such as PEG, can form stable ester or amide linkages as well as disulfide linkages. Conjugation of PEG with a molecule of interest, such as a TNFR1 antagonist and a TNFR2 agonist provided herein, can be achieved, for example, with dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl) Carbodiimide (EDC), HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxidehexafluorophosphate), or This may be achieved by using other art-known coupling agents, or by using N -hydroxysuccinimide (NHS) esters such as PEG NHS esters. Other methods include PEG maleimides that react with sulfhydryl functional groups on proteins or peptides, PEG pentafluorophenyl (PFP) esters that react with primary and secondary amines, thiol PEG that react with thiols on the side chains of cysteine residues, and clicks ( click) involves the use of chemical techniques. PEG azide, propargyl PEG, aminooxy PEG, hydroxy PEG, amino PEG, PEG acid, biotin PEG, PEG tosylate, and PEG with other functional groups are also commercially available, peptides and other therapeutic molecules can be used for bonding.

A common method for preparing PEG-protein conjugates is to associate a -NH ₂ functional group and monomethoxy PEG (mPEG) on a protein with an electrophilic functional group; This approach results in the formation of polymer chains covalently linked to globular proteins in the core. This property is used herein to provide PEG-centric constructs in which PEG, or chemically similar or compatible moieties, presents a plurality of binding or interacting moieties to one or a plurality of different targets. To increase drug (binding moiety) loading, multi-arm or branched chain PEG (or similar moieties or branched chain moieties) can be used. Alternatively, the drug is a small PEG dendron (see, eg, the PEG conjugation protocol on the BROADPHARM ^® website available at broadpharm.com/web/protocols.php; see also Banerjee et al. (2012) Journal of Drug Delivery , Article ID 103973). To attach a plurality, eg two different therapeutic moieties, heterobifunctional, eg heterobifunctional PEG moieties, eg with different reactive functional groups on each end, can be used. The PEG moiety may have two, three, or more different reactive functional groups. Such molecules target two different receptors on the same cell or, as described herein, on different cells, such as TNFR1 and TNFR2, by delivering two or more different ligands, or two targets that bind to different sites on the same receptor. It can be used to deliver an agent, or to bring together a receptor, eg to activate or inhibit a receptor, or to cross-link two different receptors, eg to inhibit a receptor activity. Homobifunctional PEG molecules with the same reactive functional group at each end can be used to recruit the same receptors on the same cell or, if the PEG chain length permits, on different cells. Such constructs can be used to trap circulating soluble receptors, or ligands such as TNF. Figure 3 herein provides an exemplary construct utilizing a PEG moiety to display a drug (linked reactive moiety).

To increase reactivity and flexibility, enhance ligand-protein binding and reduce steric hindrance, the construct may include one linker molecule, such as a spacer molecule, or a plurality thereof, as described herein. Such spacers include, for example, amino acid spacers such as alanine, glycine, and small peptides. Any of the linkers described herein, including rigid as well as GS linkers and other flexible linkers, can conjugate a reactive moiety, such as a TNFR1 inhibitor moiety described herein and/or a TNFR2 agonist described herein, to a multifunctional PEG molecule. can be used to make These constructs may also include active modifiers, such as an Fc region.

The use of a branched PEG moiety, with or without a linker, or a multi-armed PEG moiety described herein (see, e.g., FIG. 5) contains a TNFR1 inhibitor moiety or a TNFR2 agonist, or a combination of both. However, it can be used to provide other inhibitors and/or agonist moieties of any receptor(s) of interest and/or also to produce immunotoxins and other toxic conjugates. . Methods for the synthesis of a number of PEG moieties and variants thereof are known (see, eg, US Patent Publication No. 2010/0221213; Han et al., (2014) Sci Rep 4 :4387.

For example, in some embodiments containing a TNFR1 inhibitor and a TNFR2 agonist moiety, the construct comprises a bifunctional PEG moiety, and also intervenes the PEG moiety and the TNFR1 antagonist and TNFR2 agonist, respectively. contains linkers. The multispecific construct contains a branched PEG polymer to which the linker is attached and to which one or both of the TNFR1 inhibitor moiety and the TNFR2 moiety are attached. Suitable PEG moieties may have a molecular weight of greater than 30 kDa, such as greater than 30-40 kDa. An exemplary branched PEG molecule can be, for example, a three-armed heterobifunctional PEG molecule, which has one type of reactive functional group (RG1; eg , -NH ₂ ) and is a TNFR1 inhibitor moir. It contains one arm connected to a tee, and two arms with different types of reactive functional groups (RG2; eg , -COOH), each linked to a TNFR2 agonist. Such three-armed heterobifunctional branched PEG molecules are commercially available (eg, available from ^BROADPHARM® ). The first PEG arm may be linked to the N-terminus or C-terminus of the TNFR1 inhibitor moiety and the other two arms, if small molecules, to the N-terminus or C-terminus of the TNFR2 agonist or to the TNFR2 agonist can be connected In some embodiments, the constructs may also include an optional linker as described herein. Such linkers may be included between the PEG arms and the TNFR1 inhibitor moiety and/or the TNFR2 agonist(s). This PEG-centric bispecific construct provides monovalent for TNFR1 antagonist activity, which prevents TNFR1 receptor clustering leading to undesired agonism and provides bivalent for TNFR2 receptor clustering that enhances TNFR2 signaling. Exemplary structures for the PEG-centric bispecific TNFR1 antagonist/TNFR2 agonist constructs described herein fall within those shown in FIG. 3 .

In another embodiment, the linker between the TNFR1 antagonist and the TNFR2 agonist contains a branched PEG having a molecular weight of 30 kDa or greater. The branched PEG molecule contains one branch linked to the N-terminus of the TNFR1 antagonist and two branches each linked to the TNFR2 agonist, thereby providing a divalence for TNFR2 receptor clustering that enhances TNFR2 signaling.

3A-D depict various configurations of multispecific constructs in which PEG moieties link functional moieties. PEGylation moieties and PEGylation procedures are discussed in more detail in Section H below. Methods for preparing various PEG linkers and constructs are well known to those skilled in the art (see, eg, creativepegworks.com/pegylation_literature.php; and broadpharm.com/web/protocols.php). In FIG. 3 , each n may independently be 1 to 10, such as 1 to 7, 1 to 5, and 1 to 3, 1 or 2. In Figure 3a, each n is typically 1-3 depending on the particular ligand indicated. In other parts of Figure 3, n is generally 1 to 5. In all embodiments N may be 1. One skilled in the art will recognize other routine variations of the PEG moiety that serve as the central linker, and similar moieties can be used in place of PEG.

In Figure 3a,

R ¹ is H or lower alkyl (C1 to C5, or C1 or C2), such as CH ₃ , and n is usually 1 to 5, such as 1 or 2. The figures depict ligands or epitopes that bind to multiple targets ( ie , epitopes on a receptor, e.g., Targets (circled) 1 a,b,c represent different epitopes on the same receptor). Target 2 may be an epitope on a different receptor. In Figures 3b and 3c, circles are epitopes or receptor-targeting ligands, n is usually 1-5, typically n is 1 or 2, and is usually 1. 3D depicts a homobifunctional construct, where n is typically 1-3, usually 1 or 2, such as 1. Active modifiers such as Fc or others as described herein or known to those skilled in the art are optional. In all of these constructs, the PEG moiety provides activity modifying activity, such as half-life extension, or sterically links operative fragments that bind to the intended target (peptides, small molecules, aptamers, and others). It is usually 'inactive'.

3A depicts an exemplary bivalent construct in which PEG is the central moiety. One circle is, for example, a polypeptide agonist, antagonist or binding protein, such as an antibody or antigen-binding fragment thereof, or an aptamer (nucleic acid or peptide). Other circles represent different moieties, such as polysaccharides or receptor ligands. The bivalent structure enables clustering of targets for receptor activation. In some embodiments provided herein, targets include TNFR1 and TNFR2, and circles represent TNFR1 inhibitors and TNFR2 agonists described throughout the disclosure herein. Figure 3b depicts monovalent single ligands, such as CD3+, which can prevent cytokine release symptoms and are linked via PEG moieties to agonists, antagonists, or binding proteins to become bivalent for receptor clustering. FIG. 3C shows heterobifunctional PEG (or other such agents) to cross-link two different cell targeting agents, or two agents, such as Trastuzumab and Pertuzumab, that bind to two receptors or to different sites on the same receptor. Carriers such as) are shown. The constructs of Figures 3B and 3C are used, for example, to recruit checkpoint control receptors for either stimulation or inhibition of an immune response, or to cross-link two different receptors to achieve inhibition of receptor activity ( i.e. , CD3 vs CD450), or to deliver two different ligands, such as stimulatory and costimulatory ligands, to two different receptors on the same cell. These constructs can also serve as prodrugs that are directed to or accumulate in hypoxic regions of low pH where the linked moiety can be chemically released by protonation. For example, in the case of tumors, it may be a toxin, or it may be a locally released TNF inactivator ( ie, an aptamer or peptide).

Figure 3D depicts homobifunctional PEG to recruit the same receptors on the same or different cells depending on the chain length, or to trap circulating disease targets such as soluble receptors or ligands such as TNF. Additionally, in all of these embodiments, additional PEG side chain(s) optionally linked to another reactive group or functional group, such as a serum half-life extending moiety such as HSA, or an FcRn polypeptide may be included in these constructs.

Other structures are also devised in which X and Y refer to molecules that interact with reactive groups, such as binding moieties, targets (see Figure 4):

Other examples (eg, see FIG. 5 ) are as follows, where, as before, X and Y can be any targeting moiety or binding moiety or drug that interacts with the target:

f. additional activity modifier - a fusion protein comprising all or part of a polypeptide that increases serum half-life

The properties of the construct can be altered by adding a full-length polypeptide or portion thereof that increases the serum half-life, but does not substantially or not alter the interaction of the construct with TNFR1 and/or TNFR2. These include albuminization and other such modifications (see previous discussion with respect to half-life extenders; reviewed in Strohl (2015) BioDrugs 29(4) :215-239; also Tan et al. (2018) Current Pharmaceutical Design 24 :4932-4946) are included.

5. Prediction and elimination of immunogenicity in protein therapeutics

Many protein therapeutics, including those containing human germline sequences, such as recombinant human cytokines and human antibodies, are immunogenic, eliciting a host immune response to the therapeutic agent. As described herein, constructs provided herein, such as TNFR1 antagonist molecules and TNFR2 agonists, and multispecific constructs described and provided herein, if desired, are immunogenic or interact with pre-existing antibodies. It may be modified, such as by amino acid substitution, to remove or delete the epitope it functions on.

The construct undergoes prediction, identification and elimination of immunogenic B-cell and/or T-cell epitopes, thus reducing or eliminating any potential immunogenicity and increasing the safety, tolerability and efficacy of the therapeutic molecule. The molecule can be tested in vitro to determine immunogenicity before and after removal of immunogenic sequences. and tested in animal models in vivo .

As discussed in more detail below, protein therapeutics may contain immunogenic B-cell and/or T-cell epitopes. When the immune system recognizes a protein therapeutic as a foreign agent, an organized, undesirable immune response to the therapeutic agent is induced. Such reactions can lead to clinical complications such as, for example, rapid drug clearance, reduced drug action and efficacy, delayed infusion-like allergic reactions, hypersensitivity and, in some cases, life-threatening autoimmunity. Immune responses to protein therapeutics occur by two different mechanisms: the classical immune response and resistance disruption. The immunological distinction between self and non-self proteins determines the mechanism of the immune response, and proteins recognized as foreign are classical immune responses characterized by the formation of antigens within days to weeks, often after a single injection of a protein therapeutic. elicit a reaction This response, once formed by memory B-cells, is long-lasting and difficult to reverse. Subsequent exposure to this protein induces a secondary response characterized by significant amounts of IgG that negatively impacts therapy. Therapeutic proteins that induce a classical immune response include rhGAA (recombinant human acid alpha-glucosidase) and FVIII, as well as alternative therapies, such as monoclonal antibody (mAb) therapeutics, whereas complementarity-determining regions (CDRs) have significant immunogenic properties. By nature, the lack of central tolerance to the CDR regions leads to the production of anti-subject specific alloantibodies. Therapeutic proteins homologous to endogenous proteins typically do not elicit an immune response because of established immune tolerance, but may become immunogenic by destroying B-cell tolerance after repeated administration, such as IFN-γ, INF-β and erythropoietin. This is the case with poietin (EPO) (e.g. Baker et al. (2010) Self/ Nonself 1(4):314-322; Choi et al. (2017) Methods Mol . Biol . 1529:375-398; Dingman et al (2019) J. Pharm. Sci. 108(5):1637-1654).

Factors affecting the immunogenicity of protein therapeutics include, for example, the duration of treatment and the route and frequency of administration, and subcutaneous administration of protein therapeutics is more immunogenic than intravenous administration, and prolonged and frequent administration. Therapies used are more immunogenic. Patient-related factors such as the patient's immune status and polymorphisms in MHC (or HLA in humans) molecules also affect protein immunogenicity. For example, MHC molecules are highly polymorphic, with several different alleles for MHC II, including different subunits such as DP, DM, DOA, DOB, DQ and DR, these receptor subtypes There are differences in binding affinity to epitopes, and thus, differences in MHC subtypes between patients may affect the immune response to protein therapeutics. The patient's immune status can also affect immunogenicity, with autoimmune patients responding more strongly to protein therapeutics than immunocompromised patients. Other factors that affect immunogenicity include, for example, the presence of immunogenic epitopes recognized by MHC II, formation of aggregates in the final product, oxidation of proteins, aggregates in formulations and post-translational modifications such as glycosylation, Include the properties of the protein product. Recombinant proteins can be produced in several different cell types, including, for example, bacterial cells such as E. coli , and mammalian cells such as CHO cells. Proteins expressed in bacteria do not undergo post-translational modifications, such as glycosylation, but proteins produced in mammalian cells do, which can lead to different immunogenicity. For example, interferon β-1b, an interferon marketed under the trade name Betaseron ^® , is E. coli While produced in cells and not glycosylated, a later developed product marketed under the trade name Avonex ^® (interferon β-1a) is produced in CHO cells by recombinant DNA technology. Betaseron has a much higher immunogenicity than Avonex ^® interferon, 35% versus 5%, respectively. Such differences may be due in part to differences in glycosylation patterns, which may lead to aggregation (see, eg, Dingman et al. (2019) J. Pharm. Sci. 108(5):1637-1654).

The effect following administration of protein therapeutics is the development of high-affinity anti-therapeutic antibodies, also known as anti-drug antibodies, or ADAs. The production of ADA involves the stimulation of adaptive and non-adaptive immune responses, which are predominantly polyclonal and can produce neutralizing and non-neutralizing effects with protein therapeutics. ADAs can contain sub-classes of heavy chain constant regions (eg IgG1-4) as well as multiple isotypes (eg IgM, IgE and IgG), variable regions that bind with high affinity to protein therapeutics and thus have undergone somatic hypermutation of variable region genes. The immune response is induced by the recognition of immunogenic peptide fragments, such as B-cell and T-cell epitopes, in protein therapeutics. Thus, many protein therapeutics require deimmunization while retaining the desired therapeutic activity before they can be applied clinically (e.g., Baker et al. (2010) Self/ Nonself 1(4):314-322; Choi et al. (2017) Methods Mol . Biol . 1529:375-398).

Formation of anti-drug antibodies (ADA) to protein therapeutics is mediated by antigen presenting cells (APCs), such as dendritic cells (DCs) and macrophages, and by B and T lymphocytes. MHC class II-restricted T-cell epitopes present in the sequence of protein therapeutics can result in the development of a humoral response to protein therapeutics. For example, DCs stimulated through pattern recognition receptors (PRRs) stimulate T-cells and induce the generation of T-cell dependent high-affinity ADA responses. In the first step of the T-cell dependent antibody response, APCs phagocytose a protein therapeutic, process the antigen into a peptide epitope, and associate the epitope with a major histocompatibility complex (MHC) class II molecule on the APC cell surface to convert the epitope. Provided to naive T cells. To fully activate the T-cell needed to activate the B-cell, the T-cell receptor (TCR) must interact with the MHC II-epitope complex, which is a costimulatory molecule provided by APCs, such as CD80 and CD86. is accompanied by an additional signal from Naive B-cells are activated by interaction between IgM and IgD receptors on the B-cell surface and their cognate antigens. Antigen-specific T-cells then secrete cytokines that stimulate B-cell proliferation and maturation into plasma cells, which result in the engagement of CD40 and CD40 ligands, thereby leading to antibody production and antibody-production by B-cell clonal expansion. It provides additional signals leading to differentiation into secretory plasma cells and memory B-cells. Memory B-cells remain dormant until subsequent exposure to the therapeutic protein, while plasma cells secrete antibodies recognizing specific epitopes on the therapeutic protein presented by the APC MHC receptor. Many protein therapeutics, including recombinant human proteins, contain potent T-cell epitopes. For example, the immunogenicity of IFNβ1b was improved after mapping and elimination of single immunodominant (but not sub-dominant) T-cell epitopes via amino acid mutations.

Immunogenicity can also occur in a T-cell-independent process, whereby the antigen directly engages B-cells. High molecular weight aggregates of protein therapeutics can induce T-cell dependent and independent anti-drug antibody responses by stimulating DCs or by crosslinking B-cell receptors. For example, T-independent stimulation of B-cells produces an ADA response, where multimers capable of cross-linking the B-cell receptor (BCR) are effective enough to preclude the need for co-stimulation from T-cells. This can happen when building a structure. A correlation exists between enhanced immunogenicity and aggregated or multimeric proteins. For example, aggregated, but not monomeric, recombinant human interferon (IFN)α results in the production of IFNα-specific antibodies in human IFNα transgenic mice. Formation of aggregates is dependent on drug solubility and production process (e.g., Baker et al. (2010) Self/ Nonself 1(4):314-322; Dingman et al (2019) J. Pharm . Sci . 108(5):1637-1654; De Groot, AS and Moise (2007) Curr . Opin . Drug Discov . Devel. 10(3):332-340).

ADA responses to protein therapeutics may be in the form of neutralizing or binding antibodies. Neutralizing antibodies recognize a region required for biological activity within a protein therapeutic and directly ablate its activity. The humoral response is directed at B-cell epitopes within the protein therapeutic, resulting in the ability to neutralize the protein therapeutic. For example, human anti-mouse (HAMA) or human anti-human (HAHA) responses directed against an individual type of antibody therapeutic are neutralizing and can be generated against humanized and fully human antibodies. For example, in 30% of patients with hemophilia A, a neutralizing ADA develops on administered recombinant FVIII, thereby abrogating its hemostatic efficacy, and in 89-100% of patients with Pompe disease receiving rhGAA, anti -rhGAA neutralizing antibodies destroy therapeutic efficacy. Binding antibodies indirectly affect the efficacy of a protein therapeutic by altering the pharmacokinetic properties of the protein therapeutic and reducing systemic exposure, for example, by promoting rapid clearance of the protein. For example, long-term use of adalimumab causes the development of ADA in -28% of patients, resulting in lower adalimumab concentrations and poorer clinical outcomes (e.g., Baker et al. (2010) Self/ Nonself 1(4):314-322; Dingman et al. (2019) J. Pharm. Sci. 108(5):1637-1654).

ADA levels can be assessed and monitored before, during, and after treatment. A variety of assays are available. These assays include bridging immunoassays, which involve labeling a drug and detecting an ADA forming a bridge between two labeled drug molecules. Bridging assays can be used for all antibody classes and with any type of sample. Ligand-binding assays (LBAs) used to detect binding to a target include surface plasmon resonance (SPR), electrochemiluminescence, and biolayer interferometry, and can also be used to detect ADAs. Protein specific assays can be used, such as the Bethesda assay, which has been used to determine the concentration of neutralizing anti-FVIII antibodies. Anti-PEG antibodies can also be measured in assays in which biotin-PEG is conjugated to magnetic beads, and the amount of anti-PEG antibodies bound to the beads is measured using a sensor that detects changes in the size of the complex. Drug-resistance assays overcome limitations induced by the presence of drugs in a sample and improve quantification of ADA. These include, for example, pH shift antigen binding assays, acid dissociation assays, temperature shift assays and electrochemiluminescence assays. An enzyme-linked immunosorbent assay (ELISA) can be used to detect ADA, wherein a protein therapeutic is applied to a plate and incubated with the sample to detect bound ADA. ELISA for the detection of ADA may be limited due to the lack of standards for ADA. Other methods include immuno-PCR, an extension of the bridging assay, in which complexes are labeled with biotin, which is detected using an anti-biotin antibody conjugated to DNA. DNA is then amplified using PCR and quantified to assess ADA levels. Immuno-LC/MS can be used to detect ADA in plasma samples, which must be stimulated by tagging the drug with biotin, or by saturating the ADA binding by mixing an excess of drug into the sample ( See, eg, Dingman et al. (2019) J. Pharm . Sci . 108(5):1637-1654).

Prediction and removal of immunogenic epitopes from protein therapeutics ( i.e. , deimmunization) can increase the efficacy and safety of therapeutics and avoid side effects that can lead to drug failure in clinical trials. For example, depletion of T-cell epitopes from protein therapeutics by deimmunization has been successful in clinical trials of protein therapeutics, particularly antibodies. These results indicate the importance of T-cell epitopes in the generation of the ADA response, and indicate that deimmunization provides safer, less immunogenic therapeutics (e.g., Baker et al . (2010) Self/ Nonself 1(4): 314-322). These methods can be used to detect or identify or predict the immunogenicity of constructs provided herein, and can be used to identify amino acid mutations to eliminate or reduce immunogenicity or immune response in a subject. Constructs that have been modified to reduce or eliminate immunogenicity are provided. Deimmunization of protein therapeutics involves identification of highly immunogenic B-cell and/or T-cell epitopes and deletion of the identified epitopes by mutagenic substitution of key amino acid residues. As discussed below, preclinical prediction and evaluation of immunogenic regions within protein therapeutics sequences can be achieved using in silico tools focused on epitope mapping in animal models, in vitro methods such as epitope mapping, MHC/HLA affinity assays and T- cell proliferation assays, and the use of in vivo assays. To increase the efficiency of protein therapeutics deimmunization, computational epitope prediction tools are used. in silico Tools include databases and algorithms for rapidly predicting immunogenic sequences in peptide libraries. Results can then be ascertained, and the specific immunogenic effect of the epitope on B-cells or T-cells can be further assessed using in vitro assays. The effect on the immune response to protein therapeutics can be evaluated using in vivo assays in animal models such as transgenic mice and non-human primates.

Once an immunogenic epitope is identified, the amino acid sequence of a therapeutic agent can be modified to remove the epitope. Removal methods include random or site-directed mutagenesis to remove immunogenic sequences ( ie , to deimmunize epitopes). After mutagenesis, immunogenic sequences are re-evaluated to confirm that they are no longer immunogenic. Insilico simplifies this process There are tools; For example, programs are available that sequentially replace each amino acid in an immunogenic sequence with one of the other 19 naturally occurring amino acids (particularly alanine) and then re-evaluate the immunogenicity of the sequence. In this way, non-immunogenic sequences are efficiently narrowed down to the most promising candidates prior to peptide synthesis and in vitro and/or in vivo re-evaluation of immunogenicity. However, predictive and mutagenic deletion of immunogenic epitopes is not necessarily sufficient for protein deimmunization, as the protein will retain its folded, stable and active conformation to retain its therapeutic efficacy. Therefore, epitope-deleting mutants that are compatible with the structure and function of the protein must be selected.

The methods and approaches discussed below are used to predict, identify and remove epitopes from constructs provided herein.

a. B-cells and T-cells epitope

Interactions between antigens and antibodies are important for the induction of the humoral immune response against invading pathogens. Specific antibodies recognize specific antigens present in distinct regions known as antigenic determinants, ie B-cell epitopes. B-cell epitopes are solvent-exposed and surface-accessible, and contain clusters of amino acids that are recognized and bound by secreted antibodies, the B-cell receptor (BCR), which induces a cellular or humoral immune response. Contains membrane-bound immunoglobulin.

Identification of B-cell epitopes is part of the development of antibodies and other protein-based therapeutics. B-cell epitopes are classified based on their spatial structure into contiguous (also known as linear) epitopes containing sequential residues and discontinuous (also known as conformational) epitopes that are non-linear and conformational. Discontinuous B-cell epitopes contain groups of solvent-exposed amino acid residues that are not completely sequential, but come together in close proximity when the protein/antigen is folded into its three-dimensional form. Approximately 90% of B-cell epitopes are morphological. Linear B-cell epitopes can be recognized by antibodies after antigenic denaturation, but conformational epitopes are no longer recognized when the antigen is denatured. The minimum amino acid sequence, or contact residue width, required for proper folding of a discontinuous B-cell epitope falls in the range of approximately 20 to 400 residues in native proteins. The majority of identified linear B-cell epitopes are considered to be components of morphological B-cell epitopes, and more than 70% of discontinuous B-cell epitopes contain 1-5 linear segments, each 1-6 segments in length. amino acids (see, e.g., Potocnakova et al. (2016) Journal of Immunology Research , Article ID 6760830; Sanchez-Trincado et al. (2017) Journal of Immunology Research , Article ID 2680160).

T-cell epitopes are linear and bind to major histocompatibility complex (MHC) molecules through interactions of amino acid side chains with binding pockets in the MHC epitope-binding groove. The presence or absence of a specific side chain determines whether an epitope binds to MHC and, if so, how tightly it adheres (eg, De Groot, AS and Moise, L. (2007) Curr . Opin. Drug Discov . Devel . 10(3):332-340). T-cells have a T-cell receptor (TCR) that recognizes antigens displayed on the surface of antigen presenting cells (APCs) and bound to MHC molecules. T-cell epitopes are presented by MHC class I (MHC I) and II (MHC II) molecules recognized by CD8 ⁺ and CD4 ⁺ T-cells, respectively; Thus, CD8 ⁺ and CD4 ⁺ T-cell epitopes exist. CD8 ⁺ T-cells recognize CD8 ⁺ T-cell epitopes and then make cytotoxic T lymphocytes (CTLs), whereas primed CD4 ⁺ T cells make helper T (Th) cells that boost the immune response or suppress immunity Generates regulatory T (Treg) cells (see, eg, Sanchez-Trincado et al. (2017) Journal of Immunology Research , Article ID 2680160).

b. in silico epitope Prediction method

²로 고리로 조직됨을 보여주고, 에피토프 서열이 티로신, 트립토판, 노출된 측쇄를 갖는 전하를 띈 극성 아미노산이 풍부하고 또한 특이적 아미노산 쌍이 풍부함을 보여준다. 그러나, 에피토프 및 비-에피토프 잔기 사이의 차이는 유의미하지 않으며, 아미노산 조성물 단독은 에피토프 및 비-에피토프의 차별화에 불충분함이 입증된 바 있다. 에피토프 맵핑 기술 및 생물정보학의 조합이 면역정보학의 발달로 이어져 왔는데, 이것은 항체, B-세포, T-세포 및 알레르기 유발 항원의 구조의 식별, MHC 결합의 예측, 에피토프의 모형화 및 면역 네트워크의 분석을 위해 면역학에서 전산 방법의 사용을 수반한다 (예를 들어, Potocnakova 등 (2016) Journal of Immunology Research, Article ID 6760830 참고).Experimental research and in silico The analysis showed that the majority of epitopes were 15–25 residues in length and 600–1000 in area.

² shows that it is organized in a ring, and the epitope sequence is rich in tyrosine, tryptophan, charged polar amino acids with exposed side chains, and also rich in specific amino acid pairs. However, it has been demonstrated that the difference between epitope and non-epitope residues is not significant, and amino acid composition alone is insufficient to differentiate between epitope and non-epitope. The combination of epitope mapping techniques and bioinformatics has led to the development of immunoinformatics, which identifies the structure of antibodies, B-cells, T-cells and allergens, predicts MHC binding, models epitopes and analyzes immune networks. (2016) Journal of Immunology Research , Article ID 6760830).

i. B-cell epitope in silico prediction

B-cell epitope prediction identifies immunogenic epitopes so that they can be displaced or de-immunized, for example for therapeutic protein production. Databases of known B-cell epitopes have been developed and multifaceted databases such as the Immune Epitope Database (IEDB) and IEDB-3D (available at iedb.org) and AntiJen (ddg-pharmfac.net/antijen/AntiJen/ available at antijenhomepage.htm); B-cell oriented databases such as BciPep (available at imtech.res.in/raghava/bcipep/info.html), Epitome (available at rostlab.org/services/epitome/) and structural databases of allergenic proteins ( SDAP; available at fermi.utmb.edu/); and single pathogenic organism-oriented databases such as the HIV Molecular Immunology Database (available at hiv.lanl.gov/content/immunology/index.html), FLAVIdB (available at cvc.dfci.harvard.edu/flavi/) and influenza sequences. and epitope database (ISED; available at influenza.cdc.go.kr). Other B-cell epitope databases include the Morphological Epitope Database (CED; available at immunenet.cn/ced/), the Protein Data Bank (PDB; available at rcsb.org) and the Structural Epitope Database (SEDB; sedb.bicpu.edu). available at .in) (see, eg, Potocnakova et al. (2016) Journal of Immunology Research , Article ID 6760830).

Several algorithms are available for predicting B-cell epitopes in sequence or structure. Algorithms have been developed that have relied on the identification of linear epitopes via trend scales, but by developing methods based on machine learning, such as hidden Markov models (HMMs), recurrent neural networks (RNNs) and support vaccine machines (SVMs), Improved. In silico B-cell epitope prediction tools include predicting continuous/linear B-cell epitopes, and predicting discontinuous/morphological B-cell epitopes. Prediction of discrete B-cell epitopes requires information on amino acid statistics, spatial information and surface exposure. Web-available tools for continuous/linear B-cell epitope prediction include, for example, ABCPred (available at crdd.osdd.net/raghava/abcpred/), APCPred (available at omictools.com/apcpred-tool), BCPRED (BCPred and FBCPred, available at ailab.ist.psu.edu/bcpred/), BepiPred (available at cbs.dtu.dk/services/BepiPred/), LBtope (crdd.osdd.net/raghava/lbtope/ (available at crdd.osdd.net/raghava/bcepred/), BcePred (available at crdd.osdd.net/raghava/bcepred/), EPMLR (available at bioinfo.tsinghua.edu.cn/epitope/EPMLR/), BEST (available at support vaccine machine tool B -Cellular epitope prediction; available at biomine.cs.vcu.edu/datasets/BEST/), COBEpro (available at scratch.proteomics.ics.uci.edu/), PEOPLE (available at iedb.org/) and Includes SVMTrip (available at sysbio.unl.edu/SVMTriP/). Web-enabled tools for discrete/conformational B-cell epitope prediction include, for example, CEP (available at bioinfo.ernet.in/cep.htm), DiscoTope (cbs.dtu.dk/services/DiscoTope-2.0/ ), BEpro (formerly known as PEPITO; available at pepito.proteomics.ics.uci.edu/), ElliPro (available at tools.immuneepitope.org/ellipro/), SEPPA (improvements of the Protein Antigen Server) spatial epitope predictions; available at badd.tongji.edu.cn/seppa/), EPITOPIA (available at epitopia.tau.ac.il/), CBTOPE (available at crdd.osdd.net/raghava/cbtope/) ), EPCES (available at sysbio.unl.edu/EPCES/), EPSVR (Antigenic Epitope Prediction Using Support Vector Regression Server; available at sysbio.unl.edu/EPSVR/), EPMeta (available at sysbio.unl.edu/ Available at EPMeta/), PEASE (Predicting Epitopes Using Antibody Sequences; available at ofranlab.org/PEASE), EpiPred (available at opig.stats.ox.ac.uk/webapps/sabdab-sabpred/EpiPred.php) available), 3DEX (3D-Epitope-Explorer; not available online), PEPOP (available at pepop.sys2diag.cnrs.fr/), PEPOP 2.0 (available at sys2diag.cnrs.fr/index.php?page=pepop (available at curie.utmb.edu/episearch.html) and EpiSearch (available at curie.utmb.edu/episearch.html) (e.g., Potocnakova et al. (2016) Journal of Immunology Research , Article ID 6760830; Sanchez-Trincado et al. (2017) Journal of Immunology Research , Article ID 2680160; Sun et al. (2013) Comput . Math Method M. , Article ID 943636).

Sequence-based and binding site prediction methods can also be used to predict B-cell epitopes. Sequence-based prediction tools rely on the primary sequence of an antigen and utilize a tendency measure to measure the probability of each residue being part of an epitope. Sequence-based prediction tools include BEST, which predicts morphological B-cell epitopes. Binding site prediction tools that aim to identify binding sites for conformational B-cell epitopes on antibodies include, for example, ProMate, ConSurf, PINUP, and PIER (see, e.g., Sun et al. (2013) Comput . Math See Method M. , Article ID 943636).

Conformational B-cell epitopes in proteins or antigens with known 3D-structures can be identified using mimotope-based epitope prediction methods. Mimotopes are peptides selected from a library of peptides randomly selected for their ability to bind antibodies elevated against native antigens. Mimotope-based methods require input of antibody affinity-selective peptides ( ie , mimotopes) and the 3D-structure of the selected antigen. Epitope prediction methods based on mimotopes derived from phage display experiments are available, using statistical features of mimotopes to map mimotopes to overlapping localized patches on the antigenic surface, or mapping mimotopes to antigenic sequences via alignment. , which can indicate B-cell epitope locations. To identify affinity-selective peptides, ie mimotopes, random peptides are displayed on the surface of filamentous phage, and peptides that bind monoclonal antibodies with a certain degree of affinity are selected, eluted and amplified. This selection process is repeated a total of 3 to 5 times to narrow down the peptides to those with the highest affinity. Mimotopes and epitopes can elicit an immune response by combining the same paratope of a monoclonal antibody and thus have similar functionality. The selected mimotopes share high sequence similarity, indicating the presence of specific key binding motifs and physiochemical preferences during interaction with the antibody. Thus, mapping the mimotope back to the source antigen may help to more accurately locate the genuine epitope. Mimotopes have similar physicochemical properties and spatial organization to native antigens, but show little sequence similarity. Databases providing information on mimotopes include, for example, ASPD (available at mgs.bionet.nsc.ru/mgs/gnw/aspd), RELIC peptides (available on request), PepBank (pepbank.mgh.harvard edu) and MimoDB (available at immunet.cn/mimodb). in silico Mimotope-based prediction tools are essential for mapping mimotopes back to the surface of source antigens in order to locate the best aligned sequences and predict possible epitope regions. In silico based on mimotope analysis B-cell epitope prediction tools include, for example, MIMOX (available at immunet.cn/mimox/), MimoPro (available at informatics.nenu.edu.cn/MimoPro), Pep-3D-Search (available at kyc.nenu .edu.cn/Pep3DSearch), MIMOP/MimCons (available on request), MIMOP/MimAlign (available on request), LocaPep (available at atenea.monstes.upm.es/#soft), EpiSearch (available on request) (available at curie.utmb.edu/episearch.html), PepMapper (available at informatics.nenu.edu.cn/PepMapper/), FINDMAP (available online), EPIMAP (available online), MEPS (available capsur Available at .it/meps, 3DEX (3D-Epitope-Explorer; not available online), Mapitope (available at pepitope.tau.ac.il) and SiteLight (not available online) (e.g. Potocnakova, etc. (2016) Journal of Immunology Research , Article ID 6760830; Sanchez-Trincado et al. (2017) Journal of Immunology Research , Article ID 2680160; Sun et al. (2013) Comput . Math Method M. , Article ID 943636).

ii. T-cell epitope in silico prediction

Linear T-cell epitopes bind to MHC through interactions with their amino acid side chains and binding pockets in the MHC epitope-binding groove, and the presence or absence of specific side chains determines whether a T-cell epitope binds to MHC and how tightly it binds. decide whether to combine For in silico predictions, databases exist that provide libraries of existing epitopes, such as, for example, IEDB, Epitome, and SEDB, which provide information on 2D T-cell epitopes. other in silico T-cell epitope databases include the CED, AntiJen, Bcipep, and HLA epitope registries. There are several webpages and programs available that can be used to analyze sequences on protein therapeutic candidates and predict immunogenic epitopes. For example, a number of MHC-binding motif-based tools are available that scan protein sequences for potential T-cell epitopes. These T-cell epitope mapping tools include, for example, EpiMatrix, IEDB, SYFPEITHI, MHC Thread, MHCPred, MHCPred 2.0, EpiJen, NetMHC, NetCTL, nHLAPred, SVMHC, and Bimas (e.g., De Groot, AS and Moise, L. (2007) Curr. Opin . Drug Discov . Devel . 10(3):332-340). For example, the MHCPred algorithm provides information on the MHC binding potential of amino acid sequences for various alleles, EpiMatrix/JanusMatrix predicts allele-specific binding of protein therapeutics to MHC class II receptors, and T-cell Binding at the receptor interface can be assessed. Other algorithms and programs for epitope prediction include, for example, ProPred, MMBPred and Protean 3D. Epitope prediction should be combined with in vitro methods and activity assessments such that any modification to remove immunogenic sequences retains the activity of the therapeutic protein (see, e.g., Dingman et al. (2019) J. Pharm . Sci. 108( 5):1637-1654).

iii. peptide- MHC Class II binding prediction

Structure-based methods for identifying T-cell epitopes rely on modeling peptide-MHC structures and evaluating interactions using, for example, molecular dynamics simulations. Structure-based methods are computationally intensive and have lower predictive performance than data-driven methods. Structure-based T-cell epitope prediction tools include EpiDOCK (available at epidock.ddg-pharmfac.net). Data-driven methods for peptide-MHC binding prediction are based on peptide sequences known to bind MHC molecules; Peptide sequences are available in epitope databases including, for example, IEDB, EPIMHC, AntiJen and others described herein and known in the art. Peptide-MHC binding predictions can be based on sequence motifs (SMs), which contain frequently occurring amino acids (fixed residues) at specific positions known to bind MHC molecules. Motif matrices (MM) assess the contribution of all residues, such as non-fixed residues, to the binding of MHC molecules, but do not account for binding affinity. A quantitative affinity matrix (QAM) predicts binding affinity as well as peptide-MHC binding. A quantitative structure-activity relationship (QSAR) additive model predicts the binding affinity of peptides to MHC as the sum of the amino acid contributions at each position plus the contributions of adjacent side chain interactions. Machine learning (ML), the most popular and powerful approach, uses algorithms trained on datasets of peptides that either bind or do not bind MHC molecules, and examples of ML-based discrimination models include artificial neural networks (ANNs). , including those based on support antivirus machines (SVMs), decision trees (DTs) and hidden Markov models (HMMs) (see e.g. Sanchez-Trincado et al. (2017) Journal of Immunology Research , Article ID 2680160) .

In silico model to predict immunogenicity of protein therapeutics Includes a peptide-MHC class II algorithm, which predicts, with reasonable accuracy, the ability of a peptide sequence to bind to MHC class II. This algorithm allows rapid selection of libraries of sequences. However, in silico and in vitro MHC class II binding assays lead to high levels of false positives in which the identified immunogenic peptides fail to stimulate T-cell responses in vitro and in vivo . This assay does not take into account other factors that influence the formation of epitopes, such as protein processing, recognition by the T-cell receptor (TCR) and T-cell resistance to peptides. To address this, in vitro T-cell assays are used. in silico The combination of assays and in vitro assays is very useful for the identification of epitopes and the design of peptide variants with epitope-depleted protein sequences with reduced ability for MHC binding (e.g., Baker et al. (2010) Self/ Nonself 1(4):314-322).

Prediction of T-cell epitopes through peptide-MHC binding models is also complicated by MHC polymorphisms; In humans, MHC molecules are known as human leukocyte antigens (HLA), and there are hundreds of allelic variants of class I and class II HLA molecules that bind different peptides and require specific models for the prediction of peptide-MHC binding ( For example, see Sanchez-Trincado et al. (2017) Journal of Immunology Research , Article ID 2680160). T-cell epitope prediction tools based on the peptide-MHC binding model include, for example, EpiDOCK, MotifScan, Rankpep, SYFPEITHI, MAPPP, PREDIVAC, PEPVAC, EPISOPT, Vaxign, MHCPred, EpiTOP, BIMAS, TEPITOPE, Propred, Propred-1, EpiJen, IEDB-MHCI, IEDB-MHCII, IL4pred, MULTIPRED2, MHC2PRED, NetMHC, NetMHCII, NetMHCpan, NetMCHIIpan, nHLAPred, SVMHC, SVRMHC, NetCTL and WAPP (e.g. Sanchez-Trincado et al. (2017) Journal of Immunology Research , Article ID 2680160).

EpiSweep is a collection of protein design algorithms that integrate computational prediction of immunogenic T-cell epitopes with sequence-based or structure-based evaluation of the effects of epitope-deleting mutations on protein stability, structure and function, resulting in low immunogenicity and high immunogenicity. Enable selection of combinations of mutations that optimize protein therapeutics for activity and stability (e.g. Choi et al. for a step-by-step guide to application of the EpiSweep suite of deimmunization algorithms). (2017) Methods Mol . Biol . 1529:375-398, cf.).

c. in vitro epitope Prediction method

In vitro methods can be used to determine the cellular mechanisms of the immune response, identify immunogenic epitopes, and evaluate the MHC affinity, T-cell proliferation, and immunogenic effects of all protein therapeutics. For example, epitope mapping identifies immunogenic epitopes by individually analyzing peptide fragments. The peptide fragments are exposed to immune cells and immunogenicity is determined by measuring cytokines and surface markers that are indicative of an inflammatory immune response. Epitope mapping across proteins is labor intensive, and in silico programs are used in conjunction with mapping to identify regions that may be immunogenic and to narrow down epitope candidates. In vitro epitope prediction methods include, for example, structural epitope mapping methods such as X-ray crystallography, nuclear magnetic resonance and electron microscopy methods and functional epitope mapping such as antigen fragmentation/antigen binding assays, competitive binding assays, variant testing/mutation triggering, display techniques such as phage display and yeast display, and mimotope assays.

i. in vitro B-cell epitope Prediction method

Experimental methods for identifying B-cell epitopes include, for example, resolving the 3D structure of antigen-antibody complexes, screening peptide libraries for antibody binding, or performing functional assays in which antigens are mutated. and the effect on antigen-antibody interactions is analyzed. Antibody-producing B-cells have a structural epitope of about 16 to 22 residues in size and containing amino acids that contact the antibody, and a functional epitope of about 3 to 5 residues in size and influencing the affinity between the protein and the antibody. recognize The most accurate method for identifying structural epitopes is by X-ray crystallography of antigen-antibody complexes, which can be used to identify the sequence that binds to the antibody and to locate the precise location of the epitope within the protein structure, both contiguous and discontinuous epitopes. All can be identified and provide information about the strength of the bond. Structural epitope mapping identifies residues that directly contact the antibody, but does not always provide information about residues that contribute to binding strength. The freely available FTProd program can be used as a computational alternative to X-ray crystallography. Nuclear magnetic resonance (NMR) identifies structural epitopes without the need to produce crystals, but its use is limited to small proteins and peptides <25 kDa in size. NMR provides data on the structure, kinetics and binding energy of antigen-antibody complexes and is performed in solution, eliminating the need for crystal formation. Two additional methods for epitope mapping with adequate resolution include saturation transition differential NMR and antibody inhibition of hydrogen-deuterium exchange in the antigen. Electron microscopy can also be used for epitope mapping, but this is a low resolution structural method typically used for larger antigens, such as whole viral particles or viral capsids. Electron microscopy cannot detect contact residues, but can be used to confirm surface accessibility of epitopes. Cryo-electron microscopy is an alternative method in which rapidly frozen antigen-antibody complexes are observed in physiological buffer, which obviates the need for staining and fixative (eg, Dingman et al. (2019) J. Pharm. Sci . 108(5):1637-1654; Potocnakova et al. (2016) Journal of Immunology Research , Article ID 6760830).

Functional epitopes can be identified by a variety of methods including antigen fragmentation, competitive binding and transformation tests. For example, functional B-cell epitope mapping methods generally involve screening of antigen-derived proteolytic fragments or peptides for antibody binding and testing of antigen-antibody reactivity of mutant proteins that have been subjected to site-directed or random mutagenesis. Include steps. Thus, functional epitope mapping tools are used to identify and characterize residues within epitopes that are important for antibody binding. The majority of functional methods detect binding of antibodies to antigen fragments, synthetic peptides, or recombinant antigens, such as mutated variants, antigens arranged by in situ cell-free translation, and/or use selectable systems, such as phage display. It is expressed by For example, antigen fragmentation and binding assays involve the immobilization of peptides on a solid support and the use of Western blot, dot blot and/or ELISA to determine whether an epitope fragment binds to an antibody, wherein binding indicates that the peptide fragment is an immunogen. indicates that it may be Competitive binding assays that provide low-resolution mapping information about the number of potentially immunogenic epitopes on a protein by assessing whether multiple antibodies can simultaneously bind to epitopes on a protein or compete with each other for binding to the same epitope (See, eg, Dingman et al. (2019) J. Pharm. Sci . 108(5):1637-1654; Potocnakova et al. (2016) Journal of Immunology Research , Article ID 6760830).

Alteration testing, ie mutagenesis, is an epitope mapping method in which individual residues (hot spots) of a functional epitope are substituted, and the effect of the alteration on binding of the antibody to immunogenic sequences is evaluated. Hot spots, most frequently comprising Tyr, Arg and Trp residues, are active key residues that contain a fraction of the complete protein-protein interface region. Mutation of individual residues allows identification of deleterious residues that can be replaced, as long as the protein retains its structure and activity. In the case of epitope mapping via mutagenesis, peptide libraries are generated by random or site-directed mutagenesis, and the combination of display techniques and mutagenesis allows the selection of large numbers of mutated proteins. Saturation mutagenesis is another method for epitope mapping, in which amino acid residues at specific positions within the epitope are replaced with all 20 naturally occurring amino acids, and loss of antibody binding is monitored. A disadvantage of this method is that loss of immunoreactivity may be due to disruption of antigenic structures, which makes interpretation of the results difficult. Since most contacts between epitopes and antibodies occur through amino acid side chains, alanine scanning mutagenesis can be used to define the contribution of each residue side chain to antibody binding. This is done one at a time by sequential alanine substitution of each non-alanine residue, cutting the side chain to the β-carbon without adding flexibility to the protein backbone. This method identifies critical residues with side chains that make the most active contributions to paratope-epitope interactions. Computational alanine scanning can also be used to rapidly determine the effect of alanine mutations on the binding free energy in protein-protein complexes by using a simple free energy function. Combinatorial mutagenesis is based on the combinatorial randomization of distinct antigenic regions and classification of mutated residues (primary sequence proximity) to maximize the chance of identifying combined effects mediated by adjacent residues; This allows the identification of residues that are not critical for binding, but contribute to the formation of an epitope, or form multiple interactions with individually weak paratopes. Shotgun mutagenesis is a high-throughput method based on large-scale mutagenesis in which each clone carries a defined amino acid mutation (e.g., an alanine substitution) and allows direct cellular testing of naturally folded proteins for mAb reactivity. accompanies Shotgun mutagenesis allows the identification of both linear and conformational epitopes at mapping rates of 20 epitopes/month or greater (see, eg, Potocnakova et al. (2016) Journal of Immunology Research , Article ID 6760830).

Other techniques for epitope mapping include display techniques and mimotope analysis, which are inexpensive, flexible and fast. Display technologies, such as phage display and yeast display, test the binding ability of various peptides displayed on a display platform to a mAb of interest via the affinity selection method of biopanning (e.g., tethering peptides to ribosome-mRNA complexes, or bound to the surface of phage, bacterial, mammalian, insect or yeast cells) (see e.g. Potocnakova et al. (2016) Journal of Immunology Research , Article ID 6760830).

ii. in vitro T-cell epitope Prediction method

In vitro methods such as MHC or HLA binding assays and T-cell assays can be used to predict T-cell epitopes and assess T-cell responses to protein therapeutic antigens. The synthesis of hundreds or thousands of overlapping peptides for in vitro assays is an in silico epitope that can accurately model the MHC:epitope interface and predict immunogenic peptide sequences. is a limiting factor that can be overcome by the use of predictive tools (see, eg, De Groot, AS and Moise, L. (2007) Curr . Opin . Drug Discov . Devel . 10(3):332-340)

MHC / HLA combination test

T-cell epitope prediction identifies the shortest peptide sequences within the antigen that stimulate CD8 ⁺ or CD4 ⁺ T cells, which are therefore immunogenic. The immunogenicity of T-cell epitopes depends on antigen processing, peptide binding to MHC molecules and recognition by cognate TCRs; MHC-peptide binding is the most selective process and primary basis for predicting T-cell epitopes. MHC binding assays can be used to detect high affinity peptides and are often applied in conjunction with epitope mapping to identify regions of a protein that are likely to be immunogenic. In vitro MHC class II binding assays include cell-based binding assays and soluble HLA binding assays. High-throughput MHC binding assays involve incubating various doses of a peptide of interest with a control peptide and soluble MHC protein to assess binding affinity, with high affinity peptides binding more strongly to MHC and epitopes being expressed by T-cells. more likely to be recognized. For example, MHC II:epitope binding can be assessed by measuring the binding ability of a peptide exogenously added to the surface of a lymphoblastoid cell line B-cell expressing an MHC class II allele, a competition-based HLA assay It can be tailored for high-throughput screening. MHC binding assays to identify potential immunogenic epitopes are commercially available, eg, from ProImmune (see, eg, Dingman et al. (2019) J. Pharm . Sci . 108(5):1637-1654; De Groot , AS and Moise, L. (2007) Curr . Opin . Drug Discov. Devel . 10(3):332-340;

iii. in vitro T-cell assay

The presence of T-cell epitopes in protein therapeutics can be detected by assessing T-cell responses in vitro in a T-cell assay. T-cells proliferate and release cytokines when stimulated by immunogenic proteins. T-cell epitopes induce the secretion of cytokines such as IL-2, IL-4, IL-5 and IFNγ by effector T-cells, and the cytokines TGFβ and TNFα and chemokines by regulatory T-cells (Tregs). , eg induces secretion of MIP1α/1β. Proliferation of T-cells in response to immunogenic peptides/epitopes can be measured by radiolabeling with thymidine or labeling with a fluorescent dye such as carboxyfluorescein succinimidyl ester (CFSE). Flow cytometry as well as ELISA or ELISpot methods can be used to measure levels of cytokines such as IL-2 and IFN-γ secreted by T-cells to determine immunogenicity. The ELISpot method is highly sensitive and can detect individual T-cells directly derived from spleen cells or peripheral blood, as well as measure the number of antigen-specific T-cells that secrete specific cytokines. For example, ELISpot assays for measuring IL-2 and IL-4 are commercially available. Flow cytometry can also be used to measure T-cell responses, whereby T-cells that respond to specific epitopes are directly labeled with tetramers (MHC class II:epitope complexes). T-cell proliferation and cytokine release assays can be combined with T-cell phenotypes to classify the type of T-cell response that occurs. The number and phenotype of T-cells responding to an antigen can be determined by methods such as flow cytometry by identifying cell surface markers such as CD25 for effector T-cells and FoxP3 for Tregs, and/or by identifying intracellular cytokine expression. can be determined using Thus, identification of T-cell epitopes can be coupled with phenotypic studies to assess whether the immune response will be inflammatory or suppressive. Peripheral blood mononuclear cell (PBMC) assays using PBMC preparations involve several types of immune cells (e.g., CD4 ⁺ and CD8 ⁺ T-cells), better mimic the immune system in vivo , and produce protein It can be used to evaluate immunogenicity and potential immune responses without testing in humans. PBMCs are stimulated in in vitro culture with the whole Therapeutic protein or with peptides derived from the Therapeutic protein. Innate immune screening using innate lymphoid cells (ILCs) or PBMC preparations deficient in innate cell systems such as CD8 ⁺ reactive T cells can be used to distinguish between innate and adaptive immune responses to immunogenic proteins. For an in vitro T-cell assay to be useful, peptides must be tested against PBMCs derived from a large donor cohort with a wide range of MHC class II allotypes. In vitro T-cell assays can provide information on the number and potency of T-cell epitopes, which can be used during preclinical development to determine the risk of immunogenicity and to induce elimination of such epitopes by targeted amino acid substitutions. (e.g. Baker et al. (2010) Self/ Nonself 1(4):314-322; Dingman et al (2019) J. Pharm. Sci . 108(5):1637-1654; De Groot, AS and Moise, L. (2007) Curr . Opin. Drug Discov. Devel. 10(3):332-340).

d. in vivo epitope Prediction method

In vivo assessment of the immunogenicity of protein therapeutics in humans uses animal models, such as mice. In general, any human or humanized protein therapeutic can be immunogenic when administered to a non-human animal. However, animal models are useful for predicting immunogenicity, relative immunogenicity between products, comparison of drug formulations or routes of administration, determination of immunogenicity of aggregates, and elucidation of immune mechanisms. Adaptive transfer and T-cell proliferation studies in animal models can be used to determine the role of T-cells and B-cells in protein immunogenicity. Immunogenicity of human protein therapeutics can be difficult to evaluate in animals because animal MHC receptors do not directly mimic human HLA receptors and HLA and MHC genes have high inter-subject variability in HLA/MHC expression and are highly polymorphic. Because. To overcome these limitations, HLA transgenic mice have been generated that mimic human subjects and are capable of showing immunotolerance to specific proteins, which mice will tolerate the protein therapeutic being evaluated and any immunogenicity that develops is self- This is because of the destruction of tolerance and not because of the traditional immune response to foreign antigens. In vivo methods for determining the immunogenicity of protein therapeutics involve exposure of HLA-transgenic mice to either the entire protein or an epitope peptide. Several transgenic mouse strains have been generated that express common HLA gene products, such as HLA-A, HLA-B and HLA-DR molecules, and by ELISA and neutralizing antibody assays, antibodies induced by exposure to protein therapeutics. as well as to measure T-cell responses. B-cell epitopes in protein therapeutics can also be identified by immunizing HLA transgenic mice with the protein (see, eg, Dingman et al. (2019) J. Pharm. Sci . 108(5):1637-1654; De Groot , AS and Moise, L. (2007) Curr . Opin. Drug Discov . Devel . 10(3):332-340).

NOD scid that is significantly immunocompromised and lacks complement and cytokine signaling as well as most immune cells Gamma (NSG) mice can be transfected to investigate the human immune system in an in vivo model. For example, cord blood-derived hematopoietic stem cells are transplanted into a CD34 ⁺ humanized NSG mouse model to develop a functional immune system with normal T-cell and inflammatory functions. Animal models are also more useful for predicting protein immunogenicity, including non-human primates, such as rhesus monkeys and chimpanzees, because their proteins show a higher degree of homology to human proteins and , because their immune mechanisms are similar to those of humans (see, eg, Dingman et al. (2019) J. Pharm. Sci . 108(5):1637-1654).

e. Predicted B-cells and T-cells epitope eliminate( deimmunization )

As described herein, prediction and elimination ( i.e. , deimmunization) of immunogenic epitopes of protein therapeutics can increase the efficacy and safety of constructs provided herein, reduce the likelihood of adverse effects, or eliminate adverse effects. It can be prevented. For example, removal of an identified epitope, such as a B-cell epitope, can prevent the formation of ADA, thereby reducing the efficacy of an administered protein therapeutic by neutralizing the therapeutic and/or inducing rapid elimination from the body.

Deimmunization of protein therapeutics involves the identification of highly immunogenic B-cell and/or T-cell epitopes and deletion of the identified epitopes by mutagenic substitution of key amino acid residues. As discussed above, the prediction and evaluation of immunogenic regions within a protein therapeutic sequence involves the use of a variety of in silico , in vitro , and in vivo methods. Upon identification of an immunogenic epitope, the amino acid sequence of the epitope is modified by random or site-directed mutagenesis to remove the immunogenic sequence and deimmunize the epitope. For example, most contacts formed between epitopes and antibodies occur through amino acid side chains, and alanine scanning mutagenesis can be used to define the contribution of each residue side chain to antibody binding. This is done by sequential alanine substitution of each non-alanine residue, one at a time, to identify the critical residue whose side chain makes the most active contribution to the paratope-epitope interaction. However, predictive and mutagenic deletion of immunogenic epitopes is not sufficient for protein deimmunization because the protein must retain its folded and stable active conformation to maintain its therapeutic efficacy and is compatible with the protein's structure and function. Possible epitope-deleting mutations should be selected.

Insilico, which increases the efficiency of this process tools exist. For example, programs are available that sequentially replace each amino acid in an immunogenic sequence with one of the other 19 naturally occurring amino acids, and then re-evaluate the immunogenicity of the new sequence. For example, OptiMatrix is a tool that iteratively replaces all 20 amino acids at any given position in a peptide sequence, and then reanalyzes the predicted immunogenicity of the modified sequence (e.g., De Groot, AS and Moise , L. ( 2007 ) Curr . _ _ _ _ _ _ -340). EpiSweep is a suite of protein design algorithms that integrate computational predictions of immunogenic T-cell epitopes with sequence-based or structure-based assessments of the effects of epitope-deleting mutations on protein stability, structure, and function, which result in low immunogenicity and Enable selection of combinations of mutations that optimize protein therapeutics with high activity and stability (e.g., for a step-by-step guide to the application of the EpiSweep suite of deimmunization algorithms, Choi et al. (2017) Methods Mol. Biol . 1529:375-398). Computational alanine scanning can also be used to rapidly determine the effect of alanine mutations on the binding free energy in protein-protein complexes by using a simple free energy function (e.g. Potocnakova et al. (2016) Journal of Immunology Research , see Article ID 6760830).

G. pan-growth factor trap construct .

1. Receptor tyrosine Kinase ( RTK )

Receptor tyrosine kinases (RTKs) are high affinity cell surface receptors for many polypeptide growth factors, cytokines and hormones. RTKs are involved in many signaling pathways and play roles in a variety of cellular processes including cell division, proliferation, differentiation, migration and metabolism. RTKs can be activated by ligands that specifically bind to their cognate receptors. This activation in turn activates an event in a signaling pathway, for example by triggering an autocrine or paracrine cell signaling pathway, which is the activation of a second messenger that results in a specific biological effect. Approximately 20 classes of RTKs have been identified, including, for example, the epidermal growth factor receptor (EGFR) family (class I, also known as the ErbB family); insulin receptor family (class II); platelet derived growth factor receptor (PDGFR) family (class III); vascular endothelial growth factor receptor (VEGFR) family (class IV); fibroblast growth factor receptor (FGFR) family (class V); hepatocyte growth factor receptor (HGFR) family (class VIII); and the Eph receptor family (Ephs, post erythropoietin-producing human hepatocyte receptor; class IX).

RTKs are involved in regulatory pathways involved in angiogenesis, including physiological and tumor angiogenesis, and have been implicated in the regulation of cell proliferation, migration and survival. RTKs have been implicated in several diseases, including cancer and autoimmune diseases such as breast and colorectal cancers, gastric carcinomas, gliomas and mesoderm-derived tumors. Dysregulation of RTKs is associated with several cancers. For example, breast cancer is associated with amplified expression of p185-HER2. RTKs have also been implicated in eye diseases including diabetic retinopathy and macular degeneration. It has also been shown that members of the epidermal growth factor receptor (EGFR) family, as well as EGF-like growth factors (ligands), are overexpressed in synovial fibroblasts and macrophages from patients with rheumatoid arthritis (RA).

a. human epidermal growth factor receptor (HER) family

Among the RTKs associated with disease are the class I human EGFR (HER; also known as ErbB) family of receptors, which include HER1/EGFR (ErbB1), HER2 (ErbB2/Neu), HER3 (ErbB3) and HER4 (ErbB4). HER1, HER3 and HER4 are epidermal growth factor (EGF), transforming growth factor (TGF)-α, heparin-binding (HB)-EGF, amphiregulin, β-cellulin (BTC), epiregulin, epigen, and neuregulin (NRG) 1-4. HER2 does not bind any of these ligands but acts as a signal amplifier by heterodimerization with other HER family members such as HER3 and HER4 (see, for example, Jin et al. (2009) Mol . Med . 15(1-2):11-20]). HER1, HER2 and HER4 are active as tyrosine kinases, whereas HER3 is inactive as a kinase (despite having a kinase domain) and signals through the phosphatidylinositol 3-kinase pathway.

All members of the HER family have an extracellular ligand binding domain, a single transmembrane domain and a cytoplasmic tyrosine-kinase containing domain. The extracellular region of each HER family member contains four subdomains, L1, CR1, L2 and CR2, where “L” refers to the leucine-rich repeat domain and “CR” refers to the cysteine-rich region/domain (purine-like repeat also known as a domain); The four subdomains are also referred to as domains I-IV, respectively. Domains I and III are the ligand binding domains, and domains II and IV mediate binding to each other and to other members of the receptor family. Domain II contains sequences necessary for dimerization, known as dimerization arms, and domain IV contains sequences allowing for domains II/IV tethering, except for HER2, which does not undergo tethered conformation. In the absence of ligand, EGFR, HER3 and HER4 subdomains II and IV form an intramolecular auto-inhibitory tether. Upon ligand binding, the subdomain undergoes a conformational change allowing subdomains I and III to form a high affinity ligand binding pocket. Mutagenic disruption of the tether formed by subdomains II and IV or C-terminal deletion of subdomain IV has been shown to increase ligand binding affinity up to 15-fold (see, for example, Jin et al. (2009) Mol . Med . 15(1-2):11-20]).

HER family members are expressed in a variety of tissues of epithelial, mesenchymal and neural origin. Under normal physiological conditions, the activation of HER is controlled by the spatial and temporal expression of its ligand, a member of the EGF family of growth factors. Ligand binding induces the formation of multiple combinations of receptor homodimers and heterodimers, resulting in activation of native kinase domains, autophosphorylation of specific tyrosine residues in the cytoplasmic tail, recruitment and phosphorylation of several intracellular proteins, and multiple downstream signaling. induce binding to the cascade. Activated signaling pathways include the Ras-Raf-mitogen activated protein kinase mitotic pathway, the phosphatidylinositol 3-kinase-AKT cell survival pathway, and the stress activated protein kinase C and Jak/Stat pathways. Derived signaling pathways result in a variety of cellular responses including, for example, cell migration, invasion, proliferation, survival and differentiation (see, e.g., Sarup et al. (2008) Mol . Cancer Ther . 7(10):3223-3236).

b. human epidermal growth factor receptor (HER) family and its ligand Associated disease

Dysregulation of HER family members and their ligands due to overexpression or mutation has been shown to play an important role in cancer and other diseases. For example, HER1 and HER2 have been implicated in the pathogenesis and pathology of many human cancers, and alterations in these receptors are associated with more aggressive disease and poor clinical outcomes. TGF-α overexpression is associated with prostate, pancreatic, lung, ovarian and colon cancer, while NRG1 overexpression is associated with breast adenocarcinoma. HER1 overexpression is associated with glioma, head and neck, breast, bladder, prostate, kidney, and non-small cell lung cancer, and mutations in HER1 are associated with glioma, as well as lung, breast and ovarian cancer. HER2 overexpression is associated with breast, lung, pancreatic, colon, esophageal, endometrial and cervical cancer; HER3 is associated with breast, colon, gastric, prostate, and oral squamous cell carcinoma; HER4 is associated with breast and prostate cancer as well as childhood medulloblastoma (see, eg, Yarden et al. (2001) Nat. Rev. Mol . Cell Biol . 2:127-137])

The EGF family of ligands and receptors has been shown to play an important role in the development of inflammatory arthritis. For example, expression of HER2 and the presence of the EGFR ligands EGF, amphiregulin and TGF-α have been detected in RA synovial membranes. Adenoviral delivery of the human EGFR family inhibitor herstatin, an alternative splice variant of HER2, has been shown to abolish all clinical signs of collagen-induced arthritis (CIA) in mice. Herstatin interferes with dimerization and acts as a natural inhibitor of native HER1, HER2 and HER3. Patients with long-standing RA who had been previously treated with rituximab and adalimumab had a significant reduction in joint pain after treatment with the anti-EGFR/HER1 antibody cetuximab for head and neck cancer. These results indicate that HER-targeted therapy may be therapeutically useful in the treatment of autoimmune and inflammatory conditions such as rheumatoid arthritis (RA) (see, e.g., Gompels et al. (2011) Arthritis Research & Therapy 13:R161).

Macrophages are a source of TNF in chronic inflammatory RA joint tissue. Phenotypic analysis of macrophages taken from synovial tissues of RA patients revealed an abundance of HBEGF ⁺ (heparin-binding EGF-like growth factor ⁺ ) inflammatory macrophages, which express the pro-inflammatory gene NR43A (nuclear receptor subfamily 4 group A member 3 ), PLAUR (plasminogen activator, urokinase receptor), and CXCL2 , and the growth factors HB-EGF and epiregulin (EGFR family ligands). HBEGF ⁺ inflammatory macrophages also produced the pro-inflammatory cytokine IL-1 and promoted synovial fibroblast invasion in an epidermal growth factor receptor-dependent manner. Most drug treatments and EGFR inhibitors used to treat RA-targeting HBEGF ⁺ macrophages in ex vivo synovial tissue assays have been shown to effectively block macrophage-induced fibroblast responses in RA tissues in ex vivo assays, suggesting that they are not effective against RA. may provide a non-immunosuppressive treatment approach for (2019) Sci . Transl . Med . 11(491)]). This approach is advantageous over the use of traditional anti-TNF therapies, which are immunosuppressive and are often associated with the development of serious infections such as tuberculosis.

HER family signaling has also been implicated in coronary atherosclerosis, which involves migration of vascular smooth muscle cells in the intima of the arteries. Activation of the thrombin receptor is required for smooth muscle cell migration and proliferation, and activation of this G-protein coupled receptor is dependent on transactivation by HER1/EGFR in response to HB-EGF. EGFR expression is also associated with psoriasis; Expression of EGFR in normal skin is restricted to the basal layer, whereas in patients with psoriasis, EGFR and its ligand amphiregulin are highly expressed in the entire epidermal layer (see, e.g., Yarden et al. (2001) Nat. Rev. Mol . Cell Biol . 2:127-137])

Other HER-mediated diseases and conditions include neurodegenerative diseases and conditions such as multiple sclerosis, Parkinson's disease, schizophrenia and Alzheimer's disease. For example, several diseases and conditions are caused or exacerbated by exposure to one or more neuregulin (NRG) ligands, such as NRG1, including types I, II, and III, NRG2, NRG3, and/or NRG4. related to becoming Examples of NRG associated diseases include neurological or neuromuscular diseases including schizophrenia and Alzheimer's disease (eg, US Publication No. 2010/0055093).

Because of its role in cancer and other proliferative diseases, rheumatoid arthritis, neurodegenerative diseases and autoimmune diseases, HER is a target for therapeutic intervention. Anti-HER therapeutics include antibodies targeting the extracellular domain (or ectodomain) referred to herein as ECD and small molecule tyrosine kinase inhibitors. Approved therapeutics for the treatment of cancers caused by the HER protein family include monoclonal antibodies such as trastuzumab (directed HER2), pertuzumab (directed HER2), panitumumab (directed HER1/EGFR) and cetuximab (directed HER2). directed against HER1/EGFR), and small molecule tyrosine kinase inhibitors such as the HER1 kinase inhibitors gefitinib and erlotinib, and the dual HER2 kinase and HER1 kinase inhibitor lapatinib. For example, Trastuzumab is used in the treatment of HER2 overexpressing node-positive or node-negative breast cancer; Cetuximab is used for the treatment of metastatic colorectal cancer and head and neck cancer; Panitumumab is used to treat metastatic colorectal cancer; Lapatinib is used as first-line therapy for triple positive breast cancer and adjuvant therapy for patients who progressed to Trastuzumab; Erlotinib is used to treat non-small cell lung cancer and pancreatic cancer.

Anti-HER therapeutics show limited efficacy and limited duration of response. Trastuzumab (eg marketed as ^Herceptin® ) is a humanized version of a murine monoclonal antibody and targets the extracellular domain of HER2. However, the effect of trastuzumab requires high expression (at least 3-fold to 5-fold overexpression) of HER2, which results in less than 25% of breast cancer patients being treated. Of this population, a large proportion does not respond to treatment. Additionally, small molecule tyrosine kinase inhibitors often lack specificity. Except for patients who highly express HER2 and are treated with trastuzumab in combination with chemotherapy, the observed efficacy with single targeting anti-HER antibodies or small molecule tyrosine kinase inhibitors ranges from 10-15%. Treatments, particularly those directed against only one HER family member, also suffer from endogenous or acquired resistance associated with ligand activation and co-expression of other RTKs, particularly other HER family members. For example, drug resistance is often associated with upregulation of, or compensation for, other HER family members, such as HER3 and HER4, or increased expression of HER1 or HER3 ligands by tumor cells. Homodimerization and heterodimerization between members of the HER family of receptors also have implications for therapy against a single HER family receptor. Because of the limited effectiveness of available therapies, alternative anti-HER therapies are needed. Alternative and more effective therapies for targeting the HER family of RTKs and their ligands are provided herein.

2. Pan-growth factor inhibition

As described herein, resistance to single targeted anti-HER therapies, such as trastuzumab, cetuximab, gefitinib, and erlotinib, often results from co-expression and/or upregulation of other HER family members and/or It is associated with overexpression of its ligand. One strategy to reduce or overcome this resistance and improve the efficacy of HER-targeted therapies is to simultaneously inhibit multiple ligand-induced HER family members. This can be achieved, for example, by chimeric HER ligand binding molecules that act like receptor attractors and sequester multiple HER family ligands to prevent ligand dependent receptor activation and downregulate aberrant HER family activity.

a. RB242 Ligand Trap

The antagonist, designated RB242, a chimeric bispecific ligand trap that is an Fc-mediated heterodimer of EGFR (HER1) and HER3 ligand binding domains, targets all four members of the EGFR/HER family. The EGFR and HER3 ligand binding domains are dimerized by fusion of each ligand binding domain with the Fc domain of human IgG1. In RB200, the extracellular domains (ECD ) is fused to the N-terminus of the Fc fragment of human IgG1 (corresponding to residues P100-K330 of SEQ ID NO: 9), respectively, and the Gly-Arg-Met-Asp (GRMD) linker is the N-terminus of the Fc fragment. - added at the end The HER3/Fc component of RB200 contains a 6xHis tag at the COOH end for purification.

RB200 is effective for EGFR and HER3 ligands (including EGF, TGF-α, HB-EGF, amphiregulin, beta-cellulin, epiregulin, and epigen, and NRG1-α, NRG1-β1, and NRG1-β3, respectively). binds with high affinity, inhibits ligand-induced tyrosine phosphorylation of HER family members (HER1, HER2 and HER3), inhibits the proliferation of a wide range of tumor cells in vitro, and induces tumor xenografts in a nude mouse model (epidermoid carcinoma). and non-small cell lung cancer). RB200 also showed synergy with tyrosine kinase inhibitors for EGFR/HER1 and HER2 kinases such as AG-825, erlotinib, gefitinib, or lapatinib in inhibiting tumor cell proliferation in vitro. Inhibition of ligand-stimulated phosphorylation of HER1, HER2 and HER3 was more effective with RB200 compared to monoclonal antibodies targeting HER1 (C225) or HER2 (Trastuzumab and 2C4) (see, e.g., Sarup et al. (2008) Mol . Cancer Ther . 7(10):3223-3236; Gompels, etc. (2011) Arthritis Research & Therapy 13:R161).

To express the RB200 and RB242 heterodimeric chimeric fusion proteins, vectors encoding the HER1/Fc and HER3/Fc constructs were co-transfected into HEK293T cells at a ratio of 1:3 (HER1/Fc:HER3/Fc) . This results in the expression of HER1/Fc and HER3/Fc homodimers in addition to the HER1/Fc:HER3/Fc heterodimer of interest. The expressed protein was purified by a combination of Protein-A, Ni-Sepharose and EGFR-Affibody column chromatography methods. Analytical reverse-phase high performance liquid chromatography (HPLC) revealed that the RB242 heterodimer contained two homodimers and approximately 10% bound contamination (see, e.g., Sarup et al. (2008) Mol. Cancer Ther . 7(10):3223-3236). Therefore, improved methods for improving the yield and purity of heterodimers are needed.

b. RB200 and RB242 for the treatment of autoimmune diseases

As discussed elsewhere herein, a significant proportion of RA patients do not respond or stop responding to treatment with anti-TNF therapies, such as anti-TNF antibodies, associated with an increased risk of serious infections, including tuberculosis. Therefore, an alternative treatment is needed. Increased expression of EGF ligand and receptor (HER) has been documented in the synovial membrane and synovial fluid of patients with rheumatoid arthritis (RA), suggesting that therapies targeting EGFR will be used to treat RA and other autoimmune and inflammatory diseases and disorders. indicates that it can

The bispecific EGFR ligand trap RB200 (and its derivative RB242) shows a dose-dependent reduction in disease severity in collagen-induced arthritis (CIA). Mice with CIA were intraperitoneally administered with RB200 (or RB242) at a dose of 0.1 mg/kg, 1 mg/kg or 10 mg/kg on the day of disease onset (day 1) and on the 4th and 7th days of onset. Treatment with 1 mg/kg or 10 mg/kg RB200 inhibited the increase in clinical scores and paw edema in a dose-dependent manner. EGF was shown to promote angiogenesis, and RB200-treated mice showed a decrease in CD31-immunopositive staining, reflecting a decrease in synovial blood vessels and inhibition of synovial angiogenesis. Joint sections from mice treated with the PBS control showed invasion and erosion of bone by the synovium associated with significant CD31 expression as well as high numbers of infiltrating cells in the inflamed synovium. The joints of mice treated with 1 mg/kg or 10 mg/kg RB200 were protected with normal appearance, well-preserved joint structures and few CD31 positive blood vessels. These results indicate that inhibition of the EGFR mediated response may be of therapeutic use in the treatment of RA (see, e.g., Gompels et al. (2011) Arthritis Research & Therapy 13:R161).

Combination of TNF inhibition with an inhibitor of EGFR mediated signaling may increase the therapeutic efficacy of anti-TNF therapy and may be useful in the treatment of RA. Co-administration of a low dose of RB200 (0.5 mg/kg) with a suboptimal dose of etanercept (1 mg/kg) suppressed the increase in clinical scores and foot edema, and the optimal dose of etanercept (5 mg/kg) alone Complete elimination of CIA with effects similar to those observed upon administration. In contrast, low-dose RB200 alone or low-dose etanercept alone had no effect. Fluorescently labeled monoclonal antibodies against E-selectin can be used to localize endothelial activation in inflamed tissues in vivo and are a sensitive, specific and quantifiable molecular imaging technique for the evaluation of CIA. The combination of low-dose RB200 and low-dose etanercept reduced the amount of E-selectin detected in the paws to levels seen in healthy animals, whereas E-selectin was significantly reduced by CIA with low-dose RB200 alone or low-dose etanercept alone. detected in the paws of mice. There was a dose-dependent effect of RB200 alone and etanercept alone on joint structure, with progressively fewer severely destroyed joints and more joints with mild or moderate destruction, but no treatment with low-dose RB200 alone or low-dose etanercept alone. The most significant effect was observed with the combination treatment, in which 64% of the joints appeared normal compared to 0% of the mice. Combination treatment was also more effective than high-dose etanercept alone, indicating the efficacy of combining pan-EGFR and TNF-targeted therapies in promoting joint protection (see, e.g., Gompels et al. (2011) Arthritis Research & Therapy 13:R161).

c. RB242 Ligand Trap

The ligand trap, designated RB242, derived from RB200, was located in the HER3 ECD subdomain with respect to the sequence of the mature HER3 protein (SEQ ID NO: 45), the sequence of the mature EGFR protein (SEQ ID NO: 41), and the EGFR ECD subdomain with respect to Y246A, respectively. Affinity optimized Fc-mediated triple mutant EGFR:HER3 heterodimer comprising mutations T15S and G564S in domains I and IV. Compared to the parent molecule, RB200, RB242 showed an average 22-fold improvement in affinity for various ligands, including EGF, TGF-α, HB-EGF and NRG1-, and a single cultured cell and mouse model of human non-small cell lung cancer. showed improved antiproliferative activity against layered BxPC3 pancreatic cancer. RB242 also showed a 10- to 60-fold improvement in inhibition of ligand-induced HER phosphorylation compared to RB200 (see, eg, Jin et al. (2009) Mol. Med. 15(1-2):11-20]).

3. Optimized multispecific, such as bispecific, growth factor trap constructs

Provided herein are multispecific, such as bispecific, growth factor trap constructs that bind to ligands for one or more receptors and/or so long as the activity of more than one cell surface receptor is modulated. It is designed to be a pan-cell surface receptor therapeutic by specifically targeting more than one cell surface receptor such as one that interacts with the receptor. Constructs include those that target one or more HER family members as well as those that target one or more HER and additional receptors such as HER that contribute to or participate in the development of resistance to anti-HER therapies. The growth factor trap constructs provided herein are fused to one receptor, in particular a member of the HER family, such as a multimerization domain, such as the Fc of a human immunoglobulin (Ig), such as the Fc of a human IgG, EGFR/HER1, HER2 , multiple, in particular two chimeric fusion polypeptides, each containing all or part of the extracellular domain (ECD) of HER3 or HER4. The ECD or portion thereof within the chimeric fusion polypeptide may be linked directly to the Fc or indirectly via a linker such as a peptide linker. Typically, the C-terminus of an ECD polypeptide is linked to the N-terminus of a multimerization domain such as an IgG Fc.

Growth factor trap constructs herein are described, for example, in Sarup et al. (2008) Mol . Cancer Ther . 7(10):3223-3236; Gompels, etc. (2011) Arthritis Research & Therapy 13:R161; Jin et al. (2009) Mol . Med . 15(1-2):11-20; and US Patent Publication No. 2010/0055093]. The following sections describe each part of the multispecific growth factor trap construct provided herein.

a. Extracellular Domain (ECD) Polypeptides

Provided herein are multispecific, eg, bispecific, growth factor trap constructs comprising extracellular domains (ECDs) of two or more cell surface receptors (CSRs) or portion(s) thereof. In certain embodiments, the construct is a bispecific heterodimeric construct comprising two different cell surface receptors. The construct includes a first ECD polypeptide and a second ECD polypeptide, each directly or indirectly linked to the multimerization domain through a linker. In some embodiments, the first ECD polypeptide comprises the ECD of HER1/EGFR (corresponding to residues 1-621 of SEQ ID NO: 41) or a portion thereof, and the second ECD polypeptide comprises HER2 (residue 1 of SEQ ID NO: 43) -628), HER3 (corresponding to residues 1-621 of SEQ ID NO: 45) or HER4 (corresponding to residues 1-625 of SEQ ID NO: 47) or a portion thereof, in particular the ECD of HER3 or HER4 or any part thereof. In embodiments wherein the ECD polypeptide comprises less than the full-length ECD of the HER protein, it comprises at least sufficient portions of subdomains I, II and III for ligand binding and receptor dimerization. For example, the ECD can include a sufficient portion of subdomains I and III for ligand binding and/or can include a sufficient portion of the ECD to dimerize with a cell surface receptor, including a sufficient portion of subdomain II. there is. In some embodiments, the ECD contains subdomains I, II and III and at least module 1 of domain IV.

In some examples, the multispecific, such as bispecific, growth factor trap construct is a HER1/EGFR, HER2, HER3 or HER4, particularly a first ECD polypeptide containing all or part of the ECD of EGFR/HER1, and a different CSR , eg, HER2, HER3, HER4, insulin growth factor-1 receptor (IGF1-R), vascular endothelial growth factor receptor (VEGFR, eg VEGFR1), fibroblast growth factor receptor (FGFR, eg, FGFR2 or FGFR4), TNFR, platelet-derived growth factor receptor (PDGFR), hepatocyte growth factor receptor (HGFR), tyrosine kinase with immunoglobulin-like and EGF-like domain 1 (TIE, e.g., TIE-1 or TEK (TIE) -2)), a second chimeric polypeptide containing an ECD from the receptor for advanced glycation end products (RAGE), the Eph receptor, or the T-cell receptor.

In certain embodiments, the first ECD polypeptide is a full-length ECD of HER1/EGFR (corresponding to residues 1-621 of SEQ ID NO: 41) or a portion thereof (eg, in module 1 of subdomains I-III and domain IV). corresponding residues 1-501 of SEQ ID NO: 41), and the second ECD polypeptide is the full-length ECD of HER3 (corresponding to residues 1-621 of SEQ ID NO: 45) or a portion thereof (e.g., subdomain I -III and residues 1-500 of SEQ ID NO: 45 corresponding to module 1 of domain IV), wherein the ECD portion is at least a sufficient portion of subdomains I and III to bind to a ligand of the HER receptor, and a subdomain It contains a sufficient portion of ECD to dimerize with cell surface receptors, including a sufficient portion of II. The first and second ECD polypeptides form multimers, eg dimers, through interaction of their multimerization domains. The resulting multimeric construct provided herein binds additional ligands when compared to the first or second chimeric polypeptide alone or homodimer thereof and/or more than the first or second chimeric polypeptide alone or homodimer thereof. Dimerizes with many cell surface receptors. For example, a first and second ECD polypeptide form a heterodimer that binds a HER1 ligand and a HER3 ligand.

b. Modifications to the extracellular domain

In some embodiments, an ECD domain or at least one portion thereof comprises a modification that alters ligand binding, specificity or other activity or property compared to an unmodified ECD polypeptide. In such multimeric constructs, the second ECD portion may be the same ECD domain, wild-type or mutant form, or may be an ECD from any other cell surface receptor. The ECD or part thereof of each monomer is connected directly or through a linker to the multimerization domain, or directly or through a linker to a second ECD or part thereof. For example, the modification alters the ligand binding, specificity, or another activity or property of the ECD or full-length receptor containing such ECD compared to the unmodified ECD or full-length receptor, whereby the heteromultimer has an altered activity or property, such as Indicates altered ligand binding or specificity. Such modifications include any that remove, add or enhance an activity such as binding to additional ligands. An example of such a multimeric construct is a construct containing at least one HERl ECD containing a mutation in subdomain III that increases its affinity for a ligand other than EGF. This affinity increase is at least 2-fold to 10-fold, typically 100, 1000, 10 ⁴ , 10 ⁵ , 10 ⁶ fold or more.

In certain embodiments, the growth factor trap construct is a heterodimer containing a HER1 (EGFR) chimeric fusion polypeptide and a HER3 chimeric fusion polypeptide, wherein each chimeric fusion polypeptide is optionally linked to the Fc of a human IgG1 via a peptide linker. Include the ECD of the receptor. Such chimeric fusion polypeptides are referred to herein as HER1/Fc and HER3/Fc. Typically, the C-terminus of an ECD polypeptide is linked to the N-terminus of a multimerization domain such as an IgG1 Fc.

In some instances, the HERl portion is enriched for ligand binding and/or biological activity. In another example, the HER3 portion is enriched for ligand binding and/or biological activity. In another example, both the HER1 and HER3 portions are enriched for ligand binding and/or biological activity.

Exemplary modifications include, for example, S418F of HER1 (see the sequence of the mature protein set forth in SEQ ID NO: 41), which allows the HER3 ligand NRG2-β to stimulate HER1. The resulting ECD binds or interacts with at least two ligands, one for HER1, such as EGF, and a second for HER3, such as NRG2-β. Other modifications include the sequence of the mature EGFR protein (SEQ ID NO: 41) in HER3 ECD subdomain II with respect to the sequence of the mature HER3 protein (SEQ ID NO: 45), and EGFR/HER1 ECD subdomains I and IV with respect to Y246A, respectively. contains mutations T15S and G564S in , which upon binding result in an average 22-fold improvement in affinity for various ligands including EGF, TGF-α, HB-EGF and NRG1-β. Additional mutations of the HER1 ECD are the precursor HER1 shown in SEQ ID NO: 40 and corresponding to E306D/G564S, S169N/E306D/G564S and T19K/S169N/E306D/G564S in relation to the sequence of the mature HER1 polypeptide set forth in SEQ ID NO: 41. (including the signal peptide) include E330D/G588S, S193N/E330D/G588S and T43K/S193N/E330D/G588S. This mutation increases HER1 binding affinity to the ligands EGF, HB-EGF and TGF-α (see, eg, US Patent Publication No. 2010/0055093).

c. multimerization domain

In certain embodiments, the multimerization domain is an Fc domain or variant thereof that undergoes multimerization. The Fc domain can be from any immunoglobulin (Ig) molecule, including IgG, IgM or IgE. For example, the Fc domain can be from IgG1, IgG2, IgG3 or IgG4 and comprises all or part of the C _H 2 and C _H 3 domains, and optionally the hinge region. In certain instances, the Fc portion optionally comprises all or part of the hinge region, e.g., residues 99-330, 100-330, 104-330, 109-330, 111-330, 113 of SEQ ID NO:9 -330, or Fc1 of human IgG corresponding to 114-330. Also included are modified Fc domains as described in the section above that have been modified to have knob-in-holes and altered properties.

Each ECD polypeptide within the multispecific growth factor trap construct is linked directly to the Fc and indirectly through a linker, such as a chemical or polypeptide linker, to form a chimeric fusion polypeptide (ie, an ECD/Fc fusion polypeptide). The multimerization domains, such as the Fc domain of each chimeric fusion polypeptide, interact (via disulfide bonds in the case of the Fc domain) to form heteromultimers, such as heterodimers.

The linker between the ECD and the Fc portion of each chimeric fusion polypeptide can be a flexible peptide linker of various lengths and combinations, such as the hinge region of an IgG or other polypeptide composed of small amino acids such as glycine, serine, threonine, and/or alanine. may be a linker. For example, the linker can be (Gly) _n , (GGGGS) _n , (SSSSG) _n , or (AlaAlaProAla) _n where n is 1-6, GKSSGSGSESKS, GGSTSGSGKSSEGKG, GSTSGSGKSSSEGSGSTKG, GSTSGSGKPGSGEGSTKG, EGKSSGSGSESKEF, Gly -Arg-Met-Asp (GRMD), Ser-Cys-Asp-Lys-Thr (SCDKT), or Glu-Lys-Thr-Ile-Ser (EKTIS) (see SEQ ID NOs: 816-834) or elsewhere herein or any other linker known in the art to be suitable for this purpose.

d. Modifications to the Fc domain

The Fc domains in the growth factor trap constructs provided herein can improve or enhance protein expression and purity, as well as, e.g., extend half-life in vivo and/or alter immune effector function as described below, as a dominant or sole product. resulting in the formation of heterodimers.

i. Introduction of knob-in-hole

The Fc domains in the growth factor trap constructs provided herein can be engineered such that steric interactions promote stable interactions and promote the formation of heterodimers rather than homodimers from mixtures of chimeric ECD polypeptide monomers. As discussed elsewhere herein, introduction of a “knob-into-hole” (KiH; also known as “knob-into-hole”) into the C _H 3 domain of an antibody (eg, IgG) heavy chain is Optimize heterodimer production. The knob-in-hole approach involves interfacial residues asymmetrically mutating in the C _H 3 domains of the two Fc monomers in a complementary manner. Generally, “knobs” or protrusions are created by replacing amino acids with small side chains with amino acids with larger side chains, such as tyrosine or tryptophan, at the interface between the _CH 3 domains, forming a compensatory “hole”. "Or identical or knob-like sizes are created by replacing an amino acid with a larger side chain and replacing an amino acid with a smaller amino acid such as alanine or threonine. Knob and hole variants of the Fc monomer are heterodimerized by insertion of the knob into a correspondingly designed hole in the partner C _H 3 domain. The steric repulsion prevents knob-knob association and destabilizes the hole-hole homodimer.

In some embodiments, the Fc portion of a heterodimer growth factor trap construct provided herein is engineered to contain a knob-in-hole. The knob mutation can be, for example, S354C, T366Y, T366W or T394W by EU numbering, which in relation to the sequence of the human IgG1 heavy chain constant domain set forth in SEQ ID NO:9 corresponds to S237C, T249Y, T249W or T277W, respectively. The hole mutation may be, for example, Y349C, T366S, L368A, F405A, Y407T, Y407A, or Y407V by EU numbering, which in relation to the sequence of the human IgG1 heavy chain constant domain set forth in SEQ ID NO:9 is Y232C, T249S, respectively; Corresponds to L251A, F288A, Y290T, Y290A, or Y290V. The introduction of the knob-in-hole increases the yield of the heterodimer of interest and reduces the amount of homodimer impurities, e.g., when compared to RB200 and RB242, the bispecific, heterodimer growth factors provided herein Facilitates the protein purification process for trap constructs.

ii. Modifications that enhance neonatal Fc receptor (FcRn) recycling

As described elsewhere herein, fusion with an IgG Fc increases the half-life of a small protein therapeutic by taking advantage of neonatal Fc receptor (FcRn) binding and also increasing the molecular weight of the therapeutic so that it is cleared less rapidly from the body by the kidneys. . To improve pharmacokinetics and overall pharmacology, residues within the Fc region of the growth factor trap constructs provided herein can be mutated to increase the affinity for FcRn, typically greater than 30-fold, to further increase the half-life in vivo.

In some embodiments, the Fc portion of a growth factor trap construct herein is modified to enhance neonatal FcRn recycling to increase half-life in vivo. This can be effected by mutating residues at the interface of the C _H 2 and C _H 3 domains of IgG Fc that are responsible for binding to FcRn. Exemplary Fc modifications that increase binding to FcRn and can be introduced into the Fc portion of the growth factor trap constructs herein include (by EU numbering) T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M252Y/T256Q, M252F/T256D, M252Y/S254T/T256E, H433K/N4 34F/Y436H, N434F/Y436H, T250Q/ M428L, T250R/M428L, M428L/N434S, V259I/V308F, V259I/V308F/M428L, E294del/T307P/N434Y, and T256N/A378V/S383N/N434Y, and combinations thereof. Corresponding mutations by Kabat numbering and sequential numbering with respect to the sequence of the IgG1 heavy chain constant domain set forth in SEQ ID NO: 9 are presented in Table 7 (IgG1 Fc modifications that enhance FcRn binding) in the section describing Fc modifications . Other modifications known in the art that confer enhanced or increased FcRn binding are also contemplated for use herein.

Modification of the Fc portion of a growth factor trap construct provided herein to enhance FcRn binding and recycling requires a lower dose and/or less frequent dosing compared to RB200 and RB242 and improves therapeutic efficacy, thereby improving the bioavailability of therapeutics. Increases my half-life.

iii. effector function

As described herein, immune effector functions mediated by IgG Fc include complement dependent cytotoxicity (CDC), antibody dependent cell mediated cytotoxicity (ADCC; also called antibody dependent cell cytotoxicity), and antibody dependent cell mediated phagocytosis. (ADCP; also called antibody-dependent cellular phagocytosis). The Fc region of the growth factor trap constructs herein can be used as discussed below and elsewhere herein to eliminate, reduce or enhance immune effector functions including, for example, any one or more of CDC, ADCC and ADCP. may be mutated or transformed.

Because growth factors targeted by growth factor trap constructs exist as membrane proteins and free (i.e., soluble) ligands, in certain embodiments, the immune effector function of the Fc portion in the ECD/Fc fusion polypeptide, particularly ADCC, is maintained. . In an alternative embodiment, in addition to human IgG1 Fc, other Fc regions may also be included in the ECD/Fc chimeric fusion polypeptides provided herein. For example, when effector functions mediated by Fc/FcγR interactions are to be minimized, e.g., IgGs that do not properly recruit complement or effector cells and exhibit no effector functions, such as, for example, the Fc of IgG2 or IgG4. Fusion with the isotype is considered. This approach can be used where effector function is not needed or may be detrimental, for example in the context of autoimmune and inflammatory diseases and disorders.

In certain instances, an Fc portion may be modified to enhance or increase immune effector function. This can be achieved, for example, by modifications that increase binding to C1q (for CDC) and/or specific activating FcγRs (eg FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa and FcγRIIIb). An Fc region that has been modified to have increased binding to an Fc receptor may be more effective in promoting destruction of cancer cells in a patient even when linked with an ECD polypeptide. Antibodies include, for example, anti-proliferation by blocking growth pathways, intracellular signaling leading to apoptosis, enhanced downregulation and/or turnover of receptors, ADCC, ADCP, CDC and promotion of the adaptive immune response destroys tumor cells through a number of possible mechanisms. Thus, in embodiments where the growth factor trap constructs herein are used to treat cancer, the Fc portion of the construct can be modified to enhance or increase immune effector function. Table 8 (IgG1 Fc Modifications That Enhance Immune Effector Function) in Section F.4.d.i.c (Enhancement or Decrease/Elimination of Fc Immune Effector Function) include ADCC, ADCP and CDC by increasing binding to FcγR or C1q. Fc modifications that enhance immune effector function are summarized and given in relation to the sequence of the IgGl heavy chain constant domain set forth in SEQ ID NO: 9, Kabat numbering and corresponding modifications by sequential numbering. Any one or more of these modifications, alone or in various combinations, can be introduced into the IgG1 Fc portion of the growth factor trap constructs provided herein. Other modifications known in the art that confer enhanced or increased immune effector function are also contemplated for use herein. A list of the above sections describing IgG1 Fc modifications that enhance immune effector function.

In an alternative embodiment, the Fc portion of a growth factor trap construct provided herein is modified to reduce or eliminate immune effector function. This can be achieved, for example, by modifications that reduce or abolish C1q (for CDC) and/or binding to specific activating FcγRs (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa and FcγRIIIb). This could be, for example, where it is desirable to antagonize, but not kill, cells bearing the target antigen, or where undesirable or detrimental immune effector functions, such as unwanted proinflammatory cytokine release and reduction of off-target cytotoxicity, are desired. Preferable when needed. Thus, in embodiments in which the growth factor trap constructs provided herein are used for the treatment of chronic inflammatory and autoimmune diseases and disorders, such as RA, the Fc portion of the construct can be modified to reduce or eliminate immune effector function. there is.

Table 9 (IgG1 Fc modifications that enhance immune effector function) in section F.4.d.i.c (IgG1 Fc modifications that reduce or eliminate Fc immune effector function) reduce or eliminate binding to activating FcγRs and/or C1q to reduce or eliminate immune effector functions, including ADCC, ADCP and CDC, and summarized herein exemplary IgG1 Fc modifications that can be introduced into the Fc region of a growth factor trap construct. The table provides the corresponding modifications by Kabat numbering and sequential numbering with respect to the sequence of the IgG1 heavy chain constant domain set forth in SEQ ID NO:9. Any one or more of these modifications, alone or in various combinations, can be introduced into the IgG1 Fc portion of the growth factor trap constructs provided herein. Other modifications known in the art to reduce or eliminate immune effector function are also contemplated for use herein.

The Fc portion of the growth factor trap constructs provided herein can also be modified to increase binding to inhibitory FcγRs to suppress the immune response. Therapeutic antibodies with immunosuppressive Fc modifications are beneficial for the treatment of inflammatory diseases. Such mutations may be incorporated into the Fc portion of the growth factor trap constructs herein for the treatment of diseases and conditions associated with or etiology or an inflammatory component, such as, for example, RA and other inflammatory and autoimmune diseases.

Modifications that increase binding to, or confer selective binding to, inhibitory FcγRIIb and/or FcγRI, but not FcγRIIIa, can be engineered into the IgG1 Fc region in the growth factor trap constructs provided herein. Such variations include, but are not limited to, one or more of S267E, N297A, L328F, L351S, T366R, L368H, P395K, S267E/L328F, L351S/T366R/L368H/P395K, and combinations thereof by EU numbering. Table 11 (IgG1 Fc Modifications That Increase Binding to Inhibitory FcγRIIb) in Section F.4.d.i.i show the corresponding replacement by Kabat numbering and sequential numbering with respect to the sequence of the IgG1 heavy chain constant domain set forth in SEQ ID NO:9. show These modifications are summarized in the section above describing IgG1 Fc modifications that increase binding to inhibitory FcγRIIb.

4. Compositions, therapeutic uses and methods of treatment

Nucleic acid molecules encoding chimeric fusion polypeptides (ie, ECD/Fc) and growth factor trap constructs that are vectors containing the nucleic acid molecules are provided. Also provided are pharmaceutical compositions containing cells containing vectors as described herein, and growth factor trap constructs encoding any of the nucleic acid molecules, vectors or cells described herein. The growth factor trap constructs herein have been previously described, for example in Sarup et al. (2008) Mol . Cancer Ther . 7(10):3223-3236; Gompels, etc. (2011) Arthritis Research & Therapy 13:R161; Jin et al. (2009) Mol . Med . 15(1-2):11-20; and US Patent Publication No. 2010/0055093] and purified.

Bispecific, including multispecific growth factor trap constructs herein are two or more, in particular two chimeras, generated by directly or indirectly linking two or more, in particular two, of the same or different ECD polypeptides to a multimerization domain. Contains protein. In some instances where the multimerization domain is a polypeptide such as an immunoglobulin Fc, a gene fusion encoding the ECD-multimerization domain chimeric polypeptide is inserted into an appropriate expression vector. The resulting ECD-multimerizing domain chimeric proteins are transformed into recombinant expression vector(s) and host cells capable of being assembled into multimers such as dimers, particularly mammalian cells (e.g. HEK293T or CHO cells, or the present described herein or in any other suitable mammalian cell known in the art), wherein the multimerization domains interact to form a multivalent polypeptide. The resulting chimeric polypeptide and the multimers formed therefrom may be purified by any suitable method known in the art, such as, for example, affinity chromatography on a Protein A or Protein G column. Additionally or alternatively, for example, gel electrophoresis, dialysis, ion exchange chromatography, ethanol precipitation, HPLC such as reverse phase HPLC, chromatography on silica, chromatography on heparin sepharose, chromatofocusing, SDS-PAGE, and other techniques for protein purification including ammonium sulfate precipitation may be used. When two nucleic acid molecules encoding different ECD chimeric polypeptides are transformed into cells (eg, HER1/Fc and HER3/Fc), homodimers and heterodimers will form. Expression conditions can be adjusted such that heterodimer formation dominates over homodimer formation. For example, the ratio of nucleic acid molecules encoding different ECD chimeric polypeptides can be adjusted so that an excess of one nucleic acid molecule forms fewer homodimers. Additionally, as described above, introducing a knob-in-hole into an Fc monomer favors the formation of heterodimers over homodimers.

ECD chimeric polypeptides containing an Fc region may also have metal chelates or tags with other epitopes, e.g., 6xHis tag, c-myc tag, FLAG tag, maltose binding protein (MBP), glutathione-S-transferase (GST) ), or thioredoxin (TRX). The tagged domains can be used for rapid purification by metal-chelate chromatography and/or antibodies, and can be used to allow detection in Western blots, immunoprecipitation or activity depletion/blocking in bioassays.

a. pharmaceutical composition

Pharmaceutical compositions containing the multispecific, such as bispecific growth factor trap constructs, or encoding nucleic acid molecule(s) provided herein are provided. Also provided are pharmaceutical compositions comprising isolated cells comprising a nucleic acid molecule or vector provided herein. Such compositions contain a therapeutically effective amount of the growth factor trap construct. A pharmaceutical composition may be formulated in any conventional manner by mixing a selected amount of a growth factor trap construct or nucleic acid molecule with one or more physiologically acceptable carriers or excipients. A pharmaceutical composition may be used for therapeutic, prophylactic and/or diagnostic applications. The concentration of active compound in the composition will depend on rates of absorption, inactivation and excretion of the active compound, dosage schedule and dosage, as well as other factors known to those skilled in the art.

Pharmaceutical carriers or vehicles suitable for administration of the compounds provided herein include any such carrier known to those skilled in the art to be suitable for a particular mode of administration. The choice of carrier or excipient is within the skill of the administering expert and can depend on many parameters. These include, for example, the mode of administration (ie, systemic, oral, nasal, pulmonary, topical, topical or any other mode) and the disorder being treated. A pharmaceutical composition comprising a therapeutically effective amount of a multispecific, such as bispecific, growth factor trap construct or nucleic acid molecule described herein may also be presented as a lyophilized powder to be reconstituted, such as with sterile water, immediately prior to administration.

The pharmaceutical compositions provided herein may be in a variety of forms, such as solid, semi-solid, liquid, powder, aqueous or lyophilized form. The pharmaceutical compositions provided herein may be formulated for single dose (direct) administration, or for dilution or other modification. The concentration of the compound in the formulation is effective in delivering the amount upon administration, i.e., effective for the intended treatment. Typically, the composition is formulated for single dose administration. The compounds can be suspended in micronized or other suitable form, or derivatized to produce a more soluble active product. The form of the resulting mixture depends on several factors including the intended mode of administration and the solubility of the compound in the chosen carrier or vehicle. Effective concentrations are sufficient to ameliorate the target condition and can be determined empirically. To formulate a composition, a weight fraction of the compound is dissolved, suspended, dispersed or mixed in an effective concentration with a selected vehicle to alleviate or ameliorate the target condition.

Methods for producing nucleic acids encoding growth factor trap constructs provided herein include those described in Section H. Section H also describes methods of protein expression and purification, as well as vectors and cells that may be used. The compositions, formulations, dosages and methods of administration described in Section I may be suitable for the production of compositions and preparations comprising the growth factor trap constructs and encoding nucleic acid molecules described herein. Dosages and methods of administration can be determined by the administering expert and are known in the art and described elsewhere herein.

b. Therapeutic uses and methods of treatment

The bispecific, including multispecific growth factor trap constructs provided herein can be used for any purpose known to those skilled in the art for the use of such molecules. For example, growth factor trap constructs provided herein may be used for one or more of therapeutic, diagnostic, industrial and/or research purpose(s). In particular, the multispecific growth factor trap constructs provided herein can be used for the treatment of a variety of diseases and conditions involving CSRs, including RTKs, and particularly the HER family of proteins, including those described herein. HER signaling is implicated in the etiology of a variety of diseases and disorders, and any such disease or disorder thereof is contemplated for treatment with the growth factor trap constructs provided herein.

Growth factor trap constructs and encoding nucleic acid molecules provided herein, as well as pharmaceutical compositions, may be used in anti-HER therapies (e.g., trastuzumab, cetuximab, gefitinib, erlotinib, and lapatinib, and herein and/or others known in the art) are cancer and other proliferative diseases and disorders, angiogenesis-related diseases and disorders, rheumatoid arthritis and other chronic inflammatory and autoimmune diseases and disorders, as well as central nervous system (CNS) It can be used for the treatment of any condition, including, but not limited to, neurodegenerative diseases and disorders. For example, treatment using the growth factor trap constructs provided herein includes treatment of angiogenesis-related diseases and conditions, inflammatory diseases and conditions, autoimmune diseases and conditions, neurodegenerative diseases, and conditions associated with cell proliferation. However, it is not limited thereto. Such diseases and conditions include, for example, ocular disease, atherosclerosis, vascular injury, Alzheimer's disease, cancer, smooth muscle cell associated conditions, rheumatoid arthritis (RA), and various autoimmune diseases.

Dosage levels and regimens can be determined based on known dosages and regimens, extrapolated, if necessary, based on variations in the properties of the polypeptides and constructs provided herein, and/or determined empirically based on a variety of factors. there is. These factors include, for example, the body weight of the individual as well as general health, age, sex and diet, activity of the specific compound used, time of administration, rate of excretion, drug combination, severity and course of the disease, the patient's propensity for the disease and treatment. including the physician's judgment. The active ingredient is usually combined with a pharmaceutically effective carrier. The amount of active ingredient that can be combined with the carrier material to produce a single dosage form or multiple dosage form can vary depending on the host being treated and the particular mode of administration.

Dosages vary depending on the particular subject as well as the particular disorder, disease or condition being treated. Typical doses are those of known anti-HER therapies, such as trastuzumab, cetuximab, pertuzumab, and panitumumab, and antibodies including small molecule tyrosine kinase inhibitors such as gefitinib, erlotinib, and lapatinib. similar to Exemplary dosages for subjects, including humans and other animals, range from about or 0.1 to 100 mg/kg, such as 1 mg/kg to about or 30 mg/kg, such as 5 mg/kg to 25 mg/kg. Doses can be determined based on the assumption that the average human mass is about 75 kg. Dosage may be adjusted for children, infants and smaller adults.

When the patient's condition improves, a maintenance dose of the compound or composition may be administered, if necessary; The dosage, dosage form, or frequency of administration, or combinations thereof may be varied. In some cases, a subject may require intermittent treatment over an extended period of time following any recurrence of disease symptoms or based on a predetermined dosage.

Treatment of diseases and conditions using the multispecific growth factor trap constructs provided herein includes, but is not limited to, injection, subcutaneous injection, and inhalation, or intramuscular, intradermal, oral, topical, and transdermal administration, This may be achieved by any suitable route of administration using a suitable formulation as described herein.

Provided herein are methods of treating a HER-mediated or HER-associated disease or condition, comprising a multispecific growth factor trap mechanism in which HER receptors are expressed or overexpressed and which target at least one, typically two, of the HER receptors. and testing a subject with a disease to ascertain whether the selection of an agent is based on the results. In one embodiment, the disease is cancer. Examples of cancers for treatment herein include glioma, as well as pancreatic, gastric, head and neck, cervical, lung, colorectal, endometrial, prostate, esophageal, ovarian, uterine, bladder, or breast cancer. . Cancers that are treatable with the growth factor (HER ligand) trap constructs herein are generally cancers that express at least one HER receptor, typically more than one HER receptor. Such cancers can be identified by any means known in the art for detecting HER expression. For example, HER2 expression can be assessed using commercially available diagnostic/prognostic assays such as HercepTest ^™ (Dako). Paraffin-embedded tissue sections of tumor biopsies are subjected to immunohistochemistry (IHC) assays and determined according to HER2 protein staining intensity criteria. Tumors matching below the threshold score are characterized as not overexpressing HER2, whereas tumors greater than or equal to the threshold score are characterized as overexpressing HER2. In one example of treatment, a HER2 overexpressing tumor is evaluated as a candidate for treatment with a multispecific growth factor trap construct, such as any of those provided herein.

In another embodiment, the HER-mediated or HER-associated disease or condition is an inflammatory or autoimmune disorder, particularly rheumatoid arthritis. Animal models of arthritis, such as the Collagen Induced Arthritis (CIA) mouse model, can be used to test the growth factor trap constructs provided herein. For example, mice treated with a growth factor trap construct of the present invention, such as by local injection of protein, can observe a reduction in arthritis symptoms, including paw swelling, erythema, and catalepsy. A reduction in synovial angiogenesis and synovial inflammation can also be observed.

Multispecific, including bispecific, growth factor trap constructs, encoding nucleic acid molecules and pharmaceutical compositions provided herein can be used for the treatment of any disease, HER (ErbB) related disease or HER receptor mediated disease, which is Any disease, condition or disorder in which a receptor and/or ligand is implicated in some aspect of pathogenesis, pathology or development. The HER-related disease for treatment is cancer such as glioma, or pancreatic, gastric, head and neck, cervical, lung, colorectal, endometrial, prostate, esophageal, ovarian, uterine, bladder, kidney, or including breast cancer. Other conditions that may be treated include those involving the proliferation and/or migration of non-cancerous proliferative conditions such as smooth muscle cells, inflammatory or autoimmune diseases, skin disorders, and ophthalmic disorders. Diseases and conditions for treatment include, for example, rheumatoid arthritis, diabetic retinopathy, diseases of the anterior eye, psoriasis, restenosis, stenosis, atherosclerosis, hypertension from thickening of blood vessels, muscle thickening of the bladder, heart or other muscle, bladder disease, endometriosis, and obstructive airway disease, as well as one or more neuregulin (NRG) ligands, such as NRG1 (including types I, II, and III), NRG2, NRG3, and/or NRG4, or diseases or conditions associated with (eg, caused or aggravated by) exposure to other HER family ligands. Examples of NRG-associated diseases and diseases associated with other HER family ligands include schizophrenia, neurological or neuromuscular diseases including Parkinson's disease and Alzheimer's disease, cardiomyopathy, pre-eclampsia, neurological disease, and heart failure.

Examples of cancers that can be treated include carcinomas, lymphomas, blastomas, sarcomas, and leukemias or lymphoid malignancies such as squamous cell carcinoma (eg, epithelial squamous cell carcinoma), lung cancer (small cell lung cancer, non-small cell lung cancer) lung cancer, including adenocarcinoma of the lung and squamous cell carcinoma of the lung), cancer of the peritoneum, hepatocellular carcinoma, stomach cancer (including stomach cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer, colon cancer, rectal cancer, renal cell cancer, esophageal cancer, glioma, colorectal cancer, endometrial cancer, uterine cancer, salivary gland carcinoma, kidney cancer, prostate cancer, thyroid cancer, liver carcinoma, as well as head and neck cancer.

The multispecific growth factor trap constructs provided herein, upon administration, generally include a single targeted anti-HER therapy, such as trastuzumab, cetuximab, and other antibodies described herein or known in the art, as well as It can treat increased therapeutic efficacy and reduced drug resistance compared to small molecule tyrosine kinase inhibitors such as gefitinib, erlotinib and lapatinib. As described herein, resistance to single targeted anti-HER therapies is often associated with co-expression and/or upregulation of other HER family members and overexpression of their ligands. A HER-ligand binding construct provided herein that behaves as a receptor decoy and sequesters multiple HER family ligands prevents ligand dependent receptor activation and downregulates aberrant HER family activity, thereby simultaneously inhibiting multiple ligand induced HER family members. This increases therapeutic efficacy and reduces the likelihood of developing drug resistance.

5. Combination therapy

Combination therapy may be used. Combination therapy includes administration of multispecific, including bispecific, growth factor trap constructs, nucleic acid molecules, and pharmaceutical compositions provided herein with another agent or treatment, including radiation and surgery. Additional agents or therapies may be administered concurrently, before, after, or intermittently with the treatments provided herein. They may be in separate compositions or co-formulations.

Multispecific, such as bispecific, heteromultimer growth factor trap constructs, nucleic acid molecules and pharmaceutical compositions provided herein may be used as TNF antagonists/blockers, chemotherapeutic agents, single targeted anti-HER therapies (antibodies and tyrosine kinase inhibitors). including), anti-angiogenic agents, antibodies, cytotoxic agents, anti-inflammatory agents, cytokines, growth inhibitors, anti-hormonal agents, cardioprotective agents, steroids, immunostimulatory agents, immunosuppressive agents, biological or non-biological disease modifying agents -can be administered before, after, intermittently or concomitantly with one or more other therapeutic regimens or agents, including but not limited to rheumatoid drugs (DMARDs), therapeutics (including antibodies) for infectious diseases, or other therapeutic agents. . In particular, growth factor trap constructs are administered in conjunction with the TNFR1/TNFR2 axis constructs provided herein. They may also be administered in conjunction with other anti-TNF therapies, including any described in the section above or known to those skilled in the art.

TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific constructs, nucleic acids and other constructs provided herein can be administered as a regimen in combination with other anti-TNF therapies. Exemplary anti-TNF therapies that can be used in combination therapy herein include, for example, conventional synthetic DMARDs such as methotrexate (MTX), hydroxychloroquine (HCQ; ^Plaquenil® ), sulfasalazine ( ^Azulfidine® ), and leflunomide ( ^Arava® ); Biological DMARDs such as abatacept ( ^Orencia® ), anakinra ( ^Kineret® ), rituximab ( ^Rituxan® , ^Truxima® , ^MabThera® ), tocilizumab (atlizumab, ^Actemra® , ^RoActemra® ), corticosteroids steroids (e.g. dexamethasone, methylprednisolone, prednisolone, prednisone, or triamcinolone), tofacitinib ( ^Xeljanz® ), and TNF-inhibitor/anti-TNF agents such as certolizumab pegol ( ^Cimzia® ); infliximab ( ^Remicade® ), adalimumab ( ^Humira® ), golimumab ( ^Simponi® ), and etanercept ( ^Enbrel® ). Combination therapy may also include an immunotherapeutic drug such as cyclosporine, methotrexate, adriamycin or cisplatinum, and an immunotoxin.

In certain instances, growth factor trap constructs provided herein are used for the treatment of any of the chronic inflammatory, autoimmune, and/or neurodegenerative/demyelinating diseases and conditions described herein, particularly rheumatoid arthritis (RA). with any of the TNFR1 antagonist constructs, TNFR2 agonist constructs, or multispecific, such as bispecific, TNFR1 antagonist/TNFR2 agonist constructs provided herein for

In some examples, a growth factor trap construct provided herein is administered in conjunction with one or more anti-angiogenic agents. For example, an anti-angiogenic factor can be a growth factor involved in promoting angiogenesis or a small molecule or protein that binds to a growth factor receptor (eg, an antibody, Fc fusion, or cytokine). Examples of anti-angiogenic agents include antibodies that bind vascular endothelial growth factor (VEGF) or that bind VEGF-R, RNA-based therapeutics that reduce the level of VEGF or VEGF-R expression, VEGF-toxin fusions, Regeneron ) VEGF-trap, angiostatin (plasminogen fragment), antithrombin III, angiozyme, ABT-627, Bay 12-9566, benepine, bevacizumab, bisphosphonate, BMS-275291, cartilage derived inhibitor (CDI), CAI, CD59 complement fragment, CEP-7055, Col 3, combretastatin A-4, endostatin (collagen XVIII fragment), farnesyl transferase inhibitor, fibronectin fragment, GRO-beta, halofuginone, heparinase, heparin Hexasaccharide fragment, HMV833, human chorionic gonadotropin (hCG), IM-862, interferon alpha, interferon beta, interferon gamma, interferon inducible protein 10 (IP-10), interleukin-12, Kringle 5 ( plasminogen fragment), marimastat, metalloproteinase inhibitors (eg TIMPs), 2-methoxyestradiol, MMI 270 (CGS 27023A), plasminogen activator inhibitor (PAI), platelet factor -4 (PF4), prinomastat, prolactin 16 kDa fragment, proliferin-related protein (PRP), PTK 787/ZK 222594, retinoid, solimastat, squalamine, SS3304, SU5416, SU6668, SU11248, tetrahydro Cortisol-S, tetrathiomolybdate, thalidomide, thrombospondin-1 (TSP-1), TNP470, transforming growth factor beta (TGF-β), vasculostatin, vasostatin (calreticulin fragment), ZS6126, and ZD6474, but are not limited thereto.

In some examples, a growth factor trap construct provided herein is administered in conjunction with one or more tyrosine kinase inhibitors and optionally a TNFR1/TNFR2 axis construct provided herein. Examples of tyrosine kinase inhibitors include, but are not limited to, quinazolines such as PD 153035, 4-(3-chloroanilino)quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidines; 4-(phenylamino)-7H-pyrrolo(2,3-d) pyrimidine; curcumin (diferuloylmethane, 4,5-bis(4-fluoroanilino)phthalimide); tyrphostin containing a nitrothiophene moiety; PD-0183805 (Warner-Lambert); antisense molecules (eg, those that bind to ErbB encoding nucleic acids); quinoxaline (see, eg, US Pat. No. 5,804,396); Tyrphostine (see, eg, U.S. Patent No. 5,804,396); ZD6474 from Astra Zeneca; PTK-787 (Novartis/Schering AG); pan-ErbB inhibitors such as C1-1033 (Pfizer); Afinitak (ISIS 3521; Isis/Lilly); imatinib mesylate (STI571, Gleevec ^® ; Novartis); PKI 166 (Novartis); GW2016 (Glaxo SmithKline); C1-1033 (Pfizer); EKB-569 (Wyeth); cemacinib (Sugen); ZD6474 (AstraZeneca); PTK-787 (Novartis/Schering AG); INC-1 C11 (ImClone); Gefitinib ( ^Iressa® , ZD1839, AstraZeneca); and OSI-774 (marketed under the trademark ^Tarceva® , OSI Pharmaceuticals/Genentech), or any described in any of the following patent publications: U.S. Patent No. 5,804,396, and International Application Publication Nos. WO 99/09016, WO 98/ 43960, WO 97/38983, WO 99/06378, WO 99/06396, WO 96/30347, WO 96/33978, WO 96/33979, and WO 96/33980.

Other compounds useful for combination therapy include steroids such as angiostatic 4,9(11)-steroids and C21-oxygenated steroids, angiostatin, endostatin, basculostatin, canstatin and maspin, angiopoietin, bacterial polysaccharides CM101 and antibody LM609 (see, eg, US Pat. No. 5,753,230), thrombospondin (TSP-1), platelet factor 4 (PF4), interferons, metalloproteinase inhibitors, AGM-1470/TNP-470, Thalidomide, and pharmacological agents including carboxyamidotriazole (CAI), heparin or heparin fragments, anti-invasive factors, cortisone such as in the presence of retinoic acid and paclitaxel, shark cartilage extract, anionic polyamide or poly urea oligomers, oxindole derivatives, estradiol derivatives and thiazolopyrimidine derivatives.

Examples of anticancer antibodies that can be administered with the growth factor trap constructs provided herein include anti-17-IA cell surface antigen antibodies such as edrecolomab (marketed under the trademark ^Panorex® ); anti-4-1BB antibody; anti-4Dc antibody; anti-A33 antibodies such as A33 and CDP-833; anti-α1 integrin antibodies such as natalizumab; anti-α4β7 integrin antibodies such as LDP-02; anti-αVβ1 integrin antibodies such as F-200, M-200, and SJ-749; anti-αVβ3 integrin antibodies such as apsiximab, CNTO-95, Mab-17E6, and Medi-523 (marketed under the trademark Vitaxin); anti-complement factor 5 (C5) antibodies such as 5G1.1; anti-CA125 antibodies such as oregovomab (marketed under the trademark OvaRex ^® ); anti-CD3 antibodies such as visilizumab (Nuvion ^® ), and rexomab; anti-CD4 antibodies such as IDEC-151, MDX-CD4, and OKT4A; anti-CD6 antibodies such as oncolysin B and oncolysin CD6; anti-CD7 antibodies such as HB2; anti-CD19 antibodies such as B43, MT-103, and oncolysin B; anti-CD20 antibodies such as 2H7, 2H7.v16, 2H7.v114, 2H7.v115, tositumomab ( ^Bexxar® ), rituximab ( ^Rituxan® ), and ibritumomab tiuxetan ( ^Zevalin® ); anti-CD22 antibodies such as efratuzumab (Lymphocide ^® ); anti-CD23 antibodies such as IDEC-152; anti-CD25 antibodies such as basiliximab and Zenapax ^® (daclizumab); anti-CD30 antibodies such as AC10, MDX-060, and SGN-30; anti-CD33 antibodies such as gemtuzumab ozogamicin (Mylotarg ^® ), oncolysin M, and Smart Ml 95; anti-CD38 antibody; anti-CD40 antibodies such as SGN-40 and toralizumab; anti-CD40L antibodies such as 5c8, luplizumab (Antova), and IDEC-131; anti-CD44 antibodies such as vivatuzumab; anti-CD46 antibody; anti-CD52 antibodies such as ^Campath® (alemtuzumab); anti-CD55 antibodies such as SC-1; anti-CD56 antibodies such as huN901-DM1; anti-CD64 antibodies such as MDX-33; anti-CD66e antibodies such as XR-303; anti-CD74 antibodies such as IMMU-110; anti-CD80 antibodies such as galiximab and IDEC-114; anti-CD89 antibodies such as MDX-214; anti-CD123 antibody; anti-CD138 antibodies such as B-B4-DM1; anti-CD146 antibodies such as AA-98; anti-CD148 antibody; anti-CEA antibodies such as cT84.66, labetuzumab, and Pentacea ^® ; anti-CTLA-4 antibodies such as MDX-101; anti-CXCR4 antibodies; anti-EGFR antibodies such as ABX-EGF, ^Erbitux® (cetuximab), panitumumab, IMC-C225, and Merck Mab 425; anti-EpCAM antibodies such as Crucell anti-EpCAM, ING-1, and IS-IL-2; anti-ephrin B2/EphB4 antibody; anti-HER2 antibodies such as ^Herceptin® (trastuzumab), pertuzumab, and MDX-210; anti-FAP (fibroblast activation protein) antibodies such as sibrotuzumab; anti-peridin antibodies such as NXT-211; anti-FGF-1 antibody; anti-FGF-3 antibody; anti-FGF-8 antibody; anti-FGFR antibodies; anti-fibrin antibodies; anti-G250 antibodies such as WX-G250 and girentuximab ( ^Rencarex® ); anti-GD2 ganglioside antibodies such as EMD-273063 and TriGem; anti-GD3 ganglioside antibodies such as BEC2, KW-2871, and mitumomab; anti-gpIIb/IIIa antibodies such as Reopro; anti-heparinase antibodies; anti-HLA antibodies such as Oncolym, and Smart 1D10; anti-HM1.24 antibody; anti-ICAM antibodies such as ICM3; anti-IgA receptor antibodies; anti-IGF-1 antibodies such as CP-751871 and EM-164; anti-IGF-1R antibodies such as IMC-A12; anti-IL-6 antibodies such as CNTO-328 and elsilimomab; anti-IL-15 antibodies such as ^HuMax® -IL15 antibody; anti-KDR antibodies; anti-laminin 5 antibody; anti-Lewis Y antigen antibodies such as Hu3S193 and IGN-311; anti-MCAM antibodies; anti-Muc1 antibodies such as BravaRex and TriAb; anti-NCAM antibodies such as ERIC-1 and ICRT; anti-PEM antigen antibodies such as teragin and terex; anti-PSA antibodies; anti-PSCA antibodies such as IG8; anti-Ptk antibodies; anti-PTN antibodies; anti-RANKL antibodies such as AMG-162; anti-RLIP76 antibody; anti-SK-1 antigen antibodies such as Monopharm C; anti-STEAP antibody; anti-TAG72 antibodies such as CC49-SCA and MDX-220; anti-TGF-β antibodies such as CAT-152; anti-TNF-α antibodies such as CDP571, CDP870, D2E7, adalimumab ( ^Humira® ), and infliximab ( ^Remicade® ); anti-TRAIL-R1 and TRAIL-R2 antibodies; anti-VE-cadherin-2 antibody; and anti-VLA-4 antibodies, such as Antegren ^® antibodies. Anti-subject antibodies may be used including, but not limited to, the GD3 epitope antibody BEC2 and the gp72 epitope antibody 105AD7. Bispecific antibodies may also be used, including but not limited to the anti-CD3/CD20 antibody Bi20.

Examples of antibodies capable of treating autoimmune or inflammatory diseases, transplant rejection and/or GvHD that can be administered with the growth factor trap constructs provided herein include anti-α4β7 integrin antibodies such as LDP-02; anti-beta2 integrin antibodies such as LDP-01; anti-complement (C5) antibodies such as 5G1.1; anti-CD2 antibodies such as BTI-322, and MEDI-507; anti-CD3 antibodies such as OKT3, and SMART anti-CD3; anti-CD4 antibodies such as IDEC-151, MDX-CD4, and OKT4A; anti-CD11a antibody; anti-CD14 antibodies such as IC14; anti-CD18 antibody; anti-CD23 antibodies such as IDEC-152; anti-CD25 antibodies such as Genapax; anti-CD40L antibodies such as 5c8, Antova, and IDEC-131; anti-CD64 antibodies such as MDX-33; anti-CD80 antibodies such as IDEC-114; anti-CD147 antibodies such as ABX-CBL; anti-E-selectin antibodies such as CDP850; anti-gpIIb/IIIa antibodies such as ReoPro®/abxiximab; anti-ICAM-3 antibodies such as ICM3; anti-ICE antibodies such as VX-740; anti-FcγR1 antibodies such as MDX-33; anti-IgE antibodies such as rhuMAb-E25; anti-IL-4 antibodies such as SB-240683; anti-IL-5 antibodies such as SB-240563, and SCH55700; anti-IL-8 antibodies such as ABX-IL8; anti-interferon gamma antibodies; anti-TNFα antibodies such as CDP571, CDP870, D2E7, adalimumab, infliximab, and MAK-195F; and anti-VLA-4 antibodies such as antegren. Examples of other Fc-containing molecules that can be co-administered to treat autoimmune or inflammatory diseases, transplant rejection and GvHD include, but are not limited to, the TNFRII receptor/Fc fusion Enbrel ^® (etanercept), and Regeneron's IL-1 trap. Not limited.

Examples of antibodies that can be co-administered to treat infectious diseases include anti-anthrax antibodies such as ABthrax; anti-CMV antibodies such as CytoGam and sevirumab; anti-Cryptosporidium antibodies such as CryptoGAM and sporidin-G; anti-Helicobacter antibodies such as pyloran; anti-hepatitis B antibodies such as HepeX-B and Nabi-HB; anti-HIV antibodies such as HRG-214; anti-RSV antibodies such as felbizumab, HNK-20, palivazumab, and RespiGam; and anti-staphylococcal antibodies such as Aurexis, Aurograb, BSYX-A110, and SE-Mab.

In some instances, a growth factor trap construct described herein is administered in conjunction with one or more chemotherapeutic agents. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cyclophosphamide ( ^CYTOXAN® ); alkyl sulfonates such as busulfan, improsulfan and piposulfan; androgens such as calosterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenergics such as aminoglutethimide, mitotane, and trilostane; anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; Antibiotics such as aclasinomycin, actinomycin, anthramycin, azaserin, bleomycin, cactinomycin, calicheamicin, carubicin, carminomycin, carzinophylline, chromomycin, dactinomycin, daunol Bicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olibomycin , peplomycin, porphyromycin, puromycin, quelamycin, rodorubicin, streptonigryn, streptozocin, tubersidin, ubenimex, ginostatin, and zorubicin; For example, tamoxifen, raloxifene, aromatase inhibitor 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, kenoxyfen, LY 117018, onapristone, and toremifene (Pareston) estrogen; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, and trimetrexate; aziridines such as benzodepa, carboquone, meturedepa, and uredepa; ethylenimine and methylmelamine, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine; folic acid supplements such as folinic acid; Nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobemvicine, phenesterine, prednimu Steen, Trophosphamide, and Uracil Mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; proteins such as arginine deiminase and asparaginase; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, camofur, cytarabine, dideoxyuridine, doxifuridine, enocitabine, floxuridine, and 5-FU; taxanes such as paclitaxel ( ^TAXOL® , Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel ( ^TAXOTERE® , Rhone-Poulenc Rorer, Antony, France); topoisomerase inhibitors such as RFS 2000; thymidylate synthase inhibitors such as Tomudex; additional chemotherapeutic agents including aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; dedatrexate; dephosphamide; demecolsine; diaziquone; difluoromethylornithine (DMFO); eplonithine; elliptinium acetate; Etogluside; gallium nitrate; hydroxyurea; lentinan; Ronidamine; mitoguazone; mitoxantrone; furdamole; nitracrine; pentostatin; phenamet; pirarubicin; pophyllic acid; 2-ethylhydrazide; procarbazine; ^PSK® ; Razoxic acid; sizopyran; spirogermanium; tenuazinic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gastosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novanthrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; retinoic acid; esperamycin; capecitabine; and topoisomerase inhibitors; eg irinotecan Pharmaceutically acceptable salts, acids or derivatives of any of the above may also be used.

Chemotherapeutic agents can be administered as prodrugs. Examples of prodrugs that can be administered with the growth factor trap constructs described herein include, for example, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid modified prodrugs, glycosylated prodrugs, prodrugs containing beta-lactams, prodrugs containing optionally substituted phenoxy acetamides, or prodrugs containing optionally substituted phenylacetamides, and 5-fluorocytosine and 5-fluoro rocytosine and other 5-fluorouridine prodrugs that can be converted to more active cytotoxic free drugs.

In some instances, multispecific growth factor trap constructs described herein are administered with one or more immunomodulatory agents. Such agents may increase or decrease the production of one or more cytokines, up- or down-regulate self antigen presentation, mask MHC antigens, or promote the proliferation, differentiation, migration, or activation of one or more types of immune cells. can Examples of immunomodulatory agents are non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin, ibuprofen, celecoxib, diclofenac, etodolac, fenoprofen, indomethacin, ketorolac, oxaprozin, nabumetone , sulindac, tolmetin, rofecoxib, naproxen, ketoprofen, and nabumetone; steroids such as glucocorticoids, dexamethasone, cortisone, hydroxycortisone, methylprednisolone, prednisone, prednisolone and triamcinolone; eicosanoids such as prostaglandins, thromboxanes, and leukotrienes; topical steroids such as anthralin, calcipotriene, clobetasol, and tazarotene; cytokines such as TGFβ, IFNα, IFNβ, IFNγ, IL-2, IL-4, IL-10; cytokines, chemokines, or BAFF, B7, CCR2, CCR5, CD2, CD3, CD4, CD6, CD7, CD8, CD11, CD14, CD15, CD17, CD18, CD20, CD23, CD28, CD40, CD40L, CD44, CD45, CD52, CD64, CD80, CD86, CD147, CD152, Complement Factor (C5, D) CTLA-4, Eotaxin, Fas, ICAM, ICOS, IFNα, IFNβ, IFNγ, IFNAR, IgE, IL-1, IL-2, IL-2R, IL-4, IL-5R, IL-6, IL-8, IL-9 IL-12, IL-13, IL-13R1, IL-15, IL-18R, IL-23, integrin, LFA -1, LFA-3, MHC, selectin, TGFβ, TNFα, TNFβ, TNFR1, TNFR2, and antibodies to T-cell receptors, soluble receptors, and receptor antagonists, including receptor-Fc fusions (which include etanercept (Enbrel ^® ), including adalimumab (Humira ^® ), and infliximab (Remicade ^® ); heterologous anti-lymphocyte globulin; and other immunomodulatory molecules such as 2-amino-6-aryl-5 substituted pyrimidines, anti-substitute antibodies for MHC binding peptides and MHC fragments, azathioprine, brequinar, bromocriptine, cyclo Phosphamide, cyclosporine A, D-penicylamine, deoxyspergualine, FK506, glutaraldehyde, gold, hydroxychloroquine, leflunomide, malononitriloamide (e.g. leflunomide ), methotrexate, minocycline, mizoribine, mycophenolate mofetil, rapamycin, and sulfasalazine.

In some examples, multispecific growth factor trap constructs described herein are administered in conjunction with one or more cytokines. Examples of cytokines include, but are not limited to, lymphokines, monokines, and conventional polypeptide hormones. Among the cytokines are interferons such as interferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF); Interleukins (ILs), such as IL-1, IL-1alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10 , IL-11, IL-12 and IL-15; tumor necrosis factors such as TNF-alpha or TNF-beta; and other polypeptide factors including kit ligand (KL).

In some instances, multispecific growth factor trap constructs described herein are administered in conjunction with one or more cytokines or other agents that stimulate cells of the immune system and enhance desired effector function(s). For example, agents that stimulate natural killer (NK) cells, including but not limited to IL-2, can be administered with the multispecific growth factor trap constructs described herein. In another embodiment, agents that stimulate macrophages, including but not limited to C5a, and formyl peptides such as N-formyl-methionyl-leucyl-phenylalanine (see, for example, Beigier-Bompadre et al. (2003) Scand . J. Immunol . 57:221-228) can be administered with the multispecific growth factor trap constructs described herein. Agents that stimulate neutrophils, including but not limited to G-CSF and GM-CSF, can also be administered with the multispecific growth factor trap constructs described herein. Agents that promote the transport of these immunostimulatory cytokines can be administered with the multispecific growth factor trap constructs described herein. Additional agents include, but are not limited to, interferon gamma, IL-3 and IL-7, which can promote one or more effector functions. In some instances, multispecific growth factor trap constructs described herein are administered with one or more cytokines or other agents that inhibit effector cell function.

In some instances, the multispecific growth factor trap constructs described herein are aminoglycoside antibiotics (e.g., apramycin, abecacin, bambermycin, butyrosine, dibecacin, gentamicin, kanamycin, neo amycin, netylmycin, paromomycin, ribostamycin, sisomycin, and spectinomycin), aminocyclitols (e.g. spectinomycin), amphenicol antibiotics (e.g. azideamphenicol, chloramphenicol, florphenicol, and thiamphenicol), ansamycin antibiotics (e.g., lipamide and rifampin), carbapenems (e.g., imipenem, meropenem, and panipenem), cephalosporins (e.g., cefaclor, cefadroxil, cefamandole, ceftrizine, cefazedone, cefozofran, ceffimizole, cefpyramide, cefpyrom, cefprozil, cefuroxime, cefixim, cefalexin, and cefradine), cefamycins (e.g., cefbuperazone, cefoxitin, cefminox, cefmetazole, and cefotetan), lincosamides (e.g., clindamycin, and lincomycin), macrole rides (e.g., azithromycin, brefeldin A, clarithromycin, erythromycin, roxithromycin, and tobramycin), monobactams (e.g., aztreonam, carumonam, and tigemonam); Mupirocin, oxacephem (e.g., plomoxef, latamoxef, and moxalactam), penicillins (e.g., amdinocillin, amdinocillin pyroxyl, amoxicillin, bacampicillin, benzylpenicillinnan, Benzylpenicillin sodium, epicillin, penbenicillin, floxacillin, phenamecillin, phenetamate hydriodide, penicillin o-benetamine, penicillin O, penicillin V, penicillin V benzoate, penicillin V hydrabamine, penicillin mepiccycline, and phenethicillin potassium), polypeptides (e.g., bacitracin, colistin, polymyxin B, teicoplanin, and vancomycin), quinolones (e.g., amifloxacin, cinoxacin, ciprofloxacin) , enoxacin, enrofloxacin, floxacin, flumequin, gatifloxacin, gemifloxacin, grepafloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, oxol phosphoric acid, pefloxacin, pipemidic acid, losoxacin, lufloxacin, sparfloxacin, temafloxacin, tosofloxacin, and trovafloxacin), rifampin, streptogramins (e.g., quinupristine, and defopristine), sulfonamides (e.g., sulfanilamide, and sulfamethoxazole), and tetracyclines (e.g., chlortetracycline, demeclocycline hydrochloride, demethylchlortetracycline, doxycycline, durama isin, minocycline, neomycin, oxytetracycline, streptomycin, tetracycline, and vancomycin).

In some examples, multispecific growth factor trap constructs provided herein include amphotericin B, cyclopyrox, clotrimazole, econazole, fluconazole, flucytosine, itraconazole, ketoconazole, miconazole, nystatin, ter It is administered with one or more antifungal agents including, but not limited to, binafine, terconazole, and thioconazole.

In some examples, the multispecific growth factor trap constructs described herein are protease inhibitors, reverse transcriptase inhibitors, and type I interferons, viral fusion inhibitors, neuraminidase inhibitors, acyclovir, adefovir, amantadine, cancer Prenavir, clevudine, enfuvirtide, entecavir, foscarnet, ganciclovir, idoxuridine, indinavir, lopinavir, fleconaril, ribavirin, rimantadine, ritonavir, saquinavir, trifluri Administered with one or more antiviral agents including, but not limited to, others including, but not limited to, din, vidarabine, and zidovudine.

Multispecific growth factor trap constructs provided herein can be combined with other treatment regimens. For example, in one embodiment, a patient to be treated with a multispecific growth factor trap construct provided herein may undergo radiation therapy. Radiation therapy can be administered according to protocols commonly used in the art and known to those skilled in the art. Such therapy includes, but is not limited to, cesium, iridium, iodine or cobalt radiation. Radiation therapy can be systemic or directed locally to a specific area or tissue on or within the body, such as the lungs, bladder or prostate. Radiation therapy may also include treatment with isotopically labeled molecules such as antibodies. Examples of radioimmunotherapeutic agents are those sold under the tradenames Zevalin ^® (anti-CD20 labeled Y-90), LymphoCide ^® (anti-CD22 labeled Y-90), and Bexxar ^® (anti-CD20 labeled I-131). include

Typically, radiation therapy is administered in pulses over about 1 to 2 weeks. However, radiation therapy can be administered over an extended period of time. For example, radiation therapy can be administered to patients with head and neck cancer for about 6 to about 7 weeks. Optionally, radiation therapy can be administered as a single dose or multiple sequential doses. A skilled medical practitioner can empirically determine the appropriate dose or doses of radiation therapy useful herein. In some instances, a multispecific growth factor trap construct and optionally one or more other anti-cancer therapies are used to treat cancer cells ex vivo. It is believed that such an ex vivo treatment may be useful for bone marrow transplantation, particularly autologous bone marrow transplantation. For example, treatment of cell or tissue(s) containing cancer cells with a multispecific growth factor trap construct and one or more anti-cancer therapies as described herein to deplete cancer cells prior to transplantation into a recipient; can be practically exhausted.

It is also contemplated that the multispecific growth factor trap constructs provided herein may be administered to a patient or subject in conjunction with other treatment techniques such as surgery or phototherapy.

Provided herein are methods of treating cancer, eg, by administering any of the multispecific growth factor trap constructs, nucleic acid molecules, or pharmaceutical compositions provided herein in combination with another anti-cancer agent. Anti-cancer agents may include radiation and/or chemotherapeutic agents. For example, the anti-cancer agent may be a tyrosine kinase inhibitor or antibody. Exemplary anti-cancer agents include quinazoline kinase inhibitors, antisense or siRNA or other double-stranded RNA molecules, antibodies that interact with HER family receptors, and antibodies conjugated to radionuclides, or cytotoxins. Other exemplary anticancer agents include gefitinib, lapatinib, eroltinib, panitumumab, cetuximab, trastuzumab, imatinib, platinum complexes or nucleoside analogs. Examples of cytotoxic or chemotherapeutic agents include, for example, taxanes (such as paclitaxel and docetaxel) and anthracycline antibiotics, doxorubicin/adriamycin, carminomycin, daunorubicin, aminopterin, methotrexate, methopterin, dichloro- methotrexate, mitomycin C, porphyromycin, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, podophyllotoxin, or podophyllotoxin derivatives such as etoposide or etoposide phosphate, melphalan, vinblastine, vincristine, leucidine, vindesine, leunociden, maytansinol, epothilone A or B, taxotere, taxol, estramustine, cisplatin, combretastatin and analogues, and cyclophospha contains mead. Any other anticancer antibody and chemotherapeutic agent described elsewhere herein or known in the art may also be used in combination with a multispecific growth factor trap construct, nucleic acid molecule, or pharmaceutical composition provided herein to treat cancer. considered for use.

In another example, rheumatoid arthritis (RA) is treated by administering any multispecific growth factor trap construct, nucleic acid molecule, or pharmaceutical composition provided herein in combination with another anti-rheumatic drug, such as an anti-TNF therapy. ) is provided herein. Examples of anti-TNF therapies that can be used in combination with the multispecific growth factor trap constructs, nucleic acid molecules or pharmaceutical compositions provided herein include, for example, conventional synthetic DMARDs such as methotrexate (MTX), hydrochloride oxychloroquine (HCQ; Plaquenil ^® ), sulfasalazine (Azulfidine ^® ), and leflunomide (Arava ^® ); Biological DMARDs such as abatacept ( ^Orencia® ), anakinra ( ^Kineret® ), rituximab ( ^Rituxan® , ^Truxima® , ^MabThera® ), tocilizumab (atlizumab, ^Actemra® , ^RoActemra® ), corticosteroids steroids (e.g. dexamethasone, methylprednisolone, prednisolone, prednisone, or triamcinolone), tofacitinib ( ^Xeljanz® ), and TNF-inhibitor/anti-TNF agents such as certolizumab pegol ( ^Cimzia® ); infliximab ( ^Remicade® ), adalimumab ( ^Humira® ), golimumab ( ^Simponi® ), and etanercept ( ^Enbrel® ). Combination therapy may also include an immunotherapeutic drug such as cyclosporine, methotrexate, adriamycin or cisplatinum, and an immunotoxin.

In addition, any of the multispecific growth factor trap constructs described herein may be administered with any of the TNFR1 antagonist, TNFR2 agonist, or bispecific TNFR1 antagonist/TNFR2 agonist constructs provided herein to Provided herein are methods for the treatment of chronic inflammatory, autoimmune, neurodegenerative and/or demyelinating diseases, particularly RA, described elsewhere. Optionally, an additional anti-TNF therapy, such as methotrexate, or any described above or elsewhere herein or known in the art, is also useful in the treatment of chronic inflammatory, autoimmune, neurodegenerative and/or demyelinating diseases. Any other therapy, such as immunosuppressive agents, anti-angiogenic agents, cardioprotective agents, antibodies, cytotoxic agents, anti-inflammatory agents, cytokines, growth inhibitory agents, chemotherapeutic agents, biological or non-biological disease modifying anti-rheumatic drugs (DMARDs), therapeutic agents (including antibodies) for infectious diseases, or other suitable therapeutic agents described herein or known in the art.

Angiogenesis plays an important role in the formation and maintenance of the pannus in RA. Multispecific growth factor trap constructs provided herein can be used in conjunction with other treatments to modulate angiogenesis. For example, angiogenesis inhibitors can be used in conjunction with the multispecific growth factor trap constructs provided herein to treat RA. Exemplary angiogenesis inhibitors include angiostatin, antiangiogenic antithrombin III, canstatin, cartilage derived inhibitor, fibronectin fragment, IL-12, baculostatin, and others known in the art and described elsewhere herein. However, it is not limited thereto.

In some embodiments, growth factor trap constructs provided herein are used in conjunction with TNF blockers and/or other DMARDs, such as methotrexate, and compared to standard treatment RA regimens. For example, a growth factor trap construct provided herein can be combined with etanercept and/or methotrexate, eg, suboptimal doses of etanercept and/or methotrexate. To evaluate effectiveness, the combination therapy may be etanercept and/or methotrexate monotherapy, including optimal and suboptimal doses of etanercept and/or methotrexate. Growth factor trap constructs allow lower doses of other treatments to reduce adverse or undesirable side effects.

In other embodiments, the growth factor trap constructs provided herein are combined with other anti-TNF therapies, such as adalimumab or infliximab (including suboptimal doses thereof) with or without methotrexate (including methotrexate at suboptimal doses thereof). Can be combined, and treatment efficacy is compared to treatment alone with anti-TNF therapy with or without methotrexate. In another embodiment, a growth factor trap construct provided herein is used in combination with a TNFR1 antagonist, a TNFR2 agonist, or any of the multispecific, such as bispecific, TNFR1 antagonist/TNFR2 agonist constructs provided herein. and compared to a standard of care RA regimen, such as etanercept, adalimumab, or infliximab, with or without methotrexate.

H. TNFR1 antagonists and TNFR1 antagonist/ TNFR2 agonist construct Evaluation of activity and efficacy

Where appropriate or necessary, constructs provided herein may be tested in any in vivo and/or in vitro assay known to those skilled in the art to evaluate the properties and/or suitability of the constructs for treating a particular disease, disorder or condition. It can be evaluated for activity and efficacy using assays. Such assays can also be used to monitor treatment and/or predict response or select treatment targets. Exemplary assays are described in the next section.

Generally, the antagonist constructs herein are non-competitive; These are generally constructs that lock the receptor in an inactive form, meaning that selecting high affinity is less important than selecting antagonist activity, as discussed above.

1. Disease Activity Score (DAS28)

The 28-joint disease activity score (DAS28; or disease activity score of 28 joints) is a measure of disease activity in rheumatoid arthritis (RA) and is a simplification of the original DAS score, in which 44 joints were to be counted. The 28 joints to be counted are proximal interphalangeal joints (10 joints), metacarpophalangeal joints (10 joints), wrist (2), elbow (2), shoulder (2), and knee (2). DAS28 is used in clinical trials to evaluate treatments for RA because it shows RA disease activity and response to treatment. The DAS28 is based on the number of 28 swollen and tender joints with a score ranging from 0-10, with higher values indicating higher disease activity. In addition to counting the number of tender joints and tender joints (out of 28), the DAS28 measures erythrocyte sedimentation rate (ESR) or C-reactive protein (CRP), an acute phase reactant/hematologic indicator of inflammation, as well as a 100 mm visual analogue scale (VAS). ), where a value of 0 means “no activity” and a value of 100 means “highest activity possible”. DAS28 is usually combined with other measures of disease severity, such as pain and grip strength, and assesses physical function using the Health Assessment Questionnaire (HAQ).

To calculate DAS28 values using ESR or CRP levels, the following equations are used respectively.

where TJC = number of tender joints and SJC = number of swollen joints.

Values less than 2.6 indicate remission, values less than 3.2 (higher than 2.6, less than 3.2) indicate low disease activity, values greater than 3.2 and less than or equal to 5.1 indicate moderate disease activity, and values greater than 5.1 indicate high disease activity (i.e., active disease). An improvement of greater than 1.2 (i.e., a decrease in DAS28 score/value) indicates a good response/improvement; An improvement of greater than 0.6 and less than or equal to 1.2 indicates a moderate response; A DAS28 decrease of 0.6 or less indicates no improvement (see, e.g., Prevoo et al. (1995) Arthritis & Rheumatism 38(1):44-48; Wells et al. (2009) Ann. Rheum. Dis. 68:954-960]).

Therapeutic efficacy of bispecific constructs containing a selective TNFR1 antagonist, TNFR2 agonist and/or combination thereof provided herein can be determined by calculating DAS28 (ESR) or DAS28 (CRP) before, during and after treatment. can be evaluated.

2. SOMAscan for analyte quantification ^® Proteomic analysis and other proteomic tools

The SOMAscan ^® proteomic assay (SomaLogic, Inc.; Boulder, CO.) is an aptamer-based multimerized, sensitive, quantitative, and reproducible proteomic assay tool that can detect >5,000 cells in as little as 150 μL in samples such as serum, plasma, or cerebrospinal fluid. The amount of protein analyte can be measured simultaneously. Other biological matrices can also be used, such as cell culture supernatants, cell and tissue lysates, synovial fluid, and bronchoalveolar and nasal lavage. Because of its ability to simultaneously quantify a broad range or protein targets, the SOMAscan ^® assay is optimized for protein biomarker discovery, and has several applications including, for example, non-small cell lung cancer, Alzheimer's disease, cardiovascular disease and inflammatory bowel disease. It has been used to identify biomarker signatures associated with canine disease. The SOMAscan® assay can be used, for example, to determine the protein signature of RA patients by analyzing samples taken before and after initiation of treatment with a TNFR1 antagonist, TNFR2 agonist, and bispecific construct provided herein. In this way, the patient's response can be monitored at the beginning of treatment and throughout treatment.

The SOMAscan® assay utilizes a protein capture reagent known as the Slow Off-rate Modified Aptamer (sold as SOMAmer® aptamers) reagent, which mimics amino acid side chains, has a slow off-rate, is specific to a protein target, and has high affinity. It is a short, single-stranded DNA-based protein affinity reagent constructed with chemically modified nucleotides that permit binding. This assay measures proteins in their original folded form (i.e., tertiary structure) and does not detect proteins that are unfolded and denatured (i.e., inactive). For the SOMAscan ^® assay, the SOMAmer ^® -protein binding step is followed by a series of partitioning and washing steps, proteins in a biological sample are analyzed by converting each individual protein concentration to a corresponding SOMAmer ^® reagent concentration (SOMAmer ^® based DNA signal). , quantified by standard DNA detection techniques such as microarray or qPCR. The assay exploits the dual nature of the SOMAmer reagent as a protein affinity binding reagent with a defined three-dimensional structure and as containing a unique nucleotide sequence that can be recognized by specific DNA hybridization probes.

The SOMAmer ^® reagent is prepared with three tags and contains a fluorophore linked to biotin via a photocleavable linker. Briefly, for assays, biological samples of interest are diluted and incubated with respective SOMAmer ^® reagent mixtures pre-immobilized on streptavidin (SA) coated beads. The SOMAmer ^® reagent binds to proteins in the biological sample and the beads are washed to remove unbound proteins. All non-specific complexes that form have fast off rates. Proteins that remain bound to the cognate SOMAmer ^® reagent are tagged using the NHS-Biotin reagent and a polyanionic competitive solution is added that dissolves all non-specific complexes. Protein-SOMAmer® complexes and unbound (free) ^SOMAmer® reagents are released from the streptavidin beads by cleavage of the photocleavable linker using ultraviolet light. The photocleavage eluate containing all SOMAmer ^® reagents (some bound to the biotinylated protein and some not) is incubated with the biotinylated protein and a second streptavidin-coated bead that binds the biotinylated protein-SOMAmer ^® complex and unbound material is removed in a subsequent washing step. In a final elution step, the protein-bound SOMAmer ^® reagent is released from the cognate protein using denaturing conditions and the SOMAmer ^® reagent is quantified by standard DNA quantification techniques such as hybridization to custom DNA microarrays and fluorophore tagging. Data are reported as relative fluorescence units (RFU) after normalization and correction, and the measured SOMAmer ^® reagent signal correlates with protein levels found in biological samples (see, e.g., Gold et al. (2010) PLoS ONE 5(12):e15004; Candia et al. (2017) Sci . Reports 7:14248; Tanaka et al. (2018) Aging Cell. 17:e12799]).

3. Transcriptome analysis to predict responsiveness to therapy and select subjects likely to benefit from therapy

Existing anti-TNF therapies, i.e., TNF blockers such as etanercept, infliximab and others, show non-responsiveness in about 30% of RA patients. However, there are clinical markers that can predict the efficacy of these anti-TNF therapies. Analysis of differentially expressed genes following anti-TNF therapy with etanercept was performed using global transcriptome analysis to determine RNA expression signatures in peripheral blood mononuclear cells (PBMCs). Similar transcriptome analyzes can be performed to evaluate the efficacy and/or evaluate patient responsiveness to the TNFR1 antagonist and TNFR1 antagonist/TNFR2 agonist constructs herein.

In an exemplary protocol, blood samples are obtained from patients before and after treatment, PBMCs are isolated using a Ficoll density gradient, and then CD3 ⁺ , CD14 ⁺ , CD19 ⁺ and CD56 ⁺ cell populations are measured using flow cytometry. Evaluate. Total RNA is then extracted, e.g., using the Qiagen RNeasy ^® kit, and microarray analysis (e.g., using Affymetric ^® microarray technology). Gene expression profiles can be determined at an early stage of treatment to quickly identify those who will be non-responders. For example, by using this method to identify reliable biomarkers for predicting the therapeutic efficacy of etanercept in RA patients, gene pairs with >89% predictive accuracy and gene triplets with >95% predictive accuracy identified. Included herein are genes involved in TNF signaling via, for example, the NF-κB pathway, genes involved in NF-κB independent signaling, and genes involved in cellular and oxidative stress response regulation. For example, gene triplets identified include TNFAIP3, which encodes TNFα-induced protein 3, a zinc finger protein that has been shown to inhibit NF-κB activation; PDE4B, which encodes a specific cyclic nucleotide phosphodiesterase (cAMP) involved in NF-κB independent signaling; and RAPGEF1 encoding Rap guanine nucleotide-exchange factor 1, an activator of RAS signaling. Expression of all three genes was downregulated 3 days after etanercept administration in responders compared to non-responders. Other genes evaluated include CCL4 , CXCR4 , CCL3 , PIGO , FSD1 , RUNX1 , LGALS13 , PTPRD , IL1B , ADAM12 , and HCG4P6 (see, e.g., Koczan et al. (2008) Arthritis Research & Therapy 10:R50).

To validate the results obtained by microarray analysis, the expression level of a subset of genes of interest can be measured by quantitative real-time PCR (RT-PCR) using pre-designed primers and probes. To calculate the change in gene expression of a selected gene, using the ΔΔC _T method, the threshold cycle (C _T ) value for expression of a particular mRNA in a sample is normalized to the C _T value of, for example, GAPDH mRNA in the sample, and the gene Expression change (ΔΔCT) is defined as the difference between C _T values before and after treatment (see, e.g., Koczan et al. (2008) Arthritis Research & Therapy 10:R50).

4. L929 Cytotoxicity Assay

For example, TNFR1-mediated processes and cellular responses can be determined by assessing TNF-induced apoptosis with the L929 cytotoxicity assay, in which a TNFR1 antagonist inhibits TNF-induced cytotoxicity. Briefly, L929 mouse fibroblasts are plated in microtiter plates and incubated overnight with TNFR1 antagonist, 100 pg/ml TNF and 1 mg/ml actinomycin D. Cell viability after incubation with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) TNFR1 antagonists reduce TNF-mediated cytotoxicity, increasing absorbance compared to the TNF alone control.

5. HeLa IL-8 Assay

The activity of a TNFR1 antagonist can be determined using the HeLa IL-8 assay, in which the ability of the antagonist to neutralize TNF-derived IL-8 secretion in HeLa cells is assessed. Briefly, HeLa cells were plated overnight in microtiter plates in the presence of various concentrations of TNFR1 antagonist and 300 pg/ml TNF. The supernatant is then aspirated and the concentration of IL-8 is determined using a sandwich ELISA. TNFR1 antagonist activity reduces IL-8 secretion into the supernatant compared to TNF alone control.

6. HUVEC Assay

The human umbilical vein endothelial cell (HUVEC) assay can be used to determine the activity of the TNFR1 antagonists herein. Treatment of HUVECs with TNF results in upregulation of VCAM-1 expression in the cells, which can be determined, for example, by ELISA. Because TNFR1 antagonists inhibit the actions of TNF, VCAM-1 expression is reduced in HUVECs in the presence of antagonists. The level of inhibition of TNF-induced VCAM-1 expression by TNFR1 antagonists is determined by plotting the concentration of antagonist against the percentage inhibition of VCAM-1 expression.

Following the protocol for this assay, HUVECs are cultured overnight, then with TNFR1 antagonist for 1 hour, then stimulated with TNF (1 ng/mL) for 23 hours. Cells incubated with only medium were used as a negative control, and cells incubated with only TNF were used as a positive control. The cell culture supernatant was then aspirated and the cells were washed three times with ice-cold PBS and lysed in ice-cold Tris-glycerol lysis buffer (40 mM Tris, 274 mM NaCl, 2% Triton-X-100, 20% glycerol, 50 mM NaF, 1 mM Na ₃ VO ₄ , 1 x protease inhibitor tablet per 10 mL) to dissolve and incubate on ice for 15 minutes. Cell lysates are then used for VCAM-1 sandwich ELISA. To calculate inhibition of TNF-derived VCAM-1 expression, % inhibition of maximal VCAM-1 expression (i.e. measured in positive control) = {100 - [(OD value at antagonist concentration) / (OD of positive control value)]} x 100.

EC ₅₀ values are then determined by plotting antagonist concentration against percent inhibition using available software such as, for example, GraphPad Prism software.

7. Quantification and evaluation of Treg cell activity

To determine the effect of TNFR1 antagonist and TNFR1 antagonist/TNFR2 agonist constructs on Tregs, the number of Tregs was determined by isolating peripheral blood mononuclear cells (PBMCs) from blood samples, such as CD4 ⁺ CD25 ⁺ Tregs and CD4 ⁺ CD25 ^- Unregulated T cells can be quantified before and during treatment as well as during treatment using the Ficoll-Paque method followed by monoclonal antibodies (mAbs) and paramagnetic beads or other similar methods known in the art to isolate unregulated T cells. there is. By using flow cytometry and immunostaining with mAbs against CD4 and CD25 (for Tregs) or CD4 and CTLA-4 (for non-regulatory T cells), the number of each cell type can be quantified (eg For example, see Vigna-Pιrez et al. (2005) Clin . Exp . Immunol . 141(2):372-380).

CD4 ⁺ CD25 ⁺ Tregs suppress the proliferation of CD4 ⁺ CD25 ⁻ T cells. To test Treg activity in treated patients, a cell proliferation assay can be used in which Tregs and T cells are co-cultured with phytohemagglutinin (PHA, T cell stimulating) for 48 hours. Harvest cells after addition of ³ H-TdR (tritium thymidine) during the last 12 hours of incubation and determine proliferation using a liquid scintillation counter. CD4 ⁺ CD25 ^- T cells cultured alone were used as a control, and the results are expressed as the stimulation index of cell proliferation (SI), which is calculated using the following equation.

SI = (cpm of cells with PHA) ÷ (cpm of cells cultured in media only), where cpm is the counts per minute determined by counted radioactivity (see, e.g., Vigna-Perez et al. (2005) Clin. Exp. Immunol. 141(2):372-380).

To test immunoreactivity against M. tuberculosis, PBMCs are cultured for 72 hours in complete medium in the presence of total protein extracts of the bacteria. Harvest cells after addition of ³ H-TdR (tritium thymidine) during the last 12 hours of incubation and determine proliferation using a liquid scintillation counter. Results are expressed as stimulus indices as described above. For in vivo reactivity against M. tuberculosis , a standard PPD (purified protein derivative) skin test can be used (see, eg, Vigna-Pιrez et al. (2005) Clin. Exp. Immunol. 141(2):372-380).

8. Evaluation of binding properties of TNFR1 antagonist/TNFR2 agonist constructs

Specific binding of an antibody or antibody fragment or multispecific construct, such as a construct provided herein, to TNFR1 and/or TNFR2 (eg, human TNFR1 and/or TNFR2) can be achieved by any of a variety of known methods. can be evaluated. Affinity is the TNFR1 antagonist required to achieve a half-maximal potentiation of TNFR1 and/or TNFR2 signaling (EC ₅₀ ) and the equilibrium constant of antagonist-TNFR1 and/or agonist-TNFR2 complex dissociation (K _D ) in vitro; It can be quantitatively determined by a variety of metrics including the concentration of the TNFR2 agonist or multispecific construct. The equilibrium constant K _D that describes the interaction of TNFR1 or TNFR2 with a binding agent, e.g., a construct (binder) provided herein, is a solvent separated TNFR1 or TNFR2 in which the TNFR1 binding agent construct complex or the TNFR2 binding agent complex do not interact with each other. and the chemical equilibrium constant for the dissociation reaction into binder molecules.

TNFR1 antagonists, TNFR2 agonists and multispecific constructs can also be characterized by various in vitro binding assays. Examples of experiments that can be used to determine K _D or EC ₅₀ include, for example, surface plasmon resonance (SPR, eg, BIAcore ^™ analysis), isothermal titration calorimetry, fluorescence anisotropy, and ELISA-based assays, among others. include ELISA is a particularly useful method for assaying antibody activity because it usually requires minimal concentrations of antibody. A common signal analyzed in a typical ELISA assay is luminescence, which typically binds specifically to a primary antibody (e.g., a TNFR1-antagonist or TNR1 agonist TNFR2-agonist bispecific construct provided herein) It is the result of the activity of peroxidase conjugated to the secondary antibody that

The kinetics of association and dissociation of a TNFR1 antagonist to TNFR1 or a TNFR2 agonist to TNFR2 can be characterized quantitatively, for example by monitoring the rate of antibody-antigen complex formation according to established procedures. For example, surface plasmon resonance (SPR) can be used to determine the rate constants for the formation (k _on ) and dissociation (k _off ) of antagonist-TNFR1 or agonist-TNFR2 complexes. The equilibrium constant (K _D ) can be determined from these data since the equilibrium constant of this unimolecular dissociation can be expressed as the ratio of k _off to k _on value. SPR is an advantageous technique for determining the kinetic and thermodynamic parameters of receptor-antibody (or other binding agent) interactions because it does not require modification of one component by chemical labeling in the experiment. Rather, the receptor is typically immobilized on a solid metal surface that is treated with pulses of solutions of increasing concentrations of the antibody or binding agent (ie, TNFR1 antagonist or TNFR2 agonist, or bispecific constructs thereof). Antibody-receptor binding induces a distortion of the reflection angle of incident light at the metal surface, and the change in refractive index over time as the antibody is introduced into the system is well known in the art to calculate the association and dissociation rate constants of the antibody-receptor interaction. A well-known established regression model can be fitted.

9. Antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays

Antibody Dependent Cytotoxicity (ADCC) and Complement Dependent Cytotoxicity (CDC) Assays Evaluate Immune Effector Function/Cytotoxicity of Multispecific Constructs Provided herein Containing a TNFR1 antagonist, a TNFR2 agonist and an Fc monomer or dimer can be used to Typically the Fc portion(s) are modified to eliminate or substantially reduce ADCC or ADCC and CDC effector functions (to eliminate or reduce side effects to an acceptable level). Such assays are well known in the art (see, e.g., Ying et al. (2014) mAbs 6(5):1201-1210). For example, for an exemplary ADCC assay, mesothelin-negative A431 or mesothelin-positive H9 cells are incubated with a TNFR1 antagonist, TNFR2 agonist, or multispecific construct provided herein for 30 minutes, followed by 50 Add target cells to wells containing effector cells (eg, PBMCs) at an effector to target cell ratio of :1. After 24 hour incubation, lysis of target cells is measured using the CytoTox-ONE Homogenous Membrane Integrity Assay (Promega) according to the manufacturer's protocol.

For the exemplary CDC assay, A431 and H9 cells are washed in serum-free RPMI and the density is adjusted to 1 million/mL in serum-free RPMI. 50 μL of the cell suspension is then incubated with 50 μL of TNFR1 antagonist, TNFR2 agonist or multispecific construct dilution in RPMI. The negative control contains 50 μL of cell suspension with 50 μL of RPMI and the positive control contains target cells lysed with 1% Triton X-100 in a final volume of 150 μL. Fresh human plasma was diluted with PBS (1:4) and clarified by centrifugation, then 50 μL of diluted plasma was added to each cell/construct mixture and incubated in a 96-well plate at 37 °C to complement-mediated cells. allow dissolution. After 3 hours incubation, 100 μL of the supernatant is transferred to a white plate and 100 μL of substrate from the CytoTox-ONE Homogenous Membrane Integrity Assay Kit (Promega) is added. The plate is then incubated at room temperature for 10 minutes and the fluorescence signal is read using a fluorometer with an excitation wavelength of 530 nm and an emission wavelength of 590 nm. CDC of target cells is expressed as a percentage of the experimental sample relative to the positive control.

10. Disease Model

Selective TNFR1 antagonists, TNFR2 agonists, and multispecific constructs provided herein can be used to determine their effects on autoimmunity and inflammation and other diseases or disorders mediated by or related to TNF in their pathogenesis, known to those skilled in the art. It can be evaluated in any clinically relevant disease model. Exemplary disease models include collagen induced arthritis (CIA), rheumatoid arthritis synovial mononuclear cell culture, Tg197 mouse model of arthritis, ΔARE mouse model of arthritis/IBD, mouse dextran sulfate sodium (DSS) derived model of IBD, IBD induction model and experimental autoimmune encephalomyelitis (EAE) model for multiple sclerosis. Other models are known to those skilled in the art. See, eg, Malaviya et al. (2017) Pharmacol Ther . 180:90-98] provide numerous models for testing constructs for treating inflammatory lung diseases; See Feldmann et al. (2020) Lancet 395 :1407-1409] about the use of anti-TNF therapies and models, treatments for COVID-19; See Shi et al. (2013) Crit . Care 17(6) :R301] relates to the use of anti-TNF therapy for the treatment of H1N1 and viral infection models; See Orti-Casan et al. (2019) Front. Neurosci . 13:49] describe the benefits of activating TNFR2 to treat Alzheimer's disease, demonstrating our approach that TNF blockers that inhibit TNFR1 and TNFR2 are problematic. The following is a non-exhaustive discussion of the diseases, disorders and conditions that can be treated with the constructs provided herein and exemplary models for each. These are illustrative; One skilled in the art can select an appropriate model for a particular construct and target disease, disorder or condition. Since the anti-TNFR1 and TNFR2 antagonist/agonist constructs provided herein are intended for use in targeting human TNFR1/TNFR2, they will also act to react/interact with TNFR1/TNFR2 from non-human, particularly non-primate species. is not expected to For testing, in non-human models such as rodent models, models such as mouse models are transgenic for human TNFR1 and human TNFR2. They can be used against the background of murine TNFR1/2 knockout mice for in vivo models of inflammatory and autoimmune diseases. Alternatively, severely immunocompromised mice transplanted with human CD34+ stem cells (eg, NOD/NSG mice) can be used for this purpose. Alternatively, human rheumatoid arthritis synovial cells can be transplanted into immunodeficient mice to induce RA-like inflammation (see, eg, Schinnerling et al. for a description of such a model). (2019) Front Immunol. 10 :203]).

a. Collagen Induced Arthritis (CIA)

Type II collagen-induced arthritis (CIA) can be derived in mice as a model of an autoimmune inflammatory joint disease histologically similar to RA, and is characterized by inflammatory synovitis, pannus formation, and erosion of cartilage and bone. Bovine type II collagen (B-CII) is injected intradermally into the base of the tail in the presence of complete Freund's adjuvant to induce CIA. Mice can be reimmunized after 21 days using the same protocol. To investigate the effects of the selective TNFR1 antagonists, TNFR2 agonists and multispecific constructs provided herein, 3 weeks after initial challenge with B-CII or at the onset of arthritis, selective TNFR1 antagonists, TNFR2 agonists or multispecific constructs Alternatively, the control group can be administered intraperitoneally twice weekly for 3 weeks. Mice can be sacrificed 7 weeks after initial immunization for histological analysis.

To evaluate the therapeutic effect of the construct on established disease, it can be administered daily for a total of 10 days after the onset of clinical arthritis in one or more limbs. Calipers can be used to measure foot thickness to initially monitor the degree of swelling in the affected joint. proinflammatory cytokines and chemokines such as granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-10 (IL-10), IL-1β, IL-6, IL-8, RANTES (CCL5) and Serum can be drawn from mice for the measurement of monocyte chemoattractant protein 1 (MCP-1; also known as CCL2).

In another example, primate models can be used to treat RA. Responses of tender and swollen joints (e.g., as measured by clinical arthritis scores) are monitored in subjects treated with a recombinant therapeutic TNFR1 antagonist, TNFR2 agonist or bispecific construct and control to determine treatment efficacy and treatment. can be evaluated

b. Culture of rheumatoid arthritis synovial mononuclear cells

Human rheumatoid arthritis (RA) synovial mononuclear cells (MNC) expressing TNFR1 and TNFR2 can also be used to test the therapeutic efficacy of constructs provided herein. RA synovial MNCs can be obtained from RA patients undergoing joint replacement surgery and cultured ex vivo to assess RA synovial cell cytokine production and regulation. RA synovial MNC cultures spontaneously produce inflammatory cytokines and chemokines in the absence of exogenous stimuli; Antibody-mediated neutralization of TNF and selective blockade of TNFR1 in these cultures, such as by the constructs provided herein, can inhibit proinflammatory cytokines and chemokines, such as GM-CSF, IL-10, IL-1β, IL-6, IL-6, IL- 8, can inhibit the production of RANTES (CCL5) and MCP-1 (CCL2).

In an exemplary assay, to prepare RA synovial MNC, RA synovial tissue was dissected into small pieces and incubated in RPMI 1640 with 5 mg/ml collagenase A and 0.15 mg/ml DNase at 37°C for 1 hour. After that, the digested tissue is passed through a 170 μm filter and washed three times with RPMI 1640 containing 100 units/ml of streptomycin, 100 μg of penicillin and 10% FCS. Heterogeneous RA synovial MNCs are used without passaging. For ex vivo cell culture, single cell suspensions of RA synovial MNCs were cultured in the presence or absence of TNFR1 antagonists, TNFR2 agonists, or bispecific constructs or controls at 37°C and 5% CO ₂ for 2-5 days at 96 °C. -Well flat bottom plates (2 x 10 ⁵ cells/well) are cultured in RPMI 1640 medium with 5% FCS. The supernatant is then collected and used immediately or stored at -20 °C for analysis by cytokine and chemokine ELISA (see, e.g., Schmidt et al. (2013) Arthritis & Rheumatism 65(9):2262-2273). Alternatively, cytokines in culture supernatants can be quantified by cytokine bead array analysis.

c. Tg197 mouse model of arthritis

The mouse model of erosive arthritis, the Tg197 transgenic mouse, is a well-established animal model of RA. Tg197 mice are human TNF-transgenic C57BL/6 mice that overexpress human TNF and develop symmetric polyarthritis with pannus formation, bone destruction and cartilage damage characteristic of human RA. In addition to representing features of chronic destructive joint disease, this model exhibits symptoms such as enthesitis or bilateral sacroiliac arthritis that are characteristic of other inflammatory diseases such as spondyloarthritis (Bluml et al. (2010) Arthritis & Rheumatism 62(6). ):1608-1619]). The Tg197 mouse develops arthritis with 100% penetrance and provides a rapid in vivo model for evaluating human therapeutics targeting RA. For example, the Tg197 mouse model was used to evaluate the therapeutic efficacy of infliximab (originally marketed as Remicade®), the first anti-TNF drug successfully applied clinically, and to screen potential anti-RA therapies. recommended by the FDA for

The Tg197 mouse has 5 copies of the human TNF gene construct in which the 3'-region containing the 3'-untranslated and 3'-flanking sequences is exchanged with the 3'-region of the human β-globin gene. Because a series of highly conserved UA-rich sequences in the 3′-untranslated region of TNF mRNA are important for the regulation of mRNA stability and translational efficiency, this genetic construct was microinjected into mouse zygotes to investigate deregulated TNF gene expression in vivo. create a model see, for example, Keffer et al. (1991) EMBO J. 10(13):4025-4031).

d. ΔARE mouse model of arthritis/IBD

Mice lacking the 3' AU-rich element (ARE) of TNF mRNA (TnfΔ ^ARE ) overproduce TNF at 4-8 weeks of age and histopathologically develop inflammatory bowel disease similar to Crohn's disease. Mice also show clinical signs of RA. The efficacy of the TNFR1 antagonists, TNFR2 agonists and bispecific constructs provided herein can be evaluated by evaluating the inhibitory effect on Crohn's disease-like pathology and arthritis in TnfΔ ^{A RE} mice following intraperitoneal injection (e.g. , see U.S. Patent No. 9,028,822).

e. Humanized TNF/TNFR2 mice

A limitation of the development of therapeutics targeting the TNF/TNFR2 signaling pathway is that many human anti-TNF therapeutics do not interact with murine TNF or TNFR2, and human TNF may bind to and engage murine TNFR1, but not TNFNR2, in preclinical animals. that there is no model. Humanized TNF/TNFR2 mice carrying a functional human TNF-TNFR2 (hTNF-hTNFR2) signaling module can be used to evaluate therapeutics such as agonistic and antagonistic antibody constructs human TNF or human TNFR2 in various models of autoimmunity. there is. Such TNF/TNFR2 double humanized mice can be used, for example, to evaluate the TNFR2 agonist constructs provided herein.

Humanized TNF/TNFR2 mice are described by Atretkhany et al. (2018) Proc . Natl . Acad . Sci. USA . 115(51):13051-13056. Briefly, human TNFR2 knock-in (hTNFR2KI) and human TNF knock-in (hTNFKI) mice are generated using standard genetic engineering techniques. hTNFKI mice in which the human TNF gene has replaced the mouse TNF gene are crossed with hTNFR2KI mice containing the humanized TNFR2 ligand binding region and then crossed to generate double humanized double homozygous hTNFKI x hTNFR2KI mice. To assess the role of TNFR2 signaling in specific cells, eg Tregs, two LoxP sites were inserted within the hTNFR2 locus to allow conditional Cre-mediated ablation of the extracellular portion of TNFR2. For Treg-specific deletion of TNFR2, these mice are crossed with FoxP3-Cre transgenic mice (see, for example, Atretkhany et al. (2018) Proc. Natl. Acad. Sci. USA 115(51):13051-13056).

I. TNFR1 antagonist construct and TNFR1 antagonist/ TNFR2 agonist construct Methods of producing nucleic acids that encode

TNFR1 antagonist polypeptides, TNFR2 agonist polypeptides, and TNFR1 antagonist/TNFR2 agonist polypeptide constructs provided herein, which are peptides, can be used by methods well known in the art for protein purification and recombinant protein expression, and also for recombinant antibody preparation. can be obtained by Constructs provided herein that contain moieties, such as linkers, that are not polypeptides can, where appropriate, be prepared by chemical conjugation methods. The polypeptide portion can be generated by standard recombinant techniques, for example by expression in a suitable host (bacterial if glycosylation is not desired, or in eukaryotic cells such as HEK293 and CHO cells if a glycosylated form is desired). there is. Active antibodies and antibody fragments have been produced in E. coli but often suffer from aggregation and solubility problems due to improper folding, which can be addressed by further mutagenesis of the coding sequence (see, e.g., Kunz et al. (2018) Sci Rep. 8(1) :7934).

Polypeptides can also be chemically synthesized. Fusion polypeptides can be synthesized by standard methods of recombinant production. The components of the various constructs discussed above can be synthesized individually and combined using standard methods to create the construct.

Nucleic acids encoding polypeptide constructs or polypeptide portions thereof, including modifications or variants, including truncated forms, can be prepared from nucleic acids. Modified or variant polypeptides can be engineered from nucleic acids encoding wild-type polypeptides using standard recombinant DNA methods. For example, a modified TNF polypeptide such as a TNF mutein that selectively binds TNFR1 or TNFR2 and/or selectively antagonizes TNFR1 or selectively agonizes TNFR2 can be engineered from wild-type TNF, such as by site-directed mutagenesis of the encoding DNA. It can be. Any method known to those skilled in the art may be used. The discussion and explanations below in the examples are illustrative.

One. TNFR1 antagonist and TNRF2 agonist Isolation or Preparation of Nucleic Acids Encoding Polypeptides

Nucleic acids encoding TNFR1 antagonist polypeptides, TNFR2 agonist polypeptides and TNFR1 antagonist/TNFR2 agonist polypeptide constructs can be cloned or isolated using any available method known in the art for cloning and isolating nucleic acid molecules. . Such methods include polymerase chain reaction (PCR) amplification of nucleic acids and screening of libraries, including nucleic acid hybridization screening, antibody-based screening and activity-based screening. For example, where the polypeptide is produced by recombinant means, any method known to those skilled in the art can be used for the identification of nucleic acids encoding the desired polypeptide.

Nucleic acid molecules encoding the polypeptides herein may be produced synthetically, or as desired (e.g., the heavy chains and can be readily isolated and sequenced using conventional procedures, e.g., TNFR1 antagonist or TNFR2 agonist antibodies or fragments thereof. Any cell source known to produce or express the (s) can serve as a source of such DNA. In another example, once the sequence of DNA encoding the TNFR1 antagonist or TNFR2 agonist antibody or fragment(s) thereof has been determined, gene synthesis techniques can be used to construct a nucleic acid sequence.

Nucleic acid amplification methods, including, for example, polymerase chain reaction (PCR) methods, can be used to isolate nucleic acid molecules encoding a desired polypeptide. Examples of such methods include the use of a Perkin-Elmer Cetus thermal cycler and Taq polymerase (Gene Amp). A nucleic acid-containing material can be used as a starting material from which a nucleic acid molecule encoding a desired polypeptide can be isolated. For example, DNA and mRNA preparations, cell extracts, tissue extracts, fluid samples (eg, blood, serum and saliva), samples from healthy and/or diseased subjects can be used in the amplification method. In general, sources that may be human sources may be from any eukaryotic species, including, but not limited to, vertebrate, mammalian, human, porcine, bovine, feline, avian, equine, canine, and other primate sources where appropriate. there is. A nucleic acid library can also be used as a source of starting material. Primers can be designed to amplify a desired polypeptide. For example, primers can be designed based on the expressed sequence resulting in the desired polypeptide. Primers can be designed based on reverse translation of a polypeptide amino acid sequence. Degenerate primers can be used for amplification if desired. Oligonucleotide primers that hybridize to sequences at the 3' and 5' ends of the desired sequence can be used as primers to amplify the sequence by PCR from a nucleic acid sample. Primers can be used to amplify full-length polypeptides or truncated sequences thereof, such as nucleic acids encoding the TNFR1 antagonist/TNFR2 agonist polypeptide constructs provided herein, as well as any TNFR1 antagonist and TNFR1 agonist polypeptides. Nucleic acid molecules produced by amplification can be sequenced and identified as encoding the desired polypeptide or construct.

Mutagenesis techniques can be used to generate additional modified forms of TNFR1 antagonist or TNFR2 agonist antibodies or fragment(s) thereof and to create modified forms of active modifiers such as Fc and hinge region and linker portions. DNA can also be modified. For example, insertions, deletions, additions or replacements/substitutions of nucleotides can be performed using gene synthesis and routine molecular biology techniques. The additional nucleotide sequence encodes a polypeptide comprising a linker sequence containing restriction endonuclease sites for the purpose of cloning the synthetic gene into a vector, e.g., a protein expression vector or a vector designed for amplification of a core polypeptide encoding DNA sequence. Can be bound to nucleic acid molecules. Additional nucleotide sequences specifying functional DNA elements such as promoters, enhancers and IRES sequences can be operably linked to the nucleic acid molecule encoding the polypeptide. Examples of such sequences include, but are not limited to, promoter sequences and secretory sequences designed to promote intracellular protein expression, such as heterologous signal sequences designed to promote protein secretion. Such sequences are known to those skilled in the art. Additional nucleotide sequences, such as sequences specifying protein binding regions, may also be linked polypeptide encoding nucleic acid molecules. Such regions include, but are not limited to, sequences that facilitate uptake of the polypeptide into specific target cells or alter or enhance the pharmacokinetics of the synthetic gene product.

Tags and/or other moieties may be added, for example, to aid detection or affinity purification of the polypeptide. Additional nucleotide sequences may also be linked to the polypeptide-encoding nucleic acid molecule, such as, for example, base sequences specifying epitope tags or other detectable markers. Examples of such sequences include nucleic acid sequences encoding SUMO tags, or His tags or Flag tags.

Any amino acid sequence provided herein can be cloned into a vector and used by those skilled in the art to generate a corresponding encoding nucleic acid sequence that can be expressed to generate constructs comprising the polypeptides, antibodies and antibody fragments provided herein. It can be back-translated (also called back-translation) using standard commonly used methods. For example, there are several online tools that can be used to convert protein sequences into encoding DNA sequences, such as bioinformatics.org/sms2/rev_trans.html; biophp.org/minitools/protein_to_dna/demo.php; vivo.colostate.edu/molkit/rtranslate/; ebi.ac.uk/Tools/st/emboss_backtranseq/; molbiol.ru/eng/scripts/01_19.html; and geneinfinity.org/sms/sms_backtranslation.html. Such reverse translated sequences can be inserted into any of the expression vectors provided herein for expression and production of the provided antibodies or fragments. Anti-TFR1 and anti-TNFR2 antibodies, such as TNFR1 antagonist and TNFR2 agonist constructs, can be expressed as full-length or less than full-length proteins. For example, antibody fragments such as but not limited to single domain antibodies (dAbs), scFv fragments and Fab fragments may be expressed.

The identified and isolated nucleic acids can be inserted into an appropriate cloning vector. A number of vector-host systems known in the art can be used. Possible vectors include, but are not limited to, plasmids or modified viruses, provided that the vector system is compatible with the host cell used. Such vectors include, but are not limited to, bacteriophages such as lambda derivatives, or plasmids such as pCMV4, pBR322 or pUC plasmid derivatives or the pBlueScript vector (Stratagene, La Jolla, Calif.). For example, insertion into a cloning vector can be accomplished by ligating a DNA fragment into a cloning vector with complementary cohesive ends. Insertion can be performed using the TOPO cloning vector (Invitrogen, Carlsbad, Calif.).

If the cloning vector does not have complementary restriction sites used to fragment the DNA, the ends of the DNA molecule can be modified with enzymes. Alternatively, the desired site can be created by ligating nucleotide sequences (linkers) to the ends of the DNA; Such ligated linkers may contain specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences. In an alternative method, truncated vectors and polypeptide genes can be modified by homopolymer tailing.

Recombinant molecules can be introduced into host cells through, for example, transformation, transfection, infection, electroporation and sonoporation, so that many copies of the gene sequence are produced. In certain embodiments, transformation of a host cell with a recombinant DNA molecule incorporating an isolated polypeptide gene, cDNA or synthetic DNA sequence allows generation of multiple copies of the gene. Therefore, a gene can be obtained in large quantities by growing a transformant, isolating a recombinant DNA molecule from the transformant, and, if necessary, recovering the inserted gene from the isolated recombinant DNA.

For expression of antibodies and fragments thereof, generally a nucleic acid molecule encoding the heavy chain of an antibody is cloned into a vector, and a nucleic acid molecule encoding the light chain of an antibody is cloned into a vector. Methods for producing antibodies and portions thereof are well known (see, eg, US Pat. Nos. 4,816,567, 6,331,415, and 7,923,221, and numerous other important patents). Genes can be cloned into a single vector for dual expression or into separate vectors. If desired, the vectors may also contain additional sequences encoding additional constant region(s) or hinge regions to generate other antibody forms. Vectors can be transfected and expressed in host cells. Expression can be any cellular expression system known to those skilled in the art. For example, host cells include cells that do not otherwise produce immunoglobulin proteins to obtain antibody synthesis in a recombinant host cell. For example, host cells include simian COS cells, such as Chinese Hamster Ovary (CHO) cells such as CHO-DG44 (DHFR-) and FreeStyle ^™ CHO-S cells, Invitrogen), 293FS cells, HEK293 cells, NSO cells. or other myeloma cells. Other expression vectors and host cells are described herein.

Constructs provided herein, including TNFR1 antagonist, TNFR2 agonist and TNFR1 antagonist/TNFR2 agonist constructs, can comprise an antigen-binding fragment, e.g., a single domain antibody (dAb), Fab, Fab', Fab hinge, F( ab')2, short-chain Fv (scFv), scFv tandem, Fv, dsFv, scFv hinge, scFv hinge (ΔE), diabodies, Fd and Fd' fragments, including but not limited to full-length constructs or less than full-length can be or manifest. There are a variety of techniques for producing antibody fragments. For example, fragments can be derived through proteolytic digestion of intact antibodies (see, eg, Morimoto and Inouye (1992) Journal of Biochemical and Biophysical Methods 24:107-117; Brennan et al. (1985) Science 229:81-83). Fragments can also be produced directly by recombinant host cells. For example, dAb, Fab, Fv and scFv antibody fragments can all be expressed and secreted in host cells such as E. coli , CHO cells or HEK293 cells, thus facilitating mass production of such fragments. . F(ab') ₂ fragments may be generated by chemically coupling Fab'-SH fragments (see, eg, Carter et al. (1992) Bio/Technology , 10:163-167), or they may be It can be isolated directly from recombinant host cell culture. In some instances, the TNFR1 antagonist construct comprises a single domain antibody (dAb; see, eg, International Application Publication Nos. WO 2004/058820, WO 2004/081026, WO 2005/035572, WO 2006/038027, WO 2007/049017, WO 2008/149144, WO 2008/149148, WO 2010/094720, WO 2011/006914, WO 2011/051217, WO 2012/172070, WO 2012/104322, and WO 2015/104322; Enever et al., (2015 ) Protein Engineering, Design & Selection 28(3) :59-66), US Application Publication Nos. 2006/0083747, 2010/0034831, and 2012/0107330; and U.S. Patent Nos. 9,028,817 and 9,028,822), single-chain Fv fragments (scFv) (see, eg, International Application Publication Nos. WO 2017/174586, and WO 2008/113515; also [Richter, F. Thesis, titled ["Evolution of the Antagonistic Tumor Necrosis Factor Receptor One-Specific Antibody ATROSAB," Universitat Stuttgart, 2015; available from pdfs.semanticscholar.org/d8e7/8b87d76dce36225c1d497939ef37445cfa8a.pdf). dAb, Fv and scFv fragments have intact binding sites but no constant regions; They are therefore suitable for reduced non-specific binding during in vivo use. dAb and scFv fusion proteins can be constructed to attach an effector protein (eg, an IgG Fc) to either the amino terminus or the carboxy terminus of the dAb or scFv. Antibody fragments can also be linear antibodies (see, eg, US Pat. No. 5,641,870). Such linear antibody fragments may be monospecific or bispecific. Other techniques for the production of antibody fragments are known to those skilled in the art.

Upon expression, the antibody heavy and light chains, or fragment(s) thereof, are paired by interchain disulfide bonds to form a full length antibody or fragment(s) thereof. For example, for expression of a full-length Ig, a sequence encoding V _H -C _H 1-hinge-C _H 2-C _H 3 can be cloned into a first expression vector and encodes a V _L -C _L domain. A sequence may be cloned into a second expression vector. When co-expressed, full-length heavy and light chains are interconnected by disulfide bonds to generate full-length antibodies. In another example, to generate a Fab, sequences encoding fragments containing the V _H and C _H 1 regions can be cloned into a first expression vector, and sequences encoding the V _L -C _L domains can be cloned into a second expression vector. Can be cloned into expression vectors. Upon co-expression, the heavy chain pairs with the light chain to produce a Fab monomer. The sequences of the C _H 1, hinge, C _H 2 and/or C _H 3 regions of the various IgG subtypes are known to those skilled in the art (see, eg, US Publication No. 2008/0248028; see also SEQ ID NO: 9, 11, 13, and 15). Similarly, sequences of C _L , lambda or kappa are also known (eg, US Publication No. 2008/0248028; see also SEQ ID NOs: 17-22)

In addition to recombinant production, the TNFR1 antagonist polypeptides, TNFR2 agonist polypeptides and TNFR1 antagonist/TNFR2 agonist polypeptide constructs provided herein can be produced by direct peptide synthesis using well known solid phase techniques. In vitro protein synthesis can be performed using manual techniques or automation. Automated synthesis can be achieved using, for example, the Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer; Foster City, CA) according to manufacturer-supplied instructions. The various fragments of a polypeptide can be individually chemically synthesized and combined using chemical methods.

2. Generation of Mutant or Modified Nucleic Acids and Encoding Polypeptides

Modifications provided herein can be made by standard recombinant DNA techniques, such as are common to those skilled in the art. Any method known in the art for mutating any one or more amino acids in a target protein or polypeptide can be used. Methods include standard site-directed mutagenesis encoding nucleic acid molecules (eg, using kits such as the QuikChange kit available from Stratagene) or solid phase polypeptide synthesis methods.

3. Vectors and Cells

For recombinant expression of one or more of the desired polypeptides, such as any TNFR1 antagonist or TNFR2 agonist polypeptide, or TNFR1 antagonist/TNFR2 agonist polypeptide construct described herein, a nucleic acid containing all or part of the nucleotide sequence encoding the polypeptide The molecule can be inserted into an appropriate expression vector, i.e., a vector containing the necessary elements for transcription and translation of the inserted polypeptide coding sequence. Also provided are vectors containing nucleic acid molecules encoding the polypeptides. After insertion of the nucleic acid molecule(s), the vector is typically used to transform a host cell, eg to amplify the nucleic acid for replication and/or expression thereof. In these examples, vectors suitable for high levels of expression are used. In other cases, a vector is selected that is compatible with the display of the polypeptide expressed on the cell surface. The choice of vector may depend on the desired application. Many expression vectors are available and known to those skilled in the art for the expression of anti-TNFR1 and anti-TNFR2 antibodies or portions thereof, such as antigen-binding fragments. Such selections are within the level of skill of those skilled in the art. In general, expression vectors may include transcriptional promoters and optionally enhancers, translational signals and transcriptional and translational termination signals. Expression vectors used for stable transformation typically have selectable markers that allow selection and maintenance of transformed cells. Optionally, a high copy number origin of replication can be used to amplify the copy number of a vector in a cell. Vectors may also contain additional nucleotide sequences operably linked to nucleic acid molecules (eg, His tags, flag tags) to which they are normally ligated. For applications with antibodies, vectors generally include sequences encoding the constant regions. Thus, antibodies or portions thereof may also be expressed as protein fusions. For example, fusion proteins can be created to add additional functions to a polypeptide. Examples of fusion proteins are the fusion of a signal sequence, an epitope tag such as for localization, e.g. His ₆ tag or myc tag, or a tag for purification, e.g. GST tag, and/or directing protein secretion and/or membrane binding. It includes, but is not limited to, sequences that The fusion proteins herein also include the fusion of a TNFR1 antagonist and/or TNFR2 agonist to a modified Fc region, the hinge region of an IgG, and/or a peptide linker, such as a GS linker.

A variety of host-vector systems can be used to express protein coding sequences. These include mammalian cell systems infected with viruses (eg vaccinia virus, adenovirus and other viruses); insect cell systems infected with viruses (eg, baculovirus); microorganisms such as yeast containing yeast vectors; and bacteria transformed with bacteriophage, DNA, plasmid DNA or cosmid DNA. The choice between eukaryotic and bacterial systems depends on the desired post-translational modification, such as glycosylation. The expression elements of vectors vary in intensity and specificity. Depending on the host-vector system used, one of several suitable transcription and translation elements may be used.

Any method known to those skilled in the art can be used to insert a DNA fragment into a vector that encodes a polypeptide, e.g., an antibody fragment provided herein or a TNFR1 antagonist, or TNFR2 agonist, as well as appropriate transcriptional/translational control signals. Expression vectors containing nucleic acid molecules can be constructed. These methods may include in vitro recombinant DNA and synthetic techniques, and in vivo recombinants (genetic recombination). For example, insertion into a cloning vector can be accomplished by ligating a DNA fragment into a cloning vector with complementary cohesive ends. If the cloning vector does not have complementary restriction sites used to fragment the DNA, the ends of the DNA molecule can be modified with enzymes. Alternatively, the desired site can be created by ligating nucleotide sequences (linkers) to the ends of the DNA; Such ligated linkers may contain specific chemically synthesized nucleic acids encoding restriction endonuclease recognition sequences.

For example, expression of construct polypeptides such as the TNFR1 antagonist, TNFR2 agonist and TNFR1 antagonist/TNFR2 agonist constructs herein may be controlled by any promoter/enhancer known in the art. Suitable bacterial promoters are well known in the art and are described herein below. Other suitable promoters for mammalian cells, yeast cells and insect cells are well known in the art and some are exemplified below. The choice of promoter used to direct expression of the heterologous nucleic acid depends on the particular application. Promoters that may be used include, but are not limited to: eukaryotic expression vectors containing the SV40 early promoter (see, e.g., Benoist and Chambon (1981) Nature 290:304-310), 3' long terminal repeat The promoters contained in the fetal Lux sarcoma virus (see, e.g., Yamamoto et al. (1980) Cell 22 :787-797), the herpes thymidine kinase promoter (see, eg, Wagner et al. (1981) Proc. Natl . Acad . Sci . USA . 78 :1441-1445), the regulatory sequence of the metallothionein gene (see, eg, Brinster et al. (1982) Nature 296:39-42), and the cytomegalovirus (CMV) promoter; Prokaryotic expression vectors such as the β-lactamase promoter (see, eg, Jay et al. (1981) Proc . Natl . Acad . Sci . USA . 78 :5543), or the tac promoter (see, e.g., DeBoer et al. (1983) Proc . Natl . Acad . Sci . USA . 80 :21-25); See also "Useful Proteins from Recombinant Bacteria": in Scientific American 242 :79-94 (1980); The nopaline synthetase promoter is used in plant expression vectors (see, eg, Herrara-Estrella et al. (1984) Nature 303:209-213), the cauliflower mosaic virus 35S RNA promoter (see, eg, Gardner et al. (1981 ) Nucleic Acids Res . Promoter elements from yeast and other fungi such as the Gal4 promoter, alcohol dehydrogenase promoter, phosphoglycerol kinase promoter, alkaline phosphatase promoter, and the following animal transcriptional regulatory regions that exhibit tissue specificity and are used in transgenic animals: in pancreatic acinar cells The active elastase I gene control region (see, eg, Swift et al. (1984) Cell 38 :639-646; Ornitz et al. (1986) Cold Spring Harbor Symp . Quant . Biol . 50 :399-409; MacDonald (1987) Hepatology 7 :425-515), insulin gene control region active in pancreatic beta cells (see, eg, Hanahan et al. (1985) Nature 315:115-122), immunoglobulins active in lymphoid cells Gene control regions (see e.g. Grosschedl et al. (1984) Cell 38 :647-658; Adams et al. (1985) Nature 318 :533-538; Alexander et al. (1987) Mol . Cell Biol . 7 :1436-1444), mouse mammary tumor virus control region active in testis, mammary, lymphoid and mast cells (see, e.g., Leder et al. (1986) Cell 45 :485-495), the albumin gene control region active in the liver (see, eg, Pinckert et al. (1987) Genes and Devel. 1 :268-276), the alpha-fetoprotein gene control region active in the liver (see, eg, Krumlauf et al. (1985) Mol . Cell. Biol . 5 :1639-1648; Hammer et al. (1987) Science 235:53-58), the alpha-1 antitrypsin gene control region active in the liver (see, eg, Kelsey et al. (1987) Genes and Devel . 1 :161-171), the beta globin gene control region active in myeloid cells (see, e.g., Magram et al. (1985) Nature 315 :338-340; Kollias et al. (1986) Cell 46 :89-94), myelin base protein gene control region active in oligodendrocyte cells of the brain (reference, for example, Readhead et al. (1987) Cell 48 :703-712), active in skeletal muscle the phosphorus myosin light chain-2 gene control region (see, eg, Shani (1985) Nature 314:283-286), and the gonadotropin-releasing hormone gene control region active in hypothalamic gonadotropins (see, eg, For example, Mason et al. (1986) Science 234:1372-1378).

Expression vectors typically contain a transcription unit or expression cassette containing all additional elements necessary for expression of the construct or portion(s) thereof in a host cell. A typical expression cassette includes a promoter operably linked to a nucleic acid encoding a construct such as an antibody fragment, domain, derivative or analog thereof, or other polypeptides described herein (eg, TNF muteins and fusion proteins), and the entire It contains signals necessary for efficient polyadenylation of transcripts, ribosome binding sites, and translational termination. Additional elements of the cassette may include enhancers. Cassettes also typically include transcriptional termination regions downstream of structural genes to provide efficient termination. The termination region may be from the same gene as the promoter sequence or may be from a different gene. For example, a vector is operable for a nucleic acid encoding a desired polypeptide or domain, a fragment, derivative or homologue thereof, one or more origins of replication, and optionally one or more selectable markers (eg, antibiotic resistance genes). contains a promoter linked to the

The expression system can have markers that provide gene amplification, such as thymidine kinase and dihydrofolate reductase. Expression systems that do not involve gene amplification are also suitable, such as using baculovirus vectors in insect cells that have a nucleic acid sequence encoding a polypeptide under the direction of a polyhedral promoter or other strong baculovirus promoter.

For purposes herein, the Fc region of an IgG antibody, typically encoding a TNFR1 antagonist or TNFR2 agonist polypeptide and comprising an IgG hinge sequence and/or a (Gly ₄ Ser) _n , where n is an amount equal to or greater than 1-5 Encoding a modified Fc operably linked to a nucleic acid encoding an interstitial linker such as a short peptide linker such as a GS linker, including a glycine-rich flexible linker such as an integer), and other linkers described herein or known to those skilled in the art. A vector containing a sequence of nucleotides is provided. The vector may include sequences for one or all of C _H 1 , C _H 2 , hinge, C _H 3 or C _H 4 and/or C _L . Typically, like the expression of Fabs, vectors contain sequences for C _H 1 or C _L (kappa or lambda light chains). For example, V _H -C _H 1 and V _L -C _L sequences can be inserted into an expression vector suitable for expression of Fab molecules. The sequence of the constant region or hinge region is known to those skilled in the art (see, eg, US Publication No. 2008/0248028). Examples of such sequences are provided herein.

Typically, the vectors will be plasmids, viral vectors, or others known in the art for use in the expression of polypeptides in vivo or in vitro. For example, constructs provided herein, such as nucleic acids encoding TNFR1 antagonist and TNFR2 agonist polypeptide constructs, are expressed in mammalian cells, including, for example, Chinese Hamster Ovary (CHO) cells.

Exemplary eukaryotic vectors include well-known and readily available vectors such as, for example, pCMV (Agilent Technologies), pCDNA3.1 (Invitrogen (Thermo Fisher Scientific)), pCBL (from Creative BioLabs, see, eg, Figure 1). include Other eukaryotic vectors can be used as eukaryotic expression vectors, for example those containing any regulatory elements from eukaryotic viruses. Included herein are vectors derived from, for example, SV40 vectors, papillomavirus vectors, and Epstein-Bar viruses. Exemplary eukaryotic vectors include, for example, pMSG, pAV009/A+, pMT010/A+, pMAMneo-5, baculovirus pDSCE, and the CMV promoter, SV40 early promoter, SV40 late promoter, metallothionein promoter, murine mammary tumor Any other vector that allows protein expression under the direction of a viral promoter, a roux sarcoma virus promoter, a polyhedral promoter, or other promoters shown to be effective for expression in eukaryotes.

Viral vectors such as adenovirus, retrovirus or vaccinia virus vectors may be used. In some instances the vector is a defective or attenuated retroviral or other viral vector (see eg US Pat. No. 4,980,286). For example, retroviral vectors can be used (see, e.g., Miller et al. (1993) Meth . Enzymol . 217:581-599]). These retroviral vectors have been modified to delete retroviral sequences that are not required for viral genome packaging and integration into host cell DNA. In some instances, a virus armed with a nucleic acid encoding a polypeptide of the present disclosure is capable of promoting replication and spread within a target tissue. The virus can also be a lytic virus or a non-lytic virus in which the virus selectively replicates under a tissue-specific promoter. As the virus replicates, co-expression of viral genes and polypeptides will facilitate the spread of the virus in vivo.

For bacterial expression, vectors include the well known and widely disseminated vectors pBR322, pUC, pSKF, pET23D, and fusion vectors such as MBP (Sigma-Aldrich), GST (Sigma-Aldrich) and LacZ containing vectors. Exemplary plasmid vectors for transformation of E. coli cells include, for example, the pQE expression vector (available from ^Qiagen® , Valencia, CA; see also literature published by Qiagen® describing the system). The pQE vector has a phage T5 promoter (recognized by E. coli RNA polymerase) and a dual lac operator suppressor module to enable efficient translation in E. coli , a synthetic ribosome binding site (RBS II), a 6XHis tag encoding sequence, t ₀ and Provides tightly controlled, high-level expression of recombinant proteins in the T1 transcription terminator, the ColE1 origin of replication and the beta-lactamase gene to confer ampicillin resistance. The pQE vector allows placement of a 6xHis tag at the N- or C-terminus of the recombinant protein. These plasmids include pQE 32, pQE 30 and pQE 31, which provide multiple cloning sites for all three reading frames and expression of N-terminal 6xHis tagged proteins. Other exemplary plasmid vectors for transformation of E. coli cells include, for example, pET expression vectors (see, eg, US Pat. No. 4,952,496; available from NOVAGEN, Madison, WI; also described systems see literature published by NOVAGEN). Such plasmids include pET 11a, which contains a T7lac promoter, a T7 terminator, an inducible E. coli lac operator and a lac suppressor gene; pET 12a-c containing the T7 promoter, T7 terminator and E. coli ompT secretion signal; and pET 15b and pET19b ( ^NOVAGEN , Madison, WI) included.

Cells containing the vectors are also provided. Generally, any cell type that can be engineered to express heterologous DNA and has a secretory pathway is suitable. Cells include eukaryotic and prokaryotic cells, and vectors are any suitable for use therein. Generally, a cell is a cell capable of glycosylation of an encoded protein. Prokaryotic and eukaryotic cells comprising the vectors are provided. Such cells include bacterial cells, yeast cells, fungal cells, Archea, plant cells, insect cells and animal, especially mammalian cells. A cell is used to produce a polypeptide by growing the cell described above under conditions in which the encoded polypeptide is expressed by the cell and recovering the expressed polypeptide. For example, for purposes herein, a polypeptide may be secreted into a medium.

The host cell strain may be selected for its ability to control the expression of the inserted sequence or to process the expressed protein in a desired manner. Such modifications of polypeptides include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing can affect the folding and/or function of a polypeptide. Different host cells, such as but not limited to Chinese Hamster Ovary (CHO) cells, such as DG44, FreeStyle ^™ CHO-S cells (Invitrogen), DXB11, CHO-K1), HeLa, MCDK, HEK293 and WI38 cells, have such post-translational activity It has a specific cellular machinery and characteristic mechanism for the protein and can be selected to ensure the correct modification and processing of the introduced protein. Generally, the cell of choice is a cell capable of introducing N-linked glycosylation to the expressed polypeptide. Thus, eukaryotic cells containing the vectors are provided. An example of a eukaryotic cell is the mammalian Chinese Hamster Ovary (CHO) cell. For example, CHO cells deficient in dihydrofolate reductase (DHFR ⁻ ), such as DG44 cells, are used to produce the polypeptides provided herein.

4. Expression

Polypeptide constructs provided herein, including TNFR1 antagonist constructs, TNFR2 agonist constructs, TNFR1 antagonist/TNFR2 agonist multispecific constructs and portions thereof, can be used in vivo and in vitro methods, recombinant and synthetic and chemical It can be produced by any method known to those skilled in the art for protein production, including methods. The desired protein may be expressed in any organism suitable for producing the required amount and form of the protein, eg, as required for administration and treatment. Expression hosts include prokaryotic and eukaryotic organisms such as E. coli , yeast, plant, insect cells, mammalian cells including human cell lines, and transgenic animals. Expression hosts may differ in the level of protein production and the type of post-translational modifications present in the expressed protein. Selection of an expression host can be made based on these and other factors known to those skilled in the art; These include regulatory and safety considerations, production costs and the need and methods of purification. Purification methods and component assembly methods are well known to those skilled in the art.

Expression in eukaryotic hosts is yeast, such as Saccharomyces Serrebiziae and Pichia Pasteuris , insect cells such as Drosophila cells and Lepidopteran cells, plants and plant cells such as tobacco, corn, rice, algae and Lemna. Eukaryotic cells for expression also include mammalian cell lines, such as Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK293) cells, or juvenile hamster kidney (BHK) cells. Eukaryotic expression hosts also include production in transgenic animals, such as production in serum, milk and eggs.

Many expression vectors are available and known to those skilled in the art and can be used to express proteins. The choice of expression vector will be influenced by the choice of host expression system. In general, expression vectors may include transcriptional promoters and optionally enhancers, translational signals and transcriptional and translational termination signals. Expression vectors used for stable transformation typically have selectable markers that allow selection and maintenance of transformed cells. Optionally, an origin of replication can be used to amplify the copy number of a vector in a cell.

The TNFR1 antagonist, TNFR2 agonist and bispecific TNFR1 antagonist/TNFR2 agonist constructs herein may also be expressed as protein fusions. For example, fusion proteins can be created to add additional functions to a polypeptide. Examples of fusion proteins include the fusion of a signal sequence, a tag such as for localization, such as a his ₆ tag or a myc tag, or a tag for purification, such as a GST fusion, and sequences directing protein secretion and/or membrane binding. including but not limited to Fusion proteins also include fusions of an Fc region of an IgG with a linker, such as the hinge sequence of an IgG and/or a glycine-serine (GS) peptide linker. Alternatively, in some embodiments, the TNFR1 antagonist, TNFR2 agonist and bispecific TNFR1 antagonist/TNFR2 agonist constructs of the present disclosure may also be fused to serum albumin.

For long-term, high-yield production of recombinant proteins, stable expression is required. For example, a cell line stably expressing the polypeptide can be transformed with an expression vector containing a viral origin of replication or endogenous expression element and a selectable marker gene. After vector introduction, cells are allowed to grow for 1-2 days in rich medium before being switched to selective medium. The purpose of the selectable marker is to confer resistance to selection, and its presence enables the growth and recovery of cells that successfully express the introduced sequence. Resistant cells of the stably transformed cells can be propagated using tissue culture techniques appropriate to the cell type.

Any number of selection systems can be used to recover transformed cell lines. These include herpes simplex virus (HSV) thymidine kinase (TK) (see, eg, Wigler et al., (1977) Cell 11:223-232) and adenine phosphoribosyltransferase (APRT) (eg, , see Lowy, I. et al. (1980) Cell, 22:817-23) genes, which may be used in TK ^- or APRT ^- cells, respectively. In addition, antimetabolite, antibiotic or herbicide resistance can be used as selection criteria. For example, dihydrofolate reductase (DHFR), which confers resistance to methotrexate (see, for example, Wigler et al. (1980) Proc . Natl . Acad . Sci . USA . 77:3567-70); npt conferring resistance to the aminoglycoside neomycin and G-418 (see, for example, Colbere-Garapin et al. (1981) J. Mol . Biol . 150:1-14]); als or pats conferring resistance to chlorsulfuron and phosphinotricin acetyltransferases, respectively, may be used. Additional selectable genes have been described, such as trpB, which allows cells to utilize indole instead of tryptophan, or hisD, which allows cells to utilize histinol instead of histidine (see, for example, Hartman, SC and RC Mulligan (1988) Proc. Natl . Acad . Sci . USA 85:8047-8051) . Visible markers such as (but not limited to) anthocyanins, beta glucuronidase and its substrate GUS, luciferase and its substrate luciferin can also identify transformants and enable transient or stable protein expression due to specific vector systems. can be used to quantify the amount of (see, for example, Rhodes et al. (1995) Methods Mol. Biol. 55:121-131]).

a. prokaryotic cell

Prokaryotes, particularly E. coli , provide systems for producing large amounts of protein. Prokaryotic expression systems are generally used for the production of non-glycosylated products. Transformation of E. coli protocols are well known to those skilled in the art. Expression vectors for E. coli may contain an inducible promoter; Such promoters are useful for expressing proteins that drive high levels of protein expression and are slightly toxic to the host cell. Examples of inducible promoters include, for example, the lac promoter, the trp promoter, the hybrid tac promoter, the T7 and SP6 RNA promoters, and the temperature regulated λPL promoter.

Polypeptides and fusion protein constructs provided herein, such as any provided herein, can be expressed in the cytoplasmic environment of E. coli . The cytoplasm is a reducing environment and for some molecules insoluble inclusion bodies may form. Proteins can be resolubilized using reducing agents such as dithiothreitol and β-mercaptoethanol, and denaturants such as guanidine-HCl and urea. Another approach is to express proteins in the periplasmic space of bacteria that provide an oxidizing environment and chaperonin-like and disulfide isomerases and can induce soluble protein production. Typically, a leader sequence is fused to the protein to be expressed, which directs the protein to the periplasm. The leader is then removed by signal peptidases inside the periplasm. Exemplary pathways that translocate expressed proteins to the periplasm are the Sec pathway, the SRP pathway and the TAT pathway. Examples of periplasmic targeting leader sequences include the pelB leader from the pectate lyase gene, the StII leader sequence and the DsbA leader sequence, and the leader derived from the alkaline phosphatase gene. In some cases, periplasmic expression may result in leakage of the expressed protein into the culture medium. Protein secretion allows quick and simple purification from the culture supernatant. Proteins that are not secreted can be obtained from the periplasm by osmotic lysis. Similar to cytoplasmic expression, in some cases proteins can become insoluble and denaturants and reducing agents can be used to promote solubilization and refolding. Induction and growth temperature can also affect expression levels and solubility; Typically, temperatures between 25 °C and 37 °C are used. Typically, bacteria produce non-glycosylated proteins. Thus, if a protein requires glycosylation for function, glycosylation can be added in vitro after purification from the host cell.

b. yeast cells

yeast, such as Saccharomyces cerevisiae , Shizoscaromyces Pombe , Yarrowia lipolytica , Kluyveromyces lactis , and Pichia Pasteuris is a well-known expression host that can be used to produce proteins such as any of those described herein. Yeast can be transformed with stable chromosomal integration or episomal replication vectors by homologous recombination. In general, inducible promoters are used to regulate gene expression. Examples of such promoters include GAL1, GAL7, and GAL5, and metallothionein promoters such as CUP1, AOX1 or other Pichia or other yeast promoters. Expression vectors often include selectable markers such as LEU2, TRP1, HIS3 and URA3 for selection and maintenance of the transformed DNA. Proteins expressed in yeast are often soluble. Co-expression with chaperonins such as Bip and protein disulfide isomerase can improve expression levels and solubility. In addition, proteins expressed in yeast are secretion signal peptide fusions from Saccharomyces cerevisiae , such as yeast mating type alpha-factor secretion signal and yeast cell surface proteins such as Aga2p mating adhesion receptor, or Aluxula Adenine Ivorans ( Arxula fusion with adeninivoran s) glucoamylase can be used to direct secretion. Protease cleavage sites, such as the Kex-2 protease, can be engineered to remove fused sequences from expressed polypeptides as they exit the secretory pathway. Yeast can also glycosylate at Asn-X-Ser/Thr motifs.

c. insects and insect cells

Insect cells, particularly using baculovirus expression, are useful for expressing polypeptides, including antibodies or fragments thereof. Insect cells express high levels of proteins and are capable of most post-translational modifications used by higher eukaryotes. Baculoviruses have a limited host range that improves safety and reduces regulatory problems of eukaryotic expression. Typically, expression vectors use promoters for high-level expression, such as the polyhedrin promoter and the p10 promoter of baculovirus. Baculovirus systems include baculoviruses such as autographa Californica nuclear polyhedron disease virus (AcNPV), and Bombyx mori nuclear polyhedron disease virus (BmNPV), and insect cell lines such as Spodoptera Sf9 from Prugiperda , TN from Trichoplutus age , Pseudaletia A7S derived from Unifuncta , and Danus including DpN1 derived from Flexipus . For high level expression, the nucleotide sequence of the molecule to be expressed is fused immediately downstream of the polyhedrin initiation codon of the virus. To generate baculovirus recombinants capable of expressing human antibodies, dual expression flyers such as pAcUW51 (PharMingen) can be used. Mammalian secretion signals are correctly processed in insect cells and can be used to secrete the expressed proteins into the culture medium. Cell lines Pseudaletia unifuncta (A7S) and Danus Flexipus (DpN1) produces proteins with glycosylation patterns similar to mammalian cell systems. Exemplary insect cells are cells that have been altered to reduce immunogenicity, including cells with "mammalianized" baculovirus expression vectors and cells lacking the enzyme FT3.

An alternative expression system for insect cells is to use stably transformed cells. cell lines such as Schneider 2 (S2) and Kc cells ( Drosophila melanogaster ), and C7 cells ( Aedes albopictus ) can be used for expression. The Drosophila metallothionein promoter can be used to drive high-level expression in the presence of heavy metal induction with cadmium or copper. Consistent levels of expression can be induced using the baculovirus immediate early gene promoter IE1. Typical expression vectors include the pIE1-3 and pI31-4 transfer vectors (Novagen). Expression vectors are typically maintained using selectable markers such as neomycin and hygromycin.

d. mammalian expression cells

Mammalian expression systems can be used to express polypeptides, including constructs herein, including TNFR1 antagonists, TNFR2 agonists, bispecific TNFR1 antagonist/TNFR2 agonist constructs, and fusions thereof provided herein. Expression constructs are introduced into mammalian cells by viral infection, such as adenovirus, or by direct DNA transfer, such as using liposomes, calcium phosphate and DEAE-dextran, and by physical means, such as electroporation and microinjection. can be conveyed Expression vectors for mammalian cells typically include an mRNA cap site, a TATA box, a translation initiation sequence (Kozak consensus sequence) and a polyadenylation element. An internal ribosome entry site (IRES) element may be added to allow bicistronic expression with other genes as selectable markers. Such vectors often include transcriptional promoter-enhancers for high-level expression, such as, for example, the SV40 promoter-enhancer, the human cytomegalovirus (CMV) promoter and the long terminal repeats of Roux sarcoma virus (RSV). These promoter-enhancers are active in many cell types. Tissue and cell type promoters and enhancer regions can also be used for expression. Exemplary promoter/enhancer regions include genes such as elastase I, insulin, immunoglobulin, mouse mammary tumor virus, albumin, alpha-fetoprotein, alpha-1 antitrypsin, beta-globin, myelin base protein, myosin light chain-2, and those from the gonadotropin releasing hormone gene control.

Selectable markers can be used to select and maintain cells with expression constructs. Examples of selectable marker genes include hygromycin B phosphotransferase, adenosine deaminase, xanthine-guanine phosphoribosyltransferase, aminoglycoside phosphotransferase, dihydrofolate reductase (DHFR), and thymidine kinase (TK). For example, expression can be performed in the presence of methotrexate to select only cells expressing the DHFR gene. Modified anti-TNFR antibodies and antigen-binding fragments thereof can be produced using, for example, the NEO ^R /G418 system, the dihydrofolate reductase (DHFR) system or the glutamine synthetase (GS) system. The GS system expresses both heavy and light chains using joint expression vectors such as pEE12/pEE6. Fusion with cell surface signaling molecules such as TCR-ξ and Fc _ε RI-γ can direct the expression of proteins in an active state at the cell surface.

Many cell lines are available for mammalian expression, including mouse, rat, human, monkey, chicken and hamster cells. Exemplary cell lines include BHK (eg, BHK-21 cells), 293-F, CHO, CHO Express (CHOX; ExcellGene), Balb/3T3, HeLa, MT2, mouse NS0 (non-secreting), and other myeloma cell lines. , hybridoma and heterohybridoma cell lines, lymphocytes, fibroblasts, Sp2/0, COS, NIH3T3, HEK293, 293S, 2B8, and HKB cells. Cell lines adapted to serum-free media can also be used, which facilitates the purification of secreted proteins from the cell culture media. Examples include CHO-S cells ( ^Invitrogen® , Carlsbad, CA, cat # 11619-012) and the serum-free EBNA-1 cell line (eg, Pham et al. (2003) Biotechnol . Bioeng . 84:332-342]). Cell lines suitable for growth in special media optimized for maximal expression are also available. For example, DG44 CHO cells are adapted to grow in suspension culture in chemically defined animal product free media.

e. plant

Transgenic plant cells and plants can be used to express polypeptides and proteins as described herein. Expression constructs are typically delivered to plants using direct DNA transfer, such as microprojectile bombardment and PEG-mediated transfer to protoplasts, and using Agrobacterium-mediated transformation. Expression vectors can include promoter and enhancer sequences, transcriptional termination elements, and translational control elements. Expression vectors and transformation techniques are generally divided into dicotyledonous hosts such as Arabidopsis and tobacco and monocotyledonous hosts such as corn and rice. Examples of plant promoters used for expression include, for example, the cauliflower mosaic virus promoter (CaMV 35S), the nopaline synthetase promoter, the ribose bisphosphate carboxylase promoter, and ubiquitin (e.g., corn ubiquitin-1 ( ubi -1 )) and the UBQ3 promoter. Selectable markers such as hygromycin, phosphomannose isomerase and neomycin phosphotransferase are often used to facilitate selection and maintenance of transformed cells. Transformed plant cells can be maintained in culture as cells, aggregates (callus tissue) or regenerated into whole plants. Transgenic plant cells may also include algae engineered to produce the polypeptide. Plants have different glycosylation patterns than mammalian cells, which can influence the choice of proteins produced in these hosts.

5. Refining

Host cells transformed with nucleic acids encoding the polypeptide constructs provided herein can be cultured under conditions suitable for expression and recovery of the encoded protein from cell culture. Proteins produced by recombinant cells are usually secreted, but may be contained within the cell depending on the sequence and/or vector used. As will be appreciated by those skilled in the art, expression vectors containing nucleic acid molecules encoding the polypeptides provided herein can be designed with signal sequences that facilitate direct secretion of the expressed polypeptides across prokaryotic or eukaryotic cell membranes.

Methods for purifying the polypeptide from host cells depend on the host cell and expression system chosen. In the case of secreted molecules, the protein is usually purified from the culture medium after removing the cells. For intracellular expression, the cells can be lysed and the protein can be purified from the extract. When transgenic organisms such as transgenic plants and animals are used for expression, lysed cell extracts can be prepared using tissues or organs as starting materials. Additionally, transgenic animal production can include production of the polypeptide in milk or eggs, which can be harvested, and if necessary, the protein can be extracted and further purified using standard methods in the art.

Polypeptides, such as TNFR1 antagonist constructs, TNFR2 agonist constructs, TNFR1 antagonist/TNFR2 agonist bispecific constructs, and other constructs provided herein, and components thereof, can be purified using protein purification techniques known to those skilled in the art. It can be. These include, but are not limited to, SDS-PAGE, size fractionation and size exclusion chromatography, ammonium sulfate precipitation, chelation chromatography, column chromatography, HPLC, dialysis, and ion exchange chromatography such as anion exchange, and combinations thereof. don't Affinity purification techniques can also be used. Constructs herein may be purified using methods developed for the purification of antibodies and antibody fragments. An example of a method for purifying antibodies and antibody fragments is a method comprising column chromatography, wherein the solid supported column material is conjugated to Protein G, a cell surface associated protein from Streptococcus that binds immunoglobulins with high affinity. Connected. Antibodies and antibody fragments can also be purified by methods comprising Protein A chromatography, wherein Protein A binds immunoglobulins such as IgG with high affinity and is associated with the cell surface of Staphylococcus aureus . Protein is bound to the solid support column. Other immunoglobulin binding bacterial proteins that can be used to purify antibodies and antibody fragments include Protein A/G, a recombinant fusion protein that combines the IgG binding domains of Protein A and Protein G; and protein L, a surface protein from Peptostreptococcus (see, e.g., Bjorck (1988) J. Immunol . 140(4):1194-1197; Kastern et al. (1992) J. Biol . Chem . 267(18):12820-12825; Eliasson et al. (1988) J. Biol . Chem . 263:4323-4327]). The construct is substantially pure, and is typically at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% pure. Purity can be assessed by standard methods such as SDS-PAGE and Coomassie blue staining.

When antibodies and fragments thereof and related polypeptides are expressed in large amounts by transformed bacteria, typically after promoter induction, expression may be constitutive, but the polypeptide may form insoluble aggregates. Protocols for purification of polypeptide inclusion bodies are known to those skilled in the art. For example, in one method, the cell suspension is centrifuged to pellet the inclusion bodies, and the pellet containing the inclusion bodies is dissolved in a buffer such as 20 mM Tris-HCL, pH 7.2, 1 mM EDTA, 150 mM NaCl and 2 mM % Triton-X 100, non-ionic detergent, which does not harm the inclusion body but dissolves contaminants. Washing steps can be repeated to remove as much cell debris as possible. The remaining pellet of inclusion bodies is resuspended in an appropriate buffer such as 20 mM sodium phosphate, pH 6.8, 150 mM NaCl. Other suitable buffers as well as alternative purification protocols are known or can be developed by those skilled in the art.

Other methods for purification, antibodies and fragments thereof, and constructs provided herein may be used or developed. It can be purified from bacterial periplasm. For polypeptides exported to the periplasm of the bacteria in which the polypeptide is produced, the periplasmic fraction of the bacteria can be isolated by cold osmotic shock in addition to other methods known to those skilled in the art. For example, in one method, to isolate recombinant polypeptide from the periplasm, bacterial cells are centrifuged to form a pellet. The pellet is resuspended in buffer containing 20% sucrose. To lyse the cells, the bacteria are centrifuged and the pellet is resuspended in ice-cold 5 mM MgSO ₄ and kept in an ice bath for about 10 minutes. The cell suspension is centrifuged and the supernatant is decanted and stored. Recombinant polypeptide present in the supernatant can be separated from host proteins by standard separation techniques well known to those skilled in the art. These methods include, but are not limited to, the following steps: solubility fractionation, size differential filtration, and column chromatography.

Expression constructs can also be engineered to include nucleic acids encoding affinity tags, for example, for detection or purification of operably linked expression products upon expression of the encoded polypeptide. Affinity tags include, for example, small ubiquitin-like modifier (SUMO) tags, myc epitopes, GST fusions or His ₆ for affinity purification with SUMO, myc antibodies, glutathione resins and Ni-resins, respectively. A nucleic acid encoding such a tag can be linked to a nucleic acid encoding a polypeptide construct provided herein. Tags can facilitate purification and/or detection of soluble proteins. For example, a TNFR1 antagonist polypeptide construct or portion thereof can be expressed as a recombinant protein with one or more additional polypeptide domains added to facilitate protein purification. Purification facilitating domains include metal chelating peptides, such as histidine-tryptophan modules allowing purification on immobilized metals, protein A domains allowing purification on immobilized immunoglobulins, and domains utilized in the FLAGS extension/affinity purification system. However, it is not limited thereto. (Immunex Corp., Seattle, WA). A cleavable linker sequence such as a Factor Xa cleavable recognition site (Ile-Glu-Gly-Arg engineered to include a Nru I restriction site, see eg EP 92115607A) or an enterokinase (Invitrogen (Thermo Fisher Scientific), San Diego , CA) facilitates purification between the purification domain and the encoded expression polypeptide. Exemplary expression vectors encode the expression of a fusion protein containing a TNFR1 antagonist and/or TNFR2 agonist polypeptide and an enterokinase cleavage site. A small ubiquitin-like modifier (SUMO) tag facilitates purification in immobilized metal ion affinity chromatography (IMIAC) and an enterokinase cleavage site provides a cleavage site for purification of the polypeptide from the fusion protein.

Purity can be assessed by any method known in the art including gel electrophoresis, orthogonal HPLC methods, staining and spectrophotometric techniques. Expressed and purified polypeptides can be analyzed using any assay or method known to those skilled in the art, for example any of the methods described herein. These include assays based on physical and/or functional properties of the polypeptide, including but not limited to assays by gel electrophoresis, immunoassays, binding assays, and assays of TNF-mediated TNFR1 and/or TNFR2 activity.

6. Additional variations

Modified TNFR1 antagonist constructs, TNFR2 agonist constructs, bispecific TNFR1 antagonist/TNFR2 agonist constructs, and other constructs and components thereof as described above have pharmacological properties, including pharmacokinetic and pharmacodynamic properties. It contains ingredients that change (active modifiers). Constructs, such as TNFR1 inhibitor polypeptides and small molecules, and portions of TNFR2 agonist polypeptides and small molecules are not limited to polymers or polymeric moieties such as polyethylene glycol (PEG) moieties or dextran, or human serum albumin (HSA). or sialylated to reduce immunogenicity and/or increase half-life in serum and other bodily fluids. A polypeptide may also be linked to a purification tag such as a His tag or SUMO sequence. Additional modifications include, for example, glycosylation, carboxylation, hydroxylation, phosphorylation or other known modifications. Glycosylation can be incorporated in vivo, in vitro, or through a combination of in vivo and in vitro methods using a suitable expression system, such as a mammalian expression system, e.g., the polypeptide is expressed in a prokaryotic cell and It is further modified in vitro using the enzymatic transglycosylation. Other modifications can be made in vitro or by generating the modified polypeptide, for example in a suitable host that produces such modifications.

Such modified or active modifiers and modifications are unfused or unmodified polypeptides in which the modified polypeptide incorporates the function of the modification and contains at least 96%, 97%, 98%, 99% of the activity compared to the unfused or unmodified polypeptide. is affected and selected to retain at least some, typically at least 50%, 60%, 70%, 80%, 90% or 95% compared to For example, a TNFR1 antagonist construct retains the ability to inhibit TNF-mediated signaling through TNFR1.

Coupling of a polypeptide, such as a TNFR1 antagonist or TNFR2 agonist, with another polypeptide can be effected either directly or indirectly through a linker. In one example, linking can be by chemical linkage, such as through heterobifunctional agents, or thiol linkages, or other such linkages. Fusions can also be performed by recombinant expression. A fusion of a polypeptide, such as a TNFR1 antagonist or TNFR2 agonist, to another polypeptide may be at the N- or C-terminus of the TNFR1 antagonist or TNFR2 agonist polypeptide. Non-limiting examples of polypeptides that can be used in fusion proteins with TNFR1 antagonist or TNFR2 agonist polypeptides provided herein include, for example, GST (glutathione S-transferase) polypeptides, Fc domains from immunoglobulin G, albumin, heterologous signal sequences and combinations thereof.

Encoded constructs can be produced by standard recombinant techniques. For example, DNA fragments encoding different polypeptide parts can be prepared by, for example, blunt or staggered ends for ligation, restriction enzyme digestion to provide appropriate ends, appropriately filled in sticky ends, and avoiding undesirable ligation. can be ligated together in-frame according to conventional techniques using alkaline phosphatase treatment and enzymatic ligation. Fusion genes can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene segments can be performed using anchor primers that create complementary overhangs between two consecutive gene segments that can subsequently be annealed and re-amplified to create chimeric genes. Many expression vectors already encoding fusion moieties (eg, GST polypeptides) are commercially available. A nucleic acid encoding a polypeptide can be cloned into such an expression vector such that the fusion moiety is ligated in-frame to the TNFR1 antagonist, TNFR2 agonist, and multispecific, eg, bispecific, constructs provided herein.

a. PEGization

Polyethylene glycol (PEG) is used in biomaterials, biotechnology and medicine; It is a biocompatible, non-toxic, water-soluble polymer that is generally non-immunogenic. In the field of drug delivery, PEG derivatives are used for covalent attachment (i.e., "PEGylation") to proteins to reduce immunogenicity, proteolytic and renal clearance, increase serum half-life, and enhance solubility (e.g. , Zalipsky (1995) Adv . Drug Del . Rev. 16:157-182). PEG is attached to low molecular weight, relatively hydrophobic drugs to increase solubility, reduce toxicity and alter biodistribution. Conjugation to a linear or branched chain PEG moiety increases the molecular mass and hydrodynamic radius of the polypeptide and reduces the rate of glomerular filtration by the kidney. Typically, PEGylated drugs are administered as solutions, such as by injection. In the constructs herein, the PEGylation moiety and other such polymers may be part of the linker portion of the construct.

A relevant application is the synthesis of cross-linked degradable PEG networks or formulations for use in drug delivery, as many of the same chemistries used in the design of degradable soluble drug vehicles can also be used in the design of degradable gels. (See, e.g., Sawhney et al. (1993) Macromolecules 26:581-587). Intermolecular complexes can be formed by mixing two complementary polymer solutions. These complexes are stabilized by electrostatic interactions (polyanion-polycation) and/or hydrogen bonding (polyacid-polybase) between the polymers involved and/or hydrophobic interactions between the polymers in the medium (e.g. , Krupers et al. (1996) Eur . Polym . J. 32:785-790]). For example, a mixed solution of polyacrylic acid (PAAc) and polyethylene oxide (PEO) under appropriate conditions forms a complex mainly based on hydrogen bonds. Dissociation of these complexes under physiological conditions has been used for delivery of free (i.e., non-PEGylated) drugs. Complexes of complementary polymers were formed from homopolymers and copolymers.

As with PEGylated therapeutic proteins, numerous reagents for PEGylation are known. These reagents include N-hydroxysuccinimidyl (NHS) activated PEG, succinimidyl mPEG, mPEG2-N-hydroxysuccinimide, mPEG succinimidyl alpha-methylbutanoate, mPEG succinimidyl propionate , mPEG succinimidyl butanoate, mPEG carboxymethyl 3-hydroxybutanoic acid succinimidyl ester, homodifunctional PEG-succinimidyl propionate, homobifunctional PEG propionaldehyde, homobifunctional PEG moiety Tyraldehyde, PEG maleimide, PEG hydrazide, p-nitrophenyl-carbonate PEG, mPEG-benzotriazole carbonate, propionaldehyde PEG, mPEG butryaldehyde, branched mPEG2 butyraldehyde, mPEG acetyl, mPEG piperidone, mPEG Methylketone, mPEG "linkerless" maleimide, mPEG vinyl sulfone, mPEG thiol, mPEG orthopyridylthioester, mPEG orthopyridyl disulfide, Fmoc-PEG-NHS, Boc-PEG-NHS, vinylsulfone PEG-NHS, acrylic late PEG-NHS, fluorescein PEG-NHS, and biotin PEG-NHS (see, e.g., Veronese et al. (1997) J. Bioactive Compatible Polymers 12:197-207; and numerous US and worldwide patents]). In one example, polyethylene glycol has a molecular weight ranging from about 3 kDa to about 50 kDa, typically from about 5 kDa to about 30 kDa. Covalent attachment of PEG to a drug (known as "PEGylation") can be achieved by known chemical synthesis techniques. For example, PEGylation of a protein can be achieved by reacting NHS activated PEG with the protein under appropriate reaction conditions.

Numerous reactions have been described for PEGylation, but the most commonly applicable reactions to proteins are directed, use mild reaction conditions, and do not require extensive downstream processing to remove toxic catalysts or by-products. For example, monomethoxy PEG (mPEG) has only one reactive terminal hydroxyl, so its use limits some of the heterogeneity of the resulting PEG-protein product mixture. Activation of the hydroxyl group at the end of the polymer, opposite the terminal methoxy group, is usually required to achieve efficient protein PEGylation, the purpose of which is to make the derivatized PEG more susceptible to nucleophilic attack. The attacking nucleophile is usually the epsilon-amino group of the lysyl residue, but other amines may also react if local conditions are favorable (eg, the N-terminal alpha-amine of histidine or a cyclic amine). A more direct attachment is possible in proteins containing a single lysine or cysteine. The latter residue can be targeted by PEG-maleimide for thiol specific modification. Alternatively, PEG hydrazide can react with periodate oxidized protein and be reduced in the presence of NaCNBH ₃ . More specifically, PEGylated CMP sugars can react with proteins in the presence of appropriate glycosyl-transferases. One technique is the "PEGylation" technique in which multiple polymer molecules are linked to the polypeptide in question. When using this technique, the immune system has difficulty recognizing the epitopes on the polypeptide surface responsible for antibody formation, reducing the immune response. For polypeptides introduced directly into the body's circulatory system to exert a specific physiological effect (i.e. pharmaceuticals), typical potential immune responses are IgG and/or IgM responses, whereas polypeptides inhaled through the respiratory system (i.e. industrial polypeptides) have potential may cause an IgE response (i.e., an allergic reaction). One of the theories explaining the reduced immune response is that the polymer molecule(s) protect epitope(s) on the polypeptide surface that are responsible for the immune response leading to antibody formation. Another theory, or at least a partial factor, is that the heavier the conjugate, the more the immune response is reduced.

For example, upon PEGylation of a polypeptide construct and a polypeptide component provided herein, the PEG moiety is conjugated to the polypeptide via a covalent attachment. Techniques for PEGylation include the use of specialized linkers and coupling chemistries (see, eg, Harris (2002) Adv. Drug Deliv . Rev. 54:459-476), single conjugation sites (eg, molecules), Use of topographic PEG; see, eg, Veronese et al., (2002) Bioorg . Med . Chem . Lett . 12:177-180), site-specific PEGylation and/or mono-PEGylation (eg, literature [Chapman et al. (1999) Nature Biotech . 17:780-783), site-directed enzyme PEGylation (see, eg, Sato (2002) Adv . Drug Deliv . Rev. 54:487-504). Methods and techniques described in the art can produce proteins with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 PEG or PEG derivatives attached to a single protein molecule. (See, eg, US Patent Publication No. 2006/0104968).

b. albuminization

Polypeptides provided herein, such as TNFR1 antagonist constructs, TNFR2 agonist constructs, and multispecific TNFR1 antagonist/TNFR2 agonist constructs, can be fused to albumin (i.e., "albuminized") and have properties such as half-life, stability, It can be fused to human serum albumin (HSA) for human therapy to increase bioavailability and distribution and/or to improve pharmacological properties such as pharmacokinetics of the polypeptide. A number of products related to human serum albumin (HSA) have been approved for use as therapeutics, including the treatment of cancer and type 2 diabetes (see, for example, AlQahtani et al. (2019) Biomed and Pharmacotherapy 113:108750; Roscoe et al (2018) Mol . Pharmaceutics 151:15046-5047; Strohl, WR (2015) BioDrugs 4:215-239). In some instances, a mature HSA protein lacking a signal sequence and an activation sequence is fused to a protein of interest. In some instances, human serum albumin, such as human serum albumin (HSA), is conjugated to the polypeptide. An exemplary HSA protein is set forth in SEQ ID NO:35.

Fusions with HSA are provided herein. These are generally via a short peptide linker, such as, but not limited to, a glycine-serine (GS) linker, such as (GSGS) _n or (GGGGS) _n , where n = 1-5 or 6, to TNFR1 antagonists (e.g., , dAb, scFv, Fab or other antigen binding fragment as provided herein) or fusion to the N- or C-terminus of a TNFR2 agonist (eg, TNF mutein) with HSA. Exemplary TNFR1 antagonist-HSA fusions are set forth in SEQ ID NOs: 709, 713, 717, 721 and 725.

c. tablet tag

In some examples, the TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, such as bispecific, TNFR1 antagonist/TNFR2 agonist constructs and fusion proteins provided herein may contain tags for purification of the product. . Exemplary tags for purification are described elsewhere in this document. For example, exemplary polypeptides herein may contain SUMO or His sequences for purification. Generally, the tag is a cutable tag.

Polypeptide constructs comprising fusion proteins provided herein may include a His purification tag, such as a 6xHis tag. His-tagged polypeptides may optionally contain fusion partners and/or expression and secretion signals. For example, an exemplary His-polypeptide fusion protein comprises a human immunoglobulin light chain kappa (κ) leader signal peptide sequence (SEQ ID NO: 835), a 6xHis tag (SEQ ID NO: 836), a SUMO sequence (SEQ ID NO: 837), and HSA (SEQ ID NO:35). In another example, an exemplary His tagged polypeptide fusion protein comprises a human immunoglobulin light chain kappa (κ) leader signal peptide sequence (SEQ ID NO: 835), a 6xHis tag (SEQ ID NO: 836), a SUMO sequence (SEQ ID NO: 837) , and IgG Fc (reference, eg, SEQ ID NOs: 10, 12, 14, 16, 27, and 30).

In some embodiments, polypeptides and fusion proteins provided herein may include a His tag and/or SUMO sequence for accumulation in inclusion bodies. For example, the His-SUMO sequence set forth in SEQ ID NO: 838 can be linked to any of the polypeptides or fusion proteins provided herein. His-SUMO tagged polypeptides may optionally contain fusion partners and/or expression and secretion signals. For example, the His-SUMO-polypeptide fusion protein comprises a human immunoglobulin light chain kappa (κ) leader signal peptide sequence (SEQ ID NO: 835), a 6xHis tag (SEQ ID NO: 836), a SUMO sequence (SEQ ID NO: 837), and HSA (SEQ ID NO:35). In another example, an exemplary His-SUMO-polypeptide fusion protein comprises a human immunoglobulin light chain kappa (κ) leader signal peptide sequence (SEQ ID NO: 835), a 6xHis tag (SEQ ID NO: 836), a SUMO sequence (SEQ ID NO: 837 ), and IgG Fc (reference, eg, SEQ ID NOs: 10, 12, 14, 16, 27, and 30).

7. Nucleic Acid Molecules and Gene Therapy

A nucleic acid molecule encoding a polypeptide construct, a fusion protein provided herein, can be used in gene therapy, such as expression in a gene therapy vector or administration as a DNA or RNA construct. Of these, some of the TNFR1 antagonist, TNFR2 agonist and bispecific TNFR1 antagonist/TNFR2 agonist constructs provided herein are provided as fusion proteins. Vectors and other delivery vehicles, as well as nucleic acid molecules encoding these constructs, are provided herein. A nucleic acid molecule is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or It can encode polypeptides with 99% sequence identity. In another embodiment, nucleic acid molecules can include those with degenerate codon sequences encoding any of the polypeptides or constructs provided herein.

A nucleic acid molecule for use in gene therapy is optionally operably linked to regulatory sequences of the nucleic acid. These include promoters, enhancers, signal sequences and other trafficking sequences, and other such regulatory sequences well known to those skilled in the art. For vectors with specific tissue specificity, regulatory sequences may be specific for tissues such as the liver for long-term gene expression or the eye for any ophthalmic application. Exemplary promoters include inducible and constitutive promoters for expression in mammalian cells. Such promoters recognized in eukaryotes such as mammalian subjects include the CMV and SV40 promoters; adenoviral promoters such as the E2 gene promoter responsive to the E7 oncoprotein; PV promoters such as the BPV p89 promoter that are responsive to the PV E2 protein; and other suitable promoters.

Polypeptides provided herein can also be delivered into cells in gene transfer vectors. Delivery vectors may also be used for the treatment of a disease or disorder, such as rheumatoid arthritis, or any other chronic inflammatory, autoimmune, neurodegenerative or demyelinating disease or disorder described herein or known in the art, to which the polypeptide is administered. Additional other therapeutic agent(s) may be encoded. Delivery vectors encoding the polypeptides provided herein can be used systemically by administering the nucleic acid molecule to a subject. For example, the transfer vector can be a viral vector, such as an adenoviral vector. Vectors encoding the polypeptides or constructs herein may also be incorporated into stem cells, and such stem cells may be administered to a subject, for example, by transplanting or engrafting the stem cells to a site for therapy. For example, mesenchymal stem cells (MSCs) can be engineered to express therapeutic polypeptides, and such MSCs can be engrafted to the site of transplantation for therapy.

Rather than delivering proteins, nucleic acids can be administered systemically or by other routes in vivo, or ex vivo by elimination of cells, including lymphocytes, introduction of the nucleic acid therein, and reintroduction into a host or compatible recipient. there is.

Polypeptides can be delivered to cells and tissues by expression of nucleic acid molecules. Polypeptides can be administered as nucleic acid molecules that encode the polypeptide, including ex vivo techniques and direct in vivo expression. Nucleic acids can be delivered to cells and tissues by any method known to those skilled in the art. The isolated nucleic acid sequence can be incorporated into a vector for further manipulation. Methods of administering a polypeptide by expression of an encoding nucleic acid molecule include administration of a recombinant vector. Vectors may be designed to remain episomal, such as containing an origin of replication, or may be designed to be incorporated into the chromosome of a cell. Nucleic acid molecules encoding the polypeptides provided herein can also be used in ex vivo gene expression therapy using non-viral vectors. For example, a cell can be engineered to express a polypeptide operably linked to regulatory sequences or at a genomic location operably linked to regulatory sequences. These cells can then be administered topically or systemically to a subject, such as a patient in need of treatment.

Gene therapy vectors may remain episomal or may be integrated into the chromosome of the treated subject. A polypeptide can be expressed by a virus, which is administered to a subject in need of treatment. Viral vectors suitable for gene therapy include adenovirus, adeno-associated virus (AAV), retrovirus, lentivirus, vaccinia virus, and other viruses mentioned above. Viral vectors can be used, including, for example, adenovirus, adeno-associated virus (AAV), poxvirus, herpesvirus, retrovirus, and others designed for gene therapy. AAV vectors with altered tropism, such as liver cells, are available. AAV vectors consist of a capsid conferring tropism and a nucleic acid encoding a polypeptide flanking an ITR.

For example, adenovirus expression techniques are well known in the art, and methods for adenovirus production and administration are also well known. Adenovirus serotypes are available, for example, from the American Breeder Council (ATCC, Rockville, MD). Adenoviral vectors can be used ex vivo, for example cells are isolated from a patient in need of treatment and transduced with a polypeptide expressing adenoviral vector. After an appropriate incubation period, the transduced cells are administered topically and/or systemically to the subject. Alternatively, the polypeptide adenovirus particles are isolated and formulated in a pharmaceutically acceptable carrier for delivery of a therapeutically effective amount to prevent, treat or ameliorate a disease or condition in a subject. Typically, adenoviral particles are delivered at a dose ranging from 1 to 10 ¹⁴ particles per kg of body weight, generally 10 ⁶ or 10 ⁸ to 10 ¹² particles per kg of subject body weight. In some circumstances, it is desirable to provide a nucleic acid source with an agent that targets cells, such as cell surface membrane proteins or antibodies specific for target cells or ligands for receptors on target cells. Polypeptides or constructs provided herein can also be targeted for delivery to specific cell types. For example, adenoviral vectors encoding the polypeptides or constructs provided herein can be used for stable expression in non-dividing cells such as liver cells (see, eg, Margaritis et al. (2004) J. Clin . Invest . 113:1025-1031]) In another example, a viral or non-viral vector encoding a polypeptide or construct of the present disclosure may be transduced into an isolated cell for subsequent delivery. Additional cell types for expression and delivery include, but are not limited to, fibroblasts and endothelial cells.

Nucleic acid molecules can be introduced into artificial chromosomes and other non-viral vectors. Artificial chromosomes such as ACES (see Lindenbaum et al., (2004) Nucleic Acids Res . 32(21):e172) can be engineered to encode and express isoforms. Briefly, mammalian artificial chromosomes (MACs) provide a means to introduce large amounts of genetic information into cells in an autonomously replicating, non-integrating format. Mammalian satellite DNA-based artificial chromosome expression (ACE) unique among MACs can be reproducibly generated in cell lines of different species and readily purified from the host cell's chromosome. The purified mammalian ACE can then be re-introduced into a variety of recipient cell lines stably maintained over long periods of time without selective pressure using the ACE system. Using this approach, specific loading of one or two gene targets in LMTK(-) and CHO cells was achieved.

Another way to introduce a nucleic acid encoding a polypeptide is to clone a YAC clone, an expression cassette for the gene of interest and a complete adenovirus genome cloned into a yeast artificial chromosome (YAC) in positive and negative selection markers (Ad2; Ketner et al. (1994) Proc . Natl. Acad . Sci . USA . 91: 6186-6190]) and a two-step gene replacement technology in yeast, starting with a plasmid containing adenoviral sequences targeting a specific region. YACs are particularly important because they allow the incorporation of larger genes. This approach can be used to construct adenovirus-based vectors containing nucleic acids encoding any of the polypeptides or constructs described herein for gene transfer into mammalian cells or whole animals.

Nucleic acids can be encapsulated in vehicles such as liposomes, introduced into cells such as bacterial cells, particularly attenuated bacteria, or introduced into viral vectors. For example, when liposomes are used, proteins that bind to cell surface membrane proteins associated with endocytosis include, for example, capsid proteins or tropic fragments thereof for specific cell types, antibodies to proteins that undergo internalization in the circulation, and proteins that target intracellular localization and enhance intracellular half-life and/or facilitate uptake.

For ex vivo and in vivo methods, nucleic acid molecules encoding the polypeptides or constructs herein are introduced into cells derived from a suitable donor or subject to be treated. Cells into which nucleic acids may be introduced for therapeutic purposes include, for example, epithelial cells; endothelial cells; keratinocytes; fibroblasts; muscle cells; hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, and granulocytes; and various stem or progenitor cells, particularly hematopoietic stem or progenitor cells such as stem cells from bone marrow, umbilical cord blood, peripheral blood, fetal liver, and other sources thereof, including but not limited to any suitable for the disease or condition being treated. Include any available cell type desired.

For ex vivo treatment, cells from a donor compatible with the subject to be treated or cells from the subject to be treated are removed, nucleic acids are introduced into these isolated cells, and the modified cells are administered to the subject. Treatment includes direct administration, e.g., as encapsulated within a porous membrane implanted in a patient (see, e.g., U.S. Pat. Nos. 4,892,538 and 5,283,187, each of which is incorporated herein by reference in its entirety). Techniques suitable for delivering nucleic acids to mammalian cells in vitro include electroporation of liposomes and cationic lipids (e.g., DOTMA, DOPE, and DC-Chol), microinjection, cell fusion, DEAE-dextran and calcium phosphate precipitation. The use of methods is included. DNA delivery methods can be used to express a polypeptide or construct provided herein in vivo. Such methods include liposomal delivery of nucleic acids and naked DNA delivery including local and systemic delivery such as using electroporation, ultrasound and calcium phosphate delivery. Other techniques include microinjection, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, and spheroplast fusion.

In vivo expression of a polypeptide or construct herein may be linked to expression of additional molecules. For example, expression of a polypeptide can be associated with expression of a cytotoxic product, such as in an engineered virus, or expressed in a cytotoxic virus. These viruses can target specific cell types that are targeted for therapeutic effects.

In vivo expression of a polypeptide or construct provided herein may involve operably linking a nucleic acid molecule encoding the polypeptide to a specific regulatory sequence, such as a cell-specific or tissue-specific promoter. Polypeptides can also be expressed from vectors that specifically infect and/or replicate in a target cell type and/or tissue. Inducible promoters can be used to selectively regulate polypeptide or construct expression.

Nucleic acid molecules, such as naked nucleic acids, or vectors, artificial chromosomes, liposomes and other vehicles can be administered to a subject by systemic administration, topical, local and other routes of administration. When systemic and in vivo, the nucleic acid molecule or vehicle containing the nucleic acid molecule can be targeted to cells. Administration can include intravenous administration, including direct injection into a distinct organ or portion thereof, such as the liver, and direct injection into a tissue, such as direct injection into the liver (see, e.g., U.S. Patent No. 9,821,114).

Administration can also be direct, such as administration of vectors or cells that are typically targeted to cells or tissues. Cells used for in vivo expression of a polypeptide or construct herein also include the patient's autologous cells. Such cells can be removed from the patient, introduced with nucleic acid for expression of the polypeptide, and then administered to the patient, for example by injection or engraftment.

J. Composition, Formulation and Dosage

A pharmaceutically acceptable vehicle, including a TNFR1 antagonist construct, a TNFR2 agonist construct, and a multispecific, such as bispecific, TNFR1 antagonist/TNFR2 agonist construct comprising a fusion protein, provided herein. Pharmaceutical compositions containing any of the provided polypeptides and constructs, or nucleic acid molecules encoding the polypeptides or constructs, are provided. Such compositions contain an amount of a polypeptide, construct or nucleic acid that can be diluted to a therapeutically effective amount or formulated for direct administration without dilution. The specific concentration of the construct or nucleic acid depends on various parameters within the skill of one of ordinary skill in the art including, for example, the indication being treated, the construct or nucleic acid, the route of administration and regimen. Routes of administration include systemic and topical routes, oral, rectal, intravenous, intramuscular, subcutaneous, mucosal, peritoneal, and any suitable route known to those skilled in the art.

Constructs provided herein, including multispecific, such as bispecific, TNFR1 antagonist/TNFR2 agonist constructs, including TNFR1 antagonist constructs, TNFR2 agonist constructs, and fusion proteins, as discussed above , or an amount selected according to various parameters of the nucleic acid molecule encoding the construct, such as a therapeutically effective amount, is formulated in a suitable vehicle for administration. Pharmaceutical compositions may be formulated in any conventional manner by mixing selected amounts of the construct or mixtures thereof with one or more physiologically acceptable carriers or excipients or vehicles. A pharmaceutical composition may be used for therapeutic, prophylactic and/or diagnostic applications. The concentration of the active compound, i.e. construct or nucleic acid, in the composition is determined by those mentioned above, as well as rates of absorption, inactivation, and excretion of the active compound, dosing schedule, and amount administered, age and size of the subject, as well as those skilled in the art. depends on various factors including other factors known to

Pharmaceutical carriers or vehicles suitable for administration of the compounds provided herein include any such carrier known to those skilled in the art to be suitable for a particular mode of administration. A pharmaceutical composition comprising a therapeutically effective amount of a construct or nucleic acid molecule described herein may also be presented as a lyophilized powder to be reconstituted with sterile water immediately prior to administration.

1. Formulation

Pharmaceutical compositions containing any of the constructs and nucleic acids provided herein may be formulated in any conventional manner by mixing selected amounts of the active compound with one or more physiologically acceptable carriers or excipients. The choice of carrier or excipient is within the skill of the administering expert and can depend on many parameters. These include, for example, the mode of administration (ie, systemic, oral, nasal, pulmonary, topical, topical or any other mode) and the disorder being treated. Generally, a pharmaceutical composition includes components that do not significantly impair the biological properties of, or enhance or improve the pharmacological properties of, the construct or nucleic acid or encoded polypeptide. The formulation may also be co-formulated with other active agents for combination therapy.

The pharmaceutical compositions provided herein can be in a variety of forms, including but not limited to solid, semi-solid, liquid, emulsion, powder, aqueous and lyophilized forms. The pharmaceutical compositions provided herein may be formulated for single dose (direct) administration, or for dilution or other regimen. The concentration of the compound in the formulation is effective for direct administration, i.e., for the intended treatment, after dilution or mixing with other compositions, or for shearing the amount upon administration. Compositions may be formulated as single or multiple dose direct doses. The compounds can be suspended in micronized or other suitable form, or derivatized to produce a more soluble active product. The form of the resulting mixture depends on several factors including the intended mode of administration and the solubility of the compound in the chosen carrier or vehicle. The resulting mixtures are solutions, suspensions, emulsions and other such mixtures, non-aqueous or aqueous mixtures, creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, drenches, sprays, It may be formulated as a suppository, bandage or other formulation suitable for systemic, topical or topical administration. For topical internal administration, such as intramuscular, parenteral or intraarticular administration, the constructs and nucleic acids may be formulated as a solution suspension in an aqueous based medium such as isotonic buffered saline or combined with a biocompatible support or bioadhesive for internal administration. can Effective concentrations are sufficient to ameliorate the target condition and can be determined empirically. To formulate a composition, a weight fraction of the compound is dissolved, suspended, dispersed or mixed in an effective concentration with a selected vehicle to alleviate or ameliorate the target condition.

Generally, pharmaceutically acceptable compositions are prepared in view of approval by regulatory or other authorities and/or in accordance with generally accepted pharmacopeias for use in animals and humans. A pharmaceutical composition may include a carrier such as a diluent, adjuvant, excipient or vehicle with which the polypeptide is administered. Such pharmaceutical carriers can be sterile liquids such as water and oils including petroleum, animal, vegetable or synthetic origin, for example peanut oil, soybean oil, mineral oil, and sesame oil. Water is a typical carrier when the pharmaceutical composition is administered intravenously. Saline, aqueous solutions of dextrose and glycerol may also be employed as liquid carriers, particularly for injectable solutions. The composition may contain diluents such as lactose, sucrose, calcium phosphate, and carboxymethylcellulose along with the active ingredient; lubricants such as magnesium stearate, calcium stearate and talc; and binders such as starch, natural gums such as gum acacia, gelatin, glucose, molasses, polyvinylpyrrolidone, cellulose and its derivatives, povidone, crospovidone, and other such binders known to those skilled in the art. Suitable pharmaceutical excipients are starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, Contains water and ethanol. If desired, the composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents. may contain. Such compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, granules, and sustained release formulations. For example, capsules and cartridges of gelatin for use in an inhaler or insufflator may be formulated containing a powder mixture of the therapeutic compound and a suitable powder base such as lactose or starch. The composition may be formulated as a suppository with conventional binders and carriers such as triglycerides. Oral dosage forms may include standard carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and pharmaceutical grades of other such agents. Formulations for oral administration may also be suitably formulated with protease inhibitors such as Bowman-Burk inhibitors, conjugated Bowman-Burk inhibitors, aprotinin and camostat. Examples of suitable pharmaceutical carriers are described in E. W. Martin, "Remington's Pharmaceutical Sciences"]. Such compositions will contain a therapeutically effective amount of the compound, usually in purified form, together with a suitable amount of carrier to provide the compound in a form for proper administration to a subject or patient.

The pharmaceutical compositions provided herein may include, for example, antioxidants, preservatives, antibacterial agents, analgesics, binders, disintegrants, colorants, diluents, excipients, extenders, glidants, solubilizers, stabilizers, tonicity agents, vehicles, among others. , viscosity agents, flavoring agents, emulsions such as oil-in-water or water-in-oil emulsions, emulsifiers and suspending agents such as acacia, agar, alginic acid, sodium alginate, bentonite, carbomer, carrageenan, carboxymethylcellulose, cellulose, cholesterol, gelatin, Hydroxyethyl Cellulose, Hydroxypropyl Cellulose, Hydroxypropyl Methylcellulose, Methylcellulose, Octoxynol-9, Oleyl Alcohol, Povidone, Propylene Glycol Monostearate, Sodium Lauryl Sulfate, Sorbitan Ester, Stearyl Alcohol, tragacanth, xanthan gum, and its derivatives, solvents, and miscellaneous ingredients such as crystalline cellulose, microcrystalline cellulose, citric acid, dextrin, dextrose, liquid glucose, lactic acid, lactose, magnesium chloride, potassium metaphosphate, and may contain other additives including starch (see generally Alfonso R. Gennaro (2000) Remington : The Science and Practice of Pharmacy , 20th Edition. Baltimore, MD: Lippincott Williams & Wilkins). Such carriers and/or additives can be formulated by conventional methods and administered to a subject in an appropriate dose. Stabilizers such as lipids, nuclease inhibitors, polymers and chelating agents can prevent degradation of the composition in the body.

The formulation should suit the mode of administration. For example, the active compounds may be formulated for parenteral administration by injection (eg, bolus injection or continuous infusion). Injectable compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles. The sterile injectable preparation may also be a sterile injectable solution, or suspension, in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,4-butanediol. Sterile fixed oils are commonly used as solvents or dispersion media. To this end, any bland fixed oil may be synthetic mono- or diglycerides, fatty acids (including oleic acid), naturally occurring vegetable oils such as sesame oil, coconut oil, peanut oil, cottonseed oil, and other oils, or synthetic fatty vehicles such as ethyl. oleates, including but not limited to, may be used. Buffers, preservatives, antioxidants and suitable ingredients may be incorporated as needed or alternatively included in the formulation.

Active compounds, such as constructs and nucleic acids provided herein, may be formulated as the sole pharmaceutically active ingredient in a composition or may be combined with other active ingredients. An active compound can be targeted for delivery, for example, by conjugation to a targeting agent such as an antibody. Liposomal suspensions, including tissue-targeted liposomes, may also be suitable as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art. For example, liposomal formulations can be prepared by methods well known to those skilled in the art, such as those described in US Pat. No. 4,522,811. Liposomal delivery can also include slow release formulations comprising pharmaceutical matrices such as collagen gels and liposomes modified with fibronectin (see, e.g., Weiner et al. (1985) J. Pharm . Sci . 74(9):922-925). The compositions provided herein may further contain one or more adjuvants to facilitate delivery, such as, but not limited to, an inert carrier or colloidal dispersion system. Representative, non-limiting examples of such inert carriers include water, isopropyl alcohol, gaseous fluorocarbons, ethyl alcohol, polyvinylpyrrolidone, propylene glycol, gel forming substances, stearyl alcohol, stearic acid, spermaceti, sorbitan mono oleate, methylcellulose, as well as suitable combinations of two or more of these.

The active compound is included in a pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect without undesirable side effects on the subject being treated. Therapeutically effective concentrations can be determined empirically by testing compounds in known in vitro and in vivo systems, such as the assays described herein. Determination of a therapeutically effective amount is within the ability of one skilled in the art. A therapeutically effective dosage can be determined using in vitro and in vivo methods as described herein. Thus, the active compounds or mixtures thereof provided herein may be in unit dosage form for administration as a pharmaceutical preparation.

2. TFNR1 antagonist construct , TNFR2 agonist construct , multispecific, for example bispecific , Administration of Constructs and Nucleic Acids

Constructs provided herein, including TNFR1 antagonist constructs, TNFR2 agonist constructs, and multispecific, such as bispecific, including fusion proteins, TNFR1 antagonist/TNFR2 agonist constructs, and constructs encoding the constructs An active compound comprising a nucleic acid molecule may be administered by any suitable route. This disclosure includes in vitro, ex vivo or in vivo by contacting a mixture, such as a bodily fluid or other tissue sample, with an active compound provided herein. For example, when administering a compound ex vivo, a bodily fluid, such as the vitreous, or a tissue sample from a subject can be contacted with the polypeptide coated on a tube or filter, such as a tube or filter, for example in a bypass machine. For in vivo administration, the active compounds may be administered in liquid, semi-liquid or solid form by any suitable route, for example, orally, nasally, pulmonary, parenterally, intravenously, intradermally, intravitreally, retinally. intraocularly, subretinally, periocularly, subcutaneously, intraarticularly, intracapsularly, intraocularly, intraventricularly, intrathecally, intramuscularly, intraperitoneally, intratracheally, rectally, or topically, or directly into an organ It can be administered by direct injection, as well as by any combination of any two or more thereof, and can be formulated in a manner suitable for each route of administration.

Routes of administration include injection or infusion by known methods, for example intravenous, intraperitoneal, intracerebral, intramuscular, subcutaneous, intraocular, intraarterial, intrathecal, inhalational or intralesional routes, topical or sustained release systems. follow Antibodies or fragments thereof are typically administered continuously by infusion or bolus injection. An active compound provided herein may be prepared in admixture with a pharmaceutically acceptable carrier, as discussed above. Techniques for formulation and administration of compounds are known to those skilled in the art. The therapeutic composition may be administered intravenously or via the nose or lungs as a liquid or powder aerosol (lyophilized). Compositions may also be administered parenterally or subcutaneously as needed. When administered systemically, the therapeutic composition should be a sterile, pyrogen-free, parenterally acceptable solution with due regard to pH, isotonicity and stability, and other conditions known to those skilled in the art.

Therapeutic formulations can be administered in many conventional dosage forms. Dosage forms of the active compounds provided herein are prepared for storage or administration by mixing the compound having the desired degree of purity with physiologically acceptable carriers, excipients, or stabilizers. These substances are nontoxic to recipients at the dosages and concentrations employed and are used in buffers such as TRIS HCl, phosphate, citrate, acetate and other organic acid salts; antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) peptides such as polyarginine; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium and/or nonionic surfactants such as those sold under the tradenames TWEEN and PLURONICS, and polyethylene glycol (PEG).

The pharmaceutical composition may contain a stabilizer. Stabilizers can be amino acids, amino acid derivatives, amines, sugars, polyols, salts or surfactants. In some instances, stable co-formulations contain a single stabilizer. In another example, a stable co-formulation contains 2, 3, 4, 5 or 6 different stabilizers. For example, the stabilizer can be a sugar or polyol such as glycerol, sorbitol, mannitol, inositol, sucrose or trehalose. In certain instances, the stabilizing agent is sucrose. In another example, the stabilizing agent is trehalose. The sugar or polyol concentration is about 100 mM to 500 mM, 100 mM to 400 mM, 100 mM to 300 mM, 100 mM to 200 mM, 200 mM to 500 mM, 200 mM to 400 mM, 200 mM to 300 mM, 250 mM mM to 500 mM, 250 mM to 400 mM, 250 mM to 300 mM, 300 mM to 500 mM, 300 mM to 400 mM, or 400 mM to 500 mM, each inclusive.

In examples, the stabilizer can be a surfactant that is polypropylene glycol, polyethylene glycol, glycerin, sorbitol, poloxamer, and polysorbate. For example, the surfactant can be polypropylene glycol, polyethylene glycol, glycerin, sorbitol, poloxamer or polysorbates such as poloxamer 188, polysorbate 20 and polysorbate 80. In a specific example, the stabilizing agent is polysorbate 80. The concentration of the surfactant as mass concentration % (w/v) in the formulation is 0.005% to 1.0%, 0.01% to 0.5%, 0.01% to 0.1%, 0.01% to 0.05%, or 0.01% to 0.02%, each inclusive. am.

For in vivo administration, the formulation must be sterile. This is easily accomplished, such as filtration through sterile filtration membranes before or after lyophilization and reconstitution. Formulations may be stored in lyophilized form or as a solution. Other vehicles may be used, such as naturally occurring vegetable oils such as sesame, peanut, or cottonseed oil, or synthetic fatty vehicles such as ethyl oleate and the like. Buffers, preservatives, antioxidants and the like may be incorporated according to accepted pharmaceutical practice.

Determination of dosage is within the skill of the physician and may be a function of the particular disorder, route of administration, and subject. Exemplary dosages include, for example, 0.1 to 100 mg/kg, such as 1 to 10 mg/kg, or an appropriate amount based on the mass of the subject being treated; The mass of an average human subject is about 70-75 kg. The polypeptide can be administered once or more than once, eg twice, three times, four times or any number of times necessary to achieve a therapeutic effect. Multiple administrations can be performed via any route or combination of routes, and can be administered hourly, every 2 hours, every 3 hours, every 4 hours or more.

The active compound may be present at, for example, about 0.1 to 10 mg/mL, such as at least or at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10 mg/mL, or a concentration in the composition of greater concentration. The volume of the solution may be from about 0.1 to about 100 mL or more, for example, at least or about at least or 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mL, or more. In some instances, the active compound is supplied in phosphate buffered saline. For example, the composition may be supplied as a 50 mL vial or other container containing 100 mg of the polypeptide or fusion protein at a concentration of 2 mg/mL in phosphate buffered saline.

The active compounds provided herein may be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective for conventional immunoglobulin and protein preparations and lyophilization and reconstitution techniques known in the art can be used.

An active compound provided herein may be provided as a controlled release or sustained release composition. Polymeric materials are known in the art for formulation of pills and capsules that can achieve controlled or sustained release (see, e.g., Controlled Drug Bioavailability, Drug Product Design and Performance , Smolen and Ball (eds.), Wiley, New York (1984); also Levy et al. (1985) Science 228:190; During etc. (1989) Ann. Neurol . 25:351; Howard et al. (1989) J. Neurosurg . 71:105; U.S. Patent Nos. 5,679,377, 5,916,597, 5,912,015, 5,989,463, and 5,128,326; and International Application Publication Nos. WO 99/015154 and WO 99/020253). Examples of polymers used in sustained release formulations are poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid) ), polyglycolide (PLG), polyanhydride, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide- co-glycolide) (PLGA), and polyorthoesters. Generally, the polymers used in sustained-release formulations are inert, free of leachable impurities, stable on storage, sterile, and biodegradable. Sustained release formulations can be produced using any technique known in the art for the production of sustained release formulations.

The constructs and nucleic acids, and their physiologically acceptable forms of salts and solvates, are intended for administration by inhalation (orally or nasally) or including, for example, oral, transdermal, pulmonary, parenteral or rectal administration. may be formulated for other routes of administration. For administration by inhalation, the active compounds may be administered in pressurized packs or aerosols from nebulizers with the use of a suitable propellant, for example dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. It can conveniently be delivered in the form of a spray presentation. For pressurized aerosols, the dosage unit can be determined by providing a valve to deliver a metered amount. For example, capsules and cartridges of gelatin for use in an inhaler or insufflator may be formulated containing a powder mixture of the therapeutic compound and a suitable powder base such as lactose or starch.

For pulmonary administration to the lungs, the constructs may be delivered in the form of an aerosol spray presentation from a nebulizer, turbo nebulizer, or microprocessor controlled metered dose oral inhaler using a suitable propellant. Generally, the particle size of the aerosol is small, in the range of 0.5 to 5 microns. For pharmaceutical compositions formulated for pulmonary administration, detergent surfactants are generally not used. Pulmonary drug delivery is a promising non-invasive systemic administration method. The lung represents an attractive route for drug delivery, primarily due to its high surface area for absorption, thin alveolar epithelium, extensive angiogenesis, lack of hepatic first-pass metabolism, and relatively low metabolic activity.

For oral administration, the pharmaceutical composition may contain, for example, pharmaceutically acceptable excipients such as binders (eg, preaged corn starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (eg lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (eg magnesium stearate, talc or silica); disintegrants (eg potato starch or sodium starch glycolate); or in the form of tablets, pills, liquid suspensions, or capsules prepared by conventional means with a wetting agent (eg sodium lauryl sulfate). Tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may contain pharmaceutically acceptable additives such as suspending agents (eg sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifiers (eg lecithin or acacia); non-aqueous vehicles (eg, almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (eg, methyl or propyl-p-hydroxybenzoate, or sorbic acid). The formulation may also include buffer salts, flavoring agents, coloring agents and sweeteners as appropriate.

Formulations for oral administration may be formulated for controlled release of the active compound. For buccal administration, the compositions may take the form of conventionally formulated tablets or lozenges. The active compound may be formulated as a depot preparation. Such long-acting formulations may be administered by implantation (eg, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the therapeutic compounds may be formulated with suitable polymeric or hydrophobic materials (e.g., as emulsions in acceptable oils) or as ion exchange resins, or sparingly soluble derivatives, e.g., as sparingly soluble salts. It can be.

The active compounds may be formulated for parenteral administration by injection (eg, bolus injection or continuous infusion). Formulations for injection may be presented in unit dosage form (eg, in ampoules or in multi-dose containers) with an added preservative. The composition may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder-lyophilized form for constitution with a suitable vehicle, eg, sterile pyrogen-free water, before use.

Pharmaceutical compositions are formulated for topical or topical application, such as for topical application to the skin (dermal) and mucous membranes, such as the eye, in the form of gels, creams and lotions, and for application to the eye or for intracapsular or intraspinal application. Such solutions, particularly those intended for ophthalmic use, may be formulated at a pH of about 5-7 with 0.01% - 10% isotonic solutions and appropriate salts. The compounds may be formulated as an aerosol for topical application such as inhalation (see, eg, U.S. Patent Nos. 4,044,126, 4,414,209 and 4,364,923, which are steroidal agents useful in the treatment of inflammatory diseases, particularly asthma). aerosol for delivery).

The concentration of the active compound in the drug composition depends on the rate of absorption, inactivation and excretion of the active compound, the dosage schedule and dosage, as well as other factors known to those skilled in the art. As further described herein, dosage can be determined empirically using comparisons of properties and activities. For example, inhibition of TNF-mediated inflammatory signaling through TNFR1 by a construct provided herein is comparable to traditional anti-TNF therapies such as adalimumab.

If desired, the compositions may be presented in a package, kit, or dispenser device that may contain one or more unit dosage forms containing the active ingredient. In some instances, the composition may be coated onto a device such as, for example, a filter or tube of a bypass machine. For example, the package contains a metal or plastic foil such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. A composition containing an active agent may be packaged as an article of manufacture containing a packaging material, a formulation provided herein, and a label indicating the disorder in which the formulation is provided. A pharmaceutical composition may be packaged in unit dosage form containing an amount of the pharmaceutical composition for a single dose or multiple doses. The packaged composition is a multispecific, such as a bispecific, TNFR1 antagonist/comprising TNFR1 antagonist construct, a TNFR2 agonist construct, and a fusion protein, which can be reconstituted (eg, with water or saline) prior to administration. A lyophilized powder of a pharmaceutical composition containing a construct provided herein, including a TNFR2 agonist construct, and a nucleic acid molecule encoding the construct provided herein.

Articles of manufacture provided herein include packaging materials. Packaging materials for use in packaging pharmaceutical products are well known to those skilled in the art (see, eg, US Pat. Nos. 5,323,907, 5,052,558 and 5,033,252). Examples of pharmaceutical packaging materials are blister packs, bottles, tubes, inhalers (eg pressurized metered dose inhalers (MDI), dry powder inhalers (DPI), nebulizers (eg jet or ultrasonic nebulizers), and other single breath liquid systems), pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for the selected formulation and intended mode of administration and treatment.

Constructs provided herein, including TNFR1 antagonist constructs, TNFR2 agonist constructs, and multispecific, such as bispecific, TNFR1 antagonist/TNFR2 agonist constructs, including fusion proteins, and constructs encoding the constructs Active compounds comprising nucleic acid molecules, pharmaceutical compositions, and combinations of active agents and other compositions comprising other therapeutic agents may be provided as a kit. A kit may include one or more components for the practice of a method, such as instructions for use, a device, an additional of reagents (eg, sterile water or saline for dilution of the composition and/or reconstitution of the lyophilized protein), and components such as tubes, containers, and syringes. Exemplary kits may include a construct provided herein and an encoding nucleic acid, optionally instructions for use, a device for administering a compound to a subject, a device for detecting a compound in a subject, a compound or its devices for detecting metabolites, and devices for administering additional therapeutic agents to a subject. Optionally, the kit may include instructions for use. Instructions typically include tangible representations describing the active compound(s) and optionally other components included in the kit, how to determine the appropriate condition of the subject, how to determine the appropriate dosage, administration regimen and method of administration. A method for administration comprising Instructions may also include instructions for monitoring the subject during the treatment session.

A kit may also include a pharmaceutical composition described herein and items for diagnosis. For example, such kits may include items for measuring the concentration, amount or activity of an active compound administered in a subject. Kits provided herein may also include a device for administering the compound(s). Any of a variety of devices known in the art for administering medication to a subject can be included in the kits provided herein. Exemplary devices include, but are not limited to, hypodermic needles, intravenous needles, and catheters. Typically, the device for administration is compatible with the desired method of administration of the active agent.

3. Administration of Nucleic Acids Encoding Polypeptides (Gene Therapy)

Among the pharmaceutical compositions are those containing nucleic acid molecules encoding the polypeptide constructs provided herein. Rather than delivering proteins, nucleic acid molecules can be administered systemically or by other routes in vivo, or ex vivo by elimination of cells, including lymphocytes, introduction of the nucleic acid molecule therein, and reintroduction into a host or compatible recipient. It can be.

Polypeptide constructs provided herein, including TNFR1 antagonist constructs, TNFR2 agonist constructs, and multispecific, such as bispecific, TNFR1 antagonist/TNFR2 agonist constructs, including fusion proteins, are useful for the expression of nucleic acid molecules. can be delivered to cells and tissues by Nucleic acids can be delivered to cells and tissues by any method known to those skilled in the art. Isolated nucleic acids can be incorporated into vectors for further manipulation. As discussed above, methods of administering a polypeptide by expression of an encoding nucleic acid molecule include administration of a recombinant vector. Vectors may be designed to remain episomal, such as containing an origin of replication, or may be designed to be incorporated into the chromosome of a cell. Polypeptides can also be used in ex vivo gene expression therapy using non-viral vectors. Suitable gene therapy vectors and delivery methods are known to those skilled in the art and are discussed in the section above.

K. Therapeutic Uses and Methods of Treatment

A pharmaceutical composition as described above is prepared and administered to a subject having a disease, disorder or condition that can be treated with a construct that inhibits and/or agonizes TNFR1 and TNFR2, respectively. Dosages vary depending on the particular subject as well as the particular disorder, disease or condition being treated. Typical doses are similar to known TNF blockers such as etanercept. Exemplary dosages for subjects, including humans and other animals, are about or 0.1 mg/kg to 100 mg/kg, such as 1 mg/kg to about or 30 mg/kg, such as 5 mg/kg to 25 mg/kg. is the range Doses can be determined based on the assumption that the average human mass is about 75 kg. Dosage may be adjusted for children, infants and smaller adults.

The TNFR1 antagonist, TNFR2 agonist, bispecific TNFR1 antagonist/TNFR2 agonist constructs and fusion proteins provided herein are useful to those skilled in the art for the use of such molecules, including the treatment of any of the diseases, disorders and conditions described herein. It can be used for any known purpose. For example, the TNFR1 antagonist, TNFR2 agonist, multispecific TNFR1 antagonist/TNFR2 agonist constructs and fusion proteins provided herein may be used for one or more of therapeutic, diagnostic, industrial and/or research purpose(s). there is. The treatment methods provided herein include methods for therapeutic use of the TNFR1 antagonists, TNFR2 agonists, multispecific TNFR1 antagonist/TNFR2 agonist constructs and fusion proteins provided herein. For example, a TNFR1 antagonist described herein can be used to antagonize TNFR1, inhibit the binding of TNF to TNFR1, and/or inhibit TNF-mediated proinflammatory signaling through TNFR1. TNFR2 agonists can be used to induce protective/anti-inflammatory TNFR2 signaling and/or to agonize TNFR2 to induce expansion, proliferation and activation of immunosuppressive TNFR2 ⁺ regulatory T-cells (Tregs). In some embodiments, the use of a combination of a TNFR1 antagonist and a TNFR2 agonist, or a bispecific TNFR1 antagonist/TNFR2 agonist construct provided herein, as described herein, is used in the treatment of chronic inflammatory and autoimmune diseases, as well as as well as providing selective inhibition of pro-inflammatory TNFR1 activity while increasing TNFR2-associated protective signaling and Treg immunosuppressive activity beneficial for the treatment of neurodegenerative and demyelinating diseases and disorders.

The TNFR1 antagonists, TNFR2 agonists, multispecific, such as bispecific, TNFR1 antagonist/TNFR2 agonist constructs and fusion proteins provided herein, alone or in combination with other agents, may have therapeutic activity. The TNFR1 antagonists, TNFR2 agonists, bispecific TNFR1 antagonist/TNFR2 agonist constructs and fusion proteins, and encoding nucleic acid molecules provided herein can be used to treat chronic inflammatory and autoimmune diseases and disorders, as well as neurodegenerative and demyelinating diseases and disorders. anti-TNF therapy (e.g., adalimumab, infliximab, etanercept, and others described herein and/or known in the art), or other disease modifying anti- rheumatic drugs (DMARDs; e.g., methotrexate, hydroxychloroquine, sulfasalazine, leflunomide, abatacept, anakinra, rituximab, tocilizumab, tofacitinib, and those described herein and/or Others known in) can be used for the treatment of any condition for which it is used. For example, a subject to whom a therapeutic molecule provided herein is administered exhibits acute or chronic inflammation of the joints, skin, lungs, and/or digestive tract and/or exhibits an autoimmune disease, rheumatoid arthritis (RA), psoriasis, psoriasis. Arthritis, juvenile idiopathic arthritis (JIA), spondyloarthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis, inflammatory bowel disease (IBD), uveitis, fibrotic disease, endometriosis, lupus, multiple sclerosis, congestive heart failure, cardiovascular disease, Myocardial infarction (MI), atherosclerosis, metabolic disease, cytokine release syndrome, septic shock, sepsis, acute respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), COVID-19, influenza, acute and chronic neurological Degenerative diseases, demyelinating diseases and disorders, stroke, Alzheimer's disease, Parkinson's disease, Behçet's disease, Dupuytren's disease, tumor necrosis factor receptor-associated periodic syndrome (TRAPS), pancreatitis, type I diabetes, chronic obstructive pulmonary disease (COPD), chronic Bronchitis, emphysema, transplant rejection, graft-versus-host disease (GvHD), respiratory disease, lung inflammation, lung diseases and conditions, asthma, cystic fibrosis, idiopathic pulmonary fibrosis, acute fulminant viral or bacterial infection, pneumonia, TNF/TNFR1 It suffers from hereditary genetic disorders, periodic fever syndrome, and cancer as agents of the causative pathology.

The constructs provided herein, when administered, can generally result in a subject exhibiting reduced or lessened side effects compared to side effects that may be observed following administration of an anti-TNF therapy. Treatment of diseases and conditions with polypeptides provided herein, such as TNFR1 antagonists, TNFR2 agonists, and bispecific constructs and fusion proteins thereof, may be performed by infusion, subcutaneous injection, and inhalation, or intramuscular, intradermal, oral, topical and by any suitable route of administration using a suitable formulation as described herein, including but not limited to transdermal administration.

As discussed elsewhere herein, existing anti-TNF therapies, such as adalimumab, are immunosuppressive due to blockade of TNF signaling through TNFR1 and TNFR2 and are effective against, for example, sepsis and serious infections such as listeriosis, Reactivation of tuberculosis, reactivation of hepatitis B/C, herpes zoster, and reactivation of invasive fungi and increased risk of other opportunistic infections. Anti-TNF agents can also aggravate severe congestive heart failure, increase susceptibility to drug-induced lupus, liver damage, psoriasis, sarcoidosis, and demyelinated central nervous system (CNS) disease, and further development of autoimmune diseases, as well as lymphoma and solid malignancies, such as non-melanoma skin cancer. Depending on the anti-TNF agent, approximately 3-33% of treated patients do not respond to treatment, and up to 46% stop responding, leading to discontinuation of treatment or dose increase. Anti-TNF therapy has failed to treat neurodegenerative diseases and CNS conditions such as Alzheimer's disease, Parkinson's disease, stroke and multiple sclerosis (MS) associated with overexpression of TNF. Due to side effects associated with the use of anti-TNF agents, non-response in some patients, continued lack of response in patients who had an initial response, treatment failure and/or worsening of a neurodegenerative disease such as MS, other therapies are needed. Such therapies are provided herein.

As described herein, selective inhibition of TNFR1 retains the potent anti-inflammatory and protective activity of TNFR2 signaling, reduces opportunistic infections and cancers, and preserves TNF-induced Treg function. Previous selective TNFR1 antagonists suffer from immunogenicity including formation of anti-drug antibodies (ADA), e.g., short serum half-life, rapid renal clearance, and/or poor pharmacokinetics and pharmacodynamics, including poor binding affinity and potency. . The therapies provided herein overcome limitations previously associated with selective TNFR1 antagonists. Examples of therapeutic improvements using polypeptides provided herein, such as TNFR1 antagonists, include, but are not limited to, lower dosages, lower and/or less frequent administration, reduced side effects, and increased therapeutic effect.

Thus, the selective TNFR1 antagonists, TNFR2 agonists, and multispecific, such as bispecific TNFR1 antagonist/TNFR2 agonist constructs, and fusion proteins provided herein are associated with reduced side effects and, if desired, higher dosing regimens. It can be used as a men, and can have improved efficacy and safety. Side effects that may be reduced, mitigated, or eliminated compared to those observed with existing anti-TNF therapeutics, such as adalimumab and others described herein and/or known in the art, include those described herein or known in the art. development of any undesirable non-therapeutic effect known to, such as sepsis, serious infection, congestive heart failure/cardiotoxicity, antibody production, cancer, autoimmune disease and/or demyelinated central nervous system (CNS) disease; including but not limited to deterioration. In some instances, compared to side effects caused by administration of existing anti-TNF therapeutics, such as adalimumab, administration of a TNFR1 antagonist, bispecific construct or fusion protein provided herein is one of the anti-TNF therapies. At least or about 99%, at least or about 95%, at least or about 90%, at least or about 85%, at least or about 80%, at least or about 75%, at least or about 75%, at least or about 90% About 70%, at least or about 65%, at least or about 60%, at least or about 55%, at least or about 50%, at least or about 45%, at least or about 40%, at least or about 35%, at least or about 30% %, at least or about 25%, at least or about 20%, at least or about 15%, or at least or about 10%.

Dosage levels and regimens can be determined based on known dosages and regimens, extrapolated, if necessary, based on variations in the properties of the polypeptides and constructs provided herein, and/or determined empirically based on a variety of factors. there is. These factors include, for example, the body weight of the individual as well as general health, age, sex and diet, activity of the specific compound used, time of administration, rate of excretion, drug combination, severity and course of the disease, the patient's propensity for the disease and treatment. including the physician's judgment. The active ingredient is typically combined with a pharmaceutically effective carrier. The amount of active ingredient that can be combined with the carrier material to produce a single dosage form or multiple dosage form can vary depending on the host being treated and the particular mode of administration.

When the patient's condition improves, a maintenance dose of the compound or composition may be administered, if necessary; The dosage, dosage form, or frequency of administration, or combinations thereof may be varied. In some cases, a subject may require intermittent treatment over an extended period of time following any recurrence of disease symptoms or based on a predetermined dosage.

This section provides exemplary uses and methods of administration for constructs comprising the polypeptides and encoding nucleic acid molecules provided herein. These described regimens are exemplary only and do not limit the application of the molecules/constructs provided herein. Identifying a disease or condition that can be treated using the TNFR1 antagonists, TNFR2 agonists, bispecific TNFR1 antagonist/TNFR2 agonist constructs, and fusion proteins, as well as encoding nucleic acid molecules, provided herein is a skill of the treating physician. is within

1. Treatment of chronic inflammatory/autoimmune diseases and disorders

As described herein, elevated levels or uncontrolled expression of TNF and deregulation of TNF signaling can lead to chronic inflammation, which can lead to the development of autoimmune diseases and tissue damage. TNF signaling through TNFR1 is primarily proinflammatory and leads to the development of chronic inflammatory and autoimmune diseases and disorders. For example, TNFR1 signaling is specifically associated with the pathogenesis of arthritis, inflammatory bowel disease (IBD), and respiratory diseases, as well as the development of osteoclasts leading to localized bone destruction and cardiotoxic effects in TNF-induced models of heart failure and myocardial infarction. related to creation. Thus, selective blockade of TNFR1 is useful for the treatment of chronic inflammatory and autoimmune diseases and conditions. TNF signaling, mainly through the anti-inflammatory agent TNFR2, is associated with neurological, cardiac, digestive and bone protective effects. The anti-inflammatory and protective effects of TNFR2 signaling include, for example, experimental colitis, heart failure/heart disease, myocardial infarction, inflammatory arthritis, infectious diseases, pancreatic regeneration, stem cell proliferation, destruction of autoreactive T-cells, and bone mass. It has been demonstrated in the regulation of osteoclastogenesis to maintain and protect against joint inflammation and erosive destruction. TNFR2 agonism also results in proliferation and expansion of immunosuppressive TNFR2 ⁺ Tregs, promotes Treg cell suppressive activity to eliminate autoreactive/effector T-cells, prevents tissue destruction, and prevents inflammatory and autoimmune diseases and conditions suppress Thus, TNFR2 agonism can also be used to treat or alleviate the symptoms of chronic inflammatory and autoimmune diseases and conditions.

The TNFR1 antagonists, TNFR2 agonists, multispecific TNFR1 antagonist/TNFR2 agonist constructs, fusion proteins, and encoding nucleic acids provided herein can be used to treat autoimmune/inflammatory diseases and disorders associated with elevated TNF levels and deregulated TNF signaling. It can be used to treat or alleviate the symptoms of Constructs, fusion proteins and nucleic acids provided herein may be used for, for example, arthritis (e.g., rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis, spondyloarthritis), inflammatory bowel disease (e.g., Crohn's disease), among others. and ulcerative colitis), uveitis, fibrotic disease (eg, Dupuytren's disease), Behcet's disease, endometriosis, lupus, ankylosing spondylitis, psoriasis, tumor necrosis factor receptor associated periodic syndrome (TRAPS), cardiovascular disease, congestive Heart Failure, Myocardial Infarction (MI), Atherosclerosis, Respiratory Disease, Asthma, Cystic Fibrosis, Chronic Obstructive Pulmonary Disease (COPD), Pancreatitis, Type I Diabetes, Metabolic Disease, Cytokine Release Syndrome, Septic Shock, Sepsis, Acute Respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), COVID-19, influenza, chronic bronchitis, emphysema, lung inflammation, idiopathic pulmonary fibrosis, transplant rejection, graft-versus-host disease (GvHD), acute fulminant virus or bacteria infections, pneumonia, hereditary genetic diseases with TNF/TNFR1 as a causative pathological agent, and periodic fever syndrome.

TNF blockade can also reduce the cytokine storm observed in some viral infections, such as the SARS virus and the infectious COVID-19. This could prevent ventilator dependence, multi-organ damage and death in patients with severe acute respiratory syndrome (SARS), such as those caused by SARS-CoV-2 and other SARS viruses/coronaviruses. TNF-induced viral syndrome (TIVS) is caused by a TNF-induced cytokine storm that is accompanied by lung damage as well as multiple organ failure. TIVS is similar to SIRS (severe inflammatory respiratory syndrome), SARS (severe acute respiratory syndrome) and sepsis (caused by bacteria). These conditions affect lung function as well as many other organs. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2, which causes COVID-19) gains access to host cells through angiotensin-converting enzyme expressed in type II surfactant-secreting alveolar cells of the lung. Severe COVID-19 is associated with a major immune inflammatory response enriched in neutrophils, lymphocytes, macrophages, and immune mediators. Deaths from COVID-19 are primarily due to diffuse alveolar damage with pulmonary edema, hyaline membrane formation, and interstitial mononuclear inflammatory infiltrates compatible with early adult respiratory distress syndrome (ARDS). TNF is present in the blood and diseased tissue of patients with COVID-19; TNF is involved in almost all acute inflammatory reactions and plays a role in amplifying inflammation. Accordingly, treatment using the constructs herein is provided.

2. Treatment of neurodegenerative and demyelinating diseases and disorders

As discussed elsewhere herein, several neurodegenerative and demyelinating diseases and conditions are associated with chronically elevated TNF levels in the central nervous system (CNS). Elevated levels of TNF and TNF signaling through TNFR1 have been implicated in initiating and sustaining neuroinflammation and promoting neuronal apoptosis, demyelination and cognitive decline. For example, in patients with Alzheimer's disease (AD), TNF promotes microglia activation, synaptic dysfunction, neuronal apoptosis, and accumulation of plaques and tangles, and elevated TNF levels prevent AD from preventing efficient plaque clearance by microglia. It inhibits the phagocytosis of amyloid beta (Aβ) in the patient's brain. In patients with Parkinson's disease (PD), elevated TNF levels result in neuroinflammation and dopamine neurotoxicity. Polymorphisms in the gene encoding TNFR1 as well as elevated levels of TNF are associated with demyelination and the development of demyelinating disorders such as multiple sclerosis (MS). Thus, selective blockade of TNF signaling through TNFR1 can be used for the treatment or alleviation of neurodegenerative and demyelinating diseases and disorders, and other conditions of the CNS.

TNF signaling through TNFR2 is associated with anti-inflammatory and neuroprotective effects. For example, activation of TNFR2 by TNF suppresses seizures, attenuates cognitive dysfunction after brain injury, and promotes remyelination and survival of neurons. Proliferation, expansion and activation of immunosuppressive Tregs following TNFR2 agonism also have neuroprotective effects. For example, TNFR2 signaling promotes Treg cell expansion and suppressive activity in inflammatory CNS demyelinating diseases such as experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Thus, TNFR2 agonism may also be used for the treatment or amelioration of neurodegenerative and demyelinating diseases and disorders, and other conditions of the CNS.

The TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, such as bispecific, TNFR1 antagonist/TNFR2 agonist constructs, fusion proteins, and nucleic acids provided herein are useful in Alzheimer's disease, Parkinson's disease, multiple sclerosis, and stroke. It can be used to treat or alleviate the symptoms of neurodegenerative and demyelinating diseases, including but not limited to, and other CNS disorders and conditions.

3. Treatment of cancer and other immunosuppressive diseases, disorders and conditions

As described herein, tumors are infiltrated by large numbers of immunosuppressive TNFR2 ⁺ Tregs, preventing the proliferation of tumor-killing CD8 ⁺ cytotoxic T lymphocytes (CTLs) (also referred to as effector T-cells (Teffs)), resulting in tumor suppression. allow for growth Antagonism of TNFR2 on lymphocytes in the tumor microenvironment (TME) restores the balance between the two types of T-cells by eliminating Tregs and allowing the activation and expansion of effector T-cells, resulting in tumor cell lysis ( See, eg, Vanamee et al. (2017) Trends in Molecular Medicine 23(11):P1037-P1046).

TNFR2 is abundantly expressed on the surface of many types of human cancer cells, including, for example, renal cell carcinoma, colon cancer, Hodgkin's lymphoma, multiple myeloma, cutaneous non-Hodgkin's lymphoma, and ovarian cancer. TNFR2 mutations in cancer are associated with gene duplication and constitutive activation. Murine bone marrow-derived suppressor cells (MDSC) also express TNFR2, and its suppression has been shown to regulate metastasis in a murine liver cancer model. In addition, immune checkpoint inhibitors cause upregulation of TNFR2 in tumor-infiltrating Tregs, leading to tumor immune evasion and drug resistance. Not all patients respond to checkpoint inhibitor therapy, patients can relapse and severe autoimmune side effects have been observed with checkpoint inhibitor therapy (see, e.g., Vanamee et al. (2017) Trends in Molecular Medicine 23(11):P1037-P1046). Thus, blockade of TNFR2 can be used in the treatment of certain types of cancer by inhibiting MDSCs, which can directly kill tumor cells and prevent the formation of metastases, through suppression of immunosuppressive Tregs, which enable the proliferation of effector T-cells. . Since TNFR2 is also expressed in normal tissues (particularly macrophages; see eg proteinatlas.org/ensg00000028137-tnfrsf1b/tissue), TNFR2 antagonists do not have ADCC activity but have FcRn activity (or enhanced FcRn activity). Dosing is individualized because a patient's tumor must have significantly higher levels of TNFR2 than adjacent normal tissue in order to receive treatment as described herein. To this end, TNFR2 antagonists will be used in conjunction with other therapies, particularly immunomodulatory therapies that otherwise result in the accumulation of regulatory T-cells in the tumor.

Thus, the TNFR2 agonist, bispecific TNFR1 antagonist/TNFR2 agonist constructs and fusion proteins provided herein are useful for treating solid cancers, hematological malignancies, and, for example, renal cell carcinoma, colon cancer, Hodgkin's lymphoma, multiple myeloma, cutaneous non-Hodgkin's lymphoma, and other hyperproliferative diseases and disorders including, but not limited to, ovarian cancer.

4. Combination therapy

Combination therapies include TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, such as bispecific, TNFR1 antagonist/TNFR2 agonist constructs provided herein in combination with another agent or treatment including radiation and surgery; fusion proteins, and administration of nucleic acids. Additional agents or therapies may be administered concurrently, before, after, or intermittently with the treatments provided herein. They may be in separate compositions or co-formulations.

The TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, such as bispecific, TNFR1 antagonist/TNFR2 agonist constructs, fusion proteins, and nucleic acids provided herein are antagonist/blockers, antibodies, cytotoxic agents, anti -Inflammatory agents, cytokines, growth factors, growth inhibitory agents, cardioprotective agents, immunosuppressive agents, chemotherapeutic agents, biological or non-biological disease modifying anti-rheumatic drugs (DMARDs), therapeutic agents (including antibodies) for infectious diseases, or other It may be administered before, after, or concomitantly with one or more other therapeutic regimens or agents, including but not limited to therapeutic agents. The TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, such as bispecific, TNFR1 antagonist/TNFR2 agonist constructs, and nucleic acids provided herein may be used as a first-line treatment or as an anti-TNF therapeutic in acute or chronic treatment It can be administered to patients as a second-line therapy that is not as effective as a second-line therapy. Exemplary anti-TNF therapies that can be used in combination therapy herein include, for example, conventional synthetic DMARDs such as (generic and exemplary trade names): methotrexate (MTX), hydroxychloroquine (HCQ; Plaquenil ^® ), sulfasalazine ( ^Azulfidine® ), and leflunomide ( ^Arava® ); Biological DMARDs such as abatacept ( ^Orencia® ), anakinra ( ^Kineret® ), rituximab ( ^Rituxan® , ^Truxima® , ^MabThera® ), tocilizumab (atlizumab, ^Actemra® , ^RoActemra® ), corticosteroids steroids (e.g. dexamethasone, methylprednisolone, prednisolone, prednisone, or triamcinolone), tofacitinib ( ^Xeljanz® ), and TNF-inhibitor/anti-TNF agents such as certolizumab pegol ( ^Cimzia® ); infliximab ( ^Remicade® ), adalimumab ( ^Humira® ), golimumab ( ^Simponi® ), and etanercept ( ^Enbrel® ). Combination therapy may also include an immunotherapeutic drug such as cyclosporine, methotrexate, adriamycin or cisplatinum, and an immunotoxin.

Examples of anti-inflammatory drugs and agents useful in combination therapy include non-steroidal anti-inflammatory drugs (NSAIDs) including salicylates, such as aspirin, conventional NSAIDs such as ibuprofen, naproxen, ketroprofen, nabumetone , piroxicam, diclofenac, or indomethacin, and Cox-2 selective inhibitors such as celecoxib (marketed under the trade name ^Celebrex® ), or rotecoxine (marketed under the trade name ^Vioxx® ). Other compounds useful for combination therapy include antimetabolites such as methotrexate and leflunomide; corticosteroids or other steroids such as cortisone, dexamethasone, or prednisone; analgesics such as acetaminophen; aminosalicylates such as mesalamine; and cytotoxic agents such as azathioprine (marketed under the trade name ^Imuran® ), cyclophosphamide (marketed under the trade name ^Cytoxan® ), and cyclosporine A.

Additional agents that may be used in combination therapy include, for example, biological response modifiers including anti-inflammatory cytokines such as IL-10; B-cell targeting agents such as anti-CD20 antibodies (eg rituximab); compound targeting T antigen; adhesion molecule blockers; chemokine receptor antagonists; kinase inhibitors such as inhibitors of mitogen activated protein (MAP) kinase, c-Jun N-terminal kinase (JNK), or NFκB; and peroxisome proliferator-activated receptor-gamma (PPAR-γ) ligands. Additional agents that may be used in combination therapy include immunosuppressive agents. Immunosuppressive agents include, for example, tacrolimus or FK-506; mycophenolic acid; calcineurin inhibitor (CNI); CSA; and sirolimus, or other agents known to suppress the immune system.

Polypeptides and constructs provided herein can also be used in combination with agents administered to treat cardiovascular disease and/or administered during procedures to treat cardiovascular disease, such as anticoagulants. Exemplary anticoagulants include heparin, warfarin, acenocumarol, phenindione, EDTA, citrate, oxalate, and direct thrombin inhibitors such as argatroban, lepirudin, bivalirudin, and ximelagatran, but Not limited to this.

Polypeptides and constructs provided herein can be administered with antibodies for the treatment of autoimmune or inflammatory diseases, transplant rejection or GvHD. Examples of such antibodies include anti-α4β7 integrin antibodies such as LDP-02; anti-beta2 integrin antibodies such as LDP-01; anti-complement (C5) antibodies such as 5G1.1; anti-CD2 antibodies such as BTI-322 and MEDI-507; anti-CD3 antibodies such as OKT3 and SMART anti-CD3; anti-CD4 antibodies such as IDEC-151, MDX-CD4 and OKT4A; anti-CD11a antibody; anti-CD14 antibodies such as IC14; anti-CD18 antibody; anti-CD23 antibodies such as IDEC 152; anti-CD25 antibodies such as daclizumab; anti-CD40L antibodies such as 5c8, luplizumab and IDEC-131; anti-CD64 antibodies such as MDX-33; anti-CD80 antibodies such as IDEC-114; anti-CD147 antibodies such as ABX-CBL; anti-E-selectin antibodies such as CDP850; anti-gpIIb/IIIa antibodies such as ReoPro ^® /abxiximab; anti-ICAM-3 antibodies such as ICM3; anti-ICE antibodies such as VX-740; anti-FcγR1 antibodies such as MDX-33; anti-IgE antibodies such as rhuMAb-E25; anti-IL-4 antibodies such as SB-240683; anti-IL-5 antibodies such as SB-240563 and SCH55700; anti-IL-8 antibodies such as ABX-IL8; anti-interferon gamma antibodies; anti-TNFα antibodies such as CDP571, CDP870, D2E7, infliximab and MAK-195F; and anti-VLA-4 antibodies such as ^Antegren® . Examples of other Fc-containing molecules that can be co-administered to treat autoimmune or inflammatory diseases, transplant rejection and GvHD include the TNFR2-Fc fusion protein Enbrel ^® (etanercept), and Regeneron's IL-1 trap, but Not limited.

Examples of antibodies that can be co-administered to treat infectious diseases include anti-anthrax antibodies such as ABthrax; anti-CMV antibodies such as CytoGam and sevirumab; anti-Cryptosporidium antibodies such as CryptoGAM and sporidin-G; anti-Helicobacter antibodies such as pyloran; anti-hepatitis B antibodies such as HepeX-B and Nabi-HB; anti-HIV antibodies such as HRG-214; anti-RSV antibodies such as felbizumab, HNK-20, palivazumab, and RespiGam; and anti-staphylococcal antibodies such as Aurexis, Aurograb, BSYX-A110, and SE-Mab.

In some examples, the TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, such as bispecific, TNFR1 antagonist/TNFR2 agonist constructs, fusion proteins, and nucleic acids provided herein are combined with one or more chemotherapeutic agents. are administered together Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cyclophosphamide ( ^CYTOXAN® ); alkyl sulfonates such as busulfan, improsulfan and piposulfan; androgens such as calosterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenergics such as aminoglutethimide, mitotane, and trilostane; anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; Antibiotics such as aclasinomycin, actinomycin, anthramycin, azaserin, bleomycin, cactinomycin, calicheamicin, carubicin, carminomycin, carzinophylline, chromomycin, dactinomycin, daunol Bicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olibomycin , peplomycin, porphyromycin, puromycin, quelamycin, rodorubicin, streptonigryn, streptozocin, tubersidin, ubenimex, ginostatin, and zorubicin; For example, tamoxifen, raloxifene, aromatase inhibitor 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, kenoxyfen, LY 117018, onapristone, and toremifene (Pareston) estrogen; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, and trimetrexate; aziridines such as benzodepa, carboquone, meturedepa, and uredepa; ethylenimine and methylmelamine, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine; folic acid supplements such as folinic acid; Nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobemvicine, phenesterine, prednimu Steen, Trophosphamide, and Uracil Mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; proteins such as arginine deiminase and asparaginase; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, camofur, cytarabine, dideoxyuridine, doxifuridine, enocitabine, floxuridine, and 5-FU; taxanes such as paclitaxel ( ^TAXOL® , Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel ( ^TAXOTERE® , Rhone-Poulenc Rorer, Antony, France); topoisomerase inhibitors such as RFS 2000; thymidylate synthase inhibitors such as Tomudex; additional chemotherapeutic agents including aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; dedatrexate; dephosphamide; demecolsine; diaziquone; difluoromethylornithine (DMFO); eplonithine; elliptinium acetate; Etogluside; gallium nitrate; hydroxyurea; lentinan; Ronidamine; mitoguazone; mitoxantrone; furdamole; nitracrine; pentostatin; phenamet; pirarubicin; pophyllic acid; 2-ethylhydrazide; procarbazine; polysaccharide K (PSK, crestin); Razoxic acid; sizopyran; spirogermanium; tenuazinic acid; triaziquone; 2,2', 2"-trichlorotriethylamine;urethane;vindesine;dacarbazine;mannomustine;mitobronitol;mitolactol;pipobroman;gastosine; arabinoside ("Ara-C");cyclophosphamide;thiotepa;chlorambucil;gemcitabine;6-thioguanine;mercaptopurine;methotrexate; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novanthrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; retinoic acid; esperamycin; capecitabine; and topoisomerase inhibitors; eg irinotecan Pharmaceutically acceptable salts, acids or derivatives of any of the above may also be used.

Chemotherapeutic agents can be administered as prodrugs. Examples of prodrugs that can be administered with the TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, such as bispecific, TNFR1 antagonist/TNFR2 agonist constructs, fusion proteins, and nucleic acids provided herein include: For example, a phosphate-containing prodrug, a thiophosphate-containing prodrug, a sulfate-containing prodrug, a peptide-containing prodrug, a D-amino acid modified prodrug, a glycosylated prodrug, a beta-lactam-containing prodrug, an optionally substituted phenox, cyanoacetamide-containing prodrugs, or optionally substituted phenylacetamide-containing prodrugs, and 5-fluorocytosine and other 5-fluorouris that can be converted to 5-fluorocytosine and more active cytotoxic free drugs. Dean prodrugs include, but are not limited to. TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, e.g., bispecific, TNFR1 antagonist/TNFR2 agonist constructs, e.g., together with an in vivo cleavable linker from which the active form of the construct is released It can be provided as a prodrug by linking them to a targeting agent that targets a specific tissue or location of

In some examples, TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, such as bispecific, TNFR1 antagonist/TNFR2 agonist constructs, fusion proteins, and nucleic acids provided herein include but are not limited to is administered with one or more antibiotics that are not: aminoglycoside antibiotics (e.g., apramycin, abecacin, bambermycin, butyrosin, dibecacin, gentamicin, kanamycin, neomycin, netylmycin, paromo amycin, ribostamycin, sisomycin, and spectinomycin), aminocyclitols (e.g. spectinomycin), amphenicol antibiotics (e.g. azideamphenicol, chloramphenicol, florfe Nicol, and thiamphenicol), ansamycin antibiotics (eg, lipamide and rifampin), carbapenems (eg, imipenem, meropenem, and panipenem); Cephalosporins (e.g., cefaclor, cefadroxil, cefamandole, ceftrizine, cefazedone, cefozofran, ceffimisole, cefpyramide, cefpyrom, cefprozil, cefuroxime, cefixime, cephalexin, and cefradine), cefamycin (eg, cefbuperazone, cefoxitin, cefminox, cefmetazole, and cefotetan); lincosamides (eg, clindamycin, and lincomycin); macrolides (e.g., azithromycin, brefeldin A, clarithromycin, erythromycin, loxithromycin, and tobramycin), monobactams (e.g., aztreonam, carumonam, and tigemonam ); mupirocin; oxacephem (eg, plomoxef, ratamoxef, and moxalactam); Penicillins (e.g. amdinocillin, amdinocillin pyrocyl, amoxicillin, bacampicillin, benzylpenicillinnan, benzylpenicillin sodium, epicillin, penbenicillin, floxacillin, phenamecillin, penetamate hydrioda id, penicillin o-bennetamine, penicillin O, penicillin V, penicillin V benzoate, penicillin V hydrabamine, penimepicyline, and phenethicillin potassium); polypeptides (eg, bacitracin, colistin, polymyxin B, teicoplanin, and vancomycin); Quinolones (e.g., amifloxacin, cinoxacin, ciprofloxacin, enoxacin, enrofloxacin, fleoxacin, flumequin, gatifloxacin, gemifloxacin, grepafloxacin, lomefloxacin, moxifloxacin , nalidixic acid, norfloxacin, ofloxacin, oxolinic acid, pefloxacin, pipemidic acid, losoxacin, lufloxacin, sparfloxacin, temafloxacin, tosafloxacin, and trovafloxacin) ; rifampin; streptogramins (eg, quinupristine, and depopristine); sulfonamides (eg, sulfanilamide, and sulfamethoxazole); and tetracyclines (e.g., chlortetracycline, demeclocycline hydrochloride, demethylchlortetracycline, doxycycline, duramycin, minocycline, neomycin, oxytetracycline, streptomycin, tetracycline, and vancomycin).

In some examples, the TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, such as bispecific, TNFR1 antagonist/TNFR2 agonist constructs, fusion proteins, and nucleic acids provided herein are amphotericin B, ciclopyl can be administered with one or more antifungal agents including but not limited to lox, clotrimazole, econazole, fluconazole, flucytosine, itraconazole, ketoconazole, miconazole, nystatin, terbinafine, terconazole, and thioconazole. can In some examples, the TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, such as bispecific, TNFR1 antagonist/TNFR2 agonist constructs, and nucleic acids provided herein are protease inhibitors, reverse transcriptase inhibitors, and I Interferons, viral fusion inhibitors, neuraminidase inhibitors, acyclovir, adefovir, amantadine, amprenavir, clevudine, enfuvirtide, entecavir, foscarnet, ganciclovir, idoxuridine, indinavir is administered with one or more antiviral agents, including but not limited to others including, but not limited to, le, lopinavir, fleconaril, ribavirin, rimantadine, ritonavir, saquinavir, trifluridine, vidarabine, and zidovudine. .

TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, such as bispecific, TNFR1 antagonist/TNFR2 agonist constructs, fusion proteins, and nucleic acids provided herein are administered with the growth factor trap constructs described below It can also be administered with any of the therapeutic anti-TNF agents and treatments set forth below for combination therapy with the growth factor trap construct. Combination therapy can also include the growth factor trap constructs provided herein.

Pharmaceutical compositions containing TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, such as bispecific, TNFR1 antagonist/TNFR2 agonist constructs, fusion proteins, and nucleic acids provided herein are provided herein or It can be used to treat any disease, disorder and condition known in the art. Diseases, disorders and conditions include one or more chronic inflammatory, autoimmune, neurodegenerative or demyelinating diseases or conditions. Also provided are combinations of polypeptides and constructs provided herein for the treatment of chronic inflammatory, autoimmune, neurodegenerative or demyelinating diseases or conditions, and another treatment or compound. The TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, such as bispecific, TNFR1 antagonist/TNFR2 agonist constructs, fusion proteins, and nucleic acids, and additional agent(s) provided herein may be used together or sequentially or packaged as separate compositions for intermittent administration. Alternatively, they may be provided in a single composition for administration as a single composition, or in two compositions for administration. The combination may be packaged as a kit, optionally with additional reagents, instructions for use, vials and other containers, syringes, and other items for treatment.

L. Examples

The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

Example One

candidate 1 TNFR1 Expression and evaluation of antagonist molecules

Expression and purification of anti-TNFR1 molecules

Candidate monovalent TNFR1 antagonist molecules are expressed in mammalian cells, under the control of the CMV promoter, using the expression plasmid shown in Figure 1, where TE19080L is the inserted fragment. For the expression of protein therapeutics such as the constructs herein, mammalian cells such as Chinese Hamster Ovary (CHO) or Human Embryonic Kidney 293 (HEK293) cells are used to provide post-translational modifications, including glycosylation. , which can be important for proper protein structure, function and activity. Expression in bacterial, yeast or insect cells either results in no glycosylation (in the case of bacterial cells) or a different glycosylation pattern compared to expression in mammalian cells (in the case of yeast or insect cells). Expression in bacteria can also contaminate protein therapeutics with bacterial endotoxins, which can activate innate immune cells, complicate analysis of cell-based assays, and cause pyrogenic effects upon administration in vivo.

Expression plasmid construction as well as transient and stable cell line expression are described in Vazquez-Lombardi et al. (2018) Nat. Protoc . 13(1):99-117. Five exemplary TNFR1 antagonist molecules are produced for initial evaluation. The TNFR1 antagonist molecule includes (GGGGS) an H398-derived scFv containing one V _L and one V _H domain of H398 linked together by _{a three-} peptide linker (SEQ ID NO: 678); TNFR1 antagonist domain antibody (dAb) DOM1h-574-16 (SEQ ID NO: 57); TNFR1 antagonist dAb DOM1h-549 (SEQ ID NO: 58); (GGGGS) TNFR1 antagonist dAb DOM1h-574 from DMS5541 (described elsewhere herein) fused to the anti-serum albumin dAb (albudAb) of DMS5541 (DOM7h-11-3; SEQ ID NO: 52) by a _three- peptide linker fusion protein (SEQ ID NO: 704), containing -208 (SEQ ID NO: 54); and an anti-TNFR1 dAb specified DOM1h-131-206 (SEQ ID NO: 59) fused to an anti-serum albumin dAb (albudAb or DOM7h-11-3; SEQ ID NO: 52) of DMS5541 by a (GGGGS) _three- peptide linker. One, a fusion protein (SEQ ID NO: 705). Insertion of the H398 scFv, and the (GGGGS) _three linkers in the DOM1h-574-208 and DOM1h-131-206 fusion proteins, respectively, provide more flexibility between the V _H and V _L domains, and the two dAbs, and It improves the manufacturing process by increasing stability and resistance to denaturation. The sequences of each of these TNFR1 antagonist molecules are provided in Table 12 below, some with various linker sequences (see also Enever et al., (2015) Protein Engineering, Design & Selection 28(3) :59-66; These documents describe dAbs and variants thereof that can be used for further modifications and additions of linkers and modifiers).

Also, two heavy chains selected from any of SEQ ID NOs: 53-83 and 503-671, such as those set forth in SEQ ID NOs: 57-59, and at least 95%, 96%, 97%, 98%, 99% sequence identity thereto Nanobodies, including variants thereof, and constructs containing the nanobodies are provided. Constructs comprising identical heavy chains are provided. An example of these is the TNFR1 dAb designated DOM1h-131-206. In addition, constructs containing any such dAb, such as those provided directly to human serum albumin or more commonly via a linker such as a GS linker, or as an Fc fusion or in any other construct described herein Provided.

These and other such dAbs and TNFR1 binding molecules can be modified to increase specificity for TNFR1 by eliminating any antagonistic activity to TNFR2, increase or add TNFR2 agonist activity, and/or reduce immunogenic epitopes. or modified to eliminate and/or linked to activity modifiers, such Fc units and modified Fc units/modified Fc dimers, and/or serum half-life extending moieties.

The HEK293 cell line is used for transient expression, in vitro evaluation of the expressed antagonist and high affinity for TNFR1 (eg, K _d < 50 nM, or < 10 nM, or < or 5 nM), and TNFR1 signaling After identification of molecules with desired properties, such as potent inhibition of transduction (eg, IC ₅₀ <50 nM, or <10 nM, or <5 nM), stable cell lines are prepared from the derivatives of CHO cells. Typically, this is a picomolar (pM) affinity, such as about or about 19 pM affinity or 20 pM, 15 pM, 10 pM, 5 pM, 2 pM, or 1 pM affinity.

Transient expression is optimized in CHO DG44 cells (e.g., CHO-DG44 (DHFR ^- ) and FreeStyle ^TM CHO-S cells, Invitrogen) to generate transfected pools that are screened to identify or select high expressing clones do. No poly-histidine, or other purification tags are used. Instead, proteins for screening are purified from serum free media by HPLC along with other well known methods. The matrix used for the HPLC is Amsphere ^™ A3 Protein A chromatography resin (JSR Life Sciences), or other similar resin according to the manufacturer's protocol. If the protein is not at least 95% pure, as judged by size exclusion HPLC, further purification is performed (eg ion exchange or hydrophobic chromatography).

Remove endotoxin (see, eg, Vazquez-Lombardi et al. (2018) for an exemplary protocol). After protein purification, endotoxin levels are determined using a detection kit such as the QCL-1000 endpoint chromogenic LAL assay kit (Lonza). For endotoxin removal, the theoretical pi of the purified protein is determined using a sequence analysis tool (e.g., ExPASy ProtParam) and the pH low-endotoxin PBS buffer is adjusted to a pH less than but close to the theoretical pi of the purified protein. Adjust. The protein sample is then dialyzed against at least 30 volumes of pH-adjusted PBS for at least 2 hours at 4°C. An additional dialysis step is performed overnight and then again for at least 2 hours the next day. The sample is then purified using anion exchange affinity chromatography, retested to determine endotoxin levels, and the process is repeated until an acceptable level of endotoxin is achieved. The size and purity of the protein product is determined by SDS-PAGE analysis or other suitable method. Alternatively, other methods may be used, such as the Proteus Endotoxin Removal Kit and accompanying manufacturer's handbook (BIORAD, see bio-rad-anti-bodies.com/static/uploads/ifu/pur030.pdf). This step can be repeated until the desired level of endotoxin is reached, typically >0.5 endotoxin units/ml (0.5 endotoxin units/ml or less).

Screening of purified proteins

Purified TNFR1 antagonist molecule candidates can be prepared by methods described above in the detailed description, or by, for example, immunoassays (eg, ELISA), surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), or methods in the art. Screening is performed to determine binding affinity to the extracellular domain of TNFR1 using methods known in the art, such as other known kinetic interaction assays. SPR can be performed using several commercially available platforms such as, for example, the BIAcore system (GE Healthcare Life Sciences). Exemplary assays are described, eg, in Lang et al (2015) J. Biol Chem 291 :5022-5037, which describes and compares various assays for assessing binding affinity. Selected candidates include those with K _d values of <5 nM.

TNFR1 antagonists are also screened to determine whether their binding to TNFR1 is competitive or non-competitive for TNF, using methods known in the art, such as SPR. When an inhibitor binds to a receptor (eg, TNFR1) and blocks binding of a ligand (eg, TNF) by, for example, attaching to the active site, this prevents the inhibitor from "competing" with the substrate for the enzyme. Because of this, it is competitive inhibition, i.e., only the inhibitor or substrate can be bound at any given moment. In uncompetitive inhibition, the inhibitor does not block binding of the ligand to the ligand-binding site on the receptor. Instead, it attaches to other sites and blocks the receptor from responding to the bound ligand. This inhibition is said to be "non-competitive" because both inhibitor and substrate can bind simultaneously. Thus, if a ligand is added to a receptor-binding assay at a saturating concentration and it does not inhibit binding of the antibody, the two molecules are independent and non-competitive. The reverse is also valid. If both are competitive, increasing the concentration of antibody will prevent TNF from binding to the receptor. This holds true regardless of whether the binding assay is performed in cells or with receptors or ligands bound to a surface. For exemplary assays, see, eg, Frey et al. (2001) Current Protocols in Neuroscience , "Receptor Binding Techniques", available at doi.org/10.1002/0471142301.ns0104s00.

For example, the ability of TNF to bind to human TNFR1 coated on a BIAcore chip is first determined. The TNFR1 surface is then saturated with TNFR1 antagonist molecules, followed by injection of TNF, and binding of TNF to TNFR1 is reassessed. If binding is uncompetitive, both bind TNFR1; If the bonds are competitive, they will interfere with each other. If binding of TNF is unaffected or only slightly reduced, binding of the antagonist to TNFR1 is uncompetitive with respect to TNF, and if binding of TNF is abrogated or significantly reduced, binding of the antagonist to competitive with respect to TNF is considered hostile For purposes herein, competitive binders are selected.

TNFR1 antagonist molecules are described in McFarlane et al. (2002) FEBS , which result in activation of NFκB-luciferase expression (i.e., gene receptor assay) by methods known in the art, for example. Lett . 515(1-3):119-126, to determine their ability to inhibit TNFR1 signaling in the presence of TNF on cells. Cells used in these experiments are TNFR1 or TNFR2 (e.g., myeloma cell lines AMO1, U266, and L363; see, e.g., Rauert et al . (2011)) unless transfected with a TNFR1- or TNFR2-expressing plasmid. Cell Death Dis.2 ( 8):e194]) is not expressed. Alternatively, human cell lines can be used in which the TNFR1 and/or TNFR2 genes have been inactivated or knocked out using CRISPR vectors, antisense RNA expression, or other methods known in the art. TNFR1 ^- and/or TNFR2 ^- cell lines, which can be obtained from commercial sources (eg, Genoway and Synthego), can also be used. These cell lines can then be specifically transfected with TNFR1 and/or TNFR2 expression cassettes. For example, cells expressing TNFR1, TNFR2, or TNFR1 and TNFR2 can be used to assess the selectivity of an antagonist for TNFR1 and to determine the potency of inhibition of TNF signaling through TNFR1.

To determine the inhibition of TNFR1 signaling by TNFR1 antagonists, cells expressing human TNFR1 were transiently transfected with an NF-κB-luciferase reporter construct using lipofectamine and 48 hours after transfection Receptor-stimulated luciferase transcription is then measured. Cells stably expressing TNFR1 and NF-κB-luciferase were plated in 24-well plates at a density of 1 Х 10 ⁵ cells/ml culture medium until they reached 80% confluency. Incubate (approximately 24 hours). Cells are then incubated for 6 hours with 50 ng/ml of TNF and various concentrations of TNFR1 antagonist. Cells were washed twice with ice-cold PBS, 200 μl of ice-cold lysis buffer (25 mM Tris-phosphate pH 7.8, 8 mM MgCl ₂ , 1 mM DTT, 1% Triton X-100, 15% glycerol) was added and 5 NF-κB-stimulated luciferase activity is detected by incubation on ice for 10 minutes. The cell extract is then scraped into a 1.5 ml Eppendorf tube, centrifuged to pellet cell debris, and 100 μl of the supernatant is used to measure luciferase induction using a luminometer. The IC ₅₀ is then calculated by plotting relative luminescence units (RLU) versus TNFR1 antagonist concentration and using curve fitting software such as GraphPad Prism. Other similar assays for assessing inhibition of TNFR1 signaling include assays that measure the induction of phospho-IκBα, indicative of activation of the classical NF-κB pathway, in cells expressing TNFR1 treated with TNF ( See, eg, Rauert et al (2011) Cell Death Dis. 2(8):e194).

As determined above, TNFR1 antagonist candidates exhibiting inhibition of TNFR1 signaling of at least 80% and an IC ₅₀ value approximately equal to K _d (ie, <5 nM) are selected for further optimization.

Example 2

Optimization of selected candidate monovalent TNFR1 antagonist molecules

Optimization of affinity for TNFR1 and potency of inhibition of TNFR1 signaling

A candidate TNFR1 antagonist molecule that meets the selection criteria outlined above (ie, high affinity for TNFR1 and potent inhibition of TNFR1 signaling, with a K _d and IC ₅₀ value of <5 nM), has an affinity for TNFR1; and optimized to increase the potency of inhibition of TNFR1 signaling. This includes random mutagenesis, site-directed _mutagenesis , molecular _modeling , and/or error-prone PCR (PCR). For example, this is done by mutagenizing the remaining amino acids of the VH domain while conserving the amino acids most important for binding, preparing a phage library, and screening for selected, high affinity binding variants to TNFR1 (see (2017) Frontiers Immunol . 8 :986). According to this method, a phage display library for selection of such variants is produced. In a first step, computational and experimental alanine scanning mutagenesis identifies sites in the complementarity-determining regions (CDRs) that allow for mutagenesis while maintaining antigen binding. Next, the most permissive CDR positions are mutated, based on natural antibody diversity, using degenerate codons to encode the wild-type residues and the few most frequently occurring residues at each CDR position. This mutagenesis approach generates antibody libraries with variants with a broad number of CDR mutations, including antibody domains with a single mutation and other domains with dozens of mutations. In a final step, the library (approximately 10 million variants) displayed on the surface of the yeast is sorted to identify the CDR mutations that increase the affinity the most.

half-life extension

The optimized molecule is then, as described above in the detailed description, for example by fusion with an IgG Fc domain, particularly a modified Fc domain, or human serum albumin (HSA), or by PEGylation, Linked to a half-life extending moiety, an in vivo serum half-life of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, eg, 10-12 days, is achieved in SCID mice. The modified molecule is then retested in the in vitro assay described above to ensure that high affinity binding to TNFR1 and potent inhibition of TNFR1 signaling is maintained. Production of successful candidates is expanded for further in vitro and in vivo assays.

in vitro assay

Phagocytosis Assay

As discussed and described in the detailed description, existing TNF blockers that inhibit TNFR1 activity also inhibit TNFR2. There are two types of black box warnings from regulatory agencies for TNF blockers. The first is the susceptibility of anti-TNF (TNF blocker) treated patients to infection. These infections include bacteria, mycobacteria (e.g., tuberculosis), fungi (e.g., histoplasmosis, aspergillosis, candidiasis, coccidioidomycosis, blastomycosis, and pneumocystis), viruses (e.g., hepatitis B), and other opportunistic pathogens (organisms that normally do not cause disease in healthy people, but can cause serious illness when a person's immune system (resistance) is weakened). and site. Another issued black box warning relates to pediatric malignancies (eg see online.epocrates.com/u/10b3301/Humira/Black+Box+Warnings). As discussed in the detailed description, this vulnerability results from total blockage of signaling by TNFR1 and TNFR2 when their ligand, TNF, is blocked. Suppression of this innate immune effect of TNF is mediated by TNFR2 (see, for example, Ahmad et al. (2018) Front. Immunol. 9 :2572) and is primarily mediated by transmembrane forms of TNF that preferentially activate TNFR2. (See, eg, Miller et al. (2015) Journal of Immunology 195(6) :2633-2647).

Anti-TNF therapeutics such as adalimumab, infliximab, and etanercept have been shown to have an inhibitory effect on IFN-γ induced phagocytic maturation in phorbol myristate acetate-differentiated human THP-1 cells. Adalimumab and infliximab, but not etanercept, inhibit phagocytic maturation in primary human peripheral blood monocyte-derived macrophages in the presence or absence of IFN-γ (see, e.g., Harris et al. (2008) J. Infect Dis.198 : 1842-1850) . In view of the foregoing, an advantage of certain TNFR1 inhibitor constructs is the preservation of TNFR2 function, and thus macrophage function, which macrophages are important for eliminating opportunistic organisms. Opportunistic agents include the mycobacteria that cause tuberculosis (see, eg, Fraga et al. (2018) Curr . Issues Mol . Biol . 25 :169-198). Macrophage function is disrupted in autoimmune disease in patients treated with TNF blockers. TNFR1-specific antagonists only inhibit TNFR1 function, thereby avoiding disruption of TNFR2 function required for normal macrophage function. Such TNFR1-specific antagonists can be identified by assays for anti-TNF effects on macrophage phagocytosis. Among the identified TNFR1-specific antagonists are also those that increase macrophage activity by stimulating TNFR2 function (TNFR1 antagonists and TNFR2 agonists).

mycobacterium tuberculosis on the macrophage response to infection. TNFR1 Assays to determine the effect of antagonists

TNF plays an important role in mediating inflammatory host responses to various pathogens, including Mycobacterium tuberculosis , and TNF also plays a role in the immunopathology of tuberculosis (TB). Mycobacterial infection induces the secretion of TNF by macrophages. TNF enhances the ability of macrophages to engulf and kill mycobacteria. TNF also stimulates macrophage apoptosis, increasing killing and presentation of mycobacterial antigens by dendritic cells. TNF is also required for granuloma formation and maintenance, and neutralization of TNF in mice chronically infected with M. tuberculosis disrupts granuloma integrity, exacerbating infection and increasing mortality. TNF blockers, such as adalimumab and infliximab, increase susceptibility to infection with various pathogens, including M. tuberculosis, and suppress mycobacteria-containing phagocytic maturation in human macrophages, thereby preventing latent tuberculosis. Increases the risk of reactivation. Vascular maturation (i.e., phagocytic acidification and fusion with lysosomes) is essential for the presentation of mycobacterial antigens to T-cells and initiation of the adaptive immune response (see, eg, Harris et al. (2008) J. Infect. Dis. 198 :1842-1850]).

To identify and/or characterize a TNFR1 antagonist, the effect of a TNFR1 antagonist provided herein on the macrophage response to infection with M. tuberculosis is evaluated. Mycobacteria-containing phagocytic maturation was assayed in human macrophages and described by Harris et al. (2008) J. Infect. Dis . 198 :1842-1850] compared to phagocytic maturation in TNF blockers, such as adalimumab. Of interest are TNFR1 antagonists that do not increase susceptibility to infection compared to known TNF blockers, such as adalimumab.

Preparation of THP-1 cells and monocyte-derived macrophages (MDM)

Human THP-1 cells are cultured in RPMI 1640 (Invitrogen) with 10% fetal bovine serum (FBS; Gibco). Cells were differentiated into macrophage-like cells by treatment with 100 nmol/L of phorbol myristate acetate (PMA) for 24 hours and then cultured in normal medium for 3 days. To prepare human monocyte-derived macrophages (MDM), peripheral blood mononuclear cells (PBMC) are isolated from the blood of healthy donors using density gradient centrifugation in Histopaque ^® -1077 (Sigma). Monocytes were isolated by attaching to gelatin-coated culture dishes and cultured overnight in RPMI 1640 with 5% human AB serum (Sigma). Adherent cells are removed with 10 mmol/L EDTA in PBS and grown on coverslips in 12-well plates for 10 days. THP-1 cells and MDM are grown on coverslips at a concentration of 2Х10 ⁵ cells/well.

Preparation of mycobacteria

Green Fluorescent Protein (GFP)-tagged Mycobacterium bovis Bacillus Calmeter-Guerin (GFP-BCG), and attenuated M. tuberculosis strain H37Ra and its virulent counterpart, H37Rv, were mixed with 0.5% Tween, 0.2 % glycerol, and 10% albumin-dextrose-catalase supplement (BD) in Middlebrook 7H9 broth. Mycobacteria are grown to log phase prior to use and resuspended in RPMI 1640 with 10% FBS prior to infection. M. tuberculosis strain H37Ra is fluorescently labeled with PKH67 (Sigma), and strain H37Rv is labeled with fluorescein isothiocyanate (FITC, 1 mg/mL; Sigma) according to the manufacturer's protocol.

Determination of phagocytic maturation

Mycobacteria can inhibit the fusion of phagosomes with lysosomes, preventing acidification and recruitment of lysosomal hydrolases. This blockage of phagocytic maturation can be overcome by pre-treating macrophages with IFN-γ. Treatment of cells infected with mycobacteria with TNF (5 ng/mL) also enhances phagocytic acidification. To determine the effect of TNFR1 antagonists and TNF blockers on the fusion of mycobacterial phagosomes with lysosomes in macrophages, PMA-differentiated THP-1 cells or human peripheral blood MDMs were treated with or without IFN-γ treatment to GFP- BCG, or PKH67-labeled M. tuberculosis strain H37Ra, or FITC-labeled M. tuberculosis strain H37Rv, and phagosome-lysosomal fusion as a marker for acidified phagocytosis LysoTracker ^® Red (LT ), and by confocal microscopy, using CD63 and cathepsin D as phagolysosomal markers. Inhibition of IFN-γ induced phagocytic maturation/acidification is determined by colocalization of ^{LysoTracker®} Red, CD63 or cathepsin D of labeled mycobacteria. For example, a decrease in the percent colocalization of LT, or CD63, or cathepsin D with labeled mycobacteria compared to control indicates inhibition of IFN-γ induced phagocytic acidification.

TNFR1 antagonist or TNF blocker (e.g., adalimumab, infliximab, etanercept, etc; 10 μg/mL) with or without IFN-γ (200 U/mL) THP 24 hours prior to infection -1 Add to cells or MDM. As a control, cells are treated with medium alone or with 10 μg/mL human IgG1 (Calbiochem) from a myeloma patient producing IgG1. Cells were then infected with M. bovis GFP-BCG, PKH67-labeled M. tuberculosis H37Ra, or FITC-labeled M. tuberculosis H37Rv for 15 minutes and washed three times with PBS to remove unbound Remove mycobacteria and incubate for 2 hours. Multiplicity of infection (MOI) is recorded microscopically 15 min after infection of macrophages by acid-fast bacilli staining. Cells are infected with an MOI of 1 to 5 bacilli in approximately 70% of the cells.

After 2 h incubation, cells were fixed in 2% paraformaldehyde for 20 min at room temperature (RT); For strain H37Rv, cells were fixed in 4% paraformaldehyde overnight. Cells are then permeabilized with 0.1% Triton X-100 in PBS and blocked with 1% bovine serum albumin and 1% goat serum in PBS for 30 minutes at room temperature. Cells were incubated with primary antibodies (1 μg/mL mouse monoclonal antibody to CD63 (LAMP-3; Santa Cruz Biotechnology); or 10 μg/mL mouse monoclonal antibody to cathepsin D (Calbiochem)) for 1 hour at RT. ), followed by incubation with secondary antibody (4 μg/mL Alexa Fluor 488- or 568-labeled goat anti-mouse IgG; Invitrogen) for 1 hour at RT. Alternatively, prior to fixation, cells are incubated with ^{LysoTracker®} Red DND-99 (100 nmol/L; Invitrogen) for the last 60 minutes of incubation with mycobacteria. LysoTracker ^® Red DND-99 is a red-fluorescent dye for labeling and tracking acidic organelles (eg, acidified phagocytic cells) in living cells.

Coverslips are mounted on glass slides with fluorescent mounting medium (Dako) and images are recorded on a laser scanning confocal microscope, such as an Olympus FluoView ^™ 1000 and Zeiss LSM 510 laser scanning confocal microscope. Analyze and manufacture images using appropriate software and Adobe Photoshop.

Measurement of TNF

THP-1 cells are prepared as described above and infected with BCG or M. tuberculosis H37Ra with or without IFN-γ pretreatment. Levels of immunoreactive TNF in the supernatant (secreted in response to mycobacterial infection) are measured using commercial ELISA kits (R&D systems) according to the manufacturer's instructions.

TNF block (e.g. Adalimumab , infliximab or to etanercept treatment), and regulatory T-cell (Treg cell) assays and cytokine assays that distinguish specific TNFR1 inhibition

1. Conservation of FoxP3 expression

TNF blockade (with adalimumab, rituximab, or etanercept) is compared to specific TNFR1 inhibition for methylation of the FoxP3 promoter as a surrogate marker of functional regulatory T-cells. Transgenic mice constitutively expressing human TNFR1 (HuTNFR1) are used to evaluate the differential effect of TNF blockade versus specific TNFR1 inhibition on methylation of the FoxP3 promoter. Transgenic mice are prepared by standard methods and can be produced by contractor services or any method, such as Cyagen, Genoway, or Polygene.

These effects are evaluated in transgenic mice with collagen-induced arthritis (CIA), a widely used model of RA. Mice with the C57/BL6N.Q;H-2q/HuTNFR1/Hunt background will be produced by Genoway or Taconic Labs. Mice will be immunized with bovine type II collagen emulsified in complete Freud's adjuvant (CFA) as described by Tseng et al ((2019) Proc . Natl . Acad . Sci . USA . 116 :21666-21672). Assays for FoxP3 methylation are also performed as described by Tseng et al. ((2019) Proc . Natl . Acad. Sci . USA . 116 :21666-21672. Median fluorescence intensity of FoxP3 in CD4 ⁺ CD25 ⁺ cells Regulatory T-cells from mice treated with TNF blockade express lower levels of FoxP3 than regulatory T-cells with specific TNFR1 blockade, as determined by (MFI) and histograms.

2. Specific inhibition of TNFR1 versus TNF blockers spares regulatory T-cells

Using the method described by McCann et al. ((2014) Arthritis & Rheumatology 66(10):2728-2738), the number of regulatory T-cells in the lymph nodes and spleen was transduced (C57/BL6N.Q;H -2q/HuTNFR1/Hunt) CIA mice are treated with a TNF blocker and a specific inhibitor of TNFR1 and then compared.

3. Inflammatory cytokines in collagen-induced arthritis TNF blocking stand specific TNFR1 is upregulated in inhibition

Transgenic mice with collagen-induced arthritis (CIA) (C57/BL6N.Q;H-2q/HuTNFR1/Hunt) (see, e.g., McAnn et al. (2014) Arthritis & Rheumatology 66(10):2728-2738 ]) with a TNF blocker and a TNFR1-specific antagonist. Assess serum inflammatory cytokines (IFN-gamma, IL-12p70, IL-10, RANTES (CCL5); see, eg, McCann et al. (2014) Arthritis & Rheumatology 66(10):2728-2738) . Antagonists specific for blocking TNFR1 induce significantly less of one or more of IFN-gamma, IL-12p70, IL-10, or RANTES (CCL5). This is due to preservation of TNFR2 function and regulatory T-cell function in the spleen and lymph nodes.

in vivo assay

Studies are performed with humanized (HuTNFR1/HuTNF) transgenic mice. TNFR1 antagonist molecules are evaluated for efficacy in multiple autoimmune disease models. These include the models described above expressing human transgenes for TNFR1 and TNF. Alternative models for autoimmune diseases are known. For example, a model including that for RA is described in detail in Schinnerling et al. ((2019) Front. Immunol 10 :203). To establish efficacy, certain TNFR1 antagonist constructs, as well as other constructs provided herein, are tested in more than one model. Included among the models are at least the rheumatoid arthritis (RA), Crohn's disease, and multiple sclerosis (experimental autoimmune encephalitis) models (discussed in the detailed description).

Inflammatory bowel disease (IBD, including ulcerative colitis and Crohn's disease) was the first approved indication for a TNF blocker. A number of mouse models of this disease are available and have been described by Mueller ((2002) Immunology 105(1):1-8). As discussed in the detailed description, autoimmune neurodegenerative diseases, including multiple sclerosis (MS) and Alzheimer's disease, are important disease targets. Alzheimer's disease (AD) is a leading cause of dementia worldwide and represents one of the most serious health problems for the elderly. An estimated 5.4 million Americans have AD, and this number is expected to triple by 2050 if there are no medical breakthroughs to stop, prevent, or slow the disease (see, e.g., Chang et al. (2017) J. Cent. Nerv . Syst. Dis . 9:1179573517709278). Evidence indicates that the TNFR1 antagonist constructs and other constructs provided herein are therapeutic candidates because subjects undergoing prolonged treatment with TNF blockers are less likely to develop the disease (see, e.g., [Chou et al. (2016) CNS Drugs 30:1111]). Data show that up-regulated TNF expression is associated with different neurodegenerative diseases and conditions such as Alzheimer's disease, Parkinson's disease, stroke and multiple sclerosis (see, e.g., McCoy et al. (2008) J. Neuroinflammation 5(1):45]). However, existing TNF blockers do not appear to be effective in treating, ameliorating, preventing or delaying disease progression (see, eg, Tortarolo et al. (2015) J. Neurochem. 135:109-124). As described herein, various lines of evidence indicate that TNF blockers do not work in this indication because of co-inhibition of TNFR1 and TNFR2; TNFR2 has neuro-protective properties that are lost by treatment with TNF blockers. Others have attempted to approach this problem with "TNFR1 inhibitors" or "TNFR2 agonists" in various forms. None of these studies involved cross-reactivity to determine whether TNFR1 or TNFR2 were selectively targeted, unlike being one of many epitopes in the body that could be targeted.

This problem is addressed herein. First, a family of anti-TNFR1 antagonists was created and tested in the model to show that they act as predicted. Immunochemistry is then used to prove that they are selective. Several contract research laboratories (eg, Sino Biological, Inc., and LSBio) provide these services.

As discussed in the detailed description, other autoimmune and chronic inflammatory disease conditions are associated with the presence of TNF. These include type II diabetes and endometriosis. Mouse models of this disease are known, and HuTNFR1/HuTNF transgenic versions of these mice are used to demonstrate efficacy with specific anti-TNFR1 antagonists provided herein.

acute respiratory distress syndrome

Viral pathogens of the respiratory tract (eg, influenza, SARS virus/coronavirus) infect respiratory epithelial cells, and tissue-resident alveolar macrophages are the first responders to viral infection in the lung. They initiate the immune response by affecting clearance of opsonized viral particles or infected apoptotic cells through phagocytosis and release of excess inflammatory cytokines and chemokines (see, e.g., Herold et al. (2015) Eur . Resp . J. 45:1463-1478]). TNF blockers have been shown to prolong the survival of mice infected with influenza (see, eg, Shi et al. (2013) Crit . Care 17:R301). TNF blockers have been used to treat SARS-Cov 2 (see, eg, Feldmann et al. (2020) Lancet 395:1407-1409). As described in the detailed description, a known effect of TNF blockers is to reduce regulatory T-cells (Tregs), which is problematic because Tregs are natural suppressors of inflammation. Thus, TNFR1-specific inhibitors that do not interact with or agonize TNFR2, as described and provided herein, are excellent for this purpose. To test the constructs provided herein, mice are engineered to lack endogenous TNFR1 and express HuTNFR1/HuTNF. Such mice will develop acute respiratory distress syndrome induced by HuTNF/HuTNFR1 after influenza infection (as described by Shi et al. (2013) Crit . Care 17:R301). A TNFR1-specific antagonist construct that does not antagonize TNFR2 is administered. Efficacy is determined by any of several criteria:

1. Significantly reduced circulating inflammatory cytokines (eg, IFN-gamma, IL-1 alpha, IL1-beta, and IL-17) compared to TNF blockers;

2. Significantly faster recovery as measured by weight gain compared to TNF blockers (eg, Shi et al. (2013) Crit. Care 17:R301); and

3. Significantly increased survival compared to TNF blockers (eg, Shi et al. (2013) Crit. Care 17:R301).

Transgenic mice expressing human TNFR1 and human TNFR2 for use in disease models are described in Atretkhany et al. (2018) Proc . Natl . Acad . Sci . USA . 115(51):13051-13056] by standard genetic engineering methods known in the art. Specific in vivo assays for various diseases and conditions are as follows. TNFR1 provided herein using, for example, a humanized RA mouse model, such as the Collagen-Induced Arthritis (CIA) model of RA, or any other animal model of RA known in the art and/or described herein. The therapeutic effects of the antagonist molecules are evaluated and they are compared to the therapeutic effects of anti-TNF therapies such as etanercept or adalimumab. A TNFR1 antagonist molecule provided herein, or an anti-TNF therapy such as etanercept or adalimumab, is administered to the animal daily for a total of 10 days following onset of clinical arthritis in one or more limbs. The degree of swelling of the initially affected joint is monitored by measuring paw thickness using calipers. Proinflammatory cytokines and chemokines such as granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-10 (IL-10), IL-1β, IL-6, IL-8, RANTES (CCL5) and Serum is drawn from mice for the measurement of monocyte chemoattractant protein 1 (MCP-1; also known as CCL2). The regression of RA in the mouse model is then compared between mice administered with the TNFR1 antagonist molecule and mice administered with etanercept or adalimumab.

TNFR1 antagonist molecules provided herein are also tested in humanized mouse models of severe acute respiratory syndrome (SARS) and virus-induced cytokine storms. A mouse model of SARS is humanized hTNFR1 (or hTNFR1/hTNFR2) knock-in mice with SARS-CoV at various doses, eg, 10 ² , 10 ³ , 10 ⁴ , and 10 ⁵ plaque-forming units (PFU). created by infection. TNFR1 antagonist molecules are administered to infected mice, survival is assessed, and compared to survival of mice administered an anti-TNF therapy, such as adalimumab.

Viral-induced mouse models of cytokine storm include, for example, the lymphocytic choriomeningitis virus (LCMV)-induced model of cytokine storm syndrome (CSS). An LCMV-induced mouse model of CSS is generated by intraperitoneal administration of 2Х10 ⁵ PFU of LCMV-Armstrong to 8-12 week old Perforin-deficient ( Prf ^-/- ) or Prf - Tmem178 double knockout mice. To deplete monocytes/macrophages, 100 μl of clodronate-liposomes were intravenously injected into Prf ^−/− mice 2 days before and 48 and 96 hours after LCMV infection. Alternatively, 1 mg of a neutralizing anti-CSF1 antibody (clone 5A1, BioXCell) is administered 2 days prior to infection, and 0.5 mg of the antibody is administered 48 and 96 hours later. On days 3 and 8 of infection, animals are bled via submandibular venipuncture to measure serum cytokines (see, eg, Mahajan et al. (2019) J. Autoimmun. 100:62-74).

Example 3

Identification and removal of immunogenic sequences

As described herein, immunogenic sequences, such as B-cell and/or T-cell epitopes within a protein therapeutic, can be, for example, anti-drug antibodies that neutralize the therapeutic agent and/or promote its elimination from the body ( ADA) can adversely affect the activity, efficacy, and in vivo half-life of a therapeutic agent. Safety and tolerability of protein therapeutics, as immunogenic sequences can also induce undesirable immune responses leading to clinical complications, such as delayed infusion-like allergic reactions, anaphylaxis, and, in some cases, life-threatening autoimmunity. harmful to Consequently, candidate protein therapeutics are tested for immunogenicity, and to remove/replace identified immunogenic sequences, for example, by mutagenesis, to improve the in vivo efficacy and safety profile of therapeutics, and for these To ensure that the success of the drug is transferred from preclinical research to clinical practice, screening is performed.

Immunogenic sequences are identified using methods such as those described in detail or known to those skilled in the art, including the use of in silico immunogenicity prediction tools and in vitro immunogenicity tests. For example, as described elsewhere herein, a linear B-cell epitope, eg, ABCPred, APCPred, BCPRED, BepiPred, LBtope, BcePred, EPMLR, BEST, COBEpro, and SVMTriP, or as described herein / or using any other available in silico linear B-cell epitope prediction tool known to those skilled in the art. Conformational B-cell epitopes, for example, CEP, DiscoTope, BEpro, ElliPro, SEPPA, CBTOPE, EPITOPIA, EPCES, EPSVR, EPMeta, PEASE, EpiPred, 3DEX, PEPOP, PEPOP 2.0, and EpiSearch, or as described herein and/or using any other available in silico conformational B-cell epitope prediction tool known to those skilled in the art. T-cell epitopes, e.g., EpiMatrix, JanusMatrix, IEDB, SYFPEITHI, MHC Thread, MHCPred, MHCPred 2.0, EpiJen, NetMHC, NetCTL, nHLAPred, SVMHC, ProPred, MMBPred, Protean 3D, and Bimas, or as described herein and/or any other available in silico T-cell epitope prediction tool known to those skilled in the art. The following is an exemplary analysis of the human TNFR1 antagonist DMS5541 sequence (SEQ ID NO: 38) for immunogenic, linear B-cell epitopes.

Analysis of DMS5541 for immunogenic linear B-cell epitopes

The sequence of the human TNFR1 antagonist DMS5541 (SEQ ID NO: 38) is analyzed for potential immunogenicity using the SVMTriP algorithm to detect linear B-cell epitopes within the molecule. The algorithm identifies three possible epitopes in the sequence of DMS5541, as shown in Table 13 below. The results indicate that the epitope with the sequence AVKGRFTISRDNSKNTLYLQ, corresponding to residues 63 to 82 of SEQ ID NO: 38, has a high probability for immunogenicity. The three identified epitopes are then tested for immunogenicity in an in vitro B-cell assay. Any sequence positive for immunogenicity is subjected to an alanine scan. Positive amino acids/sequences are modified by substituting each amino acid in the sequence one by one with an alanine residue until the immunogenic epitope is destroyed. This results in safer and more effective TNFR1 antagonists.

As a positive control, the SVMTriP algorithm was also used to predict the known high immunogenicity of adalimumab; Adalimumab administered without methotrexate is immunogenic in approximately 50% of patients (see, eg, Ducourau et al. (2020) RMD Open 6:e001047). The SVMTriP algorithm identified at least 10 possible epitopes in the heavy chain of adalimumab, 4 of which are of high probability, thus correlating well with clinical data and validating the use of this program to predict immunogenicity.

Results from the SVMTriP analysis of DMS5541 are supported by a second algorithm, ABCPred, used to predict immunogenicity within the sequence of DMS5541. All three B-cell epitopes predicted by SVMTriP were also included in ABCPred's epitope prediction results. For example, as shown in Table 14 below, the epitopes AVKGRFTISRDNSKNT, TGRWVPFEYWGQGTLV and STDIQMTQSPSSLSAS (see table below for each residue position in SEQ ID NO: 38) overlap with the three epitopes identified by SVMTriP Contains sequences overlapping.

Example 4

human TNFR1 antagonist antibody fragments ( dAb , scFv , Fab ) Exemplary containing TNFR1 antagonist construct

Provided herein are TNFR1 antagonist constructs that selectively inhibit TNFR1 without inhibiting TNFR2. To avoid TNFR1 receptor clustering, which agonizes TNFR1, TNFR1 antagonists are monomeric and monovalent. TNFR1 antagonists contain human single domain antibodies (dAbs) specific for TNFR1. dAbs contain variable region heavy (V _H ) or variable region light (V _L ) domains. For example, the dAb can be at least or at least about 90% or 95% identical to the dAb whose amino acid sequence is set forth in any one of SEQ ID NOs: 54-672, or a dAb of any one of SEQ ID NOs: 54-672 that retains binding affinity for TNFR1. % sequence identity. Alternatively, TNFR1 antagonists contain scFvs, Fabs, or other antigen-binding fragments such as those derived from human TNFR1 antagonist antibodies, such as H398 or ATROSAB. For example, TNFR1 antagonists include the H398-derived scFvs set forth in SEQ ID NOs: 677 or 678; or an ATROSAB-derived scFv as set forth in any one of SEQ ID NOs: 673-676; or an ATROSAB-derived Fab fragment light chain and heavy chain set forth in any one of SEQ ID NOs: 679 and 680 (FabATR), respectively, or SEQ ID NOs: 681 or 682 (Fab 13.7); or a scFv or Fab fragment having at least or at least about 90% or 95% sequence identity to the scFv of any one of SEQ ID NOs: 673 to 678, or SEQ ID NOs: 679 and 680 (FabATR), respectively, or SEQ ID NOs: 681 or 682 (Fab 13.7) of any of the Fab light and heavy chains (which have affinity for TNFR1).

TNFR1 antagonists are modified to eliminate or reduce serum half-life extenders, such as IgG Fc, particularly ADCC, ADCP, and/or CDC, human serum albumin (HSA), and/or poly(ethylene)glycol (PEG) molecules. fused to Fc. For example, the C-terminus of a human anti-TNFR1 dAb, scFV, Fab or other antigen-binding fragment is fused to the N-terminus of the Fc region of a human IgG1 or IgG4 antibody via a linker. An IgG1 Fc region, such as an IgG1 Fc derived from Trastuzumab (see SEQ ID NO: 27), or an IgG4 Fc region, such as an IgG4 Fc derived from nivolumab (see SEQ ID NO: 30), is used. The linker comprises a portion of the hinge sequence of Trastuzumab containing the sequence of amino acid residue SCDKTH (corresponding to residues 222 to 227 of SEQ ID NO: 26) when the Fc is derived from Trastuzumab, or the Fc is derived from nivolumab. If present, it may contain the hinge sequence of nivolumab, which contains the sequence of amino acid residues ESKYGPPCPPCP (corresponding to residues 212 to 223 of SEQ ID NO: 29), or portions thereof that provide flexibility or other structural properties. To confer protease resistance and increase the flexibility of the fusion protein, an SCDKTH or ESKYGPPCPPCP hinge sequence can be added, for example, to (GSGS) or (GGGGS) _n (eg, residues 199 to 202 of SEQ ID NO: 707, and 116, respectively). to 120), where n is 1 to 5 or 1 to 6, or other combinations of Gly and Ser residues such as, for example, GGGGSGGGGSGGGGS (see, for example, residues 116 to 130 or SEQ ID NO: 707). Replace with a short glycine-serine (GS) peptide linker. In another embodiment, the C-terminus of the human anti-TNFR1 dAb, scFv, Fab or other antigen-binding fragment is linked to a GS linker, wherein the GS linker is linked to the N-terminus of the corresponding Fc region, Trastuzumab or Nivol. It is linked to all or part of it sufficient to provide flexibility of the lumab hinge sequence. In some embodiments, the second Fc subunit is linked to the first Fc subunit to increase the serum half-life and stability of the molecule. Because there are two Fc regions, any resulting construct is not a fusion protein because it contains one non-contiguous Fc region. In some embodiments, the N-terminus of the human TNFR1 antagonist dAb, scFV, Fab or other antigen-binding fragment is fused to the C-terminus of the serum half-life extender via a linker, as described above.

Also disclosed herein is an anti-TNFR1 antibody fused to human serum albumin (HSA) via a short peptide linker such as (GSGS) _n or GGGGS) _n (where n = 1 to 5 or 6), eg, GGGGSGGGGSGGGGS. TNFR1 antagonist fusion proteins containing dAbs, scFvs, Fabs, or other antigen-binding fragments are provided.

Also provided herein are TNFR1 antagonist molecules containing an anti-TNFR1 dAb, scFv, Fab, or other antigen-binding fragment linked to a PEG molecule that is at least 30 kDa in size.

As described herein, such constructs may be modified to reduce or eliminate immunogenicity. TNFR1 antagonist dAbs, scFvs, Fabs, or other antigen-binding fragments are analyzed by in silico, in vitro and/or in vivo methods to predict or confirm immunogenic sequences. Upon identification of an immunogenic sequence, such as a B-cell and/or T-cell epitope, the identified sequence can be modified by mutagenesis, eg, alanine scanning as described elsewhere herein, to deimmunize/deimmunize the epitope. Immunogenic sequences are removed or replaced.

The following are exemplary constructs of TNFR1 antagonist fusion proteins described and provided herein. In all embodiments comprising the Fc of trastuzumab or the Fc of nivolumab, the Fc region is optionally modified to reduce or eliminate immune effector functions, including ADCC, ADCP, and CDC, and optionally also to FcRn. Modified to enhance binding, increase the serum half-life of the fusion protein, and also optionally replace or otherwise modify or remove immunogenic sequences.

Fc modifications that reduce or eliminate immune effector function are summarized in Table 9 above, and Fc modifications that enhance FcRn binding are summarized in Table 7 above. Any one or combination of these modifications are included in the Fc region of the fusion proteins provided herein. All exemplary constructs provided herein are also made with a TNFR1 antagonist at the C terminus of the fusion protein, instead of at the N-terminus.

1a) H398 scFv - SCDKTH - Trastuzumab Fc

Provided herein are human TNFR1 antagonist fusion proteins containing scFvs derived from human TNFR1 antagonist antibody H398. The scFv contains the V _L and V _H domains of H398 linked together by a (GGGGS) _three- peptide linker. The C-terminus of the H398 scFv (SEQ ID NO: 678) is the amino acid residue SCDKTH (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27) that is fused to the N-terminus of Trastuzumab Fc region. Corresponding to residues 222-227 of SEQ ID NO: 26) is fused to a portion of the hinge sequence of Trastuzumab that contains at least the sequence of The H398 scFv-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 706):

Alternatively, the SCDKTH hinge sequence is a trastu sequence containing or having the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26) or at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues thereof. Replaced with up to the entire sequence of the hinge region of zumab.

1b) H398 scFv - GGGGSGGGGSGGGGS - Trastuzumab Fc

Provided herein are TNFR1 antagonist fusion proteins containing scFvs derived from human TNFR1 antagonist antibody H398. The scFv contains the V _L and V _H domains of H398 linked together by a (GGGGS) _three- peptide linker. The C-terminus of the H398 scFv (SEQ ID NO: 678) is fused to the GGGGSGGGGSGGGGS peptide linker, which corresponds to residues 234-450 of the Trastuzumab Fc region (SEQ ID NO: 26; see also SEQ ID NO: 27). fused to the N-terminus. The H398 scFv-GGGGSGGGGSGGGGS-Trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 707):

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker, where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker.

1c) H398 scFv - GGGGSGGGGSGGGGS - SCDKTH - Trastuzumab Fc

Provided herein are TNFR1 antagonist fusion proteins containing scFvs derived from human TNFR1 antagonist antibody H398. The scFv contains the V _L and V _H domains of H398 linked together by a (GGGGS) _three- peptide linker. The C-terminus of the H398 scFv (SEQ ID NO: 678) is the amino acid residue SCDKTH (corresponding to residues 234-450 of SEQ ID NO: 6; see also SEQ ID NO: 27) that is fused to the N-terminus of Trastuzumab Fc region. Corresponding to residues 222-227 of SEQ ID NO: 26) is fused to a GGGGSGGGGSGGGGS peptide linker that is fused to a portion of the hinge sequence of Trastuzumab comprising at least the sequence of The H398 scFv-GGGGSGGGGSGGGGS-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 708):

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker, where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker. Alternatively or additionally, the SCDKTH hinge sequence is a tra containing at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues of the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26) Replaced with up to the entire sequence of the hinge region of stuzumab.

1d) H398 scFv - GGGGSGGGGSGGGGS - HSA

Provided herein are TNFR1 antagonist fusion proteins containing scFvs derived from human TNFR1 antagonist antibody H398. The scFv contains the V _L and V _H domains of H398 linked together by a (GGGGS) _three- peptide linker. The C-terminus of H398 scFv (SEQ ID NO: 678) is fused to the GGGGSGGGGSGGGGS peptide linker, which is fused to the N-terminus of human serum albumin (HSA) without a signal peptide (corresponding to residues 19-609 of SEQ ID NO: 35). is fused to The H398 scFv-GGGGSGGGGSGGGGS-HSA fusion protein has the following sequence (SEQ ID NO: 709):

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker, where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker.

1e) H398 scFv - GGGGSGGGGSGGGGS - PEG _{30 kDa}

Provided herein are TNFR1 antagonist fusion proteins containing scFvs derived from human TNFR1 antagonist antibody H398. The scFv contains the V _L and V _H domains of H398 linked together by a (GGGGS) _three- peptide linker. The C-terminus of the H398 scFv (SEQ ID NO: 678) is fused to a GGGGSGGGGSGGGGS peptide linker covalently linked to a 30 kDa PEG molecule.

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker, where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker. Alternatively or additionally, the PEG molecule may have a molecular weight greater than 30 kDa, such as, for example, about 35 kDa, or 35 kDa, 40 kDa, 45 kDa or 50 kDa.

1f) DOM1h -574-16 - SCDKTH - Trastuzumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-574-16 (SEQ ID NO: 57). The C-terminus of DOM1h-574-16 is the amino acid residue SCDKTH (SEQ ID NO: Corresponding to residues 222-227 of 26) is fused to a portion of the hinge sequence of Trastuzumab that contains at least the sequence of The DOMlh-574-16-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 710):

Alternatively, the SCDKTH hinge sequence is trastuzumab containing the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26) or at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues thereof. Replaced with up to the entire sequence of the hinge region of

1g) DOM1h -574-16 - GGGGSGGGGSGGGGS - Trastuzumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-574-16 (SEQ ID NO: 57). The C-terminus of DOMlh-574-16 is fused to the GGGGSGGGGSGGGGS peptide linker, which is fused to the N-terminus of the Trastuzumab Fc region (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). is fused to The DOM1h-574-16-GGGGSGGGGSGGGGS-Trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 711):

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker, where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker.

1h) DOM1h -574-16 - GGGGSGGGGSGGGGS - SCDKTH - Trastuzumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-574-16 (SEQ ID NO: 57). The C-terminus of DOM1h-574-16 corresponds to residues SCDKTH (SEQ ID NO: 26 Corresponding to residues 222-227 of) is fused to a GGGGSGGGGSGGGGS peptide linker that is fused to a portion of the hinge sequence of Trastuzumab that contains at least the sequence of The DOMlh-574-16-GGGGSGGGGSGGGGS-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 712):

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker, where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker. Alternatively or additionally, the SCDKTH hinge sequence contains the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26), or at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues thereof to the entire sequence of the hinge region of trastuzumab.

1i) DOM1h -574-16 - GGGGSGGGGSGGGGS - HSA

Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-574-16 (SEQ ID NO: 57). The C-terminus of DOMlh-574-16 is fused to the GGGGSGGGGSGGGGS peptide linker, which is fused to the N-terminus of human serum albumin (HSA) without a signal peptide (corresponding to residues 19-609 of SEQ ID NO: 35). . The DOM1h-574-16-GGGGSGGGGSGGGGS-HSA fusion protein has the following sequence (SEQ ID NO: 713):

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker, where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker.

1j) DOM1h -574-16 - GGGGSGGGGSGGGGS - PEG _{30 kDa}

Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-574-16 (SEQ ID NO: 57). The C-terminus of DOM1h-574-16 is fused to a GGGGSGGGGSGGGGS peptide linker covalently linked to a 30 kDa PEG molecule.

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker, where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker. Alternatively or additionally, the PEG molecule may have a molecular weight greater than 30 kDa, such as for example 35 kDa, 40 kDa, 45 kDa or 50 kDa.

1k) DOM1h -549 - SCDKTH - Trastuzumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-549 (SEQ ID NO: 58). The C-terminus of DOM1h-549 corresponds to residues 234-450 of Trastuzumab Fc region (residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27) residue SCDKTH (residues of SEQ ID NO: 26) fused to the N-terminus. 222-227) is fused to a portion of the hinge sequence of Trastuzumab that contains at least the sequence of The DOMlh-549-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 714):

Alternatively, the SCDKTH hinge sequence is trastuzumab containing the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26) or at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues thereof. Replaced with up to the entire sequence of the hinge region of

1l) DOM1h -549 - GGGGSGGGGSGGGGS - Trastuzumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-549 (SEQ ID NO: 58). The C-terminus of DOMlh-549 is fused to the GGGGSGGGGSGGGGS peptide linker, which is fused to the N-terminus of the Trastuzumab Fc region (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). do. The DOM1h-549-GGGGSGGGGSGGGGS-Trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 715):

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker, where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker.

1m) DOM1h -549 - GGGGSGGGGSGGGGS - SCDKTH - Trastuzumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-549 (SEQ ID NO: 58). The C-terminus of DOM1h-549 corresponds to residues 234-450 of Trastuzumab Fc region (residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27) residue SCDKTH (residues of SEQ ID NO: 26) fused to the N-terminus. 222-227) is fused to a GGGGSGGGGSGGGGS peptide linker that is fused to a portion of the hinge sequence of Trastuzumab that contains at least the sequence of The DOM1h-549-GGGGSGGGGSGGGGS-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 716):

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker, where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker. Alternatively or additionally, the SCDKTH hinge sequence is at least 5, 6, 7, 8, 9 up to the entire sequence of the hinge region of Trastuzumab containing the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26). , is replaced with a moiety containing 10 or 11 adjacent residues.

1n) DOM1h -549 - GGGGSGGGGSGGGGS - HSA

Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-549 (SEQ ID NO: 58). The C-terminus of DOM1h-549 is fused to the GGGGSGGGGSGGGGS peptide linker, which is fused to the N-terminus of human serum albumin (HSA) without the signal peptide (corresponding to residues 19-609 of SEQ ID NO: 35). The DOM1h-549-GGGGSGGGGSGGGGS-HSA fusion protein has the following sequence (SEQ ID NO: 717):

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker, where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker.

1o) DOMlh -549 - GGGGSGGGGSGGGGS - PEG _{30 kDa}

Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-549 (SEQ ID NO: 58). The C-terminus of DOM1h-549 is fused to a GGGGSGGGGSGGGGS peptide linker covalently linked to a 30 kDa PEG molecule.

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker, where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker. Alternatively or additionally, the PEG molecule may have a molecular weight greater than 30 kDa, such as for example 35 kDa, 40 kDa, 45 kDa or 50 kDa.

1p) DOM1h -574-208 - SCDKTH - Trastuzumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-574-208 (SEQ ID NO: 54). The C-terminus of DOM1h-574-208 corresponds to residues SCDKTH (SEQ ID NO: 26 corresponding to residues 222-227 of The DOM1h-574-208-SCDKTH-Trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 718):

Alternatively, the SCDKTH hinge sequence is at least 5, 6, 7, 8, 9, 10 or 11 up to the entire hinge region of Trastuzumab containing the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26). replaced with at least a moiety containing adjacent residues.

1q) DOM1h -574-208 - GGGGSGGGGSGGGGS - Trastuzumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-574-208 (SEQ ID NO: 54). The C-terminus of DOM1h-574-208 is fused to the GGGGSGGGGSGGGGS peptide linker, which is fused to the N-terminus of the Trastuzumab Fc region (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). is fused to The DOM1h-574-208-GGGGSGGGGSGGGGS-Trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 719):

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker, where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker.

1r) DOM1h -574-208 - GGGGSGGGGSGGGGS - SCDKTH - Trastuzumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-574-208 (SEQ ID NO: 54). The C-terminus of DOM1h-574-208 corresponds to residues SCDKTH (SEQ ID NO: 26 Corresponding to residues 222-227 of) is fused to a GGGGSGGGGSGGGGS peptide linker that is fused to a portion of the hinge sequence of Trastuzumab that contains at least the sequence of The DOMlh-574-208-GGGGSGGGGSGGGGS-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 720):

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker, where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker. Alternatively or additionally, the SCDKTH hinge sequence is at least 5, 6, 7, 8, 9 up to the entire sequence of the hinge region of Trastuzumab containing the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26). , at least a moiety containing 10 or 11 contiguous residues.

1s) DOM1h -574-208 - GGGGSGGGGSGGGGS - HSA

Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-574-208 (SEQ ID NO: 54). The C-terminus of DOMlh-574-208 is fused to the GGGGSGGGGSGGGGS peptide linker, which is fused to the N-terminus of human serum albumin (HSA) without a signal peptide (corresponding to residues 19-609 of SEQ ID NO: 35). . The DOM1h-574-208-GGGGSGGGGSGGGGS-HSA fusion protein has the following sequence (SEQ ID NO: 721):

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker, where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker.

1t) DOM1h-574-208 - GGGGSGGGGSGGGGS - PEG _{30 kDa}

Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-574-208 (SEQ ID NO: 54). The C-terminus of DOMlh-574-208 is fused to a GGGGSGGGGSGGGGS peptide linker covalently linked to a 30 kDa PEG molecule.

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker, where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker. Alternatively or additionally, the PEG molecule may have a molecular weight of at least or greater than 30 kDa, such as for example 35 kDa, 40 kDa, 45 kDa or 50 kDa.

1u) DOM1h-131-206 - SCDKTH - Trastuzumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-131-206 (SEQ ID NO: 59). The C-terminus of DOM1h-131-206 is residue SCDKTH (SEQ ID NO: 26 corresponding to residues 222-227 of The DOM1h-131-206-SCDKTH-Trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 722):

Alternatively, the SCDKTH hinge sequence is at least 5, 6, 7, 8, 9, 10 or replaced by a moiety containing 11 adjacent residues.

1v) DOM1h-131-206 - GGGGSGGGGSGGGGS - Trastuzumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-131-206 (SEQ ID NO: 59). The C-terminus of DOM1h-131-206 is fused to the GGGGSGGGGSGGGGS peptide linker, which is fused to the N-terminus of the trastuzumab Fc region (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). is fused to The DOM1h-131-206-GGGGSGGGGSGGGGS-Trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 723):

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker, where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker.

1w) DOM1h-131-206 - GGGGSGGGGSGGGGS - SCDKTH - Trastuzumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-131-206 (SEQ ID NO: 59). The C-terminus of DOM1h-131-206 is residue SCDKTH (SEQ ID NO: 26 Corresponding to residues 222-227 of) is fused to a GGGGSGGGGSGGGGS peptide linker that is fused to a portion of the hinge sequence of Trastuzumab that contains at least the sequence of The DOM1h-131-206-GGGGSGGGGSGGGGS-SCDKTH-Trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 724):

Alternatively, the GGGGSGGGGSGGGGS linker can be another Gly-Ser (GS) linker, such as a (GSGS) _n or (GGGGS) _n linker where n = 1, 2, 3, 4, or 5 or as described herein or in the art. Replaced with any other suitable short peptide linker as known in . Alternatively or additionally, the SCDKTH hinge sequence is at least 5, 6, 7, 8, 9 up to the entire sequence of the hinge region of Trastuzumab containing the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26). , is replaced in whole or in part with 10 or 11 contiguous residues.

1z) H398 scFv - ESKYGPPCPPCP - Nivolumab Fc

Provided herein are human TNFR1 antagonist fusion proteins containing scFvs derived from human TNFR1 antagonist antibody H398. The scFv contains the V _L and V _H domains of H398 linked together by a (GGGGS) _three- peptide linker. The C-terminus of the H398 scFv (SEQ ID NO:678) corresponds to residues 224-440 of the nivolumab Fc region SEQ ID NO:29; reference, also to the hinge sequence of nivolumab containing the sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29) fused to the N-terminus of SEQ ID NO: 30). The H398 scFv-ESKYGPPCPPCP-nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 726):

Alternatively, all or some are used

1aa) H398 scFv - GGGGSGGGGSGGGGS - Nivolumab Fc

Provided herein are TNFR1 antagonist fusion proteins containing scFvs derived from human TNFR1 antagonist antibody H398. The scFv contains the V _L and V _H domains of H398 linked together by a (GGGGS) _three- peptide linker. The C-terminus of the H398 scFv (SEQ ID NO: 678) is a GGGGSGGGGSGGGGS peptide linker fused to the N-terminus of the nivolumab Fc region (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO:30). is fused to The H398 scFv-GGGGSGGGGSGGGGS-Nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 727):

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker, where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker.

1ab) H398 scFv - GGGGSGGGGSGGGGS - ESKYGPPCPPCP - Nivolumab Fc

Provided herein are TNFR1 antagonist fusion proteins containing scFvs derived from human TNFR1 antagonist antibody H398. The scFv contains the V _L and V _H domains of H398 linked together by a (GGGGS) _three- peptide linker. The C-terminus of the H398 scFv (SEQ ID NO:678) corresponds to residues 224-440 of the nivolumab Fc region SEQ ID NO:29; Reference, also fused to the GGGGSGGGGSGGGGS peptide linker fused to the hinge sequence of nivolumab containing the sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29) fused to the N-terminus of SEQ ID NO: 30). The H398 scFv-GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 728):

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker, where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker. Alternatively or additionally, a nivolumab hinge sequence containing at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29) all or part of is used.

1ac) DOM1h-574-16 - ESKYGPPCPPCP - Nivolumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-574-16 (SEQ ID NO: 57). C-terminus of DOM1h-574-16 corresponds to residues 224-440 of nivolumab Fc region SEQ ID NO: 29; reference, also to the hinge sequence of nivolumab containing the sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29) fused to the N-terminus of SEQ ID NO: 30). The DOM1h-574-16-ESKYGPPCPPCP-Nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 729):

Alternatively, all or some are used

1ad) DOM1h-574-16 - GGGGSGGGGSGGGGS - Nivolumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-574-16 (SEQ ID NO: 57). The C-terminus of DOMlh-574-16 is fused to a GGGGSGGGGSGGGGS peptide linker, which corresponds to residues 224-440 of the nivolumab Fc region (SEQ ID NO: 29; see also SEQ ID NO:30). fused at the end The DOM1h-574-16-GGGGSGGGGSGGGGS-Nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 730):

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker, where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker.

1ae) DOM1h-574-16 - GGGGSGGGGSGGGGS - ESKYGPPCPPCP - Nivolumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-574-16 (SEQ ID NO: 57). C-terminus of DOM1h-574-16 corresponds to residues 224-440 of nivolumab Fc region SEQ ID NO: 29; Reference, also fused to the GGGGSGGGGSGGGGS peptide linker fused to the hinge sequence of nivolumab containing the sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29) fused to the N-terminus of SEQ ID NO: 30). The DOM1h-574-16-GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 731):

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker, where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker. Alternatively or additionally, a nibol containing at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues up to the entire sequence of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29) At least a portion of the lumab hinge sequence is included.

1af) DOM1h-549 - ESKYGPPCPPCP - Nivolumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-549 (SEQ ID NO: 58). C-terminus of DOM1h-549 corresponds to residues 224-440 of nivolumab Fc region SEQ ID NO: 29; reference, also to the hinge sequence of nivolumab containing the sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29) fused to the N-terminus of SEQ ID NO: 30). The DOM1h-549-ESKYGPPCPPCP-nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 732):

Alternatively, all or some are included

1ag) DOM1h-549 - GGGGSGGGGSGGGGS - Nivolumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-549 (SEQ ID NO: 58). The C-terminus of DOM1h-549 is fused to the GGGGSGGGGSGGGGS peptide linker, which is fused to the N-terminus of the nivolumab Fc region (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO:30). is fused The DOM1h-549-GGGGSGGGGSGGGGS-Nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 733):

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker, where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker.

1ah) DOM1h-549 - GGGGSGGGGSGGGGS - ESKYGPPCPPCP - Nivolumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-549 (SEQ ID NO: 58). C-terminus of DOM1h-549 corresponds to residues 224-440 of nivolumab Fc region SEQ ID NO: 29; Reference, also fused to the GGGGSGGGGSGGGGS peptide linker fused to the hinge sequence of nivolumab containing the sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29) fused to the N-terminus of SEQ ID NO: 30). DOM1h-549-GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 734):

In some embodiments, the GGGGSGGGGSGGGGS linker is a GS linker, such as a (GSGS) _n or (GGGGS) _n linker, where n = 1, 2, 3, 4, or 5, or any of those described herein or known in the art. is replaced with another suitable short peptide linker of Alternatively or additionally, a nivolumab hinge sequence containing at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29) includes all or part of

1ai) DOM1h-574-208 - ESKYGPPCPPCP - Nivolumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-574-208 (SEQ ID NO: 54). C-terminus of DOM1h-574-208 corresponds to residues 224-440 of nivolumab Fc region SEQ ID NO: 29; reference, also to the hinge sequence of nivolumab containing the sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29) fused to the N-terminus of SEQ ID NO: 30). The DOM1h-574-208-ESKYGPPCPPCP-nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 735):

Alternatively, all or some may be included.

1aj) DOM1h-574-208 - GGGGSGGGGSGGGGS - Nivolumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-574-208 (SEQ ID NO: 54). The C-terminus of DOM1h-574-208 is fused to the GGGGSGGGGSGGGGS peptide linker, which corresponds to residues 224-440 of the nivolumab Fc region (SEQ ID NO: 29; see also SEQ ID NO:30). fused at the end The DOM1h-574-208-GGGGSGGGGSGGGGS-Nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 736):

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker such as

1ak) DOM1h-574-208 - GGGGSGGGGSGGGGS - ESKYGPPCPPCP - Nivolumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-574-208 (SEQ ID NO: 54). C-terminus of DOM1h-574-208 corresponds to residues 224-440 of nivolumab Fc region SEQ ID NO: 29; Reference, also fused to the GGGGSGGGGSGGGGS peptide linker fused to the hinge sequence of nivolumab containing the sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29) fused to the N-terminus of SEQ ID NO: 30). The DOM1h-574-208-GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 737):

Alternatively, the GGGGSGGGGSGGGGS linker can be a (GSGS) _n or (GGGGS) _n linker, where n = 1, 2, 3, 4, or 5, or any other suitable short chain as described herein or known in the art. replaced by a peptide linker. Alternatively or additionally, a nivolumab hinge sequence containing at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29) includes all or part of

1al) DOM1h-131-206 - ESKYGPPCPPCP - Nivolumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-131-206 (SEQ ID NO: 59). The C-terminus of DOM1h-131-206 corresponds to residues 224-440 of the nivolumab Fc region SEQ ID NO: 29; Reference, also to the hinge sequence of nivolumab containing the sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29) fused to the N-terminus of SEQ ID NO: 30). The DOM1h-131-206-ESKYGPPCPPCP-nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 738):

Alternatively, all or some are included

1am) DOM1h-131-206 - GGGGSGGGGSGGGGS - Nivolumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-131-206 (SEQ ID NO: 59). The C-terminus of DOM1h-131-206 is fused to the GGGGSGGGGSGGGGS peptide linker, which corresponds to residues 224-440 of the nivolumab Fc region (SEQ ID NO: 29; see also SEQ ID NO:30). fused at the end The DOM1h-131-206-GGGGSGGGGSGGGGS-Nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 739):

Alternatively, the GGGGSGGGGSGGGGS linker can be a (GSGS) _n or (GGGGS) _n linker, where n = 1, 2, 3, 4, or 5, or any other suitable short chain as described herein or known in the art. replaced by a peptide linker.

1an) DOM1h-131-206 - GGGGSGGGGSGGGGS - ESKYGPPCPPCP - Nivolumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-131-206 (SEQ ID NO: 59). The C-terminus of DOM1h-131-206 corresponds to residues 224-440 of the nivolumab Fc region SEQ ID NO: 29; Reference, also fused to the GGGGSGGGGSGGGGS peptide linker fused to the hinge sequence of nivolumab containing the sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29) fused to the N-terminus of SEQ ID NO: 30). The DOM1h-131-206-GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 740):

Alternatively, the GGGGSGGGGSGGGGS linker can be a (GSGS) _n or (GGGGS) _n linker, where n = 1, 2, 3, 4, or 5, or any other suitable short chain as described herein or known in the art. replaced by a peptide linker. Alternatively or additionally, a nivolumab hinge sequence containing at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29) includes all or part of

Also, HSA-linked Vhh (nanobodies) including dAbs such as DOM1h-131-206 have been synthesized, especially in short-chain form. Exemplary Vhh constructs were evaluated for binding and inhibition of TNFR1. These are described in the examples below.

Example 5

Exemplary TNFR1 antagonist constructs containing human TNFR1 antagonist TNF muteins

Provided herein are TNFR1 antagonists that selectively inhibit TNFR1 without inhibiting TNFR2. TNFR1 antagonists are monovalent to avoid TNFR1 receptor clustering that agonizes TNFR1. TNFR1 antagonists can include dominant negative inhibitors lacking the signaling capacity of TNF (DN-TNF), also known as TNF muteins, which are engineered variants of TNF with one or more mutations that abrogate signaling through TNFR1 . For example, a TNF mutein may contain one or more mutations that confer selectivity for TNFR1 but not selectivity for TNFR2. The TNFR1 selective TNF mutation is L29S, L29G, L29Y, R31E, R31N, R32Y, R32W, S86T, L29S/R32W, L29S/S86T, R32W/S86T, L29S with respect to the sequence of soluble TNF (solTNF) set forth in SEQ ID NO: 2 /R32W/S86T, R31N/R32T, R31E/S86T, R31N/R32T/S86T, E146R, V1M, R31C, C69V, Y87H, C101A, A145R, V1M/R31C/C69V/Y87H/C101A/A145R, I97T, I97 T/A145R , A84S, V85T, Q88N, T89Q, and A84S/V85T/S86T/Y87H/Q88N/T89Q, and combinations thereof.

For example, TNFR1 antagonists include mutations R32W/S86T (SEQ ID NO:685), V1M/R31C/C69V/Y87H/C101A/A145R (SEQ ID NO:701; as in XPro1595), A84S/V85T/S86T/Y87H/Q88N /T89Q (SEQ ID NO:703; as in R1antTNF), or I97T/A145R (SEQ ID NO:702; as in XENP345).

TNFR1 antagonists are fused to serum half-life extenders such as IgG Fc. For example, the C-terminus of a human TNFR1 antagonistic TNF mutein is fused to the N-terminus of the Fc region of a human IgG1 or IgG4 antibody via a linker. An IgG1 Fc region such as the IgG1 Fc from trastuzumab (see SEQ ID NO: 27) or an IgG4 Fc region such as the IgG4 from nivolumab (see SEQ ID NO: 30) is used. The linker may contain all or part of the hinge sequence of Trastuzumab, which contains at least residues SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26) when the Fc is derived from Trastuzumab, or the Fc is When derived from nivolumab, it may contain the hinge sequence of nivolumab, ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29), or a portion thereof. To confer protease resistance and increase the flexibility of the fusion protein, the SCDKTH or ESKYGPPCPPCP hinge sequence or part thereof is a short glycine-serine (GS) peptide linker such as, for example, (GSGS) _n or (GGGGS) _n ( where n = 1-5), eg GGGGSGGGGSGGGGS. In an alternative embodiment, the C-terminus of the human anti-TNFR1 TNF mutein is linked to a GS linker, the GS linker being linked to the N-terminus of the Fc region of interest sufficient to provide flexibility; or linked to all or part of the nivolumab hinge sequence. In some embodiments, the second Fc subunit can be linked to the first Fc region to increase the serum half-life and stability of the molecule. The resulting construct is not a fusion protein.

Below are exemplary constructs of TNFR1 antagonist fusion proteins containing TNFR1 selectively antagonistic TNF muteins as described and provided herein. In all embodiments containing the Fc of Trastuzumab or the Fc of Nivolumab, the Fc region is optionally modified to reduce or eliminate immune effector functions, including ADCC, ADCP and CDC, and optionally also inhibits binding to FcRn. modified to enhance the serum half-life of the fusion protein. Fc modifications that reduce or eliminate immune effector function are summarized in Table 9, and Fc modifications that enhance FcRn binding are summarized in Table 7. Any one or combination of these modifications is included in the Fc region of the fusion proteins provided herein.

2a) TNF (R32W/S86T) - SCDKTH - Trastuzumab Fc

Provided herein is a human TNFR1 antagonist fusion protein containing a TNFR1 selective antagonist TNF mutein having mutations R32W/S86T with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. The C-terminus of the TNF (R32W/S86T) mutein (SEQ ID NO: 685) is at the N-terminus of the trastuzumab Fc region (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27) It is fused to all or part of the hinge sequence of Trastuzumab, which contains at least the residues SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26) to which it is fused. The TNF(R32W/S86T)-SCDKTH-Trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 741):

Alternatively, the SCDKTH hinge sequence comprises at least 5, 6, 7, 8, 9, replaced by 10 or 11 adjacent residues.

2b) TNF (R32W/S86T) - GGGGSGGGGSGGGGS - Trastuzumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing a TNFR1 selective antagonist TNF mutein having mutations R32W/S86T with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. The C-terminus of the TNF (R32W/S86T) mutein (SEQ ID NO: 685) is fused to a GGGGSGGGGSGGGGS peptide linker, which corresponds to residues 234-450 of the Trastuzumab Fc region (SEQ ID NO: 26; also sequence number: see 27) is fused to the N-terminus. The TNF(R32W/S86T)-GGGGSGGGGSGGGGS-Trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 742):

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker such as

2c) TNF (R32W/S86T) - GGGGSGGGGSGGGGS - SCDKTH - Trastuzumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing a TNFR1 selective antagonist TNF mutein having mutations R32W/S86T with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. The C-terminus of the TNF(R32W/S86T) mutein (SEQ ID NO:685) is at the N-terminus of the Trastuzumab Fc region (corresponding to residues 234-450 of SEQ ID NO:26; see also SEQ ID NO:27) GGGGSGGGGSGGGGS peptide linker that is fused to a portion of the hinge sequence of Trastuzumab that contains at least the residues SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26) to which it is fused. The TNF(R32W/S86T)-GGGGSGGGGSGGGGS-DKTH-Trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 743):

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker such as Alternatively or additionally, the SCDKTH hinge sequence contains the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26), or at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues. It is replaced with the hinge region of trastuzumab containing a portion thereof.

2d) TNF (V1M/R31C/C69V/Y87H/C101A/A145R) - SCDKTH - Trastuzumab Fc

Provided herein is a human TNFR1 antagonist fusion protein containing a TNFR1 selective antagonist TNF mutein with mutations V1M, R31C, C69V, Y87H, C101A and A145R with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. The C-terminus of the TNF (V1M/R31C/C69V/Y87H/C101A/A145R) mutein (SEQ ID NO: 701) corresponds to residues 234-450 of the Trastuzumab Fc region (SEQ ID NO: 26; also SEQ ID NO: 26). 27) is fused to at least a portion of the hinge sequence of Trastuzumab containing residues SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26). The TNF(V1M/R31C/C69V/Y87H/C101A/A145R)-SCDKTH-Trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 744):

Alternatively, the SCDKTH hinge sequence is the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26), or a portion thereof containing at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues. It is replaced with the hinge region of trastuzumab containing

2e) TNF ( V1M / R31C / C69V / Y87H / C101A / A145R ) - GGGGSGGGGSGGGGS - Trastuzumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing a TNFR1 selective antagonist TNF mutein with mutations V1M, R31C, C69V, Y87H, C101A and A145R with respect to the sequence of soluble TNF set forth in SEQ ID NO: 2. The C-terminus of the TNF (V1M/R31C/C69V/Y87H/C101A/A145R) mutein (SEQ ID NO: 701) is fused to the GGGGSGGGGSGGGGS peptide linker, which binds to the Trastuzumab Fc region (residue 234 of SEQ ID NO: 26). corresponds to -450; see also SEQ ID NO: 27). The TNF(V1M/R31C/C69V/Y87H/C101A/A145R)-GGGGSGGGGSGGGGS-Trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 745):

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker, where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker.

2f) TNF ( V1M / R31C / C69V / Y87H / C101A / A145R ) - GGGGSGGGGSGGGGS - SCDKTH - Trastuzumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing a TNFR1 selective antagonist TNF mutein with mutations V1M, R31C, C69V, Y87H, C101A and A145R with respect to the sequence of soluble TNF set forth in SEQ ID NO: 2. The C-terminus of the TNF (V1M/R31C/C69V/Y87H/C101A/A145R) mutein (SEQ ID NO:701) corresponds to the Trastuzumab Fc region (residues 234-450 of SEQ ID NO: 26; also SEQ ID NO: 26). 27) to a GGGGSGGGGGSGGGGS peptide linker that is fused to at least a portion of the hinge sequence of Trastuzumab that contains residues SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26) that are fused to the N-terminus of . The TNF(V1M/R31C/C69V/Y87H/C101A/A145R)-GGGGSGGGGSGGGGS-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 746):

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker such as Alternatively or additionally, the SCDKTH hinge sequence is at least 5, 6, 7, 8, 9 up to the entire sequence of the hinge region of Trastuzumab containing the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26). , is replaced with a moiety containing 10 or 11 adjacent residues.

2g) TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q) - SCDKTH - Trastuzumab Fc

Provided herein is a human TNFR1 antagonist fusion protein containing a TNFR1 selective antagonist TNF mutein with mutations A84S, V85T, S86T, Y87H, Q88N and T89Q with respect to the sequence of soluble TNF set forth in SEQ ID NO: 2. The C-terminus of the TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q) mutein (SEQ ID NO: 703) corresponds to residues 234-450 of the trastuzumab Fc region (SEQ ID NO: 26; also SEQ ID NO: 703). 27) is fused to at least a portion of the hinge sequence of Trastuzumab containing residues SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26). The TNF(A84S/V85T/S86T/Y87H/Q88N/T89Q)-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 747):

Alternatively, the SCDKTH hinge sequence is at least 5, 6, 7, 8, 9, 10 or replaced by a moiety containing 11 adjacent residues.

2h) TNF ( A84S / V85T / S86T / Y87H / Q88N / T89Q ) - GGGGSGGGGSGGGGS - Trastuzumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing a TNFR1 selective antagonist TNF mutein with mutations A84S, V85T, S86T, Y87H, Q88N and T89Q with respect to the sequence of soluble TNF set forth in SEQ ID NO: 2. The C-terminus of the TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q) mutein (SEQ ID NO: 703) is fused to the GGGGSGGGGSGGGGS peptide linker, which binds to the trastuzumab Fc region (residue 234 of SEQ ID NO: 26). corresponds to -450; see also SEQ ID NO: 27). The TNF(A84S/V85T/S86T/Y87H/Q88N/T89Q)-GGGGSGGGGSGGGGS-Trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 748):

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker such as

2i) TNF ( A84S / V85T / S86T / Y87H / Q88N / T89Q ) - GGGGSGGGGSGGGGS - SCDKTH - Trastuzumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing a TNFR1 selective antagonist TNF mutein with mutations A84S, V85T, S86T, Y87H, Q88N and T89Q with respect to the sequence of soluble TNF set forth in SEQ ID NO: 2. The C-terminus of the TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q) mutein (SEQ ID NO:703) corresponds to the trastuzumab Fc region (residues 234-450 of SEQ ID NO:26; also SEQ ID NO: 27) to a GGGGSGGGGGSGGGGS peptide linker that is fused to at least a portion of the hinge sequence of Trastuzumab that contains residues SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26) that are fused to the N-terminus of . The TNF(A84S/V85T/S86T/Y87H/Q88N/T89Q)-GGGGSGGGGSGGGGS-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 749):

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker such as Alternatively or additionally, the SCDKTH hinge sequence contains the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26), or at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues. It is replaced with the hinge region of trastuzumab containing a portion thereof.

2j) TNF (I97T/A145R) - SCDKTH - Trastuzumab Fc

Provided herein is a human TNFR1 antagonist fusion protein containing a TNFR1 selective antagonist TNF mutein with mutations I97T/A145R with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. The C-terminus of the TNF (I97T/A145R) mutein (SEQ ID NO: 702) is at the N-terminus of the trastuzumab Fc region (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27) It is fused to all or part of the hinge sequence of Trastuzumab, which contains at least the residues SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26) to which it is fused. The TNF(I97T/A145R)-SCDKTH-Trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 750):

Alternatively, the SCDKTH hinge sequence is the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26), or a portion thereof containing at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues. It is replaced with the hinge region of trastuzumab containing

2k) TNF (I97T/A145R) - GGGGSGGGGSGGGGS - Trastuzumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing a TNFR1 selective antagonist TNF mutein having mutations I97T/A145R with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. The C-terminus of the TNF(I97T/A145R) mutein (SEQ ID NO:702) is fused to the GGGGSGGGGSGGGGS peptide linker, which corresponds to residues 234-450 of the trastuzumab Fc region (SEQ ID NO: 26; also sequence number: see 27) is fused to the N-terminus. The TNF(I97T/A145R)-GGGGSGGGGSGGGGS-Trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 751):

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker such as

2l) TNF (I97T/A145R) - GGGGSGGGGSGGGGS - SCDKTH - Trastuzumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing a TNFR1 selective antagonist TNF mutein having mutations I97T/A145R with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. The C-terminus of the TNF(I97T/A145R) mutein (SEQ ID NO:702) is at the N-terminus of the trastuzumab Fc region (corresponding to residues 234-450 of SEQ ID NO:26; see also SEQ ID NO:27) GGGGSGGGGSGGGGS peptide linker that is fused to a portion of the hinge sequence of Trastuzumab that contains at least the residues SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26) to which it is fused. The TNF(I97T/A145R)-GGGGSGGGGSGGGGS-SCDKTH-Trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 752):

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker such as Alternatively or additionally, the SCDKTH hinge sequence contains the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26), or at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues. It is replaced with the hinge region of trastuzumab containing a portion thereof.

2m) TNF (R32W/S86T) - ESKYGPPCPPCP - Nivolumab Fc

Provided herein is a human TNFR1 antagonist fusion protein construct containing a TNFR1 selective antagonist TNF mutein having mutations R32W/S86T with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. The C-terminus of the TNF(R32W/S86T) mutein (SEQ ID NO:685) corresponds to residues 224-440 of the nivolumab Fc region SEQ ID NO:29; Reference, also to the hinge sequence of nivolumab containing residues ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29) fused to the N-terminus of SEQ ID NO: 30). The TNF(R32W/S86T)-ESKYGPPCPPCP-Nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 753):

Alternatively, all or some are included

2n) TNF (R32W/S86T) - GGGGSGGGGSGGGGS - Nivolumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing a TNFR1 selective antagonist TNF mutein having mutations R32W/S86T with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. The C-terminus of the TNF (R32W/S86T) mutein (SEQ ID NO: 685) is fused to the GGGGSGGGGSGGGGS peptide linker, which corresponds to residues 224-440 of the nivolumab Fc region (SEQ ID NO: 29; also SEQ ID NO: 29). : 30 reference) is fused to the N-terminus. The TNF(R32W/S86T)-GGGGSGGGGSGGGGS-Nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 754):

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker such as

2o) TNF (R32W/S86T) - GGGGSGGGGSGGGGS - ESKYGPPCPPCP - Nivolumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing a TNFR1 selective antagonist TNF mutein having mutations R32W/S86T with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. The C-terminus of the TNF(R32W/S86T) mutein (SEQ ID NO:685) corresponds to residues 224-440 of the nivolumab Fc region SEQ ID NO:29; Reference, also fused to the GGGGSGGGGSGGGGS peptide linker fused to the hinge sequence of nivolumab containing the sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29) fused to the N-terminus of SEQ ID NO: 30). The TNF(R32W/S86T)-GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 755):

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker such as Alternatively or additionally, a nivolumab hinge sequence containing at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29) part of is included.

2p) TNF (V1M/R31C/C69V/Y87H/C101A/A145R) - ESKYGPPCPPCP - Nivolumab Fc

Provided herein is a human TNFR1 antagonist fusion protein containing a TNFR1 selective antagonist TNF mutein with mutations V1M, R31C, C69V, Y87H, C101A and A145R with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. The C-terminus of the TNF (V1M/R31C/C69V/Y87H/C101A/A145R) mutein (SEQ ID NO:701) corresponds to residues 224-440 of the nivolumab Fc region SEQ ID NO:29; reference, also to the hinge sequence of nivolumab containing the sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29) fused to the N-terminus of SEQ ID NO: 30). The TNF(V1M/R31C/C69V/Y87H/C101A/A145R)-ESKYGPPCPPCP-Nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 756):

Alternatively, all or some are included

2q) TNF (V1M/R31C/C69V/Y87H/C101A/A145R) - GGGGSGGGGSGGGGS - Nivolumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing a TNFR1 selective antagonist TNF mutein with mutations V1M, R31C, C69V, Y87H, C101A and A145R with respect to the sequence of soluble TNF set forth in SEQ ID NO: 2. The C-terminus of the TNF (V1M/R31C/C69V/Y87H/C101A/A145R) mutein (SEQ ID NO: 701) is fused to a GGGGSGGGGSGGGGS peptide linker, which is linked to the nivolumab Fc region (residues 224-29 of SEQ ID NO: 29). 440; see also SEQ ID NO: 30). The TNF(V1M/R31C/C69V/Y87H/C101A/A145R)-GGGGSGGGGSGGGGS-Nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 757):

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker such as

2r) TNF ( V1M / R31C / C69V / Y87H / C101A / A145R ) - GGGGSGGGGSGGGGS - ESKYGPPCPPCP - Nivolumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing a TNFR1 selective antagonist TNF mutein with mutations V1M, R31C, C69V, Y87H, C101A and A145R with respect to the sequence of soluble TNF set forth in SEQ ID NO: 2. The C-terminus of the TNF (V1M/R31C/C69V/Y87H/C101A/A145R) mutein (SEQ ID NO:701) corresponds to residues 224-440 of the nivolumab Fc region SEQ ID NO:29; Reference, also fused to the GGGGSGGGGSGGGGS peptide linker fused to the hinge sequence of nivolumab containing the sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29) fused to the N-terminus of SEQ ID NO: 30). The TNF(V1M/R31C/C69V/Y87H/C101A/A145R)-GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 758):

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker such as Alternatively or additionally, a nivolumab hinge sequence containing at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29) includes all or part of

2s) TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q) - ESKYGPPCPPCP - Nivolumab Fc

Provided herein is a human TNFR1 antagonist fusion protein containing a TNFR1 selective antagonist TNF mutein with mutations A84S, V85T, S86T, Y87H, Q88N and T89Q with respect to the sequence of soluble TNF set forth in SEQ ID NO: 2. The C-terminus of the TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q) mutein (SEQ ID NO:703) corresponds to residues 224-440 of the nivolumab Fc region SEQ ID NO:29; reference, also to the hinge sequence of nivolumab containing the sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29) fused to the N-terminus of SEQ ID NO: 30). The TNF(A84S/V85T/S86T/Y87H/Q88N/T89Q)-ESKYGPPCPPCP-Nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 759):

Alternatively, all or some are included

2t) TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q) - GGGGSGGGGSGGGGS - Nivolumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing a TNFR1 selective antagonist TNF mutein with mutations A84S, V85T, S86T, Y87H, Q88N and T89Q with respect to the sequence of soluble TNF set forth in SEQ ID NO: 2. The C-terminus of the TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q) mutein (SEQ ID NO: 703) is fused to a GGGGSGGGGSGGGGS peptide linker, which is linked to the nivolumab Fc region (residues 224-29 of SEQ ID NO: 29). 440; see also SEQ ID NO: 30). The TNF(A84S/V85T/S86T/Y87H/Q88N/T89Q)-GGGGSGGGGSGGGGS-Nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 760):

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker such as

2u) TNF ( A84S / V85T / S86T / Y87H / Q88N / T89Q ) - GGGGSGGGGSGGGGS - ESKYGPPCPPCP - Nivolumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing a TNFR1 selective antagonist TNF mutein with mutations A84S, V85T, S86T, Y87H, Q88N and T89Q with respect to the sequence of soluble TNF set forth in SEQ ID NO: 2. The C-terminus of the TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q) mutein (SEQ ID NO:703) corresponds to residues 224-440 of the nivolumab Fc region SEQ ID NO:29; Reference, also fused to the GGGGSGGGGSGGGGS peptide linker fused to the hinge sequence of nivolumab containing the sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29) fused to the N-terminus of SEQ ID NO: 30). The TNF(A84S/V85T/S86T/Y87H/Q88N/T89Q)-GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 761):

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker such as Alternatively or additionally, a nivolumab hinge sequence containing at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29) includes all or part of

2v) TNF (I97T/A145R) - ESKYGPPCPPCP - Nivolumab Fc

Provided herein is a human TNFR1 antagonist fusion protein containing a TNFR1 selective antagonist TNF mutein with mutations I97T/A145R with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. The C-terminus of the TNF(I97T/A145R) mutein (SEQ ID NO:702) corresponds to residues 224-440 of the nivolumab Fc region SEQ ID NO:29; reference, also to the hinge sequence of nivolumab containing the sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29) fused to the N-terminus of SEQ ID NO: 30). The TNF(I97T/A145R)-ESKYGPPCPPCP-Nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 762):

Alternatively, all or some are included

2w) TNF (I97T/A145R) - GGGGSGGGGSGGGGS - Nivolumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing a TNFR1 selective antagonist TNF mutein having mutations I97T/A145R with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. The C-terminus of the TNF(I97T/A145R) mutein (SEQ ID NO:702) is fused to the GGGGSGGGGSGGGGS peptide linker, which corresponds to residues 224-440 of the nivolumab Fc region (SEQ ID NO: 29; also SEQ ID NO: : 30 reference) is fused to the N-terminus. The TNF(I97T/A145R)-GGGGSGGGGSGGGGS-Nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 763):

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker such as

2x) TNF (I97T/A145R) - GGGGSGGGGSGGGGS - ESKYGPPCPPCP - Nivolumab Fc

Provided herein is a TNFR1 antagonist fusion protein containing a TNFR1 selective antagonist TNF mutein having mutations I97T/A145R with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. The C-terminus of the TNF(I97T/A145R) mutein (SEQ ID NO:702) corresponds to residues 224-440 of the nivolumab Fc region SEQ ID NO:29; Reference, also fused to the GGGGSGGGGSGGGGS peptide linker fused to the hinge sequence of nivolumab containing the sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29) fused to the N-terminus of SEQ ID NO: 30). The TNF(I97T/A145R)-GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 764):

Alternatively, the GGGGSGGGGSGGGGS linker can be a Gly-Ser linker, such as a (GSGS) _n or (GGGGS) _n linker where n = 1, 2, 3, 4, or 5, or as described herein or known in the art. Replaced with any other suitable short peptide linker such as Alternatively or additionally, a nivolumab hinge sequence containing at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29) includes all or part of

Example 6

Presentation in the context of antibodies presents difficulties in the context of inhibition of target receptors. Bivalent antibodies are more likely to activate by inducing dimerization of target receptors. In some cases this is desirable, but highly undesirable for inhibitors of TNFR1. Even transient activation of TNFR1 can induce a cytokine storm and severe toxicity. Therefore, monovalent inhibitors are needed. To achieve this, monovalent constructs are provided herein (see full description and Example 5 above). This example illustrates the activity and properties of an exemplary construct that is an N-terminal fusion protein of a single chain dAb with human serum albumin.

This example provides exemplary Nanobodies. Nanobodies, which are Vhh domain-containing proteins, contain only heavy chains and do not require cooperativity from light chains as is the case in humans and mice (Harmsen and De Haard, Appl . Microbiol Biotechnol . 77:13-22 (2007)). Since they are single chains, they are themselves small proteins (~13-15 KDa) and, because of their short half-lives, should be presented as fusion proteins, such as grafted CDRs in antibody format.

Phage libraries are described in Sabir et al. ((2014) Comptes Rendus Biologies 337 :244-249]. Ability to recover phage with high affinity binding to tumor necrosis factor receptor-1 (TNFR1) and bind to TNFR1 and binding of human tumor necrosis factor-alpha (TNF-a) to TNFR1 described in US Published Application No. US20140112929 tested for their ability to compete with

Nucleic acids encoding the respective Vhh antibodies containing single chains were expressed in CHO cells (see Sokolowska- Wedzina et al. (2014) Protein Expression and Purification 99:50-57 for description of expression vectors), respectively. was purified by HPLC chromatography. Sample 1 was a control anti-TNFR1 antibody (H398 from ThermoFisher), and Vhh1-4 were Vhh antibodies containing dAbs. The sequences (see SEQ ID NOs: 54, 1478, 58 and 59) are as follows:

The highlighted and underlined cysteine (C) of Vhh-4 and the corresponding cysteines of the other Vhh chains form a loop, where Vhh is a constrained polypeptide. Proline at amino acid residue 14 can be replaced with alanine.

Expression cassettes encoding Vhh domain antibody fragments were prepared and expressed and purified by HPLC chromatography. The table below shows the expression level of each tested Vhh antibody. Both molecules required a His tag for purification. Results are presented in the following table.

Each of Vhh 1-4 was then tested in binding studies using surface plasmon resonance (SPR) methods (Sciences GL. Biacore Assay Handbook. General Electric Company (2012); and Richter et al. (2019) MAbs 11:166-177). A competition assay of Vhh inhibition of TNF-a binding to the TNFR1 extracellular domain was also performed as described by Richter et al (2019). Inhibition of TNF-induced expression of VCAM or IL8, a comparable assay, was reported by Lin et al., (2015) J Biomed. Sci 22:53 ; and Sonnier et al. (2010) Journal of Gastrointestinal Surgery 14 :1592-1599].

A summary of the results is presented in the table below. The results show that Vhh-4 has a very high affinity (6.6 x 10 ^-13 M) for the extracellular domain of TNFR-1; It was 100% competitive for TNF binding to TNFR1 (IC50 ~1 nM) and the most efficient of the four candidates to inhibit TNF-derived VCAM-1 synthesis (0.3 nM). Of these Vhh dAb antibodies tested, Vhh-4 performed best and produced constructs comprising Vhh-4 and HSA.

The sequence below shows that a construct containing the Vhh-4:dAb portion (SEQ ID NO: 1475) is linked to human serum albumin (HSA; SEQ ID NO: 1475 residues 139-147) via a Gly-Ser linker (SEQ ID NO: 1475). 148-732) and residues 20-138 of SEQ ID NO: 1475.

Constructs containing Vhh-4 antibodies linked to HSA have been shown to have activity as TNFR1 antagonists. It blocked TNFR1 as shown by a significant decrease in IL-6 and IL-8 gene expression in THP1 cells stimulated with LPS. 3x10 ⁵ THP1 cells were treated with 5 μg, 20 μg, and 0 μg for 30 minutes as a control of the construct, and then LPS (10 ng/ml) stimulation was performed for 4 hours. RNA samples were collected and qPCR analysis was performed to analyze IL-6 and IL-8 gene expression using the housekeeping gene hypoxanthine-guanine phosphoribosyltransferase (HPRT) as an internal control. Results show that both doses significantly reduced IL-6 and IL-8 expression (n = 3, mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001).

Since variations will be apparent to those skilled in the art, it is intended that this invention be limited only by the scope of the appended claims.

SEQUENCE LISTING <110> ENOSI LIFE SCIENCES CORP. Shepard, H. Michael <120> Methods and Compositions to Treat Autoimmune Diseases and Cancer <130> 04555-5301PC <140> PCT/US21/48074 <141> 2021-08-27 <150> 63/071,313 <151> 2020-08-27 <160> 1479 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 233 <212> PRT <213> Homo sapiens <220> <223> TNF alpha (TNF) <400> 1 Met Ser Thr Glu Ser Met Ile Arg Asp Val Glu Leu Ala Glu Glu Ala 1 5 10 15 Leu Pro Lys Lys Thr Gly Gly Pro Gln Gly Ser Arg Arg Cys Leu Phe 20 25 30 Leu Ser Leu Phe Ser Phe Leu Ile Val Ala Gly Ala Thr Thr Leu Phe 35 40 45 Cys Leu Leu His Phe Gly Val Ile Gly Pro Gln Arg Glu Glu Phe Pro 50 55 60 Arg Asp Leu Ser Leu Ile Ser Pro Leu Ala Gln Ala Val Arg Ser Ser 65 70 75 80 Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro 85 90 95 Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu 100 105 110 Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser 115 120 125 Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly 130 135 140 Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala 145 150 155 160 Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro 165 170 175 Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu 180 185 190 Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu 195 200 205 Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly 210 215 220 Gln Val Tyr Phe Gly Ile Ile Ala Leu 225 230 <210> 2 <211> 157 <212> PRT <213> Homo sapiens <220> <223> Soluble form of TNF alpha (solTNF) <400> 2 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 3 <211> 455 <212> PRT <213> Homo sapiens <220> <223> TNFR1 <400> 3 Met Gly Leu Ser Thr Val Pro Asp Leu Leu Leu Pro Leu Val Leu Leu 1 5 10 15 Glu Leu Leu Val Gly Ile Tyr Pro Ser Gly Val Ile Gly Leu Val Pro 20 25 30 His Leu Gly Asp Arg Glu Lys Arg Asp Ser Val Cys Pro Gln Gly Lys 35 40 45 Tyr Ile His Pro Gln Asn Asn Ser Ile Cys Cys Thr Lys Cys His Lys 50 55 60 Gly Thr Tyr Leu Tyr Asn Asp Cys Pro Gly Pro Gly Gln Asp Thr Asp 65 70 75 80 Cys Arg Glu Cys Glu Ser Gly Ser Phe Thr Ala Ser Glu Asn His Leu 85 90 95 Arg His Cys Leu Ser Cys Ser Lys Cys Arg Lys Glu Met Gly Gln Val 100 105 110 Glu Ile Ser Ser Cys Thr Val Asp Arg Asp Thr Val Cys Gly Cys Arg 115 120 125 Lys Asn Gln Tyr Arg His Tyr Trp Ser Glu Asn Leu Phe Gln Cys Phe 130 135 140 Asn Cys Ser Leu Cys Leu Asn Gly Thr Val His Leu Ser Cys Gln Glu 145 150 155 160 Lys Gln Asn Thr Val Cys Thr Cys His Ala Gly Phe Phe Leu Arg Glu 165 170 175 Asn Glu Cys Val Ser Cys Ser Asn Cys Lys Lys Ser Leu Glu Cys Thr 180 185 190 Lys Leu Cys Leu Pro Gln Ile Glu Asn Val Lys Gly Thr Glu Asp Ser 195 200 205 Gly Thr Thr Val Leu Leu Pro Leu Val Ile Phe Phe Gly Leu Cys Leu 210 215 220 Leu Ser Leu Leu Phe Ile Gly Leu Met Tyr Arg Tyr Gln Arg Trp Lys 225 230 235 240 Ser Lys Leu Tyr Ser Ile Val Cys Gly Lys Ser Thr Pro Glu Lys Glu 245 250 255 Gly Glu Leu Glu Gly Thr Thr Thr Lys Pro Leu Ala Pro Asn Pro Ser 260 265 270 Phe Ser Pro Thr Pro Gly Phe Thr Pro Thr Leu Gly Phe Ser Pro Val 275 280 285 Pro Ser Ser Thr Phe Thr Ser Ser Ser Thr Tyr Thr Pro Gly Asp Cys 290 295 300 Pro Asn Phe Ala Ala Pro Arg Arg Glu Val Ala Pro Pro Tyr Gln Gly 305 310 315 320 Ala Asp Pro Ile Leu Ala Thr Ala Leu Ala Ser Asp Pro Ile Pro Asn 325 330 335 Pro Leu Gln Lys Trp Glu Asp Ser Ala His Lys Pro Gln Ser Leu Asp 340 345 350 Thr Asp Asp Pro Ala Thr Leu Tyr Ala Val Val Glu Asn Val Pro Pro 355 360 365 Leu Arg Trp Lys Glu Phe Val Arg Arg Leu Gly Leu Ser Asp His Glu 370 375 380 Ile Asp Arg Leu Glu Leu Gln Asn Gly Arg Cys Leu Arg Glu Ala Gln 385 390 395 400 Tyr Ser Met Leu Ala Thr Trp Arg Arg Arg Thr Pro Arg Arg Glu Ala 405 410 415 Thr Leu Glu Leu Leu Gly Arg Val Leu Arg Asp Met Asp Leu Leu Gly 420 425 430 Cys Leu Glu Asp Ile Glu Glu Ala Leu Cys Gly Pro Ala Ala Leu Pro 435 440 445 Pro Ala Pro Ser Leu Leu Arg 450 455 <210> 4 <211> 461 <212> PRT <213> Homo sapiens <220> <223> TNFR2 <400> 4 Met Ala Pro Val Ala Val Trp Ala Ala Leu Ala Val Gly Leu Glu Leu 1 5 10 15 Trp Ala Ala Ala His Ala Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr 20 25 30 Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln 35 40 45 Thr Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala Lys 50 55 60 Val Phe Cys Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp 65 70 75 80 Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys 85 90 95 Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg 100 105 110 Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu 115 120 125 Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg 130 135 140 Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val Val 145 150 155 160 Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr 165 170 175 Asp Ile Cys Arg Pro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly 180 185 190 Asn Ala Ser Met Asp Ala Val Cys Thr Ser Thr Ser Pro Thr Arg Ser 195 200 205 Met Ala Pro Gly Ala Val His Leu Pro Gln Pro Val Ser Thr Arg Ser 210 215 220 Gln His Thr Gln Pro Thr Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser 225 230 235 240 Phe Leu Leu Pro Met Gly Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly 245 250 255 Asp Phe Ala Leu Pro Val Gly Leu Ile Val Gly Val Thr Ala Leu Gly 260 265 270 Leu Leu Ile Ile Gly Val Val Asn Cys Val Ile Met Thr Gln Val Lys 275 280 285 Lys Lys Pro Leu Cys Leu Gln Arg Glu Ala Lys Val Pro His Leu Pro 290 295 300 Ala Asp Lys Ala Arg Gly Thr Gln Gly Pro Glu Gln Gln His Leu Leu 305 310 315 320 Ile Thr Ala Pro Ser Ser Ser Ser Ser Ser Ser Leu Glu Ser Ser Ala Ser 325 330 335 Ala Leu Asp Arg Arg Ala Pro Thr Arg Asn Gln Pro Gln Ala Pro Gly 340 345 350 Val Glu Ala Ser Gly Ala Gly Glu Ala Arg Ala Ser Thr Gly Ser Ser 355 360 365 Asp Ser Ser Pro Gly Gly His Gly Thr Gln Val Asn Val Thr Cys Ile 370 375 380 Val Asn Val Cys Ser Ser Ser Asp His Ser Ser Gln Cys Ser Ser Gln 385 390 395 400 Ala Ser Ser Thr Met Gly Asp Thr Asp Ser Ser Pro Ser Glu Ser Pro 405 410 415 Lys Asp Glu Gln Val Pro Phe Ser Lys Glu Glu Cys Ala Phe Arg Ser 420 425 430 Gln Leu Glu Thr Pro Glu Thr Leu Leu Gly Ser Thr Glu Glu Lys Pro 435 440 445 Leu Pro Leu Gly Val Pro Asp Ala Gly Met Lys Pro Ser 450 455 460 <210> 5 <211> 235 <212> PRT 213 <213> <220> <223> Mouse TNF alpha (TNF) <400> 5 Met Ser Thr Glu Ser Met Ile Arg Asp Val Glu Leu Ala Glu Glu Ala 1 5 10 15 Leu Pro Gln Lys Met Gly Gly Phe Gln Asn Ser Arg Arg Cys Leu Cys 20 25 30 Leu Ser Leu Phe Ser Phe Leu Leu Val Ala Gly Ala Thr Thr Leu Phe 35 40 45 Cys Leu Leu Asn Phe Gly Val Ile Gly Pro Gln Arg Asp Glu Lys Phe 50 55 60 Pro Asn Gly Leu Pro Leu Ile Ser Ser Met Ala Gln Thr Leu Thr Leu 65 70 75 80 Arg Ser Ser Ser Gln Asn Ser Ser Asp Lys Pro Val Ala His Val Val 85 90 95 Ala Asn His Gln Val Glu Glu Gln Leu Glu Trp Leu Ser Gln Arg Ala 100 105 110 Asn Ala Leu Leu Ala Asn Gly Met Asp Leu Lys Asp Asn Gln Leu Val 115 120 125 Val Pro Ala Asp Gly Leu Tyr Leu Val Tyr Ser Gln Val Leu Phe Lys 130 135 140 Gly Gln Gly Cys Pro Asp Tyr Val Leu Leu Thr His Thr Val Ser Arg 145 150 155 160 Phe Ala Ile Ser Tyr Gln Glu Lys Val Asn Leu Leu Ser Ala Val Lys 165 170 175 Ser Pro Cys Pro Lys Asp Thr Pro Glu Gly Ala Glu Leu Lys Pro Trp 180 185 190 Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp 195 200 205 Gln Leu Ser Ala Glu Val Asn Leu Pro Lys Tyr Leu Asp Phe Ala Glu 210 215 220 Ser Gly Gln Val Tyr Phe Gly Val Ile Ala Leu 225 230 235 <210> 6 <211> 156 <212> PRT 213 <213> <220> <223> Soluble form of mouse TNF alpha (TNF) <400> 6 Leu Arg Ser Ser Ser Gln Asn Ser Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn His Gln Val Glu Glu Gln Leu Glu Trp Leu Ser Gln Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Met Asp Leu Lys Asp Asn Gln Leu 35 40 45 Val Val Pro Ala Asp Gly Leu Tyr Leu Val Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Asp Tyr Val Leu Leu Thr His Thr Val Ser 65 70 75 80 Arg Phe Ala Ile Ser Tyr Gln Glu Lys Val Asn Leu Leu Ser Ala Val 85 90 95 Lys Ser Pro Cys Pro Lys Asp Thr Pro Glu Gly Ala Glu Leu Lys Pro 100 105 110 Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly 115 120 125 Asp Gln Leu Ser Ala Glu Val Asn Leu Pro Lys Tyr Leu Asp Phe Ala 130 135 140 Glu Ser Gly Gln Val Tyr Phe Gly Val Ile Ala Leu 145 150 155 <210> 7 <211> 454 <212> PRT 213 <213> <220> <223> Mouse TNFR1 <400> 7 Met Gly Leu Pro Thr Val Pro Gly Leu Leu Leu Ser Leu Val Leu Leu 1 5 10 15 Ala Leu Leu Met Gly Ile His Pro Ser Gly Val Thr Gly Leu Val Pro 20 25 30 Ser Leu Gly Asp Arg Glu Lys Arg Asp Ser Leu Cys Pro Gln Gly Lys 35 40 45 Tyr Val His Ser Lys Asn Asn Ser Ile Cys Cys Thr Lys Cys His Lys 50 55 60 Gly Thr Tyr Leu Val Ser Asp Cys Pro Ser Pro Gly Arg Asp Thr Val 65 70 75 80 Cys Arg Glu Cys Glu Lys Gly Thr Phe Thr Ala Ser Gln Asn Tyr Leu 85 90 95 Arg Gln Cys Leu Ser Cys Lys Thr Cys Arg Lys Glu Met Ser Gln Val 100 105 110 Glu Ile Ser Pro Cys Gln Ala Asp Lys Asp Thr Val Cys Gly Cys Lys 115 120 125 Glu Asn Gln Phe Gln Arg Tyr Leu Ser Glu Thr His Phe Gln Cys Val 130 135 140 Asp Cys Ser Pro Cys Phe Asn Gly Thr Val Thr Ile Pro Cys Lys Glu 145 150 155 160 Thr Gln Asn Thr Val Cys Asn Cys His Ala Gly Phe Phe Leu Arg Glu 165 170 175 Ser Glu Cys Val Pro Cys Ser His Cys Lys Lys Asn Glu Glu Cys Met 180 185 190 Lys Leu Cys Leu Pro Pro Pro Leu Ala Asn Val Thr Asn Pro Gln Asp 195 200 205 Ser Gly Thr Ala Val Leu Leu Pro Leu Val Ile Leu Leu Gly Leu Cys 210 215 220 Leu Leu Ser Phe Ile Phe Ile Ser Leu Met Cys Arg Tyr Pro Arg Trp 225 230 235 240 Arg Pro Glu Val Tyr Ser Ile Ile Cys Arg Asp Pro Val Pro Val Lys 245 250 255 Glu Glu Lys Ala Gly Lys Pro Leu Thr Pro Ala Pro Ser Pro Ala Phe 260 265 270 Ser Pro Thr Ser Gly Phe Asn Pro Thr Leu Gly Phe Ser Thr Pro Gly 275 280 285 Phe Ser Ser Pro Val Ser Ser Thr Pro Ile Ser Pro Ile Phe Gly Pro 290 295 300 Ser Asn Trp His Phe Met Pro Pro Val Ser Glu Val Val Pro Thr Gln 305 310 315 320 Gly Ala Asp Pro Leu Leu Tyr Glu Ser Leu Cys Ser Val Pro Ala Pro 325 330 335 Thr Ser Val Gln Lys Trp Glu Asp Ser Ala His Pro Gln Arg Pro Asp 340 345 350 Asn Ala Asp Leu Ala Ile Leu Tyr Ala Val Val Asp Gly Val Pro Pro 355 360 365 Ala Arg Trp Lys Glu Phe Met Arg Phe Met Gly Leu Ser Glu His Glu 370 375 380 Ile Glu Arg Leu Glu Met Gln Asn Gly Arg Cys Leu Arg Glu Ala Gln 385 390 395 400 Tyr Ser Met Leu Glu Ala Trp Arg Arg Arg Thr Pro Arg His Glu Asp 405 410 415 Thr Leu Glu Val Val Gly Leu Val Leu Ser Lys Met Asn Leu Ala Gly 420 425 430 Cys Leu Glu Asn Ile Leu Glu Ala Leu Arg Asn Pro Ala Pro Ser Ser 435 440 445 Thr Thr Arg Leu Pro Arg 450 <210> 8 <211> 474 <212> PRT 213 <213> <220> <223> Mouse TNFR2 <400> 8 Met Ala Pro Ala Ala Leu Trp Val Ala Leu Val Phe Glu Leu Gln Leu 1 5 10 15 Trp Ala Thr Gly His Thr Val Pro Ala Gln Val Val Leu Thr Pro Tyr 20 25 30 Lys Pro Glu Pro Gly Tyr Glu Cys Gln Ile Ser Gln Glu Tyr Tyr Asp 35 40 45 Arg Lys Ala Gln Met Cys Cys Ala Lys Cys Pro Pro Gly Gln Tyr Val 50 55 60 Lys His Phe Cys Asn Lys Thr Ser Asp Thr Val Cys Ala Asp Cys Glu 65 70 75 80 Ala Ser Met Tyr Thr Gln Val Trp Asn Gln Phe Arg Thr Cys Leu Ser 85 90 95 Cys Ser Ser Ser Cys Thr Thr Asp Gln Val Glu Ile Arg Ala Cys Thr 100 105 110 Lys Gln Gln Asn Arg Val Cys Ala Cys Glu Ala Gly Arg Tyr Cys Ala 115 120 125 Leu Lys Thr His Ser Gly Ser Cys Arg Gln Cys Met Arg Leu Ser Lys 130 135 140 Cys Gly Pro Gly Phe Gly Val Ala Ser Ser Arg Ala Pro Asn Gly Asn 145 150 155 160 Val Leu Cys Lys Ala Cys Ala Pro Gly Thr Phe Ser Asp Thr Thr Ser 165 170 175 Ser Thr Asp Val Cys Arg Pro His Arg Ile Cys Ser Ile Leu Ala Ile 180 185 190 Pro Gly Asn Ala Ser Thr Asp Ala Val Cys Ala Pro Glu Ser Pro Thr 195 200 205 Leu Ser Ala Ile Pro Arg Thr Leu Tyr Val Ser Gln Pro Glu Pro Thr 210 215 220 Arg Ser Gln Pro Leu Asp Gln Glu Pro Gly Pro Ser Gln Thr Pro Ser 225 230 235 240 Ile Leu Thr Ser Leu Gly Ser Thr Pro Ile Ile Glu Gln Ser Thr Lys 245 250 255 Gly Gly Ile Ser Leu Pro Ile Gly Leu Ile Val Gly Val Thr Ser Leu 260 265 270 Gly Leu Leu Met Leu Gly Leu Val Asn Cys Ile Ile Leu Val Gln Arg 275 280 285 Lys Lys Lys Pro Ser Cys Leu Gln Arg Asp Ala Lys Val Pro His Val 290 295 300 Pro Asp Glu Lys Ser Gln Asp Ala Val Gly Leu Glu Gln Gln His Leu 305 310 315 320 Leu Thr Thr Ala Pro Ser Ser Ser Ser Ser Ser Ser Leu Glu Ser Ser Ala 325 330 335 Ser Ala Gly Asp Arg Arg Ala Pro Pro Gly Gly His Pro Gln Ala Arg 340 345 350 Val Met Ala Glu Ala Gln Gly Phe Gln Glu Ala Arg Ala Ser Ser Arg 355 360 365 Ile Ser Asp Ser Ser His Gly Ser His Gly Thr His Val Asn Val Thr 370 375 380 Cys Ile Val Asn Val Cys Ser Ser Ser Ser Asp His Ser Ser Gln Cys Ser 385 390 395 400 Ser Gln Ala Ser Ala Thr Val Gly Asp Pro Asp Ala Lys Pro Ser Ala 405 410 415 Ser Pro Lys Asp Glu Gln Val Pro Phe Ser Gln Glu Glu Cys Pro Ser 420 425 430 Gln Ser Pro Cys Glu Thr Thr Glu Thr Leu Gln Ser His Glu Lys Pro 435 440 445 Leu Pro Leu Gly Val Pro Asp Met Gly Met Lys Pro Ser Gln Ala Gly 450 455 460 Trp Phe Asp Gln Ile Ala Val Lys Val Ala 465 470 <210> 9 <211> 330 <212> PRT <213> Homo sapiens <220> <223> IgG1 Heavy Chain Constant Domain <220> <221> DOMAIN <222> (1)...(98) <223> CH1 <220> <221> DOMAIN <222> (99)...(113) <223> Hinge region <220> <221> DOMAIN <222> (114)...(223) <223> CH2 <220> <221> DOMAIN <222> (224)...(230) <223> CH3 <400> 9 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 10 <211> 217 <212> PRT <213> Homo sapiens <220> <223> IgG1 Fc (CH2 and CH3 domains) <400> 10 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 11 <211> 326 <212> PRT <213> Homo sapiens <220> <223> Human IgG2 Heavy chain constant domain <220> <221> DOMAIN <222> (1)...(98) <223> CH1 <220> <221> DOMAIN <222> (99)...(110) <223> hinge region <220> <221> DOMAIN <222> (111)...(219) <223> CH2 <220> <221> DOMAIN <222> (220)...(326) <223> CH3 <400> 11 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly Lys 325 <210> 12 <211> 216 <212> PRT <213> Homo sapiens <220> <223> IgG2 Fc (CH2 and CH3 domains) <400> 12 Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 1 5 10 15 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 20 25 30 Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 35 40 45 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 50 55 60 Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln 65 70 75 80 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 85 90 95 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro 100 105 110 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 115 120 125 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 130 135 140 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 145 150 155 160 Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 165 170 175 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 180 185 190 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 195 200 205 Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 13 <211> 377 <212> PRT <213> Homo sapiens <220> <223> IgG3 heavy chain constant domain <220> <221> DOMAIN <222> (1)...(98) <223> CH1 <220> <221> DOMAIN <222> (99)...(160) <223> Hinge region <220> <221> DOMAIN <222> (161)... (270) <223> CH2 <220> <221> DOMAIN <222> (271)...(377) <223> CH3 <400> 13 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro 100 105 110 Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 115 120 125 Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135 140 Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 145 150 155 160 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 165 170 175 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 180 185 190 Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr 195 200 205 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 210 215 220 Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His 225 230 235 240 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 245 250 255 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 260 265 270 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 275 280 285 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290 295 300 Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn 305 310 315 320 Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 325 330 335 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile 340 345 350 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln 355 360 365 Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 <210> 14 <211> 217 <212> PRT <213> Homo sapiens <220> <223> IgG3 Fc (CH2 and CH3 domains) <400> 14 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 15 <211> 327 <212> PRT <213> Homo sapiens <220> <223> IgG4 Heavy chain constant domain <220> <221> DOMAIN <222> (1)...(98) <223> CH1 <220> <221> DOMAIN <222> (99)...(110) <223> Hinge region <220> <221> DOMAIN <222> (111)...(220) <223> CH2 <220> <221> DOMAIN <222> (221)...(327) <223> CH3 <400> 15 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 16 <211> 217 <212> PRT <213> Homo sapiens <220> <223> IgG4 Fc (CH2 and CH3 domains) <400> 16 Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Leu Gly Lys 210 215 <210> 17 <211> 107 <212> PRT <213> Homo sapiens <220> <223> Human Ig kappa light chain constant domain (CL) <400> 17 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 18 <211> 106 <212> PRT <213> Homo sapiens <220> <223> Immunoglobulin lambda constant 1 light chain domain (chain C) <400> 18 Gly Gln Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser 1 5 10 15 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30 Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro 35 40 45 Val Lys Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn 50 55 60 Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys 65 70 75 80 Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val 85 90 95 Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 100 105 <210> 19 <211> 106 <212> PRT <213> Homo sapiens <220> <223> Immunoglobulin lambda CONSTANT 2 light chain domain (chain C) <400> 19 Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser 1 5 10 15 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30 Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro 35 40 45 Val Lys Ala Gly Val Glu Thr Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn 50 55 60 Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys 65 70 75 80 Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val 85 90 95 Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 100 105 <210> 20 <211> 106 <212> PRT <213> Homo sapiens <220> <223> Immunoglobulin lambda CONSTANT 3 light chain domain (chain C) <400> 20 Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser 1 5 10 15 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30 Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro 35 40 45 Val Lys Ala Gly Val Glu Thr Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn 50 55 60 Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys 65 70 75 80 Ser His Lys Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val 85 90 95 Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 100 105 <210> 21 <211> 106 <212> PRT <213> Homo sapiens <220> <223> Immunoglobulin lambda CONSTANT 6 light chain domain (chain C) <400> 21 Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser 1 5 10 15 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30 Phe Tyr Pro Gly Ala Val Lys Val Ala Trp Lys Ala Asp Gly Ser Pro 35 40 45 Val Asn Thr Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn 50 55 60 Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys 65 70 75 80 Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val 85 90 95 Glu Lys Thr Val Ala Pro Ala Glu Cys Ser 100 105 <210> 22 <211> 106 <212> PRT <213> Homo sapiens <220> <223> Immunoglobulin lambda CONSTANT 7 light chain domain (chain C) <400> 22 Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser 1 5 10 15 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Val Ser Asp 20 25 30 Phe Asn Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro 35 40 45 Val Lys Val Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn 50 55 60 Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys 65 70 75 80 Ser His Arg Ser Tyr Ser Cys Arg Val Thr His Glu Gly Ser Thr Val 85 90 95 Glu Lys Thr Val Ala Pro Ala Glu Cys Ser 100 105 <210> 23 <211> 214 <212> PRT <213> artificial sequence <220> <223> Adalimumab Light Chain <400> 23 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg Tyr Asn Arg Ala Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 24 <211> 451 <212> PRT <213> artificial sequence <220> <223> Adalimumab Heavy Chain <400> 24 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 25 <211> 214 <212> PRT <213> artificial sequence <220> <223> Trastuzumab light chain <400> 25 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 26 <211> 450 <212> PRT <213> artificial sequence <220> <223> Trastuzumab heavy chain <220> <221> DOMAIN <222> (1)...(120) <223> VH <220> <221> DOMAIN <222> (121)...(218) <223> CH1 <220> <221> DOMAIN <222> (219)...(233) <223> hinge region <220> <221> DOMAIN <222> (234)...(343) <223> CH2 <220> <221> DOMAIN <222> (344)... (450) <223> CH3 <400> 26 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 27 <211> 217 <212> PRT <213> artificial sequence <220> <223> Trastuzumab Fc (CH2 and CH3 domains) <400> 27 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 28 <211> 214 <212> PRT <213> artificial sequence <220> <223> Nivolumab light chain <400> 28 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 29 <211> 440 <212> PRT <213> artificial sequence <220> <223> Nivolumab heavy chain <220> <221> DOMAIN <222> (1)...(113) <223> VH <220> <221> DOMAIN <222> (114)...(221) <223> CH1 <220> <221> DOMAIN <222> (212)...(223) <223> Hinge region <220> <221> DOMAIN <222> (224)...(333) <223> CH2 <220> <221> DOMAIN <222> (334)... (440) <223> CH3 <400> 29 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser 115 120 125 Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135 140 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 145 150 155 160 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 165 170 175 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys 180 185 190 Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp 195 200 205 Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala 210 215 220 Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 225 230 235 240 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 245 250 255 Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 260 265 270 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 275 280 285 Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 290 295 300 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 305 310 315 320 Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 325 330 335 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr 340 345 350 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 355 360 365 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 370 375 380 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 385 390 395 400 Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe 405 410 415 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 420 425 430 Ser Leu Ser Leu Ser Leu Gly Lys 435 440 <210> 30 <211> 217 <212> PRT <213> artificial sequence <220> <223> Nivolumab Fc (CH2 and CH3 domains) <400> 30 Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Leu Gly Lys 210 215 <210> 31 <211> 444 <212> PRT <213> artificial sequence <220> <223> ATROSAB heavy chain <220> <221> DOMAIN <222> (1)...(115) <223> VH <220> <221> DOMAIN <222> (116)...(218) <223> CH1 <220> <221> DOMAIN <222> (219)...(234) <223> Hinge region <220> <221> DOMAIN <222> (235)...(337) <223> CH2 <220> <221> DOMAIN <222> (338)... (444) <223> CH3 <400> 31 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe 20 25 30 Tyr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Tyr Pro Tyr Ser Gly His Ala Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Ala Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Asp Phe Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220 Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 340 345 350 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 <210> 32 <211> 219 <212> PRT <213> artificial sequence <220> <223> ATROSAB light chain <220> <221> DOMAIN <222> (1)...(113) <223> VL <220> <221> DOMAIN <222> (114)...(219) <223> CL <400> 32 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Thr Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95 Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 33 <211> 115 <212> PRT <213> artificial sequence <220> <223> H398 VH domain <400> 33 Gln Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe 20 25 30 Tyr Ile Asn Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Tyr Pro Tyr Ser Gly His Ala Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Ala Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Phe 65 70 75 80 Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Val Arg Trp Asp Phe Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr 100 105 110 Val Ser Ser 115 <210> 34 <211> 113 <212> PRT <213> artificial sequence <220> <223> H398 VL domain <400> 34 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Val Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Thr Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 35 <211> 609 <212> PRT <213> Homo sapiens <220> <223> Human Serum Albumin (HSA) <220> <221> SIGNAL <222> (1)...(18) <223> signal peptide <220> <221> PROPEP <222> (19)...(24) <223> propeptide <220> <221> PEPTIDE <222> (25)...(609) <223> albumin <400> 35 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala 20 25 30 His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu 35 40 45 Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val 50 55 60 Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp 65 70 75 80 Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95 Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala 100 105 110 Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 115 120 125 His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val 130 135 140 Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys 145 150 155 160 Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 165 170 175 Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys 180 185 190 Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu 195 200 205 Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys 210 215 220 Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val 225 230 235 240 Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser 245 250 255 Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly 260 265 270 Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile 275 280 285 Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu 290 295 300 Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp 305 310 315 320 Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser 325 330 335 Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345 350 Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 355 360 365 Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys 370 375 380 Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu 385 390 395 400 Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys 405 410 415 Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 420 425 430 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 435 440 445 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 450 455 460 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 465 470 475 480 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 485 490 495 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 500 505 510 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 515 520 525 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 530 535 540 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 545 550 555 560 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 565 570 575 Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 580 585 590 Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly 595 600 605 Leu <210> 36 <211> 365 <212> PRT <213> Homo sapiens <220> <223> Human FcRn (IgG receptor FcRn large subunit p51) <220> <221> SIGNAL <222> (1)...(23) <223> signal peptide <220> <221> DOMAIN <222> (24)...(297) <223> extracellular domain <220> <221> DOMAIN <222> (298)...(321) <223> transmembrane domain <220> <221> DOMAIN <222> (322)... (365) <223> cytoplasmic domain <400> 36 Met Gly Val Pro Arg Pro Gln Pro Trp Ala Leu Gly Leu Leu Leu Phe 1 5 10 15 Leu Leu Pro Gly Ser Leu Gly Ala Glu Ser His Leu Ser Leu Leu Tyr 20 25 30 His Leu Thr Ala Val Ser Ser Pro Ala Pro Gly Thr Pro Ala Phe Trp 35 40 45 Val Ser Gly Trp Leu Gly Pro Gln Gln Tyr Leu Ser Tyr Asn Ser Leu 50 55 60 Arg Gly Glu Ala Glu Pro Cys Gly Ala Trp Val Trp Glu Asn Gln Val 65 70 75 80 Ser Trp Tyr Trp Glu Lys Glu Thr Thr Asp Leu Arg Ile Lys Glu Lys 85 90 95 Leu Phe Leu Glu Ala Phe Lys Ala Leu Gly Gly Lys Gly Pro Tyr Thr 100 105 110 Leu Gln Gly Leu Leu Gly Cys Glu Leu Gly Pro Asp Asn Thr Ser Val 115 120 125 Pro Thr Ala Lys Phe Ala Leu Asn Gly Glu Glu Phe Met Asn Phe Asp 130 135 140 Leu Lys Gln Gly Thr Trp Gly Gly Asp Trp Pro Glu Ala Leu Ala Ile 145 150 155 160 Ser Gln Arg Trp Gln Gln Gln Asp Lys Ala Ala Asn Lys Glu Leu Thr 165 170 175 Phe Leu Leu Phe Ser Cys Pro His Arg Leu Arg Glu His Leu Glu Arg 180 185 190 Gly Arg Gly Asn Leu Glu Trp Lys Glu Pro Pro Ser Met Arg Leu Lys 195 200 205 Ala Arg Pro Ser Ser Pro Gly Phe Ser Val Leu Thr Cys Ser Ala Phe 210 215 220 Ser Phe Tyr Pro Pro Glu Leu Gln Leu Arg Phe Leu Arg Asn Gly Leu 225 230 235 240 Ala Ala Gly Thr Gly Gln Gly Asp Phe Gly Pro Asn Ser Asp Gly Ser 245 250 255 Phe His Ala Ser Ser Ser Leu Thr Val Lys Ser Gly Asp Glu His His 260 265 270 Tyr Cys Cys Ile Val Gln His Ala Gly Leu Ala Gln Pro Leu Arg Val 275 280 285 Glu Leu Glu Ser Pro Ala Lys Ser Ser Val Leu Val Val Gly Ile Val 290 295 300 Ile Gly Val Leu Leu Leu Thr Ala Ala Ala Val Gly Gly Ala Leu Leu 305 310 315 320 Trp Arg Arg Met Arg Ser Gly Leu Pro Ala Pro Trp Ile Ser Leu Arg 325 330 335 Gly Asp Asp Thr Gly Val Leu Leu Pro Thr Pro Gly Glu Ala Gln Asp 340 345 350 Ala Asp Leu Lys Asp Val Asn Val Ile Pro Ala Thr Ala 355 360 365 <210> 37 <211> 690 <212> DNA <213> artificial sequence <220> <223> DMS5541 <400> 37 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc 60 tcctgtgcag cctccggatt cacctttgat aagtattcga tggggtgggt ccgccaggct 120 ccagggaagg gtctagagtg ggtctcacag atttcggata ctgctgatcg tacatactac 180 gcacacgcgg tgaagggccg gttcaccatc tcccgcgaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgcg tgctgaggac accgcggtat attactgtgc gatatatact 300 gggcgttggg tgccttttga gtactggggt cagggaaccc tggtcaccgt ctcgagcgct 360 agcaccgaca tccagatgac ccagtctcca tcctccctgt ctgcatctgt aggagaccgt 420 gtcaccatca cttgccgggc aagtcgtccg attgggacga cgttaagttg gtaccagcag 480 aaaccaggga aagcccctaa gctcctgatc ctgtggaatt cccgtttgca aagtggggtc 540 ccatcacgtt tcagtggcag tggatctggg acagatttca ctctcaccat cagcagtctg 600 caacctgaag attttgctac gtactactgt gcgcaggctg ggacgcatcc tacgacgttc 660 ggccaaggga ccaaggtgga aatcaaacgg 690 <210> 38 <211> 230 <212> PRT <213> artificial sequence <220> <223> DMS5541 <400> 38 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Asp Ile Gln Met Thr Gln 115 120 125 Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr 130 135 140 Cys Arg Ala Ser Arg Pro Ile Gly Thr Thr Leu Ser Trp Tyr Gln Gln 145 150 155 160 Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Leu Trp Asn Ser Arg Leu 165 170 175 Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 180 185 190 Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 195 200 205 Tyr Cys Ala Gln Ala Gly Thr His Pro Thr Thr Phe Gly Gln Gly Thr 210 215 220 Lys Val Glu Ile Lys Arg 225 230 <210> 39 <211> 231 <212> PRT <213> artificial sequence <220> <223> DMS5540 (mouse specific) <400> 39 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Arg Tyr 20 25 30 Ser Met Gly Trp Leu Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Arg Ile Asp Ser Tyr Gly Arg Gly Thr Tyr Tyr Glu Asp Pro Val 50 55 60 Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ile Ser Gln Phe Gly Ser Asn Ala Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser Ala Ser Thr Asp Ile Gln Met Thr 115 120 125 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 130 135 140 Thr Cys Arg Ala Ser Arg Pro Ile Gly Thr Met Leu Ser Trp Tyr Gln 145 150 155 160 Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Leu Phe Gly Ser Arg 165 170 175 Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr 180 185 190 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr 195 200 205 Tyr Tyr Cys Ala Gln Ala Gly Thr His Pro Thr Thr Phe Gly Gln Gly 210 215 220 Thr Lys Val Glu Ile Lys Arg 225 230 <210> 40 <211> 1210 <212> PRT <213> Homo sapiens <220> <223> human EGFR (HER1) precursor <220> <221> SIGNAL <222> (1)...(24) <223> signal peptide <220> <221> DOMAIN <222> (25)...(645) <223> Extracellular domain (ECD) <220> <221> DOMAIN <222> (646)...(668) <223> transmembrane region <220> <221> DOMAIN <222> (669)...(1210) <223> cytoplasmic domain <220> <221> DOMAIN <222> (712)...(979) <223> protein kinases <400> 40 Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala 1 5 10 15 Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln 20 25 30 Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe 35 40 45 Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn 50 55 60 Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys 65 70 75 80 Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val 85 90 95 Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr 100 105 110 Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn 115 120 125 Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu 130 135 140 His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu 145 150 155 160 Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met 165 170 175 Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro 180 185 190 Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln 195 200 205 Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg 210 215 220 Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys 225 230 235 240 Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp 245 250 255 Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro 260 265 270 Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly 275 280 285 Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His 290 295 300 Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu 305 310 315 320 Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val 325 330 335 Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn 340 345 350 Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp 355 360 365 Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr 370 375 380 Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu 385 390 395 400 Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp 405 410 415 Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln 420 425 430 His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu 435 440 445 Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser 450 455 460 Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu 465 470 475 480 Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu 485 490 495 Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro 500 505 510 Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn 515 520 525 Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly 530 535 540 Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro 545 550 555 560 Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro 565 570 575 Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val 580 585 590 Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp 595 600 605 Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys 610 615 620 Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly 625 630 635 640 Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu 645 650 655 Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg Arg His 660 665 670 Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu 675 680 685 Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala Leu Leu 690 695 700 Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu Gly Ser 705 710 715 720 Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile Pro Glu Gly Glu 725 730 735 Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu Arg Glu Ala Thr Ser 740 745 750 Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met Ala Ser 755 760 765 Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser 770 775 780 Thr Val Gln Leu Ile Thr Gln Leu Met Pro Phe Gly Cys Leu Leu Asp 785 790 795 800 Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu Leu Asn 805 810 815 Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp Arg Arg 820 825 830 Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr Pro 835 840 845 Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu Gly Ala 850 855 860 Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val Pro Ile Lys Trp 865 870 875 880 Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln Ser Asp 885 890 895 Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ser 900 905 910 Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ser Ser Ile Leu Glu 915 920 925 Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr 930 935 940 Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg Pro Lys 945 950 955 960 Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met Ala Arg Asp Pro Gln 965 970 975 Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His Leu Pro Ser Pro 980 985 990 Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu Glu Asp Met Asp 995 1000 1005 Asp Val Val Asp Ala Asp Glu Tyr Leu Ile Pro Gln Gln Gly Phe Phe 1010 1015 1020 Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser Ser Leu Ser Ala 1025 1030 1035 1040 Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp Arg Asn Gly Leu Gln 1045 1050 1055 Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln Arg Tyr Ser Ser Asp 1060 1065 1070 Pro Thr Gly Ala Leu Thr Glu Asp Ser Ile Asp Asp Thr Phe Leu Pro 1075 1080 1085 Val Pro Glu Tyr Ile Asn Gln Ser Val Pro Lys Arg Pro Ala Gly Ser 1090 1095 1100 Val Gln Asn Pro Val Tyr His Asn Gln Pro Leu Asn Pro Ala Pro Ser 1105 1110 1115 1120 Arg Asp Pro His Tyr Gln Asp Pro His Ser Thr Ala Val Gly Asn Pro 1125 1130 1135 Glu Tyr Leu Asn Thr Val Gln Pro Thr Cys Val Asn Ser Thr Phe Asp 1140 1145 1150 Ser Pro Ala His Trp Ala Gln Lys Gly Ser His Gln Ile Ser Leu Asp 1155 1160 1165 Asn Pro Asp Tyr Gln Gln Asp Phe Phe Pro Lys Glu Ala Lys Pro Asn 1170 1175 1180 Gly Ile Phe Lys Gly Ser Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val 1185 1190 1195 1200 Ala Pro Gln Ser Ser Glu Phe Ile Gly Ala 1205 1210 <210> 41 <211> 1186 <212> PRT <213> Homo sapiens <220> <223> Mature human EGFR/HER1 (minus the signal peptide) <220> <221> DOMAIN <222> (1)...(621) <223> Extracellular domain (ECD) <220> <221> DOMAIN <222> (1)...(165) <223> domain I of ECD <220> <221> DOMAIN <222> (166)...(313) <223> domain II of ECD <220> <221> DOMAIN <222> (314)... (481) <223> domain III of ECD <220> <221> DOMAIN <222> (482)...(621) <223> domain IV of ECD <400> 41 Leu Glu Glu Lys Lys Val Cys Gln Gly Thr Ser Asn Lys Leu Thr Gln 1 5 10 15 Leu Gly Thr Phe Glu Asp His Phe Leu Ser Leu Gln Arg Met Phe Asn 20 25 30 Asn Cys Glu Val Val Leu Gly Asn Leu Glu Ile Thr Tyr Val Gln Arg 35 40 45 Asn Tyr Asp Leu Ser Phe Leu Lys Thr Ile Gln Glu Val Ala Gly Tyr 50 55 60 Val Leu Ile Ala Leu Asn Thr Val Glu Arg Ile Pro Leu Glu Asn Leu 65 70 75 80 Gln Ile Ile Arg Gly Asn Met Tyr Tyr Glu Asn Ser Tyr Ala Leu Ala 85 90 95 Val Leu Ser Asn Tyr Asp Ala Asn Lys Thr Gly Leu Lys Glu Leu Pro 100 105 110 Met Arg Asn Leu Gln Glu Ile Leu His Gly Ala Val Arg Phe Ser Asn 115 120 125 Asn Pro Ala Leu Cys Asn Val Glu Ser Ile Gln Trp Arg Asp Ile Val 130 135 140 Ser Ser Asp Phe Leu Ser Asn Met Ser Met Asp Phe Gln Asn His Leu 145 150 155 160 Gly Ser Cys Gln Lys Cys Asp Pro Ser Cys Pro Asn Gly Ser Cys Trp 165 170 175 Gly Ala Gly Glu Glu Asn Cys Gln Lys Leu Thr Lys Ile Ile Cys Ala 180 185 190 Gln Gln Cys Ser Gly Arg Cys Arg Gly Lys Ser Pro Ser Asp Cys Cys 195 200 205 His Asn Gln Cys Ala Ala Gly Cys Thr Gly Pro Arg Glu Ser Asp Cys 210 215 220 Leu Val Cys Arg Lys Phe Arg Asp Glu Ala Thr Cys Lys Asp Thr Cys 225 230 235 240 Pro Pro Leu Met Leu Tyr Asn Pro Thr Thr Tyr Gln Met Asp Val Asn 245 250 255 Pro Glu Gly Lys Tyr Ser Phe Gly Ala Thr Cys Val Lys Lys Cys Pro 260 265 270 Arg Asn Tyr Val Val Thr Asp His Gly Ser Cys Val Arg Ala Cys Gly 275 280 285 Ala Asp Ser Tyr Glu Met Glu Glu Asp Gly Val Arg Lys Cys Lys Lys 290 295 300 Cys Glu Gly Pro Cys Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu 305 310 315 320 Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys 325 330 335 Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe 340 345 350 Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu 355 360 365 Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln 370 375 380 Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu 385 390 395 400 Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val 405 410 415 Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile 420 425 430 Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala 435 440 445 Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr 450 455 460 Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln 465 470 475 480 Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro 485 490 495 Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val 500 505 510 Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn 515 520 525 Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn 530 535 540 Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His 545 550 555 560 Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met 565 570 575 Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val 580 585 590 Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly 595 600 605 Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr 610 615 620 Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile 625 630 635 640 Gly Leu Phe Met Arg Arg Arg His Ile Val Arg Lys Arg Thr Leu Arg 645 650 655 Arg Leu Leu Gln Glu Arg Glu Leu Val Glu Pro Leu Thr Pro Ser Gly 660 665 670 Glu Ala Pro Asn Gln Ala Leu Leu Arg Ile Leu Lys Glu Thr Glu Phe 675 680 685 Lys Lys Ile Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys 690 695 700 Gly Leu Trp Ile Pro Glu Gly Glu Lys Val Lys Ile Pro Val Ala Ile 705 710 715 720 Lys Glu Leu Arg Glu Ala Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu 725 730 735 Asp Glu Ala Tyr Val Met Ala Ser Val Asp Asn Pro His Val Cys Arg 740 745 750 Leu Leu Gly Ile Cys Leu Thr Ser Thr Val Gln Leu Ile Thr Gln Leu 755 760 765 Met Pro Phe Gly Cys Leu Leu Asp Tyr Val Arg Glu His Lys Asp Asn 770 775 780 Ile Gly Ser Gln Tyr Leu Leu Asn Trp Cys Val Gln Ile Ala Lys Gly 785 790 795 800 Met Asn Tyr Leu Glu Asp Arg Arg Leu Val His Arg Asp Leu Ala Ala 805 810 815 Arg Asn Val Leu Val Lys Thr Pro Gln His Val Lys Ile Thr Asp Phe 820 825 830 Gly Leu Ala Lys Leu Leu Gly Ala Glu Glu Lys Glu Tyr His Ala Glu 835 840 845 Gly Gly Lys Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu His 850 855 860 Arg Ile Tyr Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val 865 870 875 880 Trp Glu Leu Met Thr Phe Gly Ser Lys Pro Tyr Asp Gly Ile Pro Ala 885 890 895 Ser Glu Ile Ser Ser Ile Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro 900 905 910 Pro Ile Cys Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met 915 920 925 Ile Asp Ala Asp Ser Arg Pro Lys Phe Arg Glu Leu Ile Ile Glu Phe 930 935 940 Ser Lys Met Ala Arg Asp Pro Gln Arg Tyr Leu Val Ile Gln Gly Asp 945 950 955 960 Glu Arg Met His Leu Pro Ser Pro Thr Asp Ser Asn Phe Tyr Arg Ala 965 970 975 Leu Met Asp Glu Glu Asp Met Asp Asp Val Val Asp Ala Asp Glu Tyr 980 985 990 Leu Ile Pro Gln Gln Gly Phe Phe Ser Ser Pro Ser Thr Ser Arg Thr 995 1000 1005 Pro Leu Leu Ser Ser Leu Ser Ala Thr Ser Asn Asn Ser Thr Val Ala 1010 1015 1020 Cys Ile Asp Arg Asn Gly Leu Gln Ser Cys Pro Ile Lys Glu Asp Ser 1025 1030 1035 1040 Phe Leu Gln Arg Tyr Ser Ser Asp Pro Thr Gly Ala Leu Thr Glu Asp 1045 1050 1055 Ser Ile Asp Asp Thr Phe Leu Pro Val Pro Glu Tyr Ile Asn Gln Ser 1060 1065 1070 Val Pro Lys Arg Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn 1075 1080 1085 Gln Pro Leu Asn Pro Ala Pro Ser Arg Asp Pro His Tyr Gln Asp Pro 1090 1095 1100 His Ser Thr Ala Val Gly Asn Pro Glu Tyr Leu Asn Thr Val Gln Pro 1105 1110 1115 1120 Thr Cys Val Asn Ser Thr Phe Asp Ser Pro Ala His Trp Ala Gln Lys 1125 1130 1135 Gly Ser His Gln Ile Ser Leu Asp Asn Pro Asp Tyr Gln Gln Asp Phe 1140 1145 1150 Phe Pro Lys Glu Ala Lys Pro Asn Gly Ile Phe Lys Gly Ser Thr Ala 1155 1160 1165 Glu Asn Ala Glu Tyr Leu Arg Val Ala Pro Gln Ser Ser Glu Phe Ile 1170 1175 1180 Gly Ala 1185 <210> 42 <211> 1255 <212> PRT <213> Homo sapiens <220> <223> Human HER2 (ErbB2) precursor <220> <221> SIGNAL <222> (1)...(22) <223> signal peptide <220> <221> DOMAIN <222> (23)...(652) <223> extracellular domain (ECD) <220> <221> DOMAIN <222> (653)...(675) <223> transmembrane region <220> <221> DOMAIN <222> (676)...(1255) <223> cytoplasmic domain <220> <221> DOMAIN <222> (720)...(987) <223> protein kinase <400> 42 Met Glu Leu Ala Ala Leu Cys Arg Trp Gly Leu Leu Leu Ala Leu Leu 1 5 10 15 Pro Pro Gly Ala Ala Ser Thr Gln Val Cys Thr Gly Thr Asp Met Lys 20 25 30 Leu Arg Leu Pro Ala Ser Pro Glu Thr His Leu Asp Met Leu Arg His 35 40 45 Leu Tyr Gln Gly Cys Gln Val Val Gln Gly Asn Leu Glu Leu Thr Tyr 50 55 60 Leu Pro Thr Asn Ala Ser Leu Ser Phe Leu Gln Asp Ile Gln Glu Val 65 70 75 80 Gln Gly Tyr Val Leu Ile Ala His Asn Gln Val Arg Gln Val Pro Leu 85 90 95 Gln Arg Leu Arg Ile Val Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr 100 105 110 Ala Leu Ala Val Leu Asp Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro 115 120 125 Val Thr Gly Ala Ser Pro Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser 130 135 140 Leu Thr Glu Ile Leu Lys Gly Gly Val Leu Ile Gln Arg Asn Pro Gln 145 150 155 160 Leu Cys Tyr Gln Asp Thr Ile Leu Trp Lys Asp Ile Phe His Lys Asn 165 170 175 Asn Gln Leu Ala Leu Thr Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys 180 185 190 His Pro Cys Ser Pro Met Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser 195 200 205 Ser Glu Asp Cys Gln Ser Leu Thr Arg Thr Val Cys Ala Gly Gly Cys 210 215 220 Ala Arg Cys Lys Gly Pro Leu Pro Thr Asp Cys Cys His Glu Gln Cys 225 230 235 240 Ala Ala Gly Cys Thr Gly Pro Lys His Ser Asp Cys Leu Ala Cys Leu 245 250 255 His Phe Asn His Ser Gly Ile Cys Glu Leu His Cys Pro Ala Leu Val 260 265 270 Thr Tyr Asn Thr Asp Thr Phe Glu Ser Met Pro Asn Pro Glu Gly Arg 275 280 285 Tyr Thr Phe Gly Ala Ser Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu 290 295 300 Ser Thr Asp Val Gly Ser Cys Thr Leu Val Cys Pro Leu His Asn Gln 305 310 315 320 Glu Val Thr Ala Glu Asp Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys 325 330 335 Pro Cys Ala Arg Val Cys Tyr Gly Leu Gly Met Glu His Leu Arg Glu 340 345 350 Val Arg Ala Val Thr Ser Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys 355 360 365 Lys Ile Phe Gly Ser Leu Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp 370 375 380 Pro Ala Ser Asn Thr Ala Pro Leu Gln Pro Glu Gln Leu Gln Val Phe 385 390 395 400 Glu Thr Leu Glu Glu Ile Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro 405 410 415 Asp Ser Leu Pro Asp Leu Ser Val Phe Gln Asn Leu Gln Val Ile Arg 420 425 430 Gly Arg Ile Leu His Asn Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu 435 440 445 Gly Ile Ser Trp Leu Gly Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly 450 455 460 Leu Ala Leu Ile His His Asn Thr His Leu Cys Phe Val His Thr Val 465 470 475 480 Pro Trp Asp Gln Leu Phe Arg Asn Pro His Gln Ala Leu Leu His Thr 485 490 495 Ala Asn Arg Pro Glu Asp Glu Cys Val Gly Glu Gly Leu Ala Cys His 500 505 510 Gln Leu Cys Ala Arg Gly His Cys Trp Gly Pro Gly Pro Thr Gln Cys 515 520 525 Val Asn Cys Ser Gln Phe Leu Arg Gly Gln Glu Cys Val Glu Glu Cys 530 535 540 Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn Ala Arg His Cys 545 550 555 560 Leu Pro Cys His Pro Glu Cys Gln Pro Gln Asn Gly Ser Val Thr Cys 565 570 575 Phe Gly Pro Glu Ala Asp Gln Cys Val Ala Cys Ala His Tyr Lys Asp 580 585 590 Pro Pro Phe Cys Val Ala Arg Cys Pro Ser Gly Val Lys Pro Asp Leu 595 600 605 Ser Tyr Met Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln 610 615 620 Pro Cys Pro Ile Asn Cys Thr His Ser Cys Val Asp Leu Asp Asp Lys 625 630 635 640 Gly Cys Pro Ala Glu Gln Arg Ala Ser Pro Leu Thr Ser Ile Ile Ser 645 650 655 Ala Val Val Gly Ile Leu Leu Val Val Val Leu Gly Val Val Phe Gly 660 665 670 Ile Leu Ile Lys Arg Arg Gln Gln Lys Ile Arg Lys Tyr Thr Met Arg 675 680 685 Arg Leu Leu Gln Glu Thr Glu Leu Val Glu Pro Leu Thr Pro Ser Gly 690 695 700 Ala Met Pro Asn Gln Ala Gln Met Arg Ile Leu Lys Glu Thr Glu Leu 705 710 715 720 Arg Lys Val Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys 725 730 735 Gly Ile Trp Ile Pro Asp Gly Glu Asn Val Lys Ile Pro Val Ala Ile 740 745 750 Lys Val Leu Arg Glu Asn Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu 755 760 765 Asp Glu Ala Tyr Val Met Ala Gly Val Gly Ser Pro Tyr Val Ser Arg 770 775 780 Leu Leu Gly Ile Cys Leu Thr Ser Thr Val Gln Leu Val Thr Gln Leu 785 790 795 800 Met Pro Tyr Gly Cys Leu Leu Asp His Val Arg Glu Asn Arg Gly Arg 805 810 815 Leu Gly Ser Gln Asp Leu Leu Asn Trp Cys Met Gln Ile Ala Lys Gly 820 825 830 Met Ser Tyr Leu Glu Asp Val Arg Leu Val His Arg Asp Leu Ala Ala 835 840 845 Arg Asn Val Leu Val Lys Ser Pro Asn His Val Lys Ile Thr Asp Phe 850 855 860 Gly Leu Ala Arg Leu Leu Asp Ile Asp Glu Thr Glu Tyr His Ala Asp 865 870 875 880 Gly Gly Lys Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu Arg 885 890 895 Arg Arg Phe Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val 900 905 910 Trp Glu Leu Met Thr Phe Gly Ala Lys Pro Tyr Asp Gly Ile Pro Ala 915 920 925 Arg Glu Ile Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro 930 935 940 Pro Ile Cys Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met 945 950 955 960 Ile Asp Ser Glu Cys Arg Pro Arg Phe Arg Glu Leu Val Ser Glu Phe 965 970 975 Ser Arg Met Ala Arg Asp Pro Gln Arg Phe Val Val Ile Gln Asn Glu 980 985 990 Asp Leu Gly Pro Ala Ser Pro Leu Asp Ser Thr Phe Tyr Arg Ser Leu 995 1000 1005 Leu Glu Asp Asp Asp Met Gly Asp Leu Val Asp Ala Glu Glu Tyr Leu 1010 1015 1020 Val Pro Gln Gln Gly Phe Phe Cys Pro Asp Pro Ala Pro Gly Ala Gly 1025 1030 1035 1040 Gly Met Val His His Arg His Arg Ser Ser Ser Thr Arg Ser Gly Gly 1045 1050 1055 Gly Asp Leu Thr Leu Gly Leu Glu Pro Ser Glu Glu Glu Ala Pro Arg 1060 1065 1070 Ser Pro Leu Ala Pro Ser Glu Gly Ala Gly Ser Asp Val Phe Asp Gly 1075 1080 1085 Asp Leu Gly Met Gly Ala Ala Lys Gly Leu Gln Ser Leu Pro Thr His 1090 1095 1100 Asp Pro Ser Pro Leu Gln Arg Tyr Ser Glu Asp Pro Thr Val Pro Leu 1105 1110 1115 1120 Pro Ser Glu Thr Asp Gly Tyr Val Ala Pro Leu Thr Cys Ser Pro Gln 1125 1130 1135 Pro Glu Tyr Val Asn Gln Pro Asp Val Arg Pro Gln Pro Pro Ser Pro 1140 1145 1150 Arg Glu Gly Pro Leu Pro Ala Ala Arg Pro Ala Gly Ala Thr Leu Glu 1155 1160 1165 Arg Pro Lys Thr Leu Ser Pro Gly Lys Asn Gly Val Val Lys Asp Val 1170 1175 1180 Phe Ala Phe Gly Gly Ala Val Glu Asn Pro Glu Tyr Leu Thr Pro Gln 1185 1190 1195 1200 Gly Gly Ala Ala Pro Gln Pro His Pro Pro Pro Ala Phe Ser Pro Ala 1205 1210 1215 Phe Asp Asn Leu Tyr Tyr Trp Asp Gln Asp Pro Pro Glu Arg Gly Ala 1220 1225 1230 Pro Pro Ser Thr Phe Lys Gly Thr Pro Thr Ala Glu Asn Pro Glu Tyr 1235 1240 1245 Leu Gly Leu Asp Val Pro Val 1250 1255 <210> 43 <211> 1233 <212> PRT <213> Homo sapiens <220> <223> Mature human HER2 (ErbB2) Protein (minus the signal peptide) <220> <221> DOMAIN <222> (1)...(628) <223> Extracellular domain (ECD) <220> <221> DOMAIN <222> (1)...(172) <223> domain I of ECD <220> <221> DOMAIN <222> (173)...(319) <223> domain II of ECD <220> <221> DOMAIN <222> (320)... (488) <223> domain III of ECD <220> <221> DOMAIN <222> (489)... (628) <223> domain IV of ECD <400> 43 Thr Gln Val Cys Thr Gly Thr Asp Met Lys Leu Arg Leu Pro Ala Ser 1 5 10 15 Pro Glu Thr His Leu Asp Met Leu Arg His Leu Tyr Gln Gly Cys Gln 20 25 30 Val Val Gln Gly Asn Leu Glu Leu Thr Tyr Leu Pro Thr Asn Ala Ser 35 40 45 Leu Ser Phe Leu Gln Asp Ile Gln Glu Val Gln Gly Tyr Val Leu Ile 50 55 60 Ala His Asn Gln Val Arg Gln Val Pro Leu Gln Arg Leu Arg Ile Val 65 70 75 80 Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr Ala Leu Ala Val Leu Asp 85 90 95 Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro Val Thr Gly Ala Ser Pro 100 105 110 Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser Leu Thr Glu Ile Leu Lys 115 120 125 Gly Gly Val Leu Ile Gln Arg Asn Pro Gln Leu Cys Tyr Gln Asp Thr 130 135 140 Ile Leu Trp Lys Asp Ile Phe His Lys Asn Asn Gln Leu Ala Leu Thr 145 150 155 160 Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys His Pro Cys Ser Pro Met 165 170 175 Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser Ser Glu Asp Cys Gln Ser 180 185 190 Leu Thr Arg Thr Val Cys Ala Gly Gly Cys Ala Arg Cys Lys Gly Pro 195 200 205 Leu Pro Thr Asp Cys Cys His Glu Gln Cys Ala Ala Gly Cys Thr Gly 210 215 220 Pro Lys His Ser Asp Cys Leu Ala Cys Leu His Phe Asn His Ser Gly 225 230 235 240 Ile Cys Glu Leu His Cys Pro Ala Leu Val Thr Tyr Asn Thr Asp Thr 245 250 255 Phe Glu Ser Met Pro Asn Pro Glu Gly Arg Tyr Thr Phe Gly Ala Ser 260 265 270 Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu Ser Thr Asp Val Gly Ser 275 280 285 Cys Thr Leu Val Cys Pro Leu His Asn Gln Glu Val Thr Ala Glu Asp 290 295 300 Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys Pro Cys Ala Arg Val Cys 305 310 315 320 Tyr Gly Leu Gly Met Glu His Leu Arg Glu Val Arg Ala Val Thr Ser 325 330 335 Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys Lys Ile Phe Gly Ser Leu 340 345 350 Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp Pro Ala Ser Asn Thr Ala 355 360 365 Pro Leu Gln Pro Glu Gln Leu Gln Val Phe Glu Thr Leu Glu Glu Ile 370 375 380 Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro Asp Ser Leu Pro Asp Leu 385 390 395 400 Ser Val Phe Gln Asn Leu Gln Val Ile Arg Gly Arg Ile Leu His Asn 405 410 415 Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu Gly Ile Ser Trp Leu Gly 420 425 430 Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly Leu Ala Leu Ile His His 435 440 445 Asn Thr His Leu Cys Phe Val His Thr Val Pro Trp Asp Gln Leu Phe 450 455 460 Arg Asn Pro His Gln Ala Leu Leu His Thr Ala Asn Arg Pro Glu Asp 465 470 475 480 Glu Cys Val Gly Glu Gly Leu Ala Cys His Gln Leu Cys Ala Arg Gly 485 490 495 His Cys Trp Gly Pro Gly Pro Thr Gln Cys Val Asn Cys Ser Gln Phe 500 505 510 Leu Arg Gly Gln Glu Cys Val Glu Glu Cys Arg Val Leu Gln Gly Leu 515 520 525 Pro Arg Glu Tyr Val Asn Ala Arg His Cys Leu Pro Cys His Pro Glu 530 535 540 Cys Gln Pro Gln Asn Gly Ser Val Thr Cys Phe Gly Pro Glu Ala Asp 545 550 555 560 Gln Cys Val Ala Cys Ala His Tyr Lys Asp Pro Pro Phe Cys Val Ala 565 570 575 Arg Cys Pro Ser Gly Val Lys Pro Asp Leu Ser Tyr Met Pro Ile Trp 580 585 590 Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln Pro Cys Pro Ile Asn Cys 595 600 605 Thr His Ser Cys Val Asp Leu Asp Asp Lys Gly Cys Pro Ala Glu Gln 610 615 620 Arg Ala Ser Pro Leu Thr Ser Ile Ile Ser Ala Val Val Gly Ile Leu 625 630 635 640 Leu Val Val Val Leu Gly Val Val Phe Gly Ile Leu Ile Lys Arg Arg 645 650 655 Gln Gln Lys Ile Arg Lys Tyr Thr Met Arg Arg Leu Leu Gln Glu Thr 660 665 670 Glu Leu Val Glu Pro Leu Thr Pro Ser Gly Ala Met Pro Asn Gln Ala 675 680 685 Gln Met Arg Ile Leu Lys Glu Thr Glu Leu Arg Lys Val Lys Val Leu 690 695 700 Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys Gly Ile Trp Ile Pro Asp 705 710 715 720 Gly Glu Asn Val Lys Ile Pro Val Ala Ile Lys Val Leu Arg Glu Asn 725 730 735 Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met 740 745 750 Ala Gly Val Gly Ser Pro Tyr Val Ser Arg Leu Leu Gly Ile Cys Leu 755 760 765 Thr Ser Thr Val Gln Leu Val Thr Gln Leu Met Pro Tyr Gly Cys Leu 770 775 780 Leu Asp His Val Arg Glu Asn Arg Gly Arg Leu Gly Ser Gln Asp Leu 785 790 795 800 Leu Asn Trp Cys Met Gln Ile Ala Lys Gly Met Ser Tyr Leu Glu Asp 805 810 815 Val Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys 820 825 830 Ser Pro Asn His Val Lys Ile Thr Asp Phe Gly Leu Ala Arg Leu Leu 835 840 845 Asp Ile Asp Glu Thr Glu Tyr His Ala Asp Gly Gly Lys Val Pro Ile 850 855 860 Lys Trp Met Ala Leu Glu Ser Ile Leu Arg Arg Arg Phe Thr His Gln 865 870 875 880 Ser Asp Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe 885 890 895 Gly Ala Lys Pro Tyr Asp Gly Ile Pro Ala Arg Glu Ile Pro Asp Leu 900 905 910 Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp 915 920 925 Val Tyr Met Ile Met Val Lys Cys Trp Met Ile Asp Ser Glu Cys Arg 930 935 940 Pro Arg Phe Arg Glu Leu Val Ser Glu Phe Ser Arg Met Ala Arg Asp 945 950 955 960 Pro Gln Arg Phe Val Val Ile Gln Asn Glu Asp Leu Gly Pro Ala Ser 965 970 975 Pro Leu Asp Ser Thr Phe Tyr Arg Ser Leu Leu Glu Asp Asp Asp Met 980 985 990 Gly Asp Leu Val Asp Ala Glu Glu Tyr Leu Val Pro Gln Gln Gly Phe 995 1000 1005 Phe Cys Pro Asp Pro Ala Pro Gly Ala Gly Gly Met Val His His Arg 1010 1015 1020 His Arg Ser Ser Ser Thr Arg Ser Gly Gly Gly Asp Leu Thr Leu Gly 1025 1030 1035 1040 Leu Glu Pro Ser Glu Glu Glu Ala Pro Arg Ser Pro Leu Ala Pro Ser 1045 1050 1055 Glu Gly Ala Gly Ser Asp Val Phe Asp Gly Asp Leu Gly Met Gly Ala 1060 1065 1070 Ala Lys Gly Leu Gln Ser Leu Pro Thr His Asp Pro Ser Pro Leu Gln 1075 1080 1085 Arg Tyr Ser Glu Asp Pro Thr Val Pro Leu Pro Ser Glu Thr Asp Gly 1090 1095 1100 Tyr Val Ala Pro Leu Thr Cys Ser Pro Gln Pro Glu Tyr Val Asn Gln 1105 1110 1115 1120 Pro Asp Val Arg Pro Gln Pro Pro Ser Pro Arg Glu Gly Pro Leu Pro 1125 1130 1135 Ala Ala Arg Pro Ala Gly Ala Thr Leu Glu Arg Pro Lys Thr Leu Ser 1140 1145 1150 Pro Gly Lys Asn Gly Val Val Lys Asp Val Phe Ala Phe Gly Gly Ala 1155 1160 1165 Val Glu Asn Pro Glu Tyr Leu Thr Pro Gln Gly Gly Ala Ala Pro Gln 1170 1175 1180 Pro His Pro Pro Pro Ala Phe Ser Pro Ala Phe Asp Asn Leu Tyr Tyr 1185 1190 1195 1200 Trp Asp Gln Asp Pro Pro Glu Arg Gly Ala Pro Pro Ser Thr Phe Lys 1205 1210 1215 Gly Thr Pro Thr Ala Glu Asn Pro Glu Tyr Leu Gly Leu Asp Val Pro 1220 1225 1230 Val <210> 44 <211> 1342 <212> PRT <213> Homo sapiens <220> <223> Human HER3 (ErbB3) precursor <220> <221> SIGNAL <222> (1)...(19) <223> signal peptide <220> <221> DOMAIN <222> (20)...(643) <223> Extracellular domain (ECD) <220> <221> DOMAIN <222> (644)...(664) <223> transmembrane region <220> <221> DOMAIN <222> (665)...(1342) <223> cytoplasmic domain <220> <221> DOMAIN <222> (709)...(966) <223> protein kinase <400> 44 Met Arg Ala Asn Asp Ala Leu Gln Val Leu Gly Leu Leu Phe Ser Leu 1 5 10 15 Ala Arg Gly Ser Glu Val Gly Asn Ser Gln Ala Val Cys Pro Gly Thr 20 25 30 Leu Asn Gly Leu Ser Val Thr Gly Asp Ala Glu Asn Gln Tyr Gln Thr 35 40 45 Leu Tyr Lys Leu Tyr Glu Arg Cys Glu Val Val Met Gly Asn Leu Glu 50 55 60 Ile Val Leu Thr Gly His Asn Ala Asp Leu Ser Phe Leu Gln Trp Ile 65 70 75 80 Arg Glu Val Thr Gly Tyr Val Leu Val Ala Met Asn Glu Phe Ser Thr 85 90 95 Leu Pro Leu Pro Asn Leu Arg Val Val Arg Gly Thr Gln Val Tyr Asp 100 105 110 Gly Lys Phe Ala Ile Phe Val Met Leu Asn Tyr Asn Thr Asn Ser Ser 115 120 125 His Ala Leu Arg Gln Leu Arg Leu Thr Gln Leu Thr Glu Ile Leu Ser 130 135 140 Gly Gly Val Tyr Ile Glu Lys Asn Asp Lys Leu Cys His Met Asp Thr 145 150 155 160 Ile Asp Trp Arg Asp Ile Val Arg Asp Arg Asp Ala Glu Ile Val Val 165 170 175 Lys Asp Asn Gly Arg Ser Cys Pro Pro Cys His Glu Val Cys Lys Gly 180 185 190 Arg Cys Trp Gly Pro Gly Ser Glu Asp Cys Gln Thr Leu Thr Lys Thr 195 200 205 Ile Cys Ala Pro Gln Cys Asn Gly His Cys Phe Gly Pro Asn Pro Asn 210 215 220 Gln Cys Cys His Asp Glu Cys Ala Gly Gly Cys Ser Gly Pro Gln Asp 225 230 235 240 Thr Asp Cys Phe Ala Cys Arg His Phe Asn Asp Ser Gly Ala Cys Val 245 250 255 Pro Arg Cys Pro Gln Pro Leu Val Tyr Asn Lys Leu Thr Phe Gln Leu 260 265 270 Glu Pro Asn Pro His Thr Lys Tyr Gln Tyr Gly Gly Val Cys Val Ala 275 280 285 Ser Cys Pro His Asn Phe Val Val Asp Gln Thr Ser Cys Val Arg Ala 290 295 300 Cys Pro Pro Asp Lys Met Glu Val Asp Lys Asn Gly Leu Lys Met Cys 305 310 315 320 Glu Pro Cys Gly Gly Leu Cys Pro Lys Ala Cys Glu Gly Thr Gly Ser 325 330 335 Gly Ser Arg Phe Gln Thr Val Asp Ser Ser Asn Ile Asp Gly Phe Val 340 345 350 Asn Cys Thr Lys Ile Leu Gly Asn Leu Asp Phe Leu Ile Thr Gly Leu 355 360 365 Asn Gly Asp Pro Trp His Lys Ile Pro Ala Leu Asp Pro Glu Lys Leu 370 375 380 Asn Val Phe Arg Thr Val Arg Glu Ile Thr Gly Tyr Leu Asn Ile Gln 385 390 395 400 Ser Trp Pro Pro His Met His Asn Phe Ser Val Phe Ser Asn Leu Thr 405 410 415 Thr Ile Gly Gly Arg Ser Leu Tyr Asn Arg Gly Phe Ser Leu Leu Ile 420 425 430 Met Lys Asn Leu Asn Val Thr Ser Leu Gly Phe Arg Ser Leu Lys Glu 435 440 445 Ile Ser Ala Gly Arg Ile Tyr Ile Ser Ala Asn Arg Gln Leu Cys Tyr 450 455 460 His His Ser Leu Asn Trp Thr Lys Val Leu Arg Gly Pro Thr Glu Glu 465 470 475 480 Arg Leu Asp Ile Lys His Asn Arg Pro Arg Arg Asp Cys Val Ala Glu 485 490 495 Gly Lys Val Cys Asp Pro Leu Cys Ser Ser Gly Gly Cys Trp Gly Pro 500 505 510 Gly Pro Gly Gln Cys Leu Ser Cys Arg Asn Tyr Ser Arg Gly Gly Val 515 520 525 Cys Val Thr His Cys Asn Phe Leu Asn Gly Glu Pro Arg Glu Phe Ala 530 535 540 His Glu Ala Glu Cys Phe Ser Cys His Pro Glu Cys Gln Pro Met Glu 545 550 555 560 Gly Thr Ala Thr Cys Asn Gly Ser Gly Ser Asp Thr Cys Ala Gln Cys 565 570 575 Ala His Phe Arg Asp Gly Pro His Cys Val Ser Ser Cys Pro His Gly 580 585 590 Val Leu Gly Ala Lys Gly Pro Ile Tyr Lys Tyr Pro Asp Val Gln Asn 595 600 605 Glu Cys Arg Pro Cys His Glu Asn Cys Thr Gln Gly Cys Lys Gly Pro 610 615 620 Glu Leu Gln Asp Cys Leu Gly Gln Thr Leu Val Leu Ile Gly Lys Thr 625 630 635 640 His Leu Thr Met Ala Leu Thr Val Ile Ala Gly Leu Val Val Ile Phe 645 650 655 Met Met Leu Gly Gly Thr Phe Leu Tyr Trp Arg Gly Arg Arg Ile Gln 660 665 670 Asn Lys Arg Ala Met Arg Arg Tyr Leu Glu Arg Gly Glu Ser Ile Glu 675 680 685 Pro Leu Asp Pro Ser Glu Lys Ala Asn Lys Val Leu Ala Arg Ile Phe 690 695 700 Lys Glu Thr Glu Leu Arg Lys Leu Lys Val Leu Gly Ser Gly Val Phe 705 710 715 720 Gly Thr Val His Lys Gly Val Trp Ile Pro Glu Gly Glu Ser Ile Lys 725 730 735 Ile Pro Val Cys Ile Lys Val Ile Glu Asp Lys Ser Gly Arg Gln Ser 740 745 750 Phe Gln Ala Val Thr Asp His Met Leu Ala Ile Gly Ser Leu Asp His 755 760 765 Ala His Ile Val Arg Leu Leu Gly Leu Cys Pro Gly Ser Ser Leu Gln 770 775 780 Leu Val Thr Gln Tyr Leu Pro Leu Gly Ser Leu Leu Asp His Val Arg 785 790 795 800 Gln His Arg Gly Ala Leu Gly Pro Gln Leu Leu Leu Asn Trp Gly Val 805 810 815 Gln Ile Ala Lys Gly Met Tyr Tyr Leu Glu Glu His Gly Met Val His 820 825 830 Arg Asn Leu Ala Ala Arg Asn Val Leu Leu Lys Ser Pro Ser Gln Val 835 840 845 Gln Val Ala Asp Phe Gly Val Ala Asp Leu Leu Pro Pro Asp Asp Lys 850 855 860 Gln Leu Leu Tyr Ser Glu Ala Lys Thr Pro Ile Lys Trp Met Ala Leu 865 870 875 880 Glu Ser Ile His Phe Gly Lys Tyr Thr His Gln Ser Asp Val Trp Ser 885 890 895 Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ala Glu Pro Tyr 900 905 910 Ala Gly Leu Arg Leu Ala Glu Val Pro Asp Leu Leu Glu Lys Gly Glu 915 920 925 Arg Leu Ala Gln Pro Gln Ile Cys Thr Ile Asp Val Tyr Met Val Met 930 935 940 Val Lys Cys Trp Met Ile Asp Glu Asn Ile Arg Pro Thr Phe Lys Glu 945 950 955 960 Leu Ala Asn Glu Phe Thr Arg Met Ala Arg Asp Pro Pro Arg Tyr Leu 965 970 975 Val Ile Lys Arg Glu Ser Gly Pro Gly Ile Ala Pro Gly Pro Glu Pro 980 985 990 His Gly Leu Thr Asn Lys Lys Leu Glu Glu Val Glu Leu Glu Pro Glu 995 1000 1005 Leu Asp Leu Asp Leu Asp Leu Glu Ala Glu Glu Asp Asn Leu Ala Thr 1010 1015 1020 Thr Thr Leu Gly Ser Ala Leu Ser Leu Pro Val Gly Thr Leu Asn Arg 1025 1030 1035 1040 Pro Arg Gly Ser Gln Ser Leu Leu Ser Pro Ser Ser Gly Tyr Met Pro 1045 1050 1055 Met Asn Gln Gly Asn Leu Gly Glu Ser Cys Gln Glu Ser Ala Val Ser 1060 1065 1070 Gly Ser Ser Glu Arg Cys Pro Arg Pro Val Ser Leu His Pro Met Pro 1075 1080 1085 Arg Gly Cys Leu Ala Ser Glu Ser Ser Glu Gly His Val Thr Gly Ser 1090 1095 1100 Glu Ala Glu Leu Gln Glu Lys Val Ser Met Cys Arg Ser Arg Ser Arg 1105 1110 1115 1120 Ser Arg Ser Pro Arg Pro Arg Gly Asp Ser Ala Tyr His Ser Gln Arg 1125 1130 1135 His Ser Leu Leu Thr Pro Val Thr Pro Leu Ser Pro Pro Gly Leu Glu 1140 1145 1150 Glu Glu Asp Val Asn Gly Tyr Val Met Pro Asp Thr His Leu Lys Gly 1155 1160 1165 Thr Pro Ser Ser Arg Glu Gly Thr Leu Ser Ser Val Gly Leu Ser Ser 1170 1175 1180 Val Leu Gly Thr Glu Glu Glu Asp Glu Asp Glu Glu Tyr Glu Tyr Met 1185 1190 1195 1200 Asn Arg Arg Arg Arg His Ser Pro Pro His Pro Pro Arg Pro Ser Ser 1205 1210 1215 Leu Glu Glu Leu Gly Tyr Glu Tyr Met Asp Val Gly Ser Asp Leu Ser 1220 1225 1230 Ala Ser Leu Gly Ser Thr Gln Ser Cys Pro Leu His Pro Val Pro Ile 1235 1240 1245 Met Pro Thr Ala Gly Thr Thr Pro Asp Glu Asp Tyr Glu Tyr Met Asn 1250 1255 1260 Arg Gln Arg Asp Gly Gly Gly Pro Gly Gly Asp Tyr Ala Ala Met Gly 1265 1270 1275 1280 Ala Cys Pro Ala Ser Glu Gln Gly Tyr Glu Glu Met Arg Ala Phe Gln 1285 1290 1295 Gly Pro Gly His Gln Ala Pro His Val His Tyr Ala Arg Leu Lys Thr 1300 1305 1310 Leu Arg Ser Leu Glu Ala Thr Asp Ser Ala Phe Asp Asn Pro Asp Tyr 1315 1320 1325 Trp His Ser Arg Leu Phe Pro Lys Ala Asn Ala Gln Arg Thr 1330 1335 1340 <210> 45 <211> 1323 <212> PRT <213> Homo sapiens <220> <223> Mature human HER3 (ErbB3) protein (minus the signal peptide) <220> <221> DOMAIN <222> (1)...(621) <223> Extracellular domain (ECD) <220> <221> DOMAIN <222> (1)...(166) <223> domain I of ECD <220> <221> DOMAIN <222> (167)...(311) <223> domain II of ECD <220> <221> DOMAIN <222> (312)... (480) <223> domain III of ECD <220> <221> DOMAIN <222> (481)...(621) <223> domain IV of ECD <400> 45 Ser Glu Val Gly Asn Ser Gln Ala Val Cys Pro Gly Thr Leu Asn Gly 1 5 10 15 Leu Ser Val Thr Gly Asp Ala Glu Asn Gln Tyr Gln Thr Leu Tyr Lys 20 25 30 Leu Tyr Glu Arg Cys Glu Val Val Met Gly Asn Leu Glu Ile Val Leu 35 40 45 Thr Gly His Asn Ala Asp Leu Ser Phe Leu Gln Trp Ile Arg Glu Val 50 55 60 Thr Gly Tyr Val Leu Val Ala Met Asn Glu Phe Ser Thr Leu Pro Leu 65 70 75 80 Pro Asn Leu Arg Val Val Arg Gly Thr Gln Val Tyr Asp Gly Lys Phe 85 90 95 Ala Ile Phe Val Met Leu Asn Tyr Asn Thr Asn Ser Ser His Ala Leu 100 105 110 Arg Gln Leu Arg Leu Thr Gln Leu Thr Glu Ile Leu Ser Gly Gly Val 115 120 125 Tyr Ile Glu Lys Asn Asp Lys Leu Cys His Met Asp Thr Ile Asp Trp 130 135 140 Arg Asp Ile Val Arg Asp Arg Asp Ala Glu Ile Val Val Lys Asp Asn 145 150 155 160 Gly Arg Ser Cys Pro Pro Cys His Glu Val Cys Lys Gly Arg Cys Trp 165 170 175 Gly Pro Gly Ser Glu Asp Cys Gln Thr Leu Thr Lys Thr Ile Cys Ala 180 185 190 Pro Gln Cys Asn Gly His Cys Phe Gly Pro Asn Pro Asn Gln Cys Cys 195 200 205 His Asp Glu Cys Ala Gly Gly Cys Ser Gly Pro Gln Asp Thr Asp Cys 210 215 220 Phe Ala Cys Arg His Phe Asn Asp Ser Gly Ala Cys Val Pro Arg Cys 225 230 235 240 Pro Gln Pro Leu Val Tyr Asn Lys Leu Thr Phe Gln Leu Glu Pro Asn 245 250 255 Pro His Thr Lys Tyr Gln Tyr Gly Gly Val Cys Val Ala Ser Cys Pro 260 265 270 His Asn Phe Val Val Asp Gln Thr Ser Cys Val Arg Ala Cys Pro Pro 275 280 285 Asp Lys Met Glu Val Asp Lys Asn Gly Leu Lys Met Cys Glu Pro Cys 290 295 300 Gly Gly Leu Cys Pro Lys Ala Cys Glu Gly Thr Gly Ser Gly Ser Arg 305 310 315 320 Phe Gln Thr Val Asp Ser Ser Asn Ile Asp Gly Phe Val Asn Cys Thr 325 330 335 Lys Ile Leu Gly Asn Leu Asp Phe Leu Ile Thr Gly Leu Asn Gly Asp 340 345 350 Pro Trp His Lys Ile Pro Ala Leu Asp Pro Glu Lys Leu Asn Val Phe 355 360 365 Arg Thr Val Arg Glu Ile Thr Gly Tyr Leu Asn Ile Gln Ser Trp Pro 370 375 380 Pro His Met His Asn Phe Ser Val Phe Ser Asn Leu Thr Thr Ile Gly 385 390 395 400 Gly Arg Ser Leu Tyr Asn Arg Gly Phe Ser Leu Leu Ile Met Lys Asn 405 410 415 Leu Asn Val Thr Ser Leu Gly Phe Arg Ser Leu Lys Glu Ile Ser Ala 420 425 430 Gly Arg Ile Tyr Ile Ser Ala Asn Arg Gln Leu Cys Tyr His His Ser 435 440 445 Leu Asn Trp Thr Lys Val Leu Arg Gly Pro Thr Glu Glu Arg Leu Asp 450 455 460 Ile Lys His Asn Arg Pro Arg Arg Asp Cys Val Ala Glu Gly Lys Val 465 470 475 480 Cys Asp Pro Leu Cys Ser Ser Gly Gly Cys Trp Gly Pro Gly Pro Gly 485 490 495 Gln Cys Leu Ser Cys Arg Asn Tyr Ser Arg Gly Gly Val Cys Val Thr 500 505 510 His Cys Asn Phe Leu Asn Gly Glu Pro Arg Glu Phe Ala His Glu Ala 515 520 525 Glu Cys Phe Ser Cys His Pro Glu Cys Gln Pro Met Glu Gly Thr Ala 530 535 540 Thr Cys Asn Gly Ser Gly Ser Asp Thr Cys Ala Gln Cys Ala His Phe 545 550 555 560 Arg Asp Gly Pro His Cys Val Ser Ser Cys Pro His Gly Val Leu Gly 565 570 575 Ala Lys Gly Pro Ile Tyr Lys Tyr Pro Asp Val Gln Asn Glu Cys Arg 580 585 590 Pro Cys His Glu Asn Cys Thr Gln Gly Cys Lys Gly Pro Glu Leu Gln 595 600 605 Asp Cys Leu Gly Gln Thr Leu Val Leu Ile Gly Lys Thr His Leu Thr 610 615 620 Met Ala Leu Thr Val Ile Ala Gly Leu Val Val Ile Phe Met Met Leu 625 630 635 640 Gly Gly Thr Phe Leu Tyr Trp Arg Gly Arg Arg Ile Gln Asn Lys Arg 645 650 655 Ala Met Arg Arg Tyr Leu Glu Arg Gly Glu Ser Ile Glu Pro Leu Asp 660 665 670 Pro Ser Glu Lys Ala Asn Lys Val Leu Ala Arg Ile Phe Lys Glu Thr 675 680 685 Glu Leu Arg Lys Leu Lys Val Leu Gly Ser Gly Val Phe Gly Thr Val 690 695 700 His Lys Gly Val Trp Ile Pro Glu Gly Glu Ser Ile Lys Ile Pro Val 705 710 715 720 Cys Ile Lys Val Ile Glu Asp Lys Ser Gly Arg Gln Ser Phe Gln Ala 725 730 735 Val Thr Asp His Met Leu Ala Ile Gly Ser Leu Asp His Ala His Ile 740 745 750 Val Arg Leu Leu Gly Leu Cys Pro Gly Ser Ser Leu Gln Leu Val Thr 755 760 765 Gln Tyr Leu Pro Leu Gly Ser Leu Leu Asp His Val Arg Gln His Arg 770 775 780 Gly Ala Leu Gly Pro Gln Leu Leu Leu Asn Trp Gly Val Gln Ile Ala 785 790 795 800 Lys Gly Met Tyr Tyr Leu Glu Glu His Gly Met Val His Arg Asn Leu 805 810 815 Ala Ala Arg Asn Val Leu Leu Lys Ser Pro Ser Gln Val Gln Val Ala 820 825 830 Asp Phe Gly Val Ala Asp Leu Leu Pro Pro Asp Asp Lys Gln Leu Leu 835 840 845 Tyr Ser Glu Ala Lys Thr Pro Ile Lys Trp Met Ala Leu Glu Ser Ile 850 855 860 His Phe Gly Lys Tyr Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val 865 870 875 880 Thr Val Trp Glu Leu Met Thr Phe Gly Ala Glu Pro Tyr Ala Gly Leu 885 890 895 Arg Leu Ala Glu Val Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Ala 900 905 910 Gln Pro Gln Ile Cys Thr Ile Asp Val Tyr Met Val Met Val Lys Cys 915 920 925 Trp Met Ile Asp Glu Asn Ile Arg Pro Thr Phe Lys Glu Leu Ala Asn 930 935 940 Glu Phe Thr Arg Met Ala Arg Asp Pro Pro Arg Tyr Leu Val Ile Lys 945 950 955 960 Arg Glu Ser Gly Pro Gly Ile Ala Pro Gly Pro Glu Pro His Gly Leu 965 970 975 Thr Asn Lys Lys Leu Glu Glu Val Glu Leu Glu Pro Glu Leu Asp Leu 980 985 990 Asp Leu Asp Leu Glu Ala Glu Glu Asp Asn Leu Ala Thr Thr Thr Leu 995 1000 1005 Gly Ser Ala Leu Ser Leu Pro Val Gly Thr Leu Asn Arg Pro Arg Gly 1010 1015 1020 Ser Gln Ser Leu Leu Ser Pro Ser Ser Gly Tyr Met Pro Met Asn Gln 1025 1030 1035 1040 Gly Asn Leu Gly Glu Ser Cys Gln Glu Ser Ala Val Ser Gly Ser Ser 1045 1050 1055 Glu Arg Cys Pro Arg Pro Val Ser Leu His Pro Met Pro Arg Gly Cys 1060 1065 1070 Leu Ala Ser Glu Ser Ser Glu Gly His Val Thr Gly Ser Glu Ala Glu 1075 1080 1085 Leu Gln Glu Lys Val Ser Met Cys Arg Ser Arg Ser Arg Ser Arg Ser 1090 1095 1100 Pro Arg Pro Arg Gly Asp Ser Ala Tyr His Ser Gln Arg His Ser Leu 1105 1110 1115 1120 Leu Thr Pro Val Thr Pro Leu Ser Pro Pro Gly Leu Glu Glu Glu Asp 1125 1130 1135 Val Asn Gly Tyr Val Met Pro Asp Thr His Leu Lys Gly Thr Pro Ser 1140 1145 1150 Ser Arg Glu Gly Thr Leu Ser Ser Val Gly Leu Ser Ser Val Leu Gly 1155 1160 1165 Thr Glu Glu Glu Asp Glu Asp Glu Glu Tyr Glu Tyr Met Asn Arg Arg 1170 1175 1180 Arg Arg His Ser Pro Pro His Pro Pro Arg Pro Ser Ser Leu Glu Glu 1185 1190 1195 1200 Leu Gly Tyr Glu Tyr Met Asp Val Gly Ser Asp Leu Ser Ala Ser Leu 1205 1210 1215 Gly Ser Thr Gln Ser Cys Pro Leu His Pro Val Pro Ile Met Pro Thr 1220 1225 1230 Ala Gly Thr Thr Pro Asp Glu Asp Tyr Glu Tyr Met Asn Arg Gln Arg 1235 1240 1245 Asp Gly Gly Gly Pro Gly Gly Asp Tyr Ala Ala Met Gly Ala Cys Pro 1250 1255 1260 Ala Ser Glu Gln Gly Tyr Glu Glu Met Arg Ala Phe Gln Gly Pro Gly 1265 1270 1275 1280 His Gln Ala Pro His Val His Tyr Ala Arg Leu Lys Thr Leu Arg Ser 1285 1290 1295 Leu Glu Ala Thr Asp Ser Ala Phe Asp Asn Pro Asp Tyr Trp His Ser 1300 1305 1310 Arg Leu Phe Pro Lys Ala Asn Ala Gln Arg Thr 1315 1320 <210> 46 <211> 1308 <212> PRT <213> Homo sapiens <220> <223> Human HER4 (ErbB4) precursor <220> <221> SIGNAL <222> (1)...(25) <223> signal peptide <220> <221> DOMAIN <222> (26)...(651) <223> extracellular domain (ECD) <220> <221> DOMAIN <222> (652)...(675) <223> transmembrane region <220> <221> DOMAIN <222> (676)...(1308) <223> cytoplasmic domain <220> <221> DOMAIN <222> (718)...(985) <223> protein kinase <400> 46 Met Lys Pro Ala Thr Gly Leu Trp Val Trp Val Ser Leu Leu Val Ala 1 5 10 15 Ala Gly Thr Val Gln Pro Ser Asp Ser Gln Ser Val Cys Ala Gly Thr 20 25 30 Glu Asn Lys Leu Ser Ser Leu Ser Asp Leu Glu Gln Gln Tyr Arg Ala 35 40 45 Leu Arg Lys Tyr Tyr Glu Asn Cys Glu Val Val Met Gly Asn Leu Glu 50 55 60 Ile Thr Ser Ile Glu His Asn Arg Asp Leu Ser Phe Leu Arg Ser Val 65 70 75 80 Arg Glu Val Thr Gly Tyr Val Leu Val Ala Leu Asn Gln Phe Arg Tyr 85 90 95 Leu Pro Leu Glu Asn Leu Arg Ile Ile Arg Gly Thr Lys Leu Tyr Glu 100 105 110 Asp Arg Tyr Ala Leu Ala Ile Phe Leu Asn Tyr Arg Lys Asp Gly Asn 115 120 125 Phe Gly Leu Gln Glu Leu Gly Leu Lys Asn Leu Thr Glu Ile Leu Asn 130 135 140 Gly Gly Val Tyr Val Asp Gln Asn Lys Phe Leu Cys Tyr Ala Asp Thr 145 150 155 160 Ile His Trp Gln Asp Ile Val Arg Asn Pro Trp Pro Ser Asn Leu Thr 165 170 175 Leu Val Ser Thr Asn Gly Ser Ser Gly Cys Gly Arg Cys His Lys Ser 180 185 190 Cys Thr Gly Arg Cys Trp Gly Pro Thr Glu Asn His Cys Gln Thr Leu 195 200 205 Thr Arg Thr Val Cys Ala Glu Gln Cys Asp Gly Arg Cys Tyr Gly Pro 210 215 220 Tyr Val Ser Asp Cys Cys His Arg Glu Cys Ala Gly Gly Cys Ser Gly 225 230 235 240 Pro Lys Asp Thr Asp Cys Phe Ala Cys Met Asn Phe Asn Asp Ser Gly 245 250 255 Ala Cys Val Thr Gln Cys Pro Gln Thr Phe Val Tyr Asn Pro Thr Thr 260 265 270 Phe Gln Leu Glu His Asn Phe Asn Ala Lys Tyr Thr Tyr Gly Ala Phe 275 280 285 Cys Val Lys Lys Cys Pro His Asn Phe Val Val Asp Ser Ser Ser Cys 290 295 300 Val Arg Ala Cys Pro Ser Ser Lys Met Glu Val Glu Glu Asn Gly Ile 305 310 315 320 Lys Met Cys Lys Pro Cys Thr Asp Ile Cys Pro Lys Ala Cys Asp Gly 325 330 335 Ile Gly Thr Gly Ser Leu Met Ser Ala Gln Thr Val Asp Ser Ser Asn 340 345 350 Ile Asp Lys Phe Ile Asn Cys Thr Lys Ile Asn Gly Asn Leu Ile Phe 355 360 365 Leu Val Thr Gly Ile His Gly Asp Pro Tyr Asn Ala Ile Glu Ala Ile 370 375 380 Asp Pro Glu Lys Leu Asn Val Phe Arg Thr Val Arg Glu Ile Thr Gly 385 390 395 400 Phe Leu Asn Ile Gln Ser Trp Pro Pro Asn Met Thr Asp Phe Ser Val 405 410 415 Phe Ser Asn Leu Val Thr Ile Gly Gly Arg Val Leu Tyr Ser Gly Leu 420 425 430 Ser Leu Leu Ile Leu Lys Gln Gln Gly Ile Thr Ser Leu Gln Phe Gln 435 440 445 Ser Leu Lys Glu Ile Ser Ala Gly Asn Ile Tyr Ile Thr Asp Asn Ser 450 455 460 Asn Leu Cys Tyr Tyr His Thr Ile Asn Trp Thr Thr Leu Phe Ser Thr 465 470 475 480 Ile Asn Gln Arg Ile Val Ile Arg Asp Asn Arg Lys Ala Glu Asn Cys 485 490 495 Thr Ala Glu Gly Met Val Cys Asn His Leu Cys Ser Ser Asp Gly Cys 500 505 510 Trp Gly Pro Gly Pro Asp Gln Cys Leu Ser Cys Arg Arg Phe Ser Arg 515 520 525 Gly Arg Ile Cys Ile Glu Ser Cys Asn Leu Tyr Asp Gly Glu Phe Arg 530 535 540 Glu Phe Glu Asn Gly Ser Ile Cys Val Glu Cys Asp Pro Gln Cys Glu 545 550 555 560 Lys Met Glu Asp Gly Leu Leu Thr Cys His Gly Pro Gly Pro Asp Asn 565 570 575 Cys Thr Lys Cys Ser His Phe Lys Asp Gly Pro Asn Cys Val Glu Lys 580 585 590 Cys Pro Asp Gly Leu Gln Gly Ala Asn Ser Phe Ile Phe Lys Tyr Ala 595 600 605 Asp Pro Asp Arg Glu Cys His Pro Cys His Pro Asn Cys Thr Gln Gly 610 615 620 Cys Asn Gly Pro Thr Ser His Asp Cys Ile Tyr Tyr Pro Trp Thr Gly 625 630 635 640 His Ser Thr Leu Pro Gln His Ala Arg Thr Pro Leu Ile Ala Ala Gly 645 650 655 Val Ile Gly Gly Leu Phe Ile Leu Val Ile Val Gly Leu Thr Phe Ala 660 665 670 Val Tyr Val Arg Arg Lys Ser Ile Lys Lys Lys Arg Ala Leu Arg Arg 675 680 685 Phe Leu Glu Thr Glu Leu Val Glu Pro Leu Thr Pro Ser Gly Thr Ala 690 695 700 Pro Asn Gln Ala Gln Leu Arg Ile Leu Lys Glu Thr Glu Leu Lys Arg 705 710 715 720 Val Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys Gly Ile 725 730 735 Trp Val Pro Glu Gly Glu Thr Val Lys Ile Pro Val Ala Ile Lys Ile 740 745 750 Leu Asn Glu Thr Thr Gly Pro Lys Ala Asn Val Glu Phe Met Asp Glu 755 760 765 Ala Leu Ile Met Ala Ser Met Asp His Pro His Leu Val Arg Leu Leu 770 775 780 Gly Val Cys Leu Ser Pro Thr Ile Gln Leu Val Thr Gln Leu Met Pro 785 790 795 800 His Gly Cys Leu Leu Glu Tyr Val His Glu His Lys Asp Asn Ile Gly 805 810 815 Ser Gln Leu Leu Leu Asn Trp Cys Val Gln Ile Ala Lys Gly Met Met 820 825 830 Tyr Leu Glu Glu Arg Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn 835 840 845 Val Leu Val Lys Ser Pro Asn His Val Lys Ile Thr Asp Phe Gly Leu 850 855 860 Ala Arg Leu Leu Glu Gly Asp Glu Lys Glu Tyr Asn Ala Asp Gly Gly 865 870 875 880 Lys Met Pro Ile Lys Trp Met Ala Leu Glu Cys Ile His Tyr Arg Lys 885 890 895 Phe Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Ile Trp Glu 900 905 910 Leu Met Thr Phe Gly Gly Lys Pro Tyr Asp Gly Ile Pro Thr Arg Glu 915 920 925 Ile Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile 930 935 940 Cys Thr Ile Asp Val Tyr Met Val Met Val Lys Cys Trp Met Ile Asp 945 950 955 960 Ala Asp Ser Arg Pro Lys Phe Lys Glu Leu Ala Ala Glu Phe Ser Arg 965 970 975 Met Ala Arg Asp Pro Gln Arg Tyr Leu Val Ile Gln Gly Asp Asp Arg 980 985 990 Met Lys Leu Pro Ser Pro Asn Asp Ser Lys Phe Phe Gln Asn Leu Leu 995 1000 1005 Asp Glu Glu Asp Leu Glu Asp Met Met Asp Ala Glu Glu Tyr Leu Val 1010 1015 1020 Pro Gln Ala Phe Asn Ile Pro Pro Pro Ile Tyr Thr Ser Arg Ala Arg 1025 1030 1035 1040 Ile Asp Ser Asn Arg Ser Glu Ile Gly His Ser Pro Pro Pro Ala Tyr 1045 1050 1055 Thr Pro Met Ser Gly Asn Gln Phe Val Tyr Arg Asp Gly Gly Phe Ala 1060 1065 1070 Ala Glu Gln Gly Val Ser Val Pro Tyr Arg Ala Pro Thr Ser Thr Ile 1075 1080 1085 Pro Glu Ala Pro Val Ala Gln Gly Ala Thr Ala Glu Ile Phe Asp Asp 1090 1095 1100 Ser Cys Cys Asn Gly Thr Leu Arg Lys Pro Val Ala Pro His Val Gln 1105 1110 1115 1120 Glu Asp Ser Ser Thr Gln Arg Tyr Ser Ala Asp Pro Thr Val Phe Ala 1125 1130 1135 Pro Glu Arg Ser Pro Arg Gly Glu Leu Asp Glu Glu Gly Tyr Met Thr 1140 1145 1150 Pro Met Arg Asp Lys Pro Lys Gln Glu Tyr Leu Asn Pro Val Glu Glu 1155 1160 1165 Asn Pro Phe Val Ser Arg Arg Lys Asn Gly Asp Leu Gln Ala Leu Asp 1170 1175 1180 Asn Pro Glu Tyr His Asn Ala Ser Asn Gly Pro Pro Lys Ala Glu Asp 1185 1190 1195 1200 Glu Tyr Val Asn Glu Pro Leu Tyr Leu Asn Thr Phe Ala Asn Thr Leu 1205 1210 1215 Gly Lys Ala Glu Tyr Leu Lys Asn Asn Ile Leu Ser Met Pro Glu Lys 1220 1225 1230 Ala Lys Lys Ala Phe Asp Asn Pro Asp Tyr Trp Asn His Ser Leu Pro 1235 1240 1245 Pro Arg Ser Thr Leu Gln His Pro Asp Tyr Leu Gln Glu Tyr Ser Thr 1250 1255 1260 Lys Tyr Phe Tyr Lys Gln Asn Gly Arg Ile Arg Pro Ile Val Ala Glu 1265 1270 1275 1280 Asn Pro Glu Tyr Leu Ser Glu Phe Ser Leu Lys Pro Gly Thr Val Leu 1285 1290 1295 Pro Pro Pro Pro Tyr Arg His Arg Asn Thr Val Val 1300 1305 <210> 47 <211> 1283 <212> PRT <213> Homo sapiens <220> <223> Mature human HER4 (ErbB4) protein (minus the signal peptide) <220> <221> DOMAIN <222> (1)...(625) <223> Extracellular domain (ECD) <220> <221> DOMAIN <222> (1)...(163) <223> domain I of ECD <220> <221> DOMAIN <222> (164)... (308) <223> domain II of ECD <220> <221> DOMAIN <222> (309)... (477) <223> domain III of ECD <220> <221> DOMAIN <222> (478)... (625) <223> domain IV of ECD <400> 47 Gln Ser Val Cys Ala Gly Thr Glu Asn Lys Leu Ser Ser Leu Ser Asp 1 5 10 15 Leu Glu Gln Gln Tyr Arg Ala Leu Arg Lys Tyr Tyr Glu Asn Cys Glu 20 25 30 Val Val Met Gly Asn Leu Glu Ile Thr Ser Ile Glu His Asn Arg Asp 35 40 45 Leu Ser Phe Leu Arg Ser Val Arg Glu Val Thr Gly Tyr Val Leu Val 50 55 60 Ala Leu Asn Gln Phe Arg Tyr Leu Pro Leu Glu Asn Leu Arg Ile Ile 65 70 75 80 Arg Gly Thr Lys Leu Tyr Glu Asp Arg Tyr Ala Leu Ala Ile Phe Leu 85 90 95 Asn Tyr Arg Lys Asp Gly Asn Phe Gly Leu Gln Glu Leu Gly Leu Lys 100 105 110 Asn Leu Thr Glu Ile Leu Asn Gly Gly Val Tyr Val Asp Gln Asn Lys 115 120 125 Phe Leu Cys Tyr Ala Asp Thr Ile His Trp Gln Asp Ile Val Arg Asn 130 135 140 Pro Trp Pro Ser Asn Leu Thr Leu Val Ser Thr Asn Gly Ser Ser Gly 145 150 155 160 Cys Gly Arg Cys His Lys Ser Cys Thr Gly Arg Cys Trp Gly Pro Thr 165 170 175 Glu Asn His Cys Gln Thr Leu Thr Arg Thr Val Cys Ala Glu Gln Cys 180 185 190 Asp Gly Arg Cys Tyr Gly Pro Tyr Val Ser Asp Cys Cys His Arg Glu 195 200 205 Cys Ala Gly Gly Cys Ser Gly Pro Lys Asp Thr Asp Cys Phe Ala Cys 210 215 220 Met Asn Phe Asn Asp Ser Gly Ala Cys Val Thr Gln Cys Pro Gln Thr 225 230 235 240 Phe Val Tyr Asn Pro Thr Thr Phe Gln Leu Glu His Asn Phe Asn Ala 245 250 255 Lys Tyr Thr Tyr Gly Ala Phe Cys Val Lys Lys Cys Pro His Asn Phe 260 265 270 Val Val Asp Ser Ser Ser Cys Val Arg Ala Cys Pro Ser Ser Lys Met 275 280 285 Glu Val Glu Glu Asn Gly Ile Lys Met Cys Lys Pro Cys Thr Asp Ile 290 295 300 Cys Pro Lys Ala Cys Asp Gly Ile Gly Thr Gly Ser Leu Met Ser Ala 305 310 315 320 Gln Thr Val Asp Ser Ser Asn Ile Asp Lys Phe Ile Asn Cys Thr Lys 325 330 335 Ile Asn Gly Asn Leu Ile Phe Leu Val Thr Gly Ile His Gly Asp Pro 340 345 350 Tyr Asn Ala Ile Glu Ala Ile Asp Pro Glu Lys Leu Asn Val Phe Arg 355 360 365 Thr Val Arg Glu Ile Thr Gly Phe Leu Asn Ile Gln Ser Trp Pro Pro 370 375 380 Asn Met Thr Asp Phe Ser Val Phe Ser Asn Leu Val Thr Ile Gly Gly 385 390 395 400 Arg Val Leu Tyr Ser Gly Leu Ser Leu Leu Ile Leu Lys Gln Gln Gly 405 410 415 Ile Thr Ser Leu Gln Phe Gln Ser Leu Lys Glu Ile Ser Ala Gly Asn 420 425 430 Ile Tyr Ile Thr Asp Asn Ser Asn Leu Cys Tyr Tyr His Thr Ile Asn 435 440 445 Trp Thr Thr Leu Phe Ser Thr Ile Asn Gln Arg Ile Val Ile Arg Asp 450 455 460 Asn Arg Lys Ala Glu Asn Cys Thr Ala Glu Gly Met Val Cys Asn His 465 470 475 480 Leu Cys Ser Ser Asp Gly Cys Trp Gly Pro Gly Pro Asp Gln Cys Leu 485 490 495 Ser Cys Arg Arg Phe Ser Arg Gly Arg Ile Cys Ile Glu Ser Cys Asn 500 505 510 Leu Tyr Asp Gly Glu Phe Arg Glu Phe Glu Asn Gly Ser Ile Cys Val 515 520 525 Glu Cys Asp Pro Gln Cys Glu Lys Met Glu Asp Gly Leu Leu Thr Cys 530 535 540 His Gly Pro Gly Pro Asp Asn Cys Thr Lys Cys Ser His Phe Lys Asp 545 550 555 560 Gly Pro Asn Cys Val Glu Lys Cys Pro Asp Gly Leu Gln Gly Ala Asn 565 570 575 Ser Phe Ile Phe Lys Tyr Ala Asp Pro Asp Arg Glu Cys His Pro Cys 580 585 590 His Pro Asn Cys Thr Gln Gly Cys Asn Gly Pro Thr Ser His Asp Cys 595 600 605 Ile Tyr Tyr Pro Trp Thr Gly His Ser Thr Leu Pro Gln His Ala Arg 610 615 620 Thr Pro Leu Ile Ala Ala Gly Val Ile Gly Gly Leu Phe Ile Leu Val 625 630 635 640 Ile Val Gly Leu Thr Phe Ala Val Tyr Val Arg Arg Lys Ser Ile Lys 645 650 655 Lys Lys Arg Ala Leu Arg Arg Phe Leu Glu Thr Glu Leu Val Glu Pro 660 665 670 Leu Thr Pro Ser Gly Thr Ala Pro Asn Gln Ala Gln Leu Arg Ile Leu 675 680 685 Lys Glu Thr Glu Leu Lys Arg Val Lys Val Leu Gly Ser Gly Ala Phe 690 695 700 Gly Thr Val Tyr Lys Gly Ile Trp Val Pro Glu Gly Glu Thr Val Lys 705 710 715 720 Ile Pro Val Ala Ile Lys Ile Leu Asn Glu Thr Thr Gly Pro Lys Ala 725 730 735 Asn Val Glu Phe Met Asp Glu Ala Leu Ile Met Ala Ser Met Asp His 740 745 750 Pro His Leu Val Arg Leu Leu Gly Val Cys Leu Ser Pro Thr Ile Gln 755 760 765 Leu Val Thr Gln Leu Met Pro His Gly Cys Leu Leu Glu Tyr Val His 770 775 780 Glu His Lys Asp Asn Ile Gly Ser Gln Leu Leu Leu Asn Trp Cys Val 785 790 795 800 Gln Ile Ala Lys Gly Met Met Tyr Leu Glu Glu Arg Arg Leu Val His 805 810 815 Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Ser Pro Asn His Val 820 825 830 Lys Ile Thr Asp Phe Gly Leu Ala Arg Leu Leu Glu Gly Asp Glu Lys 835 840 845 Glu Tyr Asn Ala Asp Gly Gly Lys Met Pro Ile Lys Trp Met Ala Leu 850 855 860 Glu Cys Ile His Tyr Arg Lys Phe Thr His Gln Ser Asp Val Trp Ser 865 870 875 880 Tyr Gly Val Thr Ile Trp Glu Leu Met Thr Phe Gly Gly Lys Pro Tyr 885 890 895 Asp Gly Ile Pro Thr Arg Glu Ile Pro Asp Leu Leu Glu Lys Gly Glu 900 905 910 Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr Met Val Met 915 920 925 Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg Pro Lys Phe Lys Glu 930 935 940 Leu Ala Ala Glu Phe Ser Arg Met Ala Arg Asp Pro Gln Arg Tyr Leu 945 950 955 960 Val Ile Gln Gly Asp Asp Arg Met Lys Leu Pro Ser Pro Asn Asp Ser 965 970 975 Lys Phe Phe Gln Asn Leu Leu Asp Glu Glu Asp Leu Glu Asp Met Met 980 985 990 Asp Ala Glu Glu Tyr Leu Val Pro Gln Ala Phe Asn Ile Pro Pro Pro 995 1000 1005 Ile Tyr Thr Ser Arg Ala Arg Ile Asp Ser Asn Arg Ser Glu Ile Gly 1010 1015 1020 His Ser Pro Pro Pro Ala Tyr Thr Pro Met Ser Gly Asn Gln Phe Val 1025 1030 1035 1040 Tyr Arg Asp Gly Gly Phe Ala Ala Glu Gln Gly Val Ser Val Pro Tyr 1045 1050 1055 Arg Ala Pro Thr Ser Thr Ile Pro Glu Ala Pro Val Ala Gln Gly Ala 1060 1065 1070 Thr Ala Glu Ile Phe Asp Asp Ser Cys Cys Asn Gly Thr Leu Arg Lys 1075 1080 1085 Pro Val Ala Pro His Val Gln Glu Asp Ser Ser Thr Gln Arg Tyr Ser 1090 1095 1100 Ala Asp Pro Thr Val Phe Ala Pro Glu Arg Ser Pro Arg Gly Glu Leu 1105 1110 1115 1120 Asp Glu Glu Gly Tyr Met Thr Pro Met Arg Asp Lys Pro Lys Gln Glu 1125 1130 1135 Tyr Leu Asn Pro Val Glu Asn Pro Phe Val Ser Arg Arg Lys Asn 1140 1145 1150 Gly Asp Leu Gln Ala Leu Asp Asn Pro Glu Tyr His Asn Ala Ser Asn 1155 1160 1165 Gly Pro Pro Lys Ala Glu Asp Glu Tyr Val Asn Glu Pro Leu Tyr Leu 1170 1175 1180 Asn Thr Phe Ala Asn Thr Leu Gly Lys Ala Glu Tyr Leu Lys Asn Asn 1185 1190 1195 1200 Ile Leu Ser Met Pro Glu Lys Ala Lys Lys Ala Phe Asp Asn Pro Asp 1205 1210 1215 Tyr Trp Asn His Ser Leu Pro Pro Arg Ser Thr Leu Gln His Pro Asp 1220 1225 1230 Tyr Leu Gln Glu Tyr Ser Thr Lys Tyr Phe Tyr Lys Gln Asn Gly Arg 1235 1240 1245 Ile Arg Pro Ile Val Ala Glu Asn Pro Glu Tyr Leu Ser Glu Phe Ser 1250 1255 1260 Leu Lys Pro Gly Thr Val Leu Pro Pro Pro Pro Tyr Arg His Arg Asn 1265 1270 1275 1280 Thr Val Val <210> 48 <211> 16 <212> PRT <213> artificial sequence <220> <223> cyclic FcRnBP <400> 48 Gln Arg Phe Cys Thr Gly His Phe Gly Gly Leu Tyr Pro Cys Asn Gly 1 5 10 15 <210> 49 <211> 16 <212> PRT <213> artificial sequence <220> <223> cyclic FcRnBP <400> 49 Gln Arg Phe Cys Thr Gly His Phe Gly Gly Leu His Pro Cys Asn Gly 1 5 10 15 <210> 50 <211> 16 <212> PRT <213> artificial sequence <220> <223> linear FcRnBP <400> 50 Gln Arg Phe Val Thr Gly His Phe Gly Gly Leu Tyr Pro Ala Asn Gly 1 5 10 15 <210> 51 <211> 16 <212> PRT <213> artificial sequence <220> <223> linear FcRnBP <400> 51 Gln Arg Phe Val Thr Gly His Phe Gly Gly Leu His Pro Ala Asn Gly 1 5 10 15 <210> 52 <211> 108 <212> PRT <213> artificial sequence <220> <223> DOM7h-11-3 (albumin-binding dAb) <400> 52 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Pro Ile Gly Thr Thr 20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Leu Trp Asn Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Gln Ala Gly Thr His Pro Thr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 53 <211> 120 <212> PRT <213> artificial sequence <220> <223> DOM1m-21-23 (mouse anti-TNFR1 dAb from DMS5540) <400> 53 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Arg Tyr 20 25 30 Ser Met Gly Trp Leu Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Arg Ile Asp Ser Tyr Gly Arg Gly Thr Tyr Tyr Glu Asp Pro Val 50 55 60 Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ile Ser Gln Phe Gly Ser Asn Ala Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 54 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-208 (human anti-TNFR1 dAb from DMS5541) <400> 54 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 55 <211> 119 <212> PRT <213> artificial sequence <220> <223> GSK1995057 (anti-TNFR1 antagonistic dAb) <400> 55 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Asp Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr His Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 56 <211> 120 <212> PRT <213> artificial sequence <220> <223> GSK2862277 (anti-TNFR1 antagonistic dAb) <400> 56 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Asp Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr His Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala 115 120 <210> 57 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOMlh-574-16 anti-TNFR1 dAb <400> 57 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Asp Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 58 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOMlh-549 anti-TNFR1 dAb <400> 58 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Asp Tyr 20 25 30 Glu Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Glu Ser Gly Thr Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Arg Arg Phe Ser Ala Ser Thr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 59 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-131-206 anti-TNFR1 dAb <400> 59 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Asp Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr His Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 60 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-131-511 anti-TNFR1 dAb <400> 60 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Val Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 61 <211> 119 <212> PRT <213> artificial sequence <220> <223> variant 1-1 of DOM1h-131-511 anti-TNFR1 dAb <400> 61 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Met Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Val Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 62 <211> 119 <212> PRT <213> artificial sequence <220> <223> variant 1-2 of DOM1h-131-511 anti-TNFR1 dAb <400> 62 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Val Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 63 <211> 119 <212> PRT <213> artificial sequence <220> <223> variant 1-3 of DOM1h-131-511 anti-TNFR1 dAb <400> 63 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Val Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 64 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-131-201 anti-TNFR1 dAb <400> 64 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Val Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 65 <211> 119 <212> PRT <213> artificial sequence <220> <223> variant 1-5 of DOM1h-131-511 anti-TNFR1 dAb <400> 65 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Val Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 66 <211> 119 <212> PRT <213> artificial sequence <220> <223> variant 1-6 of DOM1h-131-511 anti-TNFR1 dAb <400> 66 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Ala Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ser Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Gly Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 67 <211> 119 <212> PRT <213> artificial sequence <220> <223> variant 1-7 of DOM1h-131-511 anti-TNFR1 dAb <400> 67 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Ala Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 68 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-131-202 anti-TNFR1 dAb <400> 68 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Asp Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 69 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-131-203 anti-TNFR1 dAb <400> 69 Glu Val Gln Leu Trp Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Asp Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Ile Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 70 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-131-204 anti-TNFR1 dAb <400> 70 Glu Val Gln Leu Ser Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Asp Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Asn Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 71 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-131-205 anti-TNFR1 dAb <400> 71 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Asp Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser 115 <210> 72 <211> 119 <212> PRT <213> artificial sequence <220> <223> variant 1-12 of DOM1h-131-511 anti-TNFR1 dAb <400> 72 Glu Val Arg Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Asp Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser 115 <210> 73 <211> 119 <212> PRT <213> artificial sequence <220> <223> variant 1-13 of DOM1h-131-511 anti-TNFR1 dAb <400> 73 Glu Val Arg Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Glu Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Asp Ser Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 74 <211> 119 <212> PRT <213> artificial sequence <220> <223> variant 1-14 of DOM1h-131-511 anti-TNFR1 dAb <400> 74 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Val Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 75 <211> 119 <212> PRT <213> artificial sequence <220> <223> variant 1-15 of DOM1h-131-511 anti-TNFR1 dAb <400> 75 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Val Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 76 <211> 119 <212> PRT <213> artificial sequence <220> <223> variant 1-18 of DOM1h-131-511 anti-TNFR1 dAb <400> 76 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Arg Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Asp Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 77 <211> 119 <212> PRT <213> artificial sequence <220> <223> variant 1-19 of DOM1h-131-511 anti-TNFR1 dAb <400> 77 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Thr Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Asp Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 78 <211> 119 <212> PRT <213> artificial sequence <220> <223> variant 1-20 of DOM1h-131-511 anti-TNFR1 dAb <400> 78 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Ser Asp Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 79 <211> 119 <212> PRT <213> artificial sequence <220> <223> variant 1-21 of DOM1h-131-511 anti-TNFR1 dAb <400> 79 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Asp Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Lys Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 80 <211> 119 <212> PRT <213> artificial sequence <220> <223> variant 1-22 of DOM1h-131-511 anti-TNFR1 dAb <400> 80 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Arg Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Val Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 81 <211> 119 <212> PRT <213> artificial sequence <220> <223> variant 1-23 of DOM1h-131-511 anti-TNFR1 dAb <400> 81 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Gln Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Val Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 82 <211> 119 <212> PRT <213> artificial sequence <220> <223> variant 1-24 of DOM1h-131-511 anti-TNFR1 dAb <400> 82 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Val Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Lys Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 83 <211> 119 <212> PRT <213> artificial sequence <220> <223> variant 1-25 of DOM1h-131-511 anti-TNFR1 dAb <400> 83 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Ala Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 84 <211> 357 <212> DNA <213> artificial sequence <220> <223> Codon Optimized Sequence 1 of DOM1h-131-206 anti-TNFR1 dAbs <400> 84 gaggttcaat tgttggaatc cggtggtgga ttggttcaac ctggtggttc tttgagattg 60 tcctgtgctg cttccggttt tactttcgct cacgagacta tggtttgggt tagacaggct 120 ccaggtaaag gattggaatg ggtttcccac attccaccag atggtcaaga tccattctac 180 gctgactccg ttaagggaag attcactatc tccagagaca actccaagaa cactttgtac 240 ttgcagatga actccttgag agctgaggat actgctgttt accactgtgc tttgttgcca 300 aagagaggac cttggtttga ttactgggga cagggaactt tggttactgt ttcttcc 357 <210> 85 <211> 366 <212> DNA <213> artificial sequence <220> <223> Codon Optimized Sequence 2 of DOM1h-131-206 anti-TNFR1 dAbs <400> 85 gagaaaagag aggttcaatt gcttgaatct ggaggaggtt tggtccagcc aggagggtcc 60 cttcgactaa gttgtgctgc cagtgggttt acgtttgctc atgaaactat ggtatgggtc 120 cgacaggcac ctggtaaagg tcttgaatgg gtttcacata tccctccaga cggtcaagac 180 ccattttacg ctgattccgt gaaaggcaga tttacaattt cacgagataa ttctaaaaac 240 accttgtact tacaaatgaa ctcattgaga gctgaggaca ctgcagttta tcactgcgct 300 ttactaccaa aacgtggacc ttggtttgat tattggggcc aaggtacgtt agtgactgtt 360 agttct 366 <210> 86 <211> 357 <212> DNA <213> artificial sequence <220> <223> Codon Optimized Sequence 3 of DOM1h-131-206 anti-TNFR1 dAbs <400> 86 gaagtgcagc ttcttgaaag tggtggaggg ctagtgcagc cagggggatc tttaagatta 60 tcatgcgctg ccagtggatt tacttttgct cacgagacga tggtctgggt gagacaagct 120 cctggaaaag gtttagagtg ggtttctcac attccacctg atggtcaaga tcctttctac 180 gcagattccg tcaaaggaag atttactatc tccagagata atagtaaaaa cactttgtac 240 ctacagatga actcacttag agccgaagat accgctgtgt accactgcgc cttgttgcca 300 aagagaggtc cttggttcga ttactggggt cagggtactc tggttacagt ctcatct 357 <210> 87 <211> 357 <212> DNA <213> artificial sequence <220> <223> Codon Optimized Sequence 4 of DOM1h-131-206 anti-TNFR1 dAbs <400> 87 gaggttcaac tgctggaatc tggtggtggt ctggtacaac cgggtggttc cctgcgtctg 60 agctgtgcag cctctggttt caccttcgct catgagacca tggtttgggt acgccaggct 120 ccgggtaaag gcctggagtg ggtaagccat atccctcctg atggtcagga cccgttctat 180 gctgattccg tcaaaggccg ttttaccatt tctcgtgaca acagcaaaaa cactctgtac 240 ctgcaaatga actccctgcg tgcagaagac acggcggttt atcactgtgc actgctgcca 300 aaacgcggcc cttggttcga ctactggggc cagggtactc tggtcactgt atcttct 357 <210> 88 <211> 119 <212> PRT <213> artificial sequence <220> <223> Codon Optimized Sequence 5 of DOM1h-131-206 anti-TNFR1 dAbs <400> 88 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Asp Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr His Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 89 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10 anti-TNFR1 dAb <400> 89 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 90 <211> 122 <212> PRT <213> artificial sequence <220> <223> TAR2h-12 anti-TNFR1 dAb <400> 90 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Ala Tyr 20 25 30 Asn Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Asp Met Tyr Gly Ala Lys Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Cys Leu Met Asp Cys Ser Gly Asp Ile Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 91 <211> 116 <212> PRT <213> artificial sequence <220> <223> TAR2h-13 anti-TNFR1 dAb <400> 91 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Pro Ala Asp 20 25 30 Glu Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Gly Trp Pro Gly Gly Ala Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Gly Arg Asn Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 92 <211> 122 <212> PRT <213> artificial sequence <220> <223> TAR2h-14 anti-TNFR1 dAb <400> 92 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Gln Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Leu Ile Asp Pro Ser Gly Gly His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asn Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Val Phe Ser Asp Trp Pro Ala Val Glu Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 93 <211> 116 <212> PRT <213> artificial sequence <220> <223> TAR2h-16 anti-TNFR1 dAb <400> 93 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Asn Tyr 20 25 30 Asp Met Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Asp Gly Thr Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gln Glu Thr Asn Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 94 <211> 124 <212> PRT <213> artificial sequence <220> <223> TAR2h-17 anti-TNFR1 dAb <400> 94 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Gly Tyr 20 25 30 Gln Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Asp Phe Thr Gly Ala His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Ser Asp Asp Leu Thr Leu Pro Glu Arg Phe Pro Phe Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 95 <211> 123 <212> PRT <213> artificial sequence <220> <223> TAR2h-18 anti-TNFR1 dAb <400> 95 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Asp Tyr 20 25 30 Asn Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Trp Ile Asp Gln Glu Gly Val Phe Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Phe Ser Ala Ala Val Met Leu Arg Thr Ser Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 96 <211> 116 <212> PRT <213> artificial sequence <220> <223> TAR2h-19 anti-TNFR1 dAb <400> 96 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe His Asp Tyr 20 25 30 Gly Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Ile Asp Gly Arg Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Arg Ile Phe Glu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 97 <211> 117 <212> PRT <213> artificial sequence <220> <223> TAR2h-20 anti-TNFR1 dAb <400> 97 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr 20 25 30 Asn Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Pro Ser Gly Asn Glu Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Ala Gly Glu Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 98 <211> 117 <212> PRT <213> artificial sequence <220> <223> TAR2h-21 anti-TNFR1 dAb <400> 98 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Glu Tyr 20 25 30 Asn Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Gly His Ser Gly Gln His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Glu Leu Asn Asn Leu Met Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 99 <211> 121 <212> PRT <213> artificial sequence <220> <223> TAR2h-22 anti-TNFR1 dAb <400> 99 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Glu Tyr 20 25 30 Asn Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Gln Glu Trp Val 35 40 45 Ser Phe Ile Ser Thr Gly Gly His Val Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Phe Ser Val Arg Phe Arg Ser Ser Ile Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 100 <211> 122 <212> PRT <213> artificial sequence <220> <223> TAR2h-23 anti-TNFR1 dAb <400> 100 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30 Thr Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Trp Ile Ala Val Asp Gly Ile His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Asp Trp Thr Ala Thr Asp Phe Ser Ile Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 101 <211> 122 <212> PRT <213> artificial sequence <220> <223> TAR2h-24 anti-TNFR1 dAb <400> 101 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Asn Tyr 20 25 30 Thr Met Leu Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Ser Ala Glu Gly Arg Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Asn Met Lys Ala Thr Asn Phe Lys Asp Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 102 <211> 122 <212> PRT <213> artificial sequence <220> <223> TAR2h-25 anti-TNFR1 dAb <400> 102 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Glu Tyr 20 25 30 Ala Met Leu Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Leu Ile Asp Arg Thr Gly Val Ile Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Arg Asp Tyr Gln Tyr His Leu Tyr Gln Asp Phe Asp Tyr Arg 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 103 <211> 122 <212> PRT <213> artificial sequence <220> <223> TAR2h-26 anti-TNFR1 dAb <400> 103 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Thr Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Met Ile Asp Pro Glu Gly Tyr His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Glu Thr Asn Arg Pro Leu Thr Tyr Lys Pro Trp Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 104 <211> 121 <212> PRT <213> artificial sequence <220> <223> TAR2h-27 anti-TNFR1 dAb <400> 104 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Asn Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Ser Gln Glu Gly His His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Phe Ser Thr Ile Ala Thr Leu Ser Leu Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 105 <211> 122 <212> PRT <213> artificial sequence <220> <223> TAR2h-29 anti-TNFR1 dAb <400> 105 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Thr Tyr 20 25 30 Asn Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ala Trp Leu Gly Ser Glu Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys His Cys Lys Ala Glu Cys Thr Gly Asp Leu Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 106 <211> 122 <212> PRT <213> artificial sequence <220> <223> TAR2h-30 anti-TNFR1 dAb <400> 106 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ala Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Ile Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Val Gly Met Glu Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Glu Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys His Ser Tyr Pro Thr Arg Gly Arg His Leu Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 107 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-32 anti-TNFR1 dAb <400> 107 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Asp Tyr Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 108 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-33 anti-TNFR1 dAb <400> 108 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe His Arg Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Ser Ser Gly Gly Ile Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Thr Gln Ala Gln Gly Leu Glu Leu Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 109 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-1 anti-TNFR1 dAb <400> 109 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Asp Tyr Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 110 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-2 anti-TNFR1 dAb <400> 110 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Asp Tyr Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 111 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-3 anti-TNFR1 dAb <400> 111 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Ala Ala Ser Gly Phe Thr Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 112 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-4 anti-TNFR1 dAb <400> 112 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 113 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-5 anti-TNFR1 dAb <400> 113 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Asp Tyr Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 114 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-6 anti-TNFR1 dAb <400> 114 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Asp Tyr Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 115 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-7 anti-TNFR1 dAb <400> 115 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Lys Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Asp Tyr Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 116 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-8 anti-TNFR1 dAb <400> 116 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Asp Tyr Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 117 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-9 anti-TNFR1 dAb <400> 117 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 118 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-10 anti-TNFR1 dAb <400> 118 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Asp Asp Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 119 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-11 anti-TNFR1 dAb <400> 119 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Asp Tyr Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 120 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-12 anti-TNFR1 dAb <400> 120 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ala 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 121 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-13 anti-TNFR1 dAb <400> 121 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Gly Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Asp Tyr Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 122 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-14 anti-TNFR1 dAb <220> <221> VARIANT <222> (20)...(20) <223> Xaa = any amino acid <400> 122 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Xaa Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 123 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-15 anti-TNFR1 dAb <400> 123 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Asp Tyr Arg Gly Arg 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 124 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-16 anti-TNFR1 dAb <400> 124 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Lys Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Asp Tyr Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 125 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-17 anti-TNFR1 dAb <400> 125 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Gly Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Asp Tyr Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 126 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-18 anti-TNFR1 dAb <400> 126 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ala 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 127 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-19 anti-TNFR1 dAb <400> 127 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Gly Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Lys Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Asp Tyr Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 128 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-20 anti-TNFR1 dAb <400> 128 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ala 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Lys Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 129 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-21 anti-TNFR1 dAb <400> 129 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Lys Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Asp Tyr Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 130 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-22 anti-TNFR1 dAb <400> 130 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Gly Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Asp Tyr Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 131 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-27 anti-TNFR1 dAb <400> 131 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Ala Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Gly Tyr Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 132 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-29 anti-TNFR1 dAb <400> 132 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Ala Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Gly Tyr Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 133 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-31 anti-TNFR1 dAb <400> 133 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ser Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Ala Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Gly Tyr Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 134 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-35 anti-TNFR1 dAb <400> 134 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu His Ala Glu Asp Ala Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Gly Tyr Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 135 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-36 anti-TNFR1 dAb <400> 135 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Leu Thr Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Ala Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Gly Tyr Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 136 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-37 anti-TNFR1 dAb <400> 136 Glu Val Gln Leu Leu Gly Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Ala Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Gly Tyr Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 137 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-38 anti-TNFR1 dAb <400> 137 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Lys Asp Ala Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Gly Tyr Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 138 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-45 anti-TNFR1 dAb <400> 138 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Pro Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Ala Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Gly Tyr Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 139 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-47 anti-TNFR1 dAb <400> 139 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Phe Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Trp Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Ala Ser Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Gly Tyr Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 140 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-48 anti-TNFR1 dAb <400> 140 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Pro Cys Ala Ala Ser Gly Phe Thr Phe Glu Trp Tyr 20 25 30 Trp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Ala Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Gly Tyr Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 141 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-57 anti-TNFR1 dAb <400> 141 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Ala Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Gly Tyr Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 142 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-56 anti-TNFR1 dAb <400> 142 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Val Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Ala Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Gly Tyr Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 143 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-58 anti-TNFR1 dAb <400> 143 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Asp Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Ala Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Gly Tyr Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 144 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-66 anti-TNFR1 dAb <400> 144 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Met Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Ala Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Gly Asn Arg Gly Leu 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 145 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-64 anti-TNFR1 dAb <400> 145 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ala 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Ala Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asp Phe Gly Tyr Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 146 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-65 anti-TNFR1 dAb <400> 146 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Gly Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Glu Leu Asn Phe Gly Tyr Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 147 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-68 anti-TNFR1 dAb <400> 147 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Ala Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Gly Asn Arg Gly Gln 100 105 110 Gly Thr Pro Val Thr Val Ser Ser 115 120 <210> 148 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-69 anti-TNFR1 dAb <400> 148 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Ala Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Gly Pro Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 149 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-67 anti-TNFR1 dAb <400> 149 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Ala Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Gly Asn Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 150 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-61 anti-TNFR1 dAb <400> 150 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Ala Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Gly Tyr Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 151 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-62 anti-TNFR1 dAb <400> 151 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Ala Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Gly Tyr Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 152 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-63 anti-TNFR1 dAb <400> 152 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Thr Ala Ile Ser Gly Ser Gly Gly Ser Thr Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Ala Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Gly Tyr Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 153 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-60 anti-TNFR1 dAb <400> 153 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Asp Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Ala Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Gly Tyr Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 154 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-55 anti-TNFR1 dAb <400> 154 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Asp Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Gln Gln Met Asn Ser Leu Arg Ala Glu Asp Ala Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Gly Tyr Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 155 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-59 anti-TNFR1 dAb <400> 155 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Ala Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Gly Tyr Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 156 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-10-70 anti-TNFR1 dAb <400> 156 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Ala Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Tyr Gly Tyr Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 157 <211> 116 <212> PRT <213> artificial sequence <220> <223> TAR2h-5 anti-TNFR1 dAb <400> 157 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Leu Tyr 20 25 30 Asn Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Ser Gln Thr Gly Arg Leu Thr Trp Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Thr Leu Glu Asp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 158 <211> 119 <212> PRT <213> artificial sequence <220> <223> TAR2h-34 anti-TNFR1 dAb <400> 158 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Pro Glu Tyr 20 25 30 Gly Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Ser His Gly Gly Glu His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gln His Pro Val Ser His Pro Lys Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 159 <211> 116 <212> PRT <213> artificial sequence <220> <223> TAR2h-35 anti-TNFR1 dAb <400> 159 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Ala Tyr 20 25 30 Asn Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Pro Ser Gly Arg Glu Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Arg Tyr Pro Asp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 160 <211> 119 <212> PRT <213> artificial sequence <220> <223> TAR2h-36 anti-TNFR1 dAb <400> 160 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Thr Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Leu Ile Asp Arg Pro Gly Asn His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Trp Gly Leu Asn Val Glu Asp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 161 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-37 anti-TNFR1 dAb <400> 161 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ile Glu Tyr 20 25 30 Asp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Met Ile Ser Ser Asp Gly Arg Leu Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Thr Trp Asp Gly Leu Asn Arg Asn Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 162 <211> 119 <212> PRT <213> artificial sequence <220> <223> TAR2h-38 anti-TNFR1 dAb <400> 162 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ile Gly Tyr 20 25 30 Asn Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Ser Pro Ser Gly Arg Glu Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Thr Leu Ser Ala Asp Gly Arg Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 163 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-39 anti-TNFR1 dAb <400> 163 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Ser Tyr 20 25 30 Asp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Asp Val Ser Gly Thr Leu Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Thr Val Glu Leu Asp Gly Leu Asp Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 164 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-40 anti-TNFR1 dAb <400> 164 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Asp Tyr 20 25 30 Asp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Asp Ser Ser Gly Ser Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Thr Ala Glu Ile Val Asn Ser Arg Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 165 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-41 anti-TNFR1 dAb <400> 165 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Lys Tyr 20 25 30 Gln Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Asp Ser Asn Gly His His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Glu Leu Asp Asn Leu Ser Ile Thr Pro Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 166 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-42 anti-TNFR1 dAb <400> 166 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Lys Tyr 20 25 30 Asn Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Pro Lys Gly Gln His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Glu Gly Met Gly Ser Asp Ala Ile Thr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 167 <211> 122 <212> PRT <213> artificial sequence <220> <223> TAR2h-43 anti-TNFR1 dAb <400> 167 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Thr Met Gly Trp Ala Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Asp Ser Asp Gly Leu His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gln Asn Pro Gln Tyr Ala Tyr Glu Ser Ser Arg Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 168 <211> 121 <212> PRT <213> artificial sequence <220> <223> TAR2h-44 anti-TNFR1 dAb <400> 168 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Leu Gln Tyr 20 25 30 Pro Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Leu Ala Pro Gly Gly Pro Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys His Pro Thr His Thr Pro His Pro Asn Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 169 <211> 122 <212> PRT <213> artificial sequence <220> <223> TAR2h-45 anti-TNFR1 dAb <400> 169 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Gly Tyr 20 25 30 Arg Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Asp Ser Glu Gly Val Leu Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Cys Ser Ser Asn Cys Asn Met Arg Asn Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 170 <211> 124 <212> PRT <213> artificial sequence <220> <223> TAR2h-47 anti-TNFR1 dAb <400> 170 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Pro Val Tyr 20 25 30 Asn Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Ala Gly Asn Gly Gln Gln Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Phe Ala Ser Lys Val Ser Pro Met Ser Leu Thr Asp Phe Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 171 <211> 124 <212> PRT <213> artificial sequence <220> <223> TAR2h-48 anti-TNFR1 dAb <400> 171 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe His Lys Tyr 20 25 30 Gly Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Asp Leu Ala Gly Leu His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Phe Ala Thr Tyr Ser Ser Gly Asn Glu Glu Gln Pro Phe Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 172 <211> 123 <212> PRT <213> artificial sequence <220> <223> TAR2h-50 anti-TNFR1 dAb <400> 172 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr 20 25 30 Asn Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Ala Gln Ser Gly Gly His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Phe Ser His Pro Asp Glu Glu Gly Thr Gln Met Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 173 <211> 116 <212> PRT <213> artificial sequence <220> <223> TAR2h-51 anti-TNFR1 dAb <400> 173 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Thr Tyr 20 25 30 Asn Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Asp Ala Gly Gly Met His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Thr Glu Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 174 <211> 116 <212> PRT <213> artificial sequence <220> <223> TAR2h-66 anti-TNFR1 dAb <220> <221> VARIANT <222> (33)...(33) <223> Xaa = any amino acid <400> 174 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Glu Tyr 20 25 30 Xaa Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Leu Ile Ser Pro Arg Gly Ser Lys Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Lys Pro Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 175 <211> 116 <212> PRT <213> artificial sequence <220> <223> TAR2h-67 anti-TNFR1 dAb <400> 175 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Asp Tyr 20 25 30 Pro Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Gly Leu Lys Gly Ile His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Leu Asn Asn Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 176 <211> 119 <212> PRT <213> artificial sequence <220> <223> TAR2h-68 anti-TNFR1 dAb <400> 176 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Asn Gly 20 25 30 Asn Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Glu Tyr Gly Thr Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Arg Asn Asp Arg Pro Gly Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 177 <211> 124 <212> PRT <213> artificial sequence <220> <223> TAR2h-70 anti-TNFR1 dAb <400> 177 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Pro Thr Glu 20 25 30 His Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Asp Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Leu His Trp Ser Ser Asp Ser Gly Pro Val His Phe Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 178 <211> 121 <212> PRT <213> artificial sequence <220> <223> TAR2h-71 anti-TNFR1 dAb <400> 178 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Asn Val 20 25 30 Asp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Ser Ala Gly Gly Glu Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Arg Met Leu Ala Asn Ser Pro Leu Ala Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 179 <211> 116 <212> PRT <213> artificial sequence <220> <223> TAR2h-72 anti-TNFR1 dAb <400> 179 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Tyr Glu 20 25 30 Pro Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Ser His Thr Gly Arg Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Arg Trp Ser Ser Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 180 <211> 119 <212> PRT <213> artificial sequence <220> <223> TAR2h-73 anti-TNFR1 dAb <400> 180 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Pro Ser Glu 20 25 30 Lys Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Asp Glu Arg Gly Ile Met Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Arg Trp Thr Phe Asn Thr Ala Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 181 <211> 119 <212> PRT <213> artificial sequence <220> <223> TAR2h-74 anti-TNFR1 dAb <400> 181 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Glu 20 25 30 Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Gly Pro Arg Gly Met Pro Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Met Asn Ser His Asp Gly Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 182 <211> 119 <212> PRT <213> artificial sequence <220> <223> TAR2h-75 anti-TNFR1 dAb <400> 182 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Ala Tyr 20 25 30 Thr Met Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Asp Pro His Gly Thr Ile Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Pro Arg Ala Ala Pro Arg Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 183 <211> 119 <212> PRT <213> artificial sequence <220> <223> TAR2h-76 anti-TNFR1 dAb <400> 183 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Ala Ser 20 25 30 Glu Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Pro Ser Gly Ser Ala Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Trp Thr Pro Gly Arg Thr Thr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 184 <211> 124 <212> PRT <213> artificial sequence <220> <223> TAR2h-77 anti-TNFR1 dAb <400> 184 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Pro Thr Glu 20 25 30 His Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Asp Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Leu His Trp Ser Ser Asp Ser Gly Pro Val His Phe Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 185 <211> 123 <212> PRT <213> artificial sequence <220> <223> TAR2h-78 anti-TNFR1 dAb <400> 185 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Lys Leu Tyr 20 25 30 Asn Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Ala Ala Ala Gly Pro Glu Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Gly Asp Ile Ser Ser Ile Pro Gln His Pro Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 186 <211> 119 <212> PRT <213> artificial sequence <220> <223> TAR2h-79 anti-TNFR1 dAb <400> 186 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Asn Val 20 25 30 Asp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Ser Ala Gly Gly Glu Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Ala Asp Ile Thr Lys Gly Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 187 <211> 121 <212> PRT <213> artificial sequence <220> <223> TAR2h-15 anti-TNFR1 dAb <400> 187 Glu Val Arg Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Lys Tyr 20 25 30 Thr Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Ser Asp Asp Gly Asn Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Pro Ile Leu Ala Pro Arg Asn Leu Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 188 <211> 119 <212> PRT <213> artificial sequence <220> <223> TAR2h-131-8 anti-TNFR1 dAb <400> 188 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 189 <211> 119 <212> PRT <213> artificial sequence <220> <223> TAR2h-131-24 anti-TNFR1 dAb <400> 189 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 190 <211> 121 <212> PRT <213> artificial sequence <220> <223> TAR2h-15-8 anti-TNFR1 dAb <400> 190 Glu Val Arg Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Gly Lys Ser 20 25 30 Thr Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Ser Asp Asp Gly Asn Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Pro Ile Leu Ala Pro Arg Asn Leu Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 191 <211> 121 <212> PRT <213> artificial sequence <220> <223> TAR2h-15-8-1 anti-TNFR1 dAb <400> 191 Glu Val Arg Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Asn Phe Gly Lys Ser 20 25 30 Thr Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Ser Asp Asp Gly Asn Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Pro Ile Leu Ala Pro Arg Asn Leu Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 192 <211> 121 <212> PRT <213> artificial sequence <220> <223> TAR2h-15-8-2 anti-TNFR1 dAb <400> 192 Glu Val Arg Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Gly Lys Gly 20 25 30 Thr Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Ser Asp Asp Gly Asn Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Pro Ile Leu Ala Pro Arg Asn Leu Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 193 <211> 122 <212> PRT <213> artificial sequence <220> <223> TAR2h-185-23 anti-TNFR1 dAb <220> <221> VARIANT <222> (62)...(62) <223> Xaa = any amino acid <400> 193 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Arg Tyr 20 25 30 Asn Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Leu Ile Asp Pro Ser Gly Gly His Thr Tyr Tyr Ala Xaa Ser Val 50 55 60 Lys Gly Arg Ser Thr Ile Ser Arg Asn Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Lys Pro Val Phe Ser Asp Trp Pro Ala Val Glu Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 194 <211> 119 <212> PRT <213> artificial sequence <220> <223> TAR2h-154-10-5 anti-TNFR1 dAb <400> 194 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Met Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Pro Gly Phe Thr Phe Glu His Glu 20 25 30 Gly Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Gly Glu Asp Gly Gln Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Ile Pro Lys Ala Gly Pro Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 195 <211> 122 <212> PRT <213> artificial sequence <220> <223> TAR2h-14-2 anti-TNFR1 dAb <400> 195 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Asp Gln Tyr 20 25 30 Asp Met Ser Trp Val Arg Arg Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Leu Ile Asp Pro Ser Gly Gly His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asn Asn Thr Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Val Phe Ser Asp Trp Pro Ala Val Glu Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 196 <211> 121 <212> PRT <213> artificial sequence <220> <223> TAR2h-151-8 anti-TNFR1 dAb <400> 196 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Tyr Gly 20 25 30 Asn Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Met Thr Thr Asp Ser Pro Pro Gly Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 197 <211> 122 <212> PRT <213> artificial sequence <220> <223> TAR2h-152-7 anti-TNFR1 dAb <400> 197 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Lys Glu 20 25 30 Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Trp Ile Ser Pro His Gly Ala His Thr Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Arg Phe Ser Tyr Tyr Pro Arg Val Ser Phe Asp Tyr Arg 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 198 <211> 116 <212> PRT <213> artificial sequence <220> <223> TAR2h-35-4 anti-TNFR1 dAb <400> 198 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Ala Tyr 20 25 30 Asn Met Phe Trp Phe Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Ala Ile Gly Pro Ser Gly Arg Glu Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Thr Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Arg Tyr Pro Asp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 199 <211> 119 <212> PRT <213> artificial sequence <220> <223> TAR2h-154-7 anti-TNFR1 dAb <400> 199 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu His Glu 20 25 30 Gly Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Gly Glu Asp Gly Gln Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Ile Pro Lys Ala Gly Pro Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 200 <211> 123 <212> PRT <213> artificial sequence <220> <223> TAR2h-80 anti-TNFR1 dAb <400> 200 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Lys Leu Tyr 20 25 30 Asn Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Ala Ala Ala Gly Pro Glu Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Gly Asp Ile Ser Ser Ile Pro Gln His Pro Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 201 <211> 119 <212> PRT <213> artificial sequence <220> <223> TAR2h-81 anti-TNFR1 dAb <400> 201 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Glu 20 25 30 Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Gly Pro Arg Gly Met Pro Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Met Asn Ser His Asp Gly Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 202 <211> 121 <212> PRT <213> artificial sequence <220> <223> TAR2h-82 anti-TNFR1 dAb <400> 202 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Ala Ser 20 25 30 Glu Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Pro Ser Gly Ser Ala Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Arg Met Leu Ala Asn Ser Pro Leu Ala Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 203 <211> 123 <212> PRT <213> artificial sequence <220> <223> TAR2h-83 anti-TNFR1 dAb <400> 203 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr 20 25 30 Asn Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Ala Gln Ser Gly Gly His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Phe Ser His Pro Asp Glu Glu Gly Thr Gln Met Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 204 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-84 anti-TNFR1 dAb <400> 204 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Asp Tyr 20 25 30 Gln Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Arg Ile Asp Arg Gly Gly Phe His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Ser Trp His Ala Asp Gln Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 205 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-85 anti-TNFR1 dAb <400> 205 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Lys Asp Tyr 20 25 30 Asn Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ala Thr Ser Gly Arg Glu Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Phe Thr Phe Gly Gly Asn Gln Asp Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 206 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-86 anti-TNFR1 dAb <400> 206 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Lys Tyr 20 25 30 Asn Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Pro Lys Gly Gln His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Glu Gly Met Gly Ser Asp Ala Ile Thr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 207 <211> 123 <212> PRT <213> artificial sequence <220> <223> TAR2h-87 anti-TNFR1 dAb <400> 207 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr 20 25 30 Asn Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Ala Gln Ser Gly Gly His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Phe Ser His Pro Asp Glu Glu Gly Thr Gln Met Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 208 <211> 122 <212> PRT <213> artificial sequence <220> <223> TAR2h-88 anti-TNFR1 dAb <400> 208 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Arg Tyr 20 25 30 Asp Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Pro Arg Gly Arg Glu Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Met Ile Asn Tyr His Gly Thr Pro Ser Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 209 <211> 122 <212> PRT <213> artificial sequence <220> <223> TAR2h-89 anti-TNFR1 dAb <220> <221> VARIANT <222> (30)...(30) <223> Xaa = Any Amino Acid <400> 209 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Xaa Asn Tyr 20 25 30 Asn Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Trp Ile Ser Gly Ala Gly His Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Val Asp Met Ala Gly Lys Leu Asn Val Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 210 <211> 122 <212> PRT <213> artificial sequence <220> <223> TAR2h-90 anti-TNFR1 dAb <400> 210 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Lys Gln Tyr 20 25 30 Asn Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Ser Pro Ser Gly Gly Glu Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Thr Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Val Asp Met Ala Gly Lys Leu Asn Val Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 211 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-91 anti-TNFR1 dAb <400> 211 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Asp Tyr 20 25 30 Gln Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Arg Ile Asp Arg Gly Gly Phe His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Ser Trp His Ala Asp Gln Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 212 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-92 anti-TNFR1 dAb <400> 212 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Val 20 25 30 Asn Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Gly Pro Ser Gly Thr Glu Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys His Ser Lys Thr Gly Ser Ala Met Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 213 <211> 119 <212> PRT <213> artificial sequence <220> <223> TAR2h-93 anti-TNFR1 dAb <400> 213 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Asn Gly 20 25 30 Asn Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Glu Tyr Gly Thr Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Arg Asn Asp Arg Pro Gly Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 214 <211> 119 <212> PRT <213> artificial sequence <220> <223> TAR2h-94 anti-TNFR1 dAb <400> 214 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Glu 20 25 30 Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Gly Pro Arg Gly Met Pro Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Met Asn Ser His Asp Gly Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 215 <211> 117 <212> PRT <213> artificial sequence <220> <223> TAR2h-95 anti-TNFR1 dAb <400> 215 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Lys Gly Ser 20 25 30 Asn Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Leu Ile Asp Gly Arg Gly Gln His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Ser Val Arg Glu Phe Asp Tyr Arg Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 216 <211> 121 <212> PRT <213> artificial sequence <220> <223> TAR2h-96 anti-TNFR1 dAb <400> 216 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Glu 20 25 30 Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Gly Pro Arg Gly Met Pro Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Arg Met Leu Ala Asn Ser Pro Leu Ala Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 217 <211> 116 <212> PRT <213> artificial sequence <220> <223> TAR2h-97 anti-TNFR1 dAb <400> 217 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Glu Ser 20 25 30 Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Thr Ala Gln Gly Gly Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Asp Val Leu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 218 <211> 117 <212> PRT <213> artificial sequence <220> <223> TAR2h-99 anti-TNFR1 dAb <400> 218 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Glu Tyr 20 25 30 Asn Met Leu Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Gly Pro Ser Gly Arg Glu Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Ser Ile Thr Leu Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 219 <211> 124 <212> PRT <213> artificial sequence <220> <223> TAR2h-100 anti-TNFR1 dAb <400> 219 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Asn Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Asp Ala Tyr Gly Thr His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Glu Gly Leu Gln Thr Ser Asp His Gly Glu Arg Ile Ser Phe Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 220 <211> 122 <212> PRT <213> artificial sequence <220> <223> TAR2h-101 anti-TNFR1 dAb <400> 220 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Gln Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Leu Ile Asp Pro Ser Gly Gly His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asn Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Val Phe Ser Asp Trp Pro Ala Val Glu Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 221 <211> 122 <212> PRT <213> artificial sequence <220> <223> TAR2h-102 anti-TNFR1 dAb <400> 221 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Gln Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Leu Ile Asp Pro Ser Gly Gly His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asn Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Val Phe Ser Asp Trp Pro Ala Val Glu Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 222 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-103 anti-TNFR1 dAb <400> 222 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Arg Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Met Ile Asp Val Pro Gly Leu His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ile Ser Gln Phe Gly Ser Asn Ala Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 223 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-104 anti-TNFR1 dAb <220> <221> VARIANT <222> (62)...(62) <223> Xaa = Any Amino Acid <220> <221> VARIANT <222> (104)... (104) <223> Xaa = Any Amino Acid <400> 223 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Xaa Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Ala Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Xaa Pro Asn Phe Gly Tyr Arg Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 224 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-105 anti-TNFR1 dAb <400> 224 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Arg Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Asp Pro Pro Ser Val His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ile Ser Gln Phe Gly Ser Asn Ala Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 225 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-106 anti-TNFR1 dAb <220> <221> VARIANT <222> (62)...(62) <223> Xaa = Any Amino Acid <400> 225 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Arg Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Met Ile Asp Val Gly Gly Ser His Thr Tyr Tyr Ala Xaa Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ile Ser Gln Phe Gly Ser Asn Ala Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 226 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-107 anti-TNFR1 dAb <220> <221> VARIANT <222> (62)...(62) <223> Xaa = Any Amino Acid <400> 226 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Arg Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Met Ile Asp Thr Gly Gly Val His Thr Tyr Tyr Ala Xaa Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ile Ser Gln Phe Gly Ser Asn Ala Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 227 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-108 anti-TNFR1 dAb <400> 227 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Arg Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Met Ile Asp Val Pro Gly Arg His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ile Ser Gln Phe Gly Ser Asn Ala Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 228 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-109 anti-TNFR1 dAb <400> 228 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Arg Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Met Ile Ala His Ala Gly Pro Glu Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ile Ser Gln Phe Gly Ser Asn Ala Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 229 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-110 anti-TNFR1 dAb <400> 229 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Arg Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Met Ile Asp Thr Arg Gly Val Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ile Ser Gln Phe Gly Ser Asn Ala Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 230 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-111 anti-TNFR1 dAb <400> 230 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Arg Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Met Ile Asp Val Pro Gly Asn His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ile Ser Gln Phe Gly Ser Asn Ala Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 231 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-112 anti-TNFR1 dAb <400> 231 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Arg Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Met Ile Asp Val Gly Gly Arg His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ile Ser Gln Phe Gly Pro Asn Ala Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 232 <211> 118 <212> PRT <213> artificial sequence <220> <223> TAR2h-113 anti-TNFR1 dAb <400> 232 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Arg Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Arg Ile Asp Ser Tyr Gly Arg Gly Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Arg Ser Pro Tyr Thr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 233 <211> 117 <212> PRT <213> artificial sequence <220> <223> TAR2h-114 anti-TNFR1 dAb <400> 233 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Asn Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Ser Thr Gln Gly Tyr His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ala Phe Thr Ser Asp Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 234 <211> 119 <212> PRT <213> artificial sequence <220> <223> TAR2h-115 anti-TNFR1 dAb <400> 234 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Asn Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Gly Pro Gly Leu Glu Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gln Gly Met Ser Lys Thr Ser Thr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 235 <211> 117 <212> PRT <213> artificial sequence <220> <223> TAR2h-116 anti-TNFR1 dAb <400> 235 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Glu Tyr 20 25 30 Tyr Met Glu Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Asp Pro Asp Gly Ser Leu Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro Arg Glu Lys Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 236 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-117 anti-TNFR1 dAb <400> 236 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Lys Tyr 20 25 30 Gln Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Asp Ser Asn Gly His His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gln Leu Ser Val Gln Gly Ser Asn Leu Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 237 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-118 anti-TNFR1 dAb <400> 237 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val His Tyr 20 25 30 Thr Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Trp Ile His Ser Asp Gly Val His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Phe Thr Trp Gly Glu Lys Lys Thr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 238 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-119 anti-TNFR1 dAb <400> 238 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Met Gly Tyr 20 25 30 Asp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Ala Lys Gly Thr Glu Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Ser Ser Gly Ser Asp Gly Leu Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 239 <211> 124 <212> PRT <213> artificial sequence <220> <223> TAR2h-120 anti-TNFR1 dAb <400> 239 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Pro Val Tyr 20 25 30 Asn Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Ala Gly Asn Gly Gln Gln Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Phe Ala Ser Lys Val Ser Pro Met Ser Leu Thr Asp Phe Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 240 <211> 124 <212> PRT <213> artificial sequence <220> <223> TAR2h-121 anti-TNFR1 dAb <400> 240 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Gln Tyr 20 25 30 Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Ser Gly Gly Met Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Ile Arg Asp Ser Thr Leu Pro Arg Gly Thr Leu Phe Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 241 <211> 121 <212> PRT <213> artificial sequence <220> <223> TAR2h-122 anti-TNFR1 dAb <400> 241 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Thr Tyr 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Leu Pro Gly Ser Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys His Ser Lys Ser Ser His Arg Gln Ser Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 242 <211> 122 <212> PRT <213> artificial sequence <220> <223> TAR2h-123 anti-TNFR1 dAb <400> 242 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Gln Tyr 20 25 30 Asp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Phe Ser Gly Tyr Glu Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Arg Gly Pro Ala Pro Met Arg Ser Leu Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 243 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-124 anti-TNFR1 dAb <400> 243 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Asp Tyr 20 25 30 Pro Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Thr Ser Met Gly Glu Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Met Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Pro Thr His Phe Pro Ile Arg Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 244 <211> 116 <212> PRT <213> artificial sequence <220> <223> TAR2h-125 anti-TNFR1 dAb <400> 244 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Lys Gln Tyr 20 25 30 Asn Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Ser Pro Ser Gly Gly Glu Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Ile Lys Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 245 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-126 anti-TNFR1 dAb <400> 245 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Met Tyr 20 25 30 Ser Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Asp Phe Asp Gly Leu His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Phe Ser Thr Ser Thr Met Ala Leu Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 246 <211> 122 <212> PRT <213> artificial sequence <220> <223> TAR2h-127 anti-TNFR1 dAb <400> 246 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Arg Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Pro Glu Tyr 20 25 30 Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Gly Thr Ala Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Met Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Tyr Arg Pro Arg Thr Gly Ser Met Leu Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 247 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-128 anti-TNFR1 dAb <400> 247 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Lys Tyr 20 25 30 Asn Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Pro Lys Gly Gln Gln Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Glu Gly Met Gly Ser Asp Ala Ile Thr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 248 <211> 122 <212> PRT <213> artificial sequence <220> <223> TAR2h-129 anti-TNFR1 dAb <400> 248 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Asp Tyr 20 25 30 Asp Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Asp Arg Lys Gly His His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Thr Thr Asp Ile Gln Arg Leu Asn Ser Ala Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 249 <211> 119 <212> PRT <213> artificial sequence <220> <223> TAR2h-130 anti-TNFR1 dAb <400> 249 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Asn Gly 20 25 30 Val Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asn Glu Asn Gly Gly Ala Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Ser Ile Glu Ser Pro Ile Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 250 <211> 119 <212> PRT <213> artificial sequence <220> <223> TAR2h-131 anti-TNFR1 dAb <400> 250 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Pro Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Pro Lys Arg Gly Pro Arg Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 251 <211> 121 <212> PRT <213> artificial sequence <220> <223> TAR2h-132 anti-TNFR1 dAb <400> 251 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Glu Ser 20 25 30 Val Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Pro Gly Gly Ser Glu Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Ala Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Arg Thr Gly Pro Pro Gly Ser Thr Val Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 252 <211> 116 <212> PRT <213> artificial sequence <220> <223> TAR2h-133 anti-TNFR1 dAb <400> 252 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Asp Glu 20 25 30 Pro Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Gly Lys Glu Gly Gln Pro Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Gly Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 253 <211> 121 <212> PRT <213> artificial sequence <220> <223> TAR2h-151 anti-TNFR1 dAb <400> 253 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Tyr Gly 20 25 30 Asn Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Met Thr Thr Asp Ser Pro Pro Gly Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 254 <211> 122 <212> PRT <213> artificial sequence <220> <223> TAR2h-152 anti-TNFR1 dAb <400> 254 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Lys Glu 20 25 30 Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Trp Ile Ser Pro His Gly Ala Leu Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Arg Phe Ser Tyr Tyr Pro Arg Val Ser Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 255 <211> 119 <212> PRT <213> artificial sequence <220> <223> TAR2h-153 anti-TNFR1 dAb <400> 255 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Asn Gly 20 25 30 Asn Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Glu Tyr Gly Thr Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Arg Asn Asp Arg Pro Gly Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 256 <211> 119 <212> PRT <213> artificial sequence <220> <223> TAR2h-154 anti-TNFR1 dAb <220> <221> VARIANT <222> (53)...(53) <223> Xaa = Any Amino Acid <400> 256 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Asn Gly 20 25 30 Asn Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Xaa Tyr Gly Thr Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Arg Asn Asp Arg Pro Gly Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 257 <211> 124 <212> PRT <213> artificial sequence <220> <223> TAR2h-159 anti-TNFR1 dAb <400> 257 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Gly Gln 20 25 30 Asp Met Arg Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Pro Ser Ser Gly Phe Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Arg Ala Lys Asp Arg Ser Val Ser Gln Met Pro Tyr Phe Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 258 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-165 anti-TNFR1 dAb <400> 258 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Met Arg Pro 20 25 30 Asp Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Lys Asp Trp Gly Asp Gln Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Ala Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ala Asp Ser Arg Ala Gln Leu Asp Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 259 <211> 121 <212> PRT <213> artificial sequence <220> <223> TAR2h-166 anti-TNFR1 dAb <400> 259 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Tyr Phe Leu Phe Arg Ala Thr Ser Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 260 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-168 anti-TNFR1 dAb <400> 260 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe His Asp Asp 20 25 30 Asp Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Pro Gly Asn Gly Tyr Val Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Arg Pro Asp Pro Thr Ser Val Phe Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 261 <211> 116 <212> PRT <213> artificial sequence <220> <223> TAR2h-171 anti-TNFR1 dAb <400> 261 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Asp Asp 20 25 30 Trp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ala Ala Tyr Gly Ile Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Ala Ala Val Tyr Tyr Cys 85 90 95 Ala Glu Ser Gly Lys Val Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 262 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-172 anti-TNFR1 dAb <400> 262 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Glu Arg 20 25 30 Pro Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Leu Ile Gly Ala Asp Gly Leu Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Phe Arg Pro Gly Leu Leu Trp Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 263 <211> 119 <212> PRT <213> artificial sequence <220> <223> TAR2h-173 anti-TNFR1 dAb <400> 263 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Gly Gln 20 25 30 Asp Met Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Asn Ala Asp Gly Met Ala Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Thr Ser Pro Thr Met Arg Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 264 <211> 118 <212> PRT <213> artificial sequence <220> <223> TAR2h-174 anti-TNFR1 dAb <400> 264 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Glu Glu 20 25 30 Tyr Met Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Leu Ile Pro His Thr Gly Asn Pro Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Ala Asn Ser Leu Leu Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 265 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-176 anti-TNFR1 dAb <400> 265 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe His Arg Cys 20 25 30 Lys Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Glu Tyr Asp Gly Arg Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Glu Cys Thr Arg Pro Tyr Gly Met Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 266 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-178 anti-TNFR1 dAb <400> 266 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Arg Tyr 20 25 30 Ser Met Gly Trp Leu Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Asp Lys Val Gly His His Thr Trp Tyr Glu Asp Pro Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ile Ser Gln Phe Gly Pro Asn Ala Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 267 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-201 anti-TNFR1 dAb <220> <221> VARIANT <222> (101)...(101) <223> Xaa = Any Amino Acid <400> 267 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Arg Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Met Ile Ala His Ala Gly Pro Glu Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ile Ser Xaa Phe Gly Ser Asn Ala Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 268 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-202 anti-TNFR1 dAb <400> 268 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Arg Tyr 20 25 30 Asn Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Asp Pro Pro Ser Val His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Ile Ser Gln Phe Gly Ser Asn Ala Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 269 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-203 anti-TNFR1 dAb <220> <221> VARIANT <222> (96)...(96) <223> Xaa = Any Amino Acid <400> 269 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Arg Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Asp Pro Pro Ser Val His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Xaa 85 90 95 Ala Glu Ile Ser Gln Phe Gly Ser Asn Ala Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 270 <211> 120 <212> PRT <213> artificial sequence <220> <223> TAR2h-204 anti-TNFR1 dAb <400> 270 Glu Val Gln Leu Phe Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Arg Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Met Ile Ala His Ala Gly Pro Glu Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ile Ser Gln Phe Gly Ser Asn Ala Leu Asp Tyr Trp Gly Arg 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 271 <211> 122 <212> PRT <213> artificial sequence <220> <223> TAR2h-185-25 anti-TNFR1 dAb <400> 271 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Arg Tyr 20 25 30 Asn Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Leu Ile Asp Pro Ser Gly Gly His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asn Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Lys Pro Val Phe Ser Asp Trp Pro Ala Val Glu Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Val Val Thr Val Ser Ser 115 120 <210> 272 <211> 119 <212> PRT <213> artificial sequence <220> <223> TAR2h-154-10 anti-TNFR1 dAb <400> 272 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Met Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu His Glu 20 25 30 Gly Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Gly Glu Asp Gly Gln Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Ile Pro Lys Ala Gly Pro Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 273 <211> 119 <212> PRT <213> artificial sequence <220> <223> TAR2h-205 anti-TNFR1 dAb <400> 273 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Gly His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Thr Gly Arg Trp Glu Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 274 <211> 119 <212> PRT <213> artificial sequence <220> <223> TAR2h-5d1 anti-TNFR1 dAb <400> 274 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Pro Val Tyr 20 25 30 Met Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Asp Ala Leu Gly Gly Arg Thr Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Thr Met Ser Asn Lys Thr His Thr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser 115 <210> 275 <211> 114 <212> PRT <213> artificial sequence <220> <223> TAR2h-5d2 anti-TNFR1 dAb <400> 275 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Ala Tyr 20 25 30 Asn Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Asn Thr Phe Gly Asn Thr Arg Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Gly Ser Arg Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser <210> 276 <211> 118 <212> PRT <213> artificial sequence <220> <223> TAR2h-5d3 anti-TNFR1 dAb <400> 276 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Tyr Arg 20 25 30 Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 35 40 45 Trp Ile Thr Arg Thr Gly Gly Thr Thr Gln Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Pro Ala Lys Leu Val Gly Val Gly Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser 115 <210> 277 <211> 118 <212> PRT <213> artificial sequence <220> <223> TAR2h-5d4 anti-TNFR1 dAb <400> 277 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Lys Tyr 20 25 30 Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 35 40 45 Gln Ile Gly Ala Lys Gly Gln Ser Thr Asp Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Lys Lys Arg Gly Glu Asn Tyr Phe Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser 115 <210> 278 <211> 119 <212> PRT <213> artificial sequence <220> <223> TAR2h-5d5 anti-TNFR1 dAb <400> 278 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Arg Tyr 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Asp Ile Ser Arg Ser Gly Arg Tyr Thr His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Arg Ile Asp Ser Ser Gln Asn Gly Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser 115 <210> 279 <211> 114 <212> PRT <213> artificial sequence <220> <223> TAR2h-5d6 anti-TNFR1 dAb <400> 279 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Tyr Lys 20 25 30 Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 35 40 45 Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Gln Lys Glu Asn Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser <210> 280 <211> 119 <212> PRT <213> artificial sequence <220> <223> TAR2h-5d7 anti-TNFR1 dAb <400> 280 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Asp Tyr 20 25 30 Ala Met Trp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Ser Ser Asn Gly Gly Ser Thr Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Arg Val Arg Lys Arg Thr Pro Glu Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser 115 <210> 281 <211> 119 <212> PRT <213> artificial sequence <220> <223> TAR2h-5d8 anti-TNFR1 dAb <400> 281 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Arg Tyr 20 25 30 Lys Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Gly Arg Asn Gly Thr Lys Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ile Tyr Thr Gly Lys Pro Ala Ala Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser 115 <210> 282 <211> 118 <212> PRT <213> artificial sequence <220> <223> TAR2h-5d9 anti-TNFR1 dAb <400> 282 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Lys Lys Tyr 20 25 30 Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 35 40 45 Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Met Leu Arg Thr Lys Asn Lys Val Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser 115 <210> 283 <211> 119 <212> PRT <213> artificial sequence <220> <223> TAR2h-5d10 anti-TNFR1 dAb <400> 283 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Arg Tyr 20 25 30 Lys Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Gly Arg Asn Gly Thr Lys Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ile Tyr Thr Gly Lys Pro Ala Ala Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser 115 <210> 284 <211> 118 <212> PRT <213> artificial sequence <220> <223> TAR2h-5d11 anti-TNFR1 dAb <400> 284 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Arg 20 25 30 Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 35 40 45 Ser Ile Ser Ser Arg Gly Arg His Thr Ser Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Arg Val Pro Gly Arg Gly Arg Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser 115 <210> 285 <211> 119 <212> PRT <213> artificial sequence <220> <223> TAR2h-5d12 anti-TNFR1 dAb <400> 285 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Arg Arg Tyr 20 25 30 Arg Met Arg Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Pro Gly Gly Lys His Thr Thr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Glu Gly Gly Ala Ser Ser Ala Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser 115 <210> 286 <211> 118 <212> PRT <213> artificial sequence <220> <223> TAR2h-5d13 anti-TNFR1 dAb <400> 286 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Tyr Gly 20 25 30 Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 35 40 45 Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Arg His Ser Ser Glu Ala Arg Gln Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser 115 <210> 287 <211> 118 <212> PRT <213> artificial sequence <220> <223> TAR2h-5d1 variant anti-TNFR1 dAb <400> 287 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Pro Val Tyr 20 25 30 Met Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Asp Ala Leu Gly Gly Arg Thr Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Thr Met Ser Asn Lys Thr His Thr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Ser 115 <210> 288 <211> 115 <212> PRT <213> artificial sequence <220> <223> TAR2h-5d2 variant anti-TNFR1 dAb <220> <221> VARIANT <222> (57)...(57) <223> Xaa = Any Amino Acid <400> 288 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Ala Tyr 20 25 30 Asn Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Asn Thr Phe Gly Asn Xaa Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Ser Arg Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser 115 <210> 289 <211> 119 <212> PRT <213> artificial sequence <220> <223> TAR2h-5d3 variant anti-TNFR1 dAb <220> <221> VARIANT <222> (30)...(30) <223> Xaa = Any Amino Acid <400> 289 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Xaa Gly Tyr 20 25 30 Arg Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Trp Ile Thr Arg Thr Gly Gly Thr Thr Gln Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Ala Lys Leu Val Gly Val Gly Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser 115 <210> 290 <211> 119 <212> PRT <213> artificial sequence <220> <223> TAR2h-5d4 variant anti-TNFR1 dAb <220> <221> VARIANT <222> (33)...(33) <223> Xaa = Any Amino Acid <400> 290 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Lys Tyr 20 25 30 Xaa Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Gly Ala Lys Gly Gln Ser Thr Asp Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Lys Lys Arg Gly Glu Asn Tyr Phe Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser 115 <210> 291 <211> 115 <212> PRT <213> artificial sequence <220> <223> TAR2h-5d6 variant anti-TNFR1 dAb <220> <221> VARIANT <222> (30)...(30) <223> Xaa = Any Amino Acid <400> 291 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Xaa Gly Tyr 20 25 30 Lys Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gln Lys Glu Asn Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser 115 <210> 292 <211> 119 <212> PRT <213> artificial sequence <220> <223> TAR2h-5d9 variant anti-TNFR1 dAb <220> <221> VARIANT <222> (33)...(33) <223> Xaa = Any Amino Acid <400> 292 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Lys Lys Tyr 20 25 30 Xaa Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Met Leu Arg Thr Lys Asn Lys Val Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser 115 <210> 293 <211> 119 <212> PRT <213> artificial sequence <220> <223> TAR2h-5d11 variant anti-TNFR1 dAb <220> <221> VARIANT <222> (30)...(30) <223> Xaa = Any Amino Acid <400> 293 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Xaa Ser Tyr 20 25 30 Arg Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Arg Gly Arg His Thr Ser Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Arg Val Pro Gly Arg Gly Arg Ser Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser 115 <210> 294 <211> 119 <212> PRT <213> artificial sequence <220> <223> TAR2h-5d13 variant anti-TNFR1 dAb <220> <221> VARIANT <222> (30)...(30) <223> Xaa = Any Amino Acid <400> 294 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Xaa Arg Tyr 20 25 30 Gly Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Arg His Ser Ser Glu Ala Arg Gln Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser 115 <210> 295 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-1 anti-TNFR1 dAb <400> 295 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Pro Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Tyr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Leu Pro Lys Arg Gly Pro Arg Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 296 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-2 anti-TNFR1 dAb <400> 296 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Pro Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Arg Phe Asp Tyr Trp Gly His Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 297 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-3 anti-TNFR1 dAb <400> 297 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Pro Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Leu Pro Lys Arg Gly Pro Arg Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 298 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-4 anti-TNFR1 dAb <400> 298 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Pro Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Arg Phe Asp Tyr Trp Gly Leu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 299 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-5 anti-TNFR1 dAb <400> 299 Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Pro Met Val Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Tyr Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Arg Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 300 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-6 anti-TNFR1 dAb <400> 300 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Pro Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Arg Phe Asp Tyr Trp Gly Leu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 301 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-7 anti-TNFR1 dAb <400> 301 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Pro Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Ala Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Arg Phe Asp Tyr Trp Gly Leu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 302 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-9 anti-TNFR1 dAb <400> 302 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Arg Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Pro Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Arg Phe Asp Tyr Trp Gly Leu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 303 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-10 anti-TNFR1 dAb <400> 303 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Pro Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Arg Phe Asp Tyr Trp Gly Leu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 304 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-11 anti-TNFR1 dAb <400> 304 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Pro Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Pro Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Arg Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 305 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-12 anti-TNFR1 dAb <400> 305 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Pro Met Val Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Leu Pro Lys Arg Gly Pro Arg Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 306 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-13 anti-TNFR1 dAb <400> 306 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Pro Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Pro Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Arg Phe Asp Tyr Trp Gly Leu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 307 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-14 anti-TNFR1 dAb <400> 307 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Pro Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Ala Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Leu Pro Lys Arg Gly Pro Arg Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 308 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-15 anti-TNFR1 dAb <400> 308 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Pro Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Ser Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Arg Phe Asp Tyr Trp Gly Leu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 309 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-16 anti-TNFR1 dAb <400> 309 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Pro Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Pro Pro Lys Arg Gly Pro Arg Phe Asp Tyr Trp Gly Leu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 310 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-17 anti-TNFR1 dAb <400> 310 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Pro Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Lys Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Arg Phe Asp Tyr Trp Gly Leu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 311 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-18 anti-TNFR1 dAb <400> 311 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Pro Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Ala 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Arg Phe Asp Tyr Trp Gly Leu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 312 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-19 anti-TNFR1 dAb <400> 312 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Pro Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Pro Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Arg Phe Asp Tyr Trp Gly Leu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 313 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-20 anti-TNFR1 dAb <400> 313 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Pro Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Arg Phe Asp Tyr Trp Gly Leu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 314 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-21 anti-TNFR1 dAb <400> 314 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Pro Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Tyr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Met Leu Pro Lys Arg Gly Pro Arg Phe Gly Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 315 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-22 anti-TNFR1 dAb <400> 315 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Pro Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Cys Ala Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Arg Phe Asp Tyr Trp Gly Leu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 316 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-23 anti-TNFR1 dAb <400> 316 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 317 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-25 anti-TNFR1 dAb <400> 317 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gln Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 318 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-26 anti-TNFR1 dAb <400> 318 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 319 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-27 anti-TNFR1 dAb <400> 319 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Tyr Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 320 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-28 anti-TNFR1 dAb <400> 320 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Gly Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 321 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-29 anti-TNFR1 dAb <220> <221> VARIANT <222> (12)...(12) <223> Xaa = Any Amino Acid <400> 321 Glu Gly Gln Leu Leu Glu Ser Gly Gly Gly Leu Xaa Gln Pro Gly Gly 1 5 10 15 Ser Leu Leu Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 322 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-30 anti-TNFR1 dAb <400> 322 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 323 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-31 anti-TNFR1 dAb <400> 323 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Pro Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 324 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-32 anti-TNFR1 dAb <400> 324 Glu Gly Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Gly Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Pro Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 325 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-33 anti-TNFR1 dAb <400> 325 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Asn Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 326 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-34 anti-TNFR1 dAb <400> 326 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Pro Lys Arg Gly Pro Gly Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 327 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-35 anti-TNFR1 dAb <400> 327 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Pro Lys Arg Gly Pro Val Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 328 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-36 anti-TNFR1 dAb <400> 328 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Tyr Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 329 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-37 anti-TNFR1 dAb <400> 329 Glu Gly Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Leu Leu Ser Cys Ala Pro Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Lys Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 330 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-38 anti-TNFR1 dAb <400> 330 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Val Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 331 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-39 anti-TNFR1 dAb <400> 331 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Leu Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 332 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-40 anti-TNFR1 dAb <400> 332 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Pro Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Leu Pro Lys Arg Gly Pro Arg Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 333 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-41 anti-TNFR1 dAb <400> 333 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Pro Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Leu Pro Lys Arg Gly Pro Arg Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 334 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-42 anti-TNFR1 dAb <400> 334 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Pro Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Arg Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 335 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-43 anti-TNFR1 dAb <400> 335 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Pro Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Gly Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Pro Leu Pro Lys Arg Gly Pro Arg Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 336 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-44 anti-TNFR1 dAb <400> 336 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Pro Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Leu Pro Lys Arg Gly Pro Arg Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 337 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-45 anti-TNFR1 dAb <400> 337 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Pro Met Val Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Met Leu Pro Lys Arg Gly Pro Arg Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 338 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-46 anti-TNFR1 dAb <400> 338 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Pro Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Arg Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 339 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-47 anti-TNFR1 dAb <220> <221> VARIANT <222> (32)...(32) <223> Xaa = Any Amino Acid <400> 339 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Xaa 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 340 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-48 anti-TNFR1 dAb <220> <221> VARIANT <222> (32)...(32) <223> Xaa = Any Amino Acid <400> 340 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Xaa 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Ile Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 341 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-49 anti-TNFR1 dAb <400> 341 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Asn Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 342 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-50 anti-TNFR1 dAb <400> 342 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Val Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 343 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-51 anti-TNFR1 dAb <400> 343 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Leu Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 344 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-52 anti-TNFR1 dAb <400> 344 Glu Val Gln Leu Leu Glu Ser Gly Gly Asn Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 345 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-53 anti-TNFR1 dAb <400> 345 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 346 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-54 anti-TNFR1 dAb <400> 346 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Lys 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 347 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-55 anti-TNFR1 dAb <400> 347 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Pro Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Ala Thr Val Ser Ser 115 <210> 348 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-56 anti-TNFR1 dAb <400> 348 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 349 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-57 anti-TNFR1 dAb <400> 349 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 350 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-58 anti-TNFR1 dAb <400> 350 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Asp 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Gln Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 351 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-59 anti-TNFR1 dAb <400> 351 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Asp 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 352 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-60 anti-TNFR1 dAb <400> 352 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 353 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-61 anti-TNFR1 dAb <400> 353 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 354 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-62 anti-TNFR1 dAb <400> 354 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 355 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-63 anti-TNFR1 dAb <400> 355 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Gln Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 356 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-64 anti-TNFR1 dAb <400> 356 Glu Val Gln Leu Ser Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 357 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-65 anti-TNFR1 dAb <400> 357 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 358 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-66 anti-TNFR1 dAb <400> 358 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Met Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 359 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-67 anti-TNFR1 dAb <400> 359 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Ser Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 360 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-68 anti-TNFR1 dAb <400> 360 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asn Asn Ser Met Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 361 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-69 anti-TNFR1 dAb <400> 361 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 362 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-70 anti-TNFR1 dAb <400> 362 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Pro Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 363 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-71 anti-TNFR1 dAb <400> 363 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Met Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 364 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-72 anti-TNFR1 dAb <400> 364 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Asp 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 365 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-73 anti-TNFR1 dAb <400> 365 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Glu Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 366 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-74 anti-TNFR1 dAb <400> 366 Asp Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Val Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 367 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-75 anti-TNFR1 dAb <400> 367 Glu Val Gln Leu Met Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Glu Leu Pro Lys Arg Gly Pro Trp Phe Asp His Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 368 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-76 anti-TNFR1 dAb <400> 368 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Ala Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Met Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 369 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-77 anti-TNFR1 dAb <400> 369 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Thr Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Met Val Thr Val Ser Ser 115 <210> 370 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-78 anti-TNFR1 dAb <400> 370 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Met Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Leu Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr His Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 371 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-79 anti-TNFR1 dAb <400> 371 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Met Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Gln Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 372 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-80 anti-TNFR1 dAb <400> 372 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Pro Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 373 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-81 anti-TNFR1 dAb <400> 373 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Ser Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 374 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-82 anti-TNFR1 dAb <400> 374 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ala Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 375 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-83 anti-TNFR1 dAb <400> 375 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 376 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-86 anti-TNFR1 dAb <400> 376 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Glu Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 377 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-87 anti-TNFR1 dAb <400> 377 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 378 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-88 anti-TNFR1 dAb <400> 378 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Pro Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 379 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-89 anti-TNFR1 dAb <400> 379 Glu Val Gln Leu Leu Glu Ser Gly Gly Asp Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 380 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-90 anti-TNFR1 dAb <400> 380 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Ser Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 381 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-91 anti-TNFR1 dAb <400> 381 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu His Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Pro Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 382 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-92 anti-TNFR1 dAb <400> 382 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Asp 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 383 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-93 anti-TNFR1 dAb <400> 383 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Pro Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 384 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-94 anti-TNFR1 dAb <400> 384 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Asp Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 385 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-95 anti-TNFR1 dAb <400> 385 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Asp 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 386 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-96 anti-TNFR1 dAb <400> 386 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr His Cys 85 90 95 Ala Val Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 387 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-97 anti-TNFR1 dAb <400> 387 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Glu Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 388 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-99 anti-TNFR1 dAb <400> 388 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile His Pro Gly Gly 1 5 10 15 Thr Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Pro Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 389 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-100 anti-TNFR1 dAb <400> 389 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Pro Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser 115 <210> 390 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-101 anti-TNFR1 dAb <400> 390 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 391 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-102 anti-TNFR1 dAb <400> 391 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 392 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-103 anti-TNFR1 dAb <400> 392 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Pro Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 393 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-104 anti-TNFR1 dAb <400> 393 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Asp Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Asp Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 394 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-105 anti-TNFR1 dAb <400> 394 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Thr Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 395 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-106 anti-TNFR1 dAb <400> 395 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Asp Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Met Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Ile Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 396 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-107 anti-TNFR1 dAb <400> 396 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Asp Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 397 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-108 anti-TNFR1 dAb <400> 397 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Thr Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 398 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-109 anti-TNFR1 dAb <400> 398 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Thr Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Leu Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Thr Ser Arg Asp Asn Ser Arg Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 399 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-110 anti-TNFR1 dAb <400> 399 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Ile Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 400 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-111 anti-TNFR1 dAb <400> 400 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser 115 <210> 401 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-112 anti-TNFR1 dAb <400> 401 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Ile Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 402 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-113 anti-TNFR1 dAb <400> 402 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Asp 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 403 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-114 anti-TNFR1 dAb <400> 403 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Asp 20 25 30 Val Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 404 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-115 anti-TNFR1 dAb <400> 404 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Pro Tyr Tyr Thr Asp Ser Val 50 55 60 Glu Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 405 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-116 anti-TNFR1 dAb <400> 405 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 406 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-117 anti-TNFR1 dAb <400> 406 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Pro Tyr Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 407 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-120 anti-TNFR1 dAb <400> 407 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 408 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-121 anti-TNFR1 dAb <400> 408 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Glu 20 25 30 Thr Met Val Trp Val Arg Arg Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 409 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-122 anti-TNFR1 dAb <400> 409 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Lys Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 410 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-123 anti-TNFR1 dAb <400> 410 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Gly Gly Val Asp Thr Tyr Tyr Ala Asp Pro Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 411 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-124 anti-TNFR1 dAb <400> 411 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Gly Gly Arg Asp Thr Tyr Tyr Ala Asp Pro Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 412 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-125 anti-TNFR1 dAb <400> 412 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Gly Gly Val Asp Thr Tyr Tyr Ala Asp Pro Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 413 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-126 anti-TNFR1 dAb <400> 413 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Gln 20 25 30 Val Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 414 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-127 anti-TNFR1 dAb <400> 414 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Gly Gly Leu Asp Thr Tyr Tyr Ala Asp Pro Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 415 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-128 anti-TNFR1 dAb <400> 415 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Ala Val Gly Ser Asp Thr Tyr Tyr Ala Asp Pro Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 416 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-129 anti-TNFR1 dAb <400> 416 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 417 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-130 anti-TNFR1 dAb <400> 417 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Asn Glu 20 25 30 Val Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Gly 115 <210> 418 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-131 anti-TNFR1 dAb <400> 418 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Gln 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 419 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-132 anti-TNFR1 dAb <400> 419 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asn Thr 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 420 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-136 anti-TNFR1 dAb <400> 420 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Pro Tyr Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 421 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-151 anti-TNFR1 dAb <400> 421 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Gly Gly Val Asp Thr Tyr Tyr Ala Asp Pro Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 422 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-180 anti-TNFR1 dAb <400> 422 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Pro Tyr Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 423 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-181 anti-TNFR1 dAb <400> 423 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Pro Tyr Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 424 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-182 anti-TNFR1 dAb <400> 424 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Pro Tyr Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 425 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-183 anti-TNFR1 dAb <400> 425 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Pro Tyr Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 426 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-184 anti-TNFR1 dAb <400> 426 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Pro Tyr Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 427 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-185 anti-TNFR1 dAb <400> 427 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Pro Tyr Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 428 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-188 anti-TNFR1 dAb <400> 428 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Gly Gly Val Asp Thr Tyr Tyr Ala Asp Pro Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 429 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-189 anti-TNFR1 dAb <400> 429 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Gly Gly Val Asp Thr Tyr Tyr Ala Asp Pro Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 430 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-190 anti-TNFR1 dAb <400> 430 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Gly Gly Val Asp Thr Tyr Tyr Ala Asp Pro Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 431 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-191 anti-TNFR1 dAb <400> 431 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Gly Gly Val Asp Thr Tyr Tyr Ala Asp Pro Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 432 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-192 anti-TNFR1 dAb <400> 432 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Gly Gly Val Asp Thr Tyr Tyr Ala Asp Pro Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 433 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-193 anti-TNFR1 dAb <400> 433 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Gly Gly Val Asp Thr Tyr Tyr Ala Asp Pro Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 434 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-194 anti-TNFR1 dAb <400> 434 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Val Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 435 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-195 anti-TNFR1 dAb <400> 435 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Val Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 436 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-196 anti-TNFR1 dAb <400> 436 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Val Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 437 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-197 anti-TNFR1 dAb <400> 437 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Gly Gly Val Asp Thr Tyr Tyr Ala Asp Pro Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 438 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-198 anti-TNFR1 dAb <400> 438 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Val Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 439 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-500 anti-TNFR1 dAb <400> 439 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe His Asp Gln 20 25 30 His Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 440 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-501 anti-TNFR1 dAb <400> 440 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Asn Asn 20 25 30 Ile Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 441 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-502 anti-TNFR1 dAb <400> 441 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Lys Asn Lys 20 25 30 Thr Met Val Trp Val Arg Gln Ala Gln Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 442 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-503 anti-TNFR1 dAb <400> 442 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys His 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 443 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-504 anti-TNFR1 dAb <400> 443 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 444 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-505 anti-TNFR1 dAb <400> 444 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Arg Gly Gly Gly Thr Tyr Tyr Thr Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Thr Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 445 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-506 anti-TNFR1 dAb <400> 445 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gln Gln Gly Glu Gly Thr Tyr Tyr Thr Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 446 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-507 anti-TNFR1 dAb <400> 446 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Gly Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 447 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-508 anti-TNFR1 dAb <400> 447 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Gly Gly Leu Gly Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 448 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-509 anti-TNFR1 dAb <400> 448 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 449 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-510 anti-TNFR1 dAb <400> 449 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Lys 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Arg Gly Glu Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 450 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-511 anti-TNFR1 dAb <400> 450 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Val Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 451 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-512 anti-TNFR1 dAb <400> 451 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 452 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-513 anti-TNFR1 dAb <400> 452 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Arg Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 453 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-514 anti-TNFR1 dAb <220> <221> VARIANT <222> (4)...(4) <223> Xaa = any amino acid <400> 453 Glu Val Gln Xaa Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 454 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-515 anti-TNFR1 dAb <400> 454 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Ser Asp Asp Gly Asn Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 455 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-516 anti-TNFR1 dAb <400> 455 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Ala Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Gly Gly Glu Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Pro Pro Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 456 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-517 anti-TNFR1 dAb <400> 456 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe His Asn Lys 20 25 30 Asp Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Gly Gly Glu Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Leu Leu Leu Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 457 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-518 anti-TNFR1 dAb <400> 457 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Met Arg 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Gly Gly Glu Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 458 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-519 anti-TNFR1 dAb <400> 458 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Gly Gly Glu Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 459 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-520 anti-TNFR1 dAb <400> 459 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Gly Gly Leu Val Thr Tyr Tyr Thr Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 460 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-521 anti-TNFR1 dAb <400> 460 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Thr 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Val Gly Gly Asp Thr Tyr Tyr Ala Asp Pro Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 461 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-522 anti-TNFR1 dAb <400> 461 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Asn Lys 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Leu Gly Leu Val Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 462 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-523 anti-TNFR1 dAb <400> 462 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Thr 20 25 30 Asn Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Glu Ile Arg Val Gly Gly Gly Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 463 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-524 anti-TNFR1 dAb <400> 463 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Lys Asn Lys 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gln Glu Gly Glu Gly Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 464 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-525 anti-TNFR1 dAb <400> 464 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Thr 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Glu Gly Ser Val Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 465 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-526 anti-TNFR1 dAb <400> 465 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Asn Lys 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Ala Thr Gly Thr Ile Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 466 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-527 anti-TNFR1 dAb <400> 466 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Lys 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Lys Gly Gln Ala Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 467 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-528 anti-TNFR1 dAb <220> <221> VARIANT <222> (4)...(4) <223> Xaa = any amino acid <400> 467 Glu Val Gln Xaa Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Arg Val Gly Gln Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 468 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-529 anti-TNFR1 dAb <400> 468 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Gly Gly Glu Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 469 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-530 anti-TNFR1 dAb <400> 469 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Gly Gly Glu Ser Thr Tyr Tyr Ala Asp Pro Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 470 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-531 anti-TNFR1 dAb <400> 470 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Asn Lys 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Ala Thr Gly Thr Ile Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 471 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-532 anti-TNFR1 dAb <400> 471 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Lys 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Lys Gly Gln Ala Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 472 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-533 anti-TNFR1 dAb <400> 472 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Lys 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Lys Gly Gln Asp Pro Phe Tyr Thr Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 473 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-534 anti-TNFR1 dAb <400> 473 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Gly Gly Leu Val Thr Tyr Tyr Thr Asp Pro Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 474 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-535 anti-TNFR1 dAb <400> 474 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Gly Gly Leu Val Thr Tyr Tyr Thr Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 475 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-536 anti-TNFR1 dAb <400> 475 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Gly Gly Leu Val Thr Tyr Tyr Thr Asp Pro Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 476 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-537 anti-TNFR1 dAb <400> 476 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Val Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 477 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-538 anti-TNFR1 dAb <400> 477 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Val Gly Gln Asp Pro Phe Tyr Ala Asp Pro Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 478 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-539 anti-TNFR1 dAb <400> 478 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Val Gly Gln Asp Pro Phe Tyr Thr Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 479 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-540 anti-TNFR1 dAb <400> 479 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Val Gly Gln Asp Pro Phe Tyr Thr Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 480 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-541 anti-TNFR1 dAb <400> 480 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Gly Gly Glu Ser Thr Tyr Tyr Thr Asp Pro Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 481 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-542 anti-TNFR1 dAb <400> 481 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Gly Gly Glu Ser Thr Tyr Tyr Thr Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 482 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-543 anti-TNFR1 dAb <400> 482 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Gly Gly Glu Ser Thr Tyr Tyr Thr Asp Pro Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 483 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-544 anti-TNFR1 dAb <400> 483 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Lys 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Lys Gly Gln Ala Thr Tyr Tyr Thr Asp Pro Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 484 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-545 anti-TNFR1 dAb <400> 484 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Lys 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Lys Gly Gln Ala Thr Tyr Tyr Thr Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 485 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-546 anti-TNFR1 dAb <400> 485 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Lys 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Lys Gly Gln Ala Thr Tyr Tyr Thr Asp Pro Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 486 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-547 anti-TNFR1 dAb <400> 486 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Val Gly Gln Asp Pro Phe Tyr Thr Asp Pro Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 487 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-548 anti-TNFR1 dAb <400> 487 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Val Gly Gln Asp Pro Phe Tyr Ala Asp Pro Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 488 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-549 anti-TNFR1 dAb <400> 488 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Lys 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Lys Gly Gln Asp Pro Phe Tyr Thr Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 489 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-550 anti-TNFR1 dAb <400> 489 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Lys 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Lys Gly Gln Asp Pro Phe Tyr Ala Asp Pro Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 490 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-551 anti-TNFR1 dAb <400> 490 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Lys 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Lys Gly Gln Asp Pro Phe Tyr Thr Asp Pro Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 491 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-552 anti-TNFR1 dAb <400> 491 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Lys 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Lys Gly Gln Asp Pro Phe Tyr Thr Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 492 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-553 anti-TNFR1 dAb <400> 492 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Lys 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Lys Gly Gln Asp Pro Phe Tyr Thr Asp Pro Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 493 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-554 anti-TNFR1 dAb <400> 493 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Val Gly Gln Asp Pro Phe Tyr Thr Asp Pro Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 494 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-555 anti-TNFR1 dAb <400> 494 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Gly Gly Glu Ser Pro Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 495 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-556 anti-TNFR1 dAb <400> 495 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Val Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 496 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-557 anti-TNFR1 dAb <400> 496 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Val Gly Gln Asp Pro Phe Tyr Ala Asp Pro Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 497 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-558 anti-TNFR1 dAb <400> 497 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Val Gly Gln Asp Pro Phe Tyr Thr Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 498 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-559 anti-TNFR1 dAb <400> 498 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Val Gly Gln Asp Pro Phe Tyr Thr Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 499 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-560 anti-TNFR1 dAb <400> 499 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Gly Gly Glu Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 500 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-561 anti-TNFR1 dAb <400> 500 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Gly Gly Glu Ser Thr Tyr Tyr Thr Asp Pro Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 501 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-562 anti-TNFR1 dAb <400> 501 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Gly Gly Glu Ser Thr Tyr Tyr Thr Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 502 <211> 119 <212> PRT <213> artificial sequence <220> <223> Tar2h-131-563 anti-TNFR1 dAb <400> 502 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Asp Gly Gly Gly Glu Ser Thr Tyr Tyr Thr Asp Pro Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 503 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574' anti-TNFR1 dAb <400> 503 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Gly His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Thr Gly His Trp Glu Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 504 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-1 anti-TNFR1 dAb <400> 504 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Gly His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Thr Gly Arg Trp Glu Pro Tyr Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 505 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-2 anti-TNFR1 dAb <400> 505 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Gly His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Thr Gly Arg Trp Glu Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 506 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-4 anti-TNFR1 dAb <400> 506 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Gly His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Thr Gly Arg Trp Glu Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 507 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-72 anti-TNFR1 dAb <400> 507 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 508 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-109 anti-TNFR1 dAb <400> 508 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 509 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-132 anti-TNFR1 dAb <400> 509 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Asp His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Arg Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 510 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-135 anti-TNFR1 dAb <400> 510 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Thr His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 511 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-138 anti-TNFR1 dAb <400> 511 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Phe Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Ala Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 512 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-156 anti-TNFR1 dAb <400> 512 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Phe Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 513 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-162 anti-TNFR1 dAb <400> 513 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Phe Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ser His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 514 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-180 anti-TNFR1 dAb <400> 514 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 515 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-14 anti-TNFR1 dAb <400> 515 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Asp Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 516 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-93 anti-TNFR1 dAb <400> 516 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Leu Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Gly Asp Arg Arg Tyr Tyr Asp Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 517 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-123 anti-TNFR1 dAb <400> 517 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Ala Asp Arg Arg Tyr Tyr Ala Asp Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 518 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-125 anti-TNFR1 dAb <400> 518 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ala Asn Thr Ala Asp Arg Arg Tyr Tyr Ala Asp Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 519 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-126 anti-TNFR1 dAb <400> 519 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ala Asn Thr Gly Asp Arg Arg Tyr Tyr Ala His Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 520 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-129 anti-TNFR1 dAb <400> 520 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Val Asn Thr Gly Asp Arg Arg Tyr Tyr Ala Asp Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 521 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-133 anti-TNFR1 dAb <400> 521 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Asp His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 522 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-137 anti-TNFR1 dAb <400> 522 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Thr Asp Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 523 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-160 anti-TNFR1 dAb <400> 523 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Leu Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Asp His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 524 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-139 anti-TNFR1 dAb <400> 524 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Leu Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Ala Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 525 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-155 anti-TNFR1 dAb <400> 525 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Leu Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 526 <211> 120 <212> PRT <213> artificial sequence <220> <223> DOM1h-509 anti-TNFR1 dAb <400> 526 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gln Tyr 20 25 30 Arg Met His Trp Val Arg Gln Ala Pro Gly Lys Ser Leu Glu Trp Val 35 40 45 Ser Ser Ile Asp Thr Arg Gly Ser Ser Thr Tyr Tyr Ala Asp Pro Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ala Val Thr Met Phe Ser Pro Phe Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 527 <211> 123 <212> PRT <213> artificial sequence <220> <223> DOM1h-510 anti-TNFR1 dAb <400> 527 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Asp Tyr 20 25 30 Gly Met Arg Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Thr Arg Thr Gly Arg Val Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Trp Arg Asn Arg His Gly Glu Tyr Leu Ala Asp Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 528 <211> 120 <212> PRT <213> artificial sequence <220> <223> DOM1h-543 anti-TNFR1 dAb <400> 528 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Met Arg Tyr 20 25 30 Arg Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Asp Ser Asn Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Arg Thr Glu Arg Ser Pro Val Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 529 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-549 anti-TNFR1 dAb <400> 529 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Asp Tyr 20 25 30 Glu Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Glu Ser Gly Thr Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Arg Arg Phe Ser Ala Ser Thr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 530 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-7 anti-TNFR1 dAb <400> 530 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Gly His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 531 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-8 anti-TNFR1 dAb <400> 531 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Gly His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 532 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-9 anti-TNFR1 dAb <400> 532 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Gly His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Met Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 533 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-10 anti-TNFR1 dAb <400> 533 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Asp Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Gly His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 534 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-11 anti-TNFR1 dAb <400> 534 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Gly His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Thr Gly Arg Trp Glu Pro Phe Asp His Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 535 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-12 anti-TNFR1 dAb <400> 535 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Asp His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Thr Gly Arg Trp Glu Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 536 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-13 anti-TNFR1 dAb <400> 536 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Asp Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Thr Gly Arg Trp Glu Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 537 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-15 anti-TNFR1 dAb <400> 537 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Asp His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 538 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-16 anti-TNFR1 dAb <400> 538 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Asp Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 539 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-17 anti-TNFR1 dAb <400> 539 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Asp His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 540 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-18 anti-TNFR1 dAb <400> 540 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Asp Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Asp Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 541 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-19 anti-TNFR1 dAb <400> 541 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Asp Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Asp His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 542 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-25 anti-TNFR1 dAb <400> 542 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Asp Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 543 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-26 anti-TNFR1 dAb <400> 543 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Asp Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 544 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-27 anti-TNFR1 dAb <400> 544 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Asp Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Lys Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 545 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-28 anti-TNFR1 dAb <400> 545 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Asp Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 546 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-29 anti-TNFR1 dAb <400> 546 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Asp Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Arg Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 547 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-30 anti-TNFR1 dAb <400> 547 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ala Asn Thr Gly Asp Arg Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 548 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-31 anti-TNFR1 dAb <400> 548 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Asn Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 549 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-32 anti-TNFR1 dAb <400> 549 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Asp Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Ala Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 550 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-33 anti-TNFR1 dAb <400> 550 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Asp Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Asp Asn Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 551 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-35 anti-TNFR1 dAb <400> 551 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ile Thr Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Asp Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Gln Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 552 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-36 anti-TNFR1 dAb <400> 552 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Asp Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 553 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-37 anti-TNFR1 dAb <400> 553 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Phe Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Asp Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 554 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-38 anti-TNFR1 dAb <400> 554 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Gly Asp Arg Arg Tyr Tyr Asp Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 555 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-39 anti-TNFR1 dAb <400> 555 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Asp Arg Arg Tyr Tyr Ala Asp Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 556 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-40 anti-TNFR1 dAb <400> 556 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Asp Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Lys Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 557 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-53 anti-TNFR1 dAb <400> 557 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Glu Arg Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 558 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-54 anti-TNFR1 dAb <400> 558 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Asn Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Asp Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Tyr Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Thr Ser 115 <210> 559 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-65 anti-TNFR1 dAb <400> 559 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ala Asn Thr Gly Asp Arg Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 560 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-66 anti-TNFR1 dAb <400> 560 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ala Asn Thr Gly Asp Arg Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Lys Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 561 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-67 anti-TNFR1 dAb <400> 561 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ala Asn Thr Gly Asp Arg Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 562 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-68 anti-TNFR1 dAb <400> 562 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ala Asn Thr Gly Asp Arg Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Arg Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 563 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-69 anti-TNFR1 dAb <400> 563 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ala Asn Thr Gly Asp Arg Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Ala Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 564 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-70 anti-TNFR1 dAb <400> 564 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Tyr Thr Gly Arg Trp Glu Pro Phe Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 565 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-71 anti-TNFR1 dAb <400> 565 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Lys Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 566 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-73 anti-TNFR1 dAb <400> 566 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Arg Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 567 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-74 anti-TNFR1 dAb <400> 567 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Ala Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 568 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-75 anti-TNFR1 dAb <400> 568 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Gly Asp Arg Arg Tyr Tyr Asp Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 569 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-76 anti-TNFR1 dAb <400> 569 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Gly Asp Arg Arg Tyr Tyr Asp Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Lys Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 570 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-77 anti-TNFR1 dAb <400> 570 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Gly Asp Arg Arg Tyr Tyr Asp Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 571 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-78 anti-TNFR1 dAb <400> 571 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Gly Asp Arg Arg Tyr Tyr Asp Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Arg Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 572 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-79 anti-TNFR1 dAb <400> 572 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Gly Asp Arg Arg Tyr Tyr Asp Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Ala Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 573 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-84 anti-TNFR1 dAb <400> 573 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Asp Arg Arg Tyr Tyr Ala Asp Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 574 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-85 anti-TNFR1 dAb <400> 574 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Asp Arg Arg Tyr Tyr Ala Asp Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Lys Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 575 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-86 anti-TNFR1 dAb <400> 575 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Asp Arg Arg Tyr Tyr Ala Asp Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 576 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-87 anti-TNFR1 dAb <400> 576 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Asp Arg Arg Tyr Tyr Ala Asp Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Arg Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 577 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-88 anti-TNFR1 dAb <400> 577 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Asp Arg Arg Tyr Tyr Ala Asp Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Ala Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 578 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-90 anti-TNFR1 dAb <400> 578 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Leu Lys Phe 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ala Asn Thr Gly Asp Arg Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Ala Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 579 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-91 anti-TNFR1 dAb <400> 579 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Leu Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Ala Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 580 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-92 anti-TNFR1 dAb <400> 580 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Phe Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Gly Asp Arg Arg Tyr Tyr Asp Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 581 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-94 anti-TNFR1 dAb <400> 581 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ala Asn Thr Gly Asp Arg Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Pro Asp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 582 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-95 anti-TNFR1 dAb <400> 582 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ala Asn Thr Gly Asp Arg Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Pro Asp Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 583 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-96 anti-TNFR1 dAb <400> 583 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Pro Asp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 584 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-97 anti-TNFR1 dAb <400> 584 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Pro Asp Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 585 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-98 anti-TNFR1 dAb <400> 585 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Gly Asp Arg Arg Tyr Tyr Asp Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Pro Asp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 586 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-99 anti-TNFR1 dAb <400> 586 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Gly Asp Arg Arg Tyr Tyr Asp Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Pro Asp Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 587 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-100 anti-TNFR1 dAb <400> 587 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Gln Ile Ser Ala Trp Gly Asp Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 588 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-101 anti-TNFR1 dAb <400> 588 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Gly Gly Gln Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 589 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-102 anti-TNFR1 dAb <400> 589 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Ser Gly Tyr Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 590 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-103 anti-TNFR1 dAb <400> 590 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Gly Gly Thr Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 591 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-104 anti-TNFR1 dAb <400> 591 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Lys Gly Thr Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 592 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-105 anti-TNFR1 dAb <400> 592 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Gln Ile Ser Glu Thr Gly Arg Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 593 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-106 anti-TNFR1 dAb <400> 593 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Asn Asn Thr Gly Ser Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 594 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-107 anti-TNFR1 dAb <400> 594 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 595 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-108 anti-TNFR1 dAb <400> 595 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Ala Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 596 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-110 anti-TNFR1 dAb <400> 596 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Ala Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 597 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-111 anti-TNFR1 dAb <400> 597 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Asp Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Arg Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 598 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-112 anti-TNFR1 dAb <400> 598 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Thr His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Ala Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 599 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-113 anti-TNFR1 dAb <400> 599 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Ala Asp Arg Arg Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Ala Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 600 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-114 anti-TNFR1 dAb <400> 600 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Leu Asn Thr Ala Asp Arg Thr Tyr Tyr Asp His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Ala Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 601 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-115 anti-TNFR1 dAb <400> 601 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Ala Asp Arg Thr Tyr Tyr Asp His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Ala Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 602 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-116 anti-TNFR1 dAb <400> 602 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Arg Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Ala Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 603 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-117 anti-TNFR1 dAb <400> 603 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Arg Tyr Tyr Asp His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Ala Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 604 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-118 anti-TNFR1 dAb <400> 604 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Tyr Thr Gly Arg Trp Val Ser Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 605 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-119 anti-TNFR1 dAb <400> 605 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Tyr Thr Gly Arg Trp Val Ser Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 606 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-120 anti-TNFR1 dAb <400> 606 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 607 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-121 anti-TNFR1 dAb <400> 607 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 608 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-122 anti-TNFR1 dAb <400> 608 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ala Asn Thr Ala Asp Arg Arg Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Ala Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 609 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-124 anti-TNFR1 dAb <400> 609 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Asp Arg Arg Tyr Tyr Ala His Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 610 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-127 anti-TNFR1 dAb <400> 610 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Ala Asp Arg Arg Tyr Tyr Ala His Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 611 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-128 anti-TNFR1 dAb <400> 611 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ala Asn Thr Ala Asp Arg Arg Tyr Tyr Ala His Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 612 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-130 anti-TNFR1 dAb <400> 612 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ala Asn Thr Gly Asp Arg Arg Tyr Tyr Ala Asp Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 613 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-131 anti-TNFR1 dAb <400> 613 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Asp His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Ala Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 614 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-134 anti-TNFR1 dAb <400> 614 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ser His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 615 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-140 anti-TNFR1 dAb <400> 615 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Phe Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ala Asp Thr Gly Asp Arg Arg Tyr Tyr Asp Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 616 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-141 anti-TNFR1 dAb <400> 616 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Phe Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Arg Tyr Tyr Asp Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 617 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-142 anti-TNFR1 dAb <400> 617 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Phe Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Gly Asp Arg Arg Tyr Tyr Asp His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 618 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-143 anti-TNFR1 dAb <400> 618 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Phe Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Gly Asp Arg Arg Tyr Tyr Asp Asp Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 619 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-144 anti-TNFR1 dAb <400> 619 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Phe Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ala Asp Thr Ala Asp Arg Arg Tyr Tyr Asp Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 620 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-145 anti-TNFR1 dAb <400> 620 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Phe Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ala Asp Thr Gly Asp Arg Arg Tyr Tyr Asp His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 621 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-146 anti-TNFR1 dAb <400> 621 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Phe Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ala Asp Thr Gly Asp Arg Arg Tyr Tyr Asp Asp Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 622 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-147 anti-TNFR1 dAb <400> 622 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Gly Pro Phe Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 623 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-148 anti-TNFR1 dAb <400> 623 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 624 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-149 anti-TNFR1 dAb <400> 624 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Gly Pro Phe Gln Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 625 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-150 anti-TNFR1 dAb <400> 625 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Gln Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 626 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-151 anti-TNFR1 dAb <400> 626 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Ala Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 627 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-152 anti-TNFR1 dAb <400> 627 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Ala Pro Phe Gln Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 628 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-153 anti-TNFR1 dAb <400> 628 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Gln Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 629 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-154 anti-TNFR1 dAb <400> 629 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Gly Asp Arg Arg Tyr Tyr Asp His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Ala Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 630 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-157 anti-TNFR1 dAb <400> 630 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Leu Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Asp His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Arg Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 631 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-158 anti-TNFR1 dAb <400> 631 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Phe Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Asp His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Arg Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 632 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-159 anti-TNFR1 dAb <400> 632 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Phe Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Asp His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 633 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-161 anti-TNFR1 dAb <400> 633 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Leu Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ser His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 634 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-163 anti-TNFR1 dAb <400> 634 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Phe Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Thr His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 635 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-164 anti-TNFR1 dAb <400> 635 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Leu Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Thr His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 636 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-165 anti-TNFR1 dAb <400> 636 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Phe Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Ala Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 637 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-166 anti-TNFR1 dAb <400> 637 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Leu Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Ala Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 638 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-167 anti-TNFR1 dAb <400> 638 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Leu Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Gly Asp Arg Arg Tyr Tyr Asp His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Ala Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 639 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-168 anti-TNFR1 dAb <400> 639 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Phe Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Gly Asp Arg Arg Tyr Tyr Asp His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Ala Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 640 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-169 anti-TNFR1 dAb <400> 640 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ala Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 641 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-170 anti-TNFR1 dAb <400> 641 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Phe Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 642 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-171 anti-TNFR1 dAb <400> 642 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ala Asp Thr Ala Asp Arg Thr Tyr Tyr Asp His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 643 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-172 anti-TNFR1 dAb <400> 643 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ala Asp Thr Ala Asp Arg Thr Tyr Tyr Asp His Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 644 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-173 anti-TNFR1 dAb <400> 644 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ala Asp Thr Ala Asp Arg Arg Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Ala Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 645 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-174 anti-TNFR1 dAb <400> 645 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Arg Tyr Tyr Ala His Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Ala Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 646 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-175 anti-TNFR1 dAb <400> 646 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ala Asp Thr Ala Asp Arg Arg Tyr Tyr Ala His Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Ala Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 647 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-176 anti-TNFR1 dAb <400> 647 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Arg Tyr Tyr Asp His Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Ala Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 648 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-177 anti-TNFR1 dAb <400> 648 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ala Asp Thr Ala Asp Arg Arg Tyr Tyr Asp His Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Ala Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 649 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-178 anti-TNFR1 dAb <400> 649 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ala Asp Thr Ala Asp Arg Arg Tyr Tyr Asp His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Ala Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 650 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-179 anti-TNFR1 dAb <400> 650 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Phe Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Arg Tyr Tyr Asp Asp Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Thr Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 651 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-188 anti-TNFR1 dAb <400> 651 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 652 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-189 anti-TNFR1 dAb <400> 652 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Phe Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Tyr Thr Gly Arg Trp Glu Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 653 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-190 anti-TNFR1 dAb <400> 653 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Phe Lys Tyr 20 25 30 Ser Met Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Tyr Thr Gly Arg Trp Ala Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 654 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-191 anti-TNFR1 dAb <400> 654 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 655 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-192 anti-TNFR1 dAb <400> 655 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Asn Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 656 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-193 anti-TNFR1 dAb <400> 656 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Asn Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 657 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-194 anti-TNFR1 dAb <400> 657 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 658 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-195 anti-TNFR1 dAb <400> 658 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Phe Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 659 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-196 anti-TNFR1 dAb <400> 659 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 660 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-201 anti-TNFR1 dAb <400> 660 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Lys Asn 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Leu 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 661 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-202 anti-TNFR1 dAb <400> 661 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Phe Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Tyr Thr Gly Arg Trp Ala Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 662 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-203 anti-TNFR1 dAb <400> 662 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Phe Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Ala Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 663 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-204 anti-TNFR1 dAb <400> 663 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Phe Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Gln Trp Ala Pro Tyr Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Arg Ala 115 <210> 664 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-205 anti-TNFR1 dAb <400> 664 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Asp Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Asn Trp Gly His Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 665 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-206 anti-TNFR1 dAb <400> 665 Glu Val Gln Leu Leu Asp Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Phe Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ser Pro Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gly Asn Thr Leu Asn 65 70 75 80 Leu Gln Met Thr Pro Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 666 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-207 anti-TNFR1 dAb <400> 666 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Met Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ser Ser Gly Phe Thr Phe Ser Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Leu Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Glu Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Ala Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 667 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-208 anti-TNFR1 dAb <400> 667 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 668 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-209 anti-TNFR1 dAb <400> 668 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Phe Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Gln Trp Ala Pro Tyr Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 669 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-211 anti-TNFR1 dAb <400> 669 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 670 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-212 anti-TNFR1 dAb <400> 670 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 671 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-213 anti-TNFR1 dAb <400> 671 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Thr Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 672 <211> 119 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-214 anti-TNFR1 dAb <400> 672 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 673 <211> 251 <212> PRT <213> artificial sequence <220> <223> ATROSAB scFv (scFv IZI06.1) <400> 673 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe 20 25 30 Tyr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Tyr Pro Tyr Ser Gly His Ala Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Ala Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Asp Phe Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ser 115 120 125 Ala Gln Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr 130 135 140 Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu 145 150 155 160 His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly 165 170 175 Gln Ser Pro Gln Leu Leu Ile Tyr Thr Val Ser Asn Arg Phe Ser Gly 180 185 190 Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 195 200 205 Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser 210 215 220 Gln Ser Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu 225 230 235 240 Ile Lys Arg Gly Gly Ser Gly Gly Ala Ala Ala 245 250 <210> 674 <211> 243 <212> PRT <213> artificial sequence <220> <223> scFV IG11 <400> 674 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe 20 25 30 Tyr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Val Pro Thr Gln Gly Glu Ala Lys Tyr Asn Asp Lys Phe 50 55 60 Lys Ala Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Asp Phe Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ser 115 120 125 Ala Gln Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr 130 135 140 Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu 145 150 155 160 His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly 165 170 175 Gln Ser Pro Gln Leu Leu Ile Tyr Thr Val Ser Asn Arg Phe Ser Gly 180 185 190 Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 195 200 205 Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser 210 215 220 Gln Ser Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu 225 230 235 240 Ile Lys Arg <210> 675 <211> 243 <212> PRT <213> artificial sequence <220> <223> scFV T12B <400> 675 His Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe 20 25 30 Tyr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Val Pro Ser Gln Gly Glu Ala Lys Tyr Asn Asp Lys Phe 50 55 60 Lys Ala Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Asp Phe Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ser 115 120 125 Ala Gln Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr 130 135 140 Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu 145 150 155 160 His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly 165 170 175 Gln Ser Pro Gln Leu Leu Ile Tyr Thr Val Ser Asn Arg Phe Ser Gly 180 185 190 Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 195 200 205 Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser 210 215 220 Gln Gly Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu 225 230 235 240 Ile Lys Arg <210> 676 <211> 242 <212> PRT <213> artificial sequence <220> <223> scFV 13.7 <400> 676 His Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe 20 25 30 Tyr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Val Pro Ser Gln Gly Glu Ala Lys Tyr Asn Asp Lys Phe 50 55 60 Lys Ala Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Asp Phe Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Asp Val Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 130 135 140 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Leu Leu 145 150 155 160 His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly 165 170 175 Lys Ala Pro Lys Leu Leu Ile Tyr Thr Val Ser Asn Arg Phe Ser Gly 180 185 190 Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 195 200 205 Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ser 210 215 220 Gln Ser Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu 225 230 235 240 Ile Lys <210> 677 <211> 231 <212> PRT <213> artificial sequence <220> <223> H398 scFV w/ AST linker <400> 677 Gln Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe 20 25 30 Tyr Ile Asn Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Tyr Pro Tyr Ser Gly His Ala Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Ala Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Phe 65 70 75 80 Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Val Arg Trp Asp Phe Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr 100 105 110 Val Ser Ser Ala Ser Thr Asp Ile Val Met Thr Gln Ser Pro Leu Ser 115 120 125 Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser 130 135 140 Gln Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Val 145 150 155 160 Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Thr Val Ser Asn 165 170 175 Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 180 185 190 Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val 195 200 205 Tyr Phe Cys Ser Gln Ser Thr His Val Pro Tyr Thr Phe Gly Gly Gly 210 215 220 Thr Lys Leu Glu Ile Lys Arg 225 230 <210> 678 <211> 243 <212> PRT <213> artificial sequence <220> <223> H398 scFv w/ (GGGGS)3 linker <400> 678 Gln Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe 20 25 30 Tyr Ile Asn Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Tyr Pro Tyr Ser Gly His Ala Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Ala Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Phe 65 70 75 80 Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Val Arg Trp Asp Phe Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr 100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser 130 135 140 Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu 145 150 155 160 His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Val Gln Lys Pro Gly 165 170 175 Gln Ser Pro Lys Leu Leu Ile Tyr Thr Val Ser Asn Arg Phe Ser Gly 180 185 190 Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 195 200 205 Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser 210 215 220 Gln Ser Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu 225 230 235 240 Ile Lys Arg <210> 679 <211> 219 <212> PRT <213> artificial sequence <220> <223> FabATR (ATROSAB Fab) light chain <400> 679 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Thr Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95 Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 680 <211> 218 <212> PRT <213> artificial sequence <220> <223> FabATR (ATROSAB Fab) heavy chain <400> 680 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe 20 25 30 Tyr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Tyr Pro Tyr Ser Gly His Ala Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Ala Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Asp Phe Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 <210> 681 <211> 219 <212> PRT <213> artificial sequence <220> <223> Fab 13.7 light chain <400> 681 Asp Val Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala 35 40 45 Pro Lys Leu Leu Ile Tyr Thr Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ser Gln Ser 85 90 95 Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 682 <211> 218 <212> PRT <213> artificial sequence <220> <223> Fab 13.7 heavy chain <400> 682 His Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe 20 25 30 Tyr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Val Pro Ser Gln Gly Glu Ala Lys Tyr Asn Asp Lys Phe 50 55 60 Lys Ala Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Asp Phe Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 <210> 683 <211> 119 <212> PRT <213> artificial sequence <220> <223> Nb 70 nanobody <400> 683 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Pro Leu Arg Leu Ser Cys Ala Ala Ser Val Arg Thr Phe Ser Ser Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Ser Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Leu His Glu Asp Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser 115 <210> 684 <211> 124 <212> PRT <213> artificial sequence <220> <223> Nb 96 nanobody <400> 684 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Thr Leu Ser Cys Thr Arg Thr Gly Phe Thr Ala Ser Thr Asn 20 25 30 Ala Tyr Gly Trp Tyr Arg Gln Gly Pro Gly Lys Lys Cys Glu Trp Val 35 40 45 Ser Tyr Met Thr Ile Pro Ser Gly Arg Thr Thr Tyr Ala Asp Ala Val 50 55 60 Lys Gly Arg Phe Ala Met Ser Arg Asp Lys Ala Lys Ser Thr Val Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Asp Val Pro Phe Ser Thr Leu Pro Ala Met Cys Thr Asn Asp Gly 100 105 110 Pro Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 685 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR1-selective antagonist TNF mutein R32W/S86T <400> 685 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Trp 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Thr Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 686 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR1-selective antagonist TNF mutein L29S <400> 686 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Ser Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 687 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR1-selective antagonist TNF mutein L29G <400> 687 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Gly Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 688 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR1-selective antagonist TNF mutein L29Y <400> 688 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Tyr Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 689 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR1-selective antagonist TNF mutein R31E <400> 689 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Glu Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 690 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR1-selective antagonist TNF mutein R31N <400> 690 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Asn Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 691 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR1-selective antagonist TNF mutein R32W <400> 691 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Trp 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 692 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR1-selective antagonist TNF mutein R32Y <400> 692 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Tyr 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 693 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR1-selective antagonist TNF mutein S86T <400> 693 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Thr Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 694 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR1-selective antagonist TNF mutein R31N/R32T <400> 694 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Asn Thr 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 695 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR1-selective antagonist TNF mutein L29S/R32W <400> 695 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Ser Asn Arg Trp 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 696 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR1-selective antagonist TNF mutein L29S/S86T <400> 696 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Ser Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Thr Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 697 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR1-selective antagonist TNF mutein L29S/R32W/S86T <400> 697 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Ser Asn Arg Trp 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Thr Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 698 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR1-selective antagonist TNF mutein R31E/S86T <400> 698 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Glu Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Thr Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 699 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR1-selective antagonist TNF mutein R31N/R32T/S86T <400> 699 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Asn Thr 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Thr Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 700 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR1-selective antagonist TNF mutein E146R <400> 700 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Arg Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 701 <211> 157 <212> PRT <213> artificial sequence <220> <223> XPro1595 <400> 701 Met Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Cys Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Val Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser His Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Ala Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Arg Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 702 <211> 157 <212> PRT <213> artificial sequence <220> <223> XENP345 <400> 702 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Thr Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Arg Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 703 <211> 157 <212> PRT <213> artificial sequence <220> <223> R1antTNF <400> 703 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ser Thr Thr His Asn Gln Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 704 <211> 242 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-208 - albudAb fusion protein <400> 704 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 130 135 140 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 145 150 155 160 Arg Pro Ile Gly Thr Thr Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys 165 170 175 Ala Pro Lys Leu Leu Ile Leu Trp Asn Ser Arg Leu Gln Ser Gly Val 180 185 190 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 195 200 205 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Gln 210 215 220 Ala Gly Thr His Pro Thr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 225 230 235 240 Lys Arg <210> 705 <211> 242 <212> PRT <213> artificial sequence <220> <223> DOM1h-131-206 dAb - albudAb fusion protein <400> 705 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Asp Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr His Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 130 135 140 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 145 150 155 160 Arg Pro Ile Gly Thr Thr Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys 165 170 175 Ala Pro Lys Leu Leu Ile Leu Trp Asn Ser Arg Leu Gln Ser Gly Val 180 185 190 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 195 200 205 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Gln 210 215 220 Ala Gly Thr His Pro Thr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 225 230 235 240 Lys Arg <210> 706 <211> 466 <212> PRT <213> artificial sequence <220> <223> H398 scFv-SCDKTH-trastuzumab Fc fusion protein <400> 706 Gln Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe 20 25 30 Tyr Ile Asn Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Tyr Pro Tyr Ser Gly His Ala Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Ala Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Phe 65 70 75 80 Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Val Arg Trp Asp Phe Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr 100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser 130 135 140 Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu 145 150 155 160 His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Val Gln Lys Pro Gly 165 170 175 Gln Ser Pro Lys Leu Leu Ile Tyr Thr Val Ser Asn Arg Phe Ser Gly 180 185 190 Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 195 200 205 Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser 210 215 220 Gln Ser Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu 225 230 235 240 Ile Lys Arg Ser Cys Asp Lys Thr His Ala Pro Glu Leu Leu Gly Gly 245 250 255 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 260 265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 275 280 285 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 290 295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 340 345 350 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 355 360 365 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 370 375 380 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 405 410 415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 420 425 430 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 435 440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 450 455 460 Gly Lys 465 <210> 707 <211> 475 <212> PRT <213> artificial sequence <220> <223> H398 scFv-GGGGSGGGGSGGGGS-Trastuzumab Fc fusion protein <400> 707 Gln Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe 20 25 30 Tyr Ile Asn Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Tyr Pro Tyr Ser Gly His Ala Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Ala Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Phe 65 70 75 80 Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Val Arg Trp Asp Phe Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr 100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser 130 135 140 Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu 145 150 155 160 His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Val Gln Lys Pro Gly 165 170 175 Gln Ser Pro Lys Leu Leu Ile Tyr Thr Val Ser Asn Arg Phe Ser Gly 180 185 190 Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 195 200 205 Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser 210 215 220 Gln Ser Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu 225 230 235 240 Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 245 250 255 Gly Ser Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 260 265 270 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 275 280 285 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 290 295 300 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 305 310 315 320 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 325 330 335 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 340 345 350 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 355 360 365 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 370 375 380 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 385 390 395 400 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 405 410 415 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 420 425 430 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 435 440 445 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 450 455 460 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470 475 <210> 708 <211> 481 <212> PRT <213> artificial sequence <220> <223> H398 scFv-GGGGSGGGGSGGGGS-SCDKTH-Trastuzumab Fc fusion protein <400> 708 Gln Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe 20 25 30 Tyr Ile Asn Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Tyr Pro Tyr Ser Gly His Ala Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Ala Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Phe 65 70 75 80 Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Val Arg Trp Asp Phe Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr 100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser 130 135 140 Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu 145 150 155 160 His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Val Gln Lys Pro Gly 165 170 175 Gln Ser Pro Lys Leu Leu Ile Tyr Thr Val Ser Asn Arg Phe Ser Gly 180 185 190 Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 195 200 205 Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser 210 215 220 Gln Ser Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu 225 230 235 240 Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 245 250 255 Gly Ser Ser Cys Asp Lys Thr His Ala Pro Glu Leu Leu Gly Gly Pro 260 265 270 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 275 280 285 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 290 295 300 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 305 310 315 320 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 325 330 335 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 340 345 350 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 355 360 365 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 370 375 380 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 385 390 395 400 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 405 410 415 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 420 425 430 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 435 440 445 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 450 455 460 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 465 470 475 480 Lys <210> 709 <211> 849 <212> PRT <213> artificial sequence <220> <223> H398 scFv-GGGGSGGGGSGGGGS-HSA fusion protein <400> 709 Gln Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe 20 25 30 Tyr Ile Asn Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Tyr Pro Tyr Ser Gly His Ala Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Ala Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Phe 65 70 75 80 Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Val Arg Trp Asp Phe Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr 100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser 130 135 140 Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu 145 150 155 160 His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Val Gln Lys Pro Gly 165 170 175 Gln Ser Pro Lys Leu Leu Ile Tyr Thr Val Ser Asn Arg Phe Ser Gly 180 185 190 Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 195 200 205 Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser 210 215 220 Gln Ser Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu 225 230 235 240 Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 245 250 255 Gly Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala 260 265 270 His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu 275 280 285 Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val 290 295 300 Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp 305 310 315 320 Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 325 330 335 Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala 340 345 350 Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 355 360 365 His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val 370 375 380 Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys 385 390 395 400 Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 405 410 415 Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys 420 425 430 Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu 435 440 445 Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys 450 455 460 Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val 465 470 475 480 Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser 485 490 495 Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly 500 505 510 Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile 515 520 525 Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu 530 535 540 Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp 545 550 555 560 Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser 565 570 575 Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 580 585 590 Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 595 600 605 Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys 610 615 620 Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu 625 630 635 640 Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys 645 650 655 Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 660 665 670 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 675 680 685 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 690 695 700 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 705 710 715 720 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 725 730 735 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 740 745 750 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 755 760 765 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 770 775 780 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 785 790 795 800 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 805 810 815 Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 820 825 830 Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly 835 840 845 Leu <210> 710 <211> 342 <212> PRT <213> artificial sequence <220> <223> DOMlh-574-16-SCDKTH-Trastuzumab Fc fusion protein <400> 710 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Asp Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ser Cys Asp Lys Thr His Ala Pro Glu 115 120 125 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 130 135 140 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 145 150 155 160 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 165 170 175 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 180 185 190 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 195 200 205 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 210 215 220 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 225 230 235 240 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 245 250 255 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 260 265 270 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 275 280 285 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 290 295 300 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 305 310 315 320 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 325 330 335 Ser Leu Ser Pro Gly Lys 340 <210> 711 <211> 351 <212> PRT <213> artificial sequence <220> <223> DOMlh-574-16-GGGGSGGGGSGGGGS-Trastuzumab Fc fusion protein <400> 711 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Asp Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 130 135 140 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 145 150 155 160 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 165 170 175 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 180 185 190 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 195 200 205 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 210 215 220 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 225 230 235 240 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 245 250 255 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 260 265 270 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 275 280 285 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 290 295 300 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 305 310 315 320 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 325 330 335 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 340 345 350 <210> 712 <211> 357 <212> PRT <213> artificial sequence <220> <223> DOMlh-574-16-GGGGSGGGGSGGGGS-SCDKTH-Trastuzumab Fc fusion protein <400> 712 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Asp Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Ser Cys Asp Lys Thr His Ala Pro Glu Leu 130 135 140 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 145 150 155 160 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 165 170 175 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 180 185 190 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 195 200 205 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 210 215 220 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 225 230 235 240 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 245 250 255 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 260 265 270 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 275 280 285 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 290 295 300 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 305 310 315 320 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 325 330 335 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 340 345 350 Leu Ser Pro Gly Lys 355 <210> 713 <211> 725 <212> PRT <213> artificial sequence <220> <223> DOMlh-574-16-GGGGSGGGGSGGGGS-HSA fusion protein <400> 713 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Asp Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys 130 135 140 Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys 145 150 155 160 Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe 165 170 175 Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr 180 185 190 Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr 195 200 205 Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr 210 215 220 Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu 225 230 235 240 Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val 245 250 255 Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu 260 265 270 Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr 275 280 285 Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala 290 295 300 Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro 305 310 315 320 Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln 325 330 335 Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys 340 345 350 Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe 355 360 365 Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu 370 375 380 Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu 385 390 395 400 Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys 405 410 415 Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu 420 425 430 Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp 435 440 445 Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp 450 455 460 Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp 465 470 475 480 Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr 485 490 495 Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys 500 505 510 Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile 515 520 525 Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln 530 535 540 Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr 545 550 555 560 Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys 565 570 575 Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr 580 585 590 Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro 595 600 605 Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg 610 615 620 Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys 625 630 635 640 Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu 645 650 655 Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu 660 665 670 Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met 675 680 685 Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys 690 695 700 Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln 705 710 715 720 Ala Ala Leu Gly Leu 725 <210> 714 <211> 342 <212> PRT <213> artificial sequence <220> <223> DOMlh-549-SCDKTH-Trastuzumab Fc fusion protein <400> 714 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Asp Tyr 20 25 30 Glu Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Glu Ser Gly Thr Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Arg Arg Phe Ser Ala Ser Thr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ser Cys Asp Lys Thr His Ala Pro Glu 115 120 125 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 130 135 140 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 145 150 155 160 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 165 170 175 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 180 185 190 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 195 200 205 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 210 215 220 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 225 230 235 240 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 245 250 255 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 260 265 270 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 275 280 285 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 290 295 300 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 305 310 315 320 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 325 330 335 Ser Leu Ser Pro Gly Lys 340 <210> 715 <211> 351 <212> PRT <213> artificial sequence <220> <223> DOMlh-549-GGGGSGGGGSGGGGS-Trastuzumab Fc fusion protein <400> 715 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Asp Tyr 20 25 30 Glu Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Glu Ser Gly Thr Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Arg Arg Phe Ser Ala Ser Thr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 130 135 140 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 145 150 155 160 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 165 170 175 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 180 185 190 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 195 200 205 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 210 215 220 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 225 230 235 240 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 245 250 255 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 260 265 270 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 275 280 285 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 290 295 300 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 305 310 315 320 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 325 330 335 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 340 345 350 <210> 716 <211> 357 <212> PRT <213> artificial sequence <220> <223> DOMlh-549-GGGGSGGGGSGGGGS-SCDKTH-Trastuzumab Fc fusion protein <400> 716 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Asp Tyr 20 25 30 Glu Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Glu Ser Gly Thr Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Arg Arg Phe Ser Ala Ser Thr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Ser Cys Asp Lys Thr His Ala Pro Glu Leu 130 135 140 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 145 150 155 160 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 165 170 175 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 180 185 190 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 195 200 205 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 210 215 220 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 225 230 235 240 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 245 250 255 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 260 265 270 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 275 280 285 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 290 295 300 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 305 310 315 320 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 325 330 335 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 340 345 350 Leu Ser Pro Gly Lys 355 <210> 717 <211> 725 <212> PRT <213> artificial sequence <220> <223> DOMlh-549-GGGGSGGGGSGGGGS-HSA fusion protein <400> 717 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Asp Tyr 20 25 30 Glu Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Glu Ser Gly Thr Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Arg Arg Phe Ser Ala Ser Thr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys 130 135 140 Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys 145 150 155 160 Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe 165 170 175 Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr 180 185 190 Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr 195 200 205 Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr 210 215 220 Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu 225 230 235 240 Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val 245 250 255 Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu 260 265 270 Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr 275 280 285 Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala 290 295 300 Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro 305 310 315 320 Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln 325 330 335 Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys 340 345 350 Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe 355 360 365 Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu 370 375 380 Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu 385 390 395 400 Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys 405 410 415 Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu 420 425 430 Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp 435 440 445 Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp 450 455 460 Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp 465 470 475 480 Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr 485 490 495 Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys 500 505 510 Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile 515 520 525 Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln 530 535 540 Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr 545 550 555 560 Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys 565 570 575 Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr 580 585 590 Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro 595 600 605 Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg 610 615 620 Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys 625 630 635 640 Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu 645 650 655 Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu 660 665 670 Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met 675 680 685 Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys 690 695 700 Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln 705 710 715 720 Ala Ala Leu Gly Leu 725 <210> 718 <211> 342 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-208-SCDKTH-Trastuzumab Fc fusion protein <400> 718 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ser Cys Asp Lys Thr His Ala Pro Glu 115 120 125 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 130 135 140 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 145 150 155 160 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 165 170 175 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 180 185 190 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 195 200 205 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 210 215 220 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 225 230 235 240 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 245 250 255 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 260 265 270 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 275 280 285 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 290 295 300 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 305 310 315 320 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 325 330 335 Ser Leu Ser Pro Gly Lys 340 <210> 719 <211> 351 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-208-GGGGSGGGGSGGGGS-Trastuzumab Fc fusion protein <400> 719 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 130 135 140 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 145 150 155 160 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 165 170 175 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 180 185 190 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 195 200 205 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 210 215 220 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 225 230 235 240 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 245 250 255 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 260 265 270 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 275 280 285 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 290 295 300 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 305 310 315 320 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 325 330 335 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 340 345 350 <210> 720 <211> 357 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-208-GGGGSGGGGSGGGGS-SCDKTH-Trastuzumab Fc fusion proteins <400> 720 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Ser Cys Asp Lys Thr His Ala Pro Glu Leu 130 135 140 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 145 150 155 160 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 165 170 175 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 180 185 190 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 195 200 205 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 210 215 220 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 225 230 235 240 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 245 250 255 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 260 265 270 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 275 280 285 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 290 295 300 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 305 310 315 320 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 325 330 335 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 340 345 350 Leu Ser Pro Gly Lys 355 <210> 721 <211> 725 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-208-GGGGSGGGGSGGGGS-HSA fusion protein <400> 721 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys 130 135 140 Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys 145 150 155 160 Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe 165 170 175 Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr 180 185 190 Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr 195 200 205 Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr 210 215 220 Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu 225 230 235 240 Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val 245 250 255 Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu 260 265 270 Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr 275 280 285 Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala 290 295 300 Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro 305 310 315 320 Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln 325 330 335 Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys 340 345 350 Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe 355 360 365 Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu 370 375 380 Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu 385 390 395 400 Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys 405 410 415 Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu 420 425 430 Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp 435 440 445 Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp 450 455 460 Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp 465 470 475 480 Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr 485 490 495 Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys 500 505 510 Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile 515 520 525 Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln 530 535 540 Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr 545 550 555 560 Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys 565 570 575 Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr 580 585 590 Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro 595 600 605 Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg 610 615 620 Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys 625 630 635 640 Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu 645 650 655 Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu 660 665 670 Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met 675 680 685 Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys 690 695 700 Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln 705 710 715 720 Ala Ala Leu Gly Leu 725 <210> 722 <211> 342 <212> PRT <213> artificial sequence <220> <223> DOM1h-131-206-SCDKTH-Trastuzumab Fc fusion protein <400> 722 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Asp Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr His Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ser Cys Asp Lys Thr His Ala Pro Glu 115 120 125 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 130 135 140 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 145 150 155 160 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 165 170 175 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 180 185 190 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 195 200 205 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 210 215 220 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 225 230 235 240 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 245 250 255 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 260 265 270 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 275 280 285 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 290 295 300 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 305 310 315 320 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 325 330 335 Ser Leu Ser Pro Gly Lys 340 <210> 723 <211> 351 <212> PRT <213> artificial sequence <220> <223> DOM1h-131-206-GGGGSGGGGSGGGGS-Trastuzumab Fc fusion protein <400> 723 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Asp Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr His Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 130 135 140 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 145 150 155 160 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 165 170 175 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 180 185 190 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 195 200 205 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 210 215 220 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 225 230 235 240 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 245 250 255 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 260 265 270 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 275 280 285 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 290 295 300 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 305 310 315 320 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 325 330 335 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 340 345 350 <210> 724 <211> 357 <212> PRT <213> artificial sequence <220> <223> DOM1h-131-206-GGGGSGGGGSGGGGS-SCDKTH-Trastuzumab Fc fusion proteins <400> 724 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Asp Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr His Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Ser Cys Asp Lys Thr His Ala Pro Glu Leu 130 135 140 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 145 150 155 160 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 165 170 175 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 180 185 190 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 195 200 205 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 210 215 220 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 225 230 235 240 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 245 250 255 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 260 265 270 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 275 280 285 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 290 295 300 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 305 310 315 320 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 325 330 335 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 340 345 350 Leu Ser Pro Gly Lys 355 <210> 725 <211> 725 <212> PRT <213> artificial sequence <220> <223> DOM1h-131-206-GGGGSGGGGSGGGGS-HSA fusion protein <400> 725 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Asp Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr His Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys 130 135 140 Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys 145 150 155 160 Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe 165 170 175 Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr 180 185 190 Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr 195 200 205 Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr 210 215 220 Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu 225 230 235 240 Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val 245 250 255 Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu 260 265 270 Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr 275 280 285 Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala 290 295 300 Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro 305 310 315 320 Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln 325 330 335 Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys 340 345 350 Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe 355 360 365 Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu 370 375 380 Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu 385 390 395 400 Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys 405 410 415 Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu 420 425 430 Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp 435 440 445 Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp 450 455 460 Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp 465 470 475 480 Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr 485 490 495 Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys 500 505 510 Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile 515 520 525 Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln 530 535 540 Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr 545 550 555 560 Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys 565 570 575 Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr 580 585 590 Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro 595 600 605 Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg 610 615 620 Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys 625 630 635 640 Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu 645 650 655 Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu 660 665 670 Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met 675 680 685 Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys 690 695 700 Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln 705 710 715 720 Ala Ala Leu Gly Leu 725 <210> 726 <211> 472 <212> PRT <213> artificial sequence <220> <223> H398 scFv-ESKYGPPCPPCP-Nivolumab Fc fusion protein <400> 726 Gln Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe 20 25 30 Tyr Ile Asn Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Tyr Pro Tyr Ser Gly His Ala Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Ala Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Phe 65 70 75 80 Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Val Arg Trp Asp Phe Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr 100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser 130 135 140 Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu 145 150 155 160 His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Val Gln Lys Pro Gly 165 170 175 Gln Ser Pro Lys Leu Leu Ile Tyr Thr Val Ser Asn Arg Phe Ser Gly 180 185 190 Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 195 200 205 Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser 210 215 220 Gln Ser Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu 225 230 235 240 Ile Lys Arg Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala 245 250 255 Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 260 265 270 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 275 280 285 Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 290 295 300 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 305 310 315 320 Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 325 330 335 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 340 345 350 Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 355 360 365 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr 370 375 380 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 385 390 395 400 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 405 410 415 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 420 425 430 Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe 435 440 445 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 450 455 460 Ser Leu Ser Leu Ser Leu Gly Lys 465 470 <210> 727 <211> 475 <212> PRT <213> artificial sequence <220> <223> H398 scFv-GGGGSGGGGSGGGGS-Nivolumab Fc fusion protein <400> 727 Gln Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe 20 25 30 Tyr Ile Asn Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Tyr Pro Tyr Ser Gly His Ala Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Ala Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Phe 65 70 75 80 Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Val Arg Trp Asp Phe Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr 100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser 130 135 140 Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu 145 150 155 160 His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Val Gln Lys Pro Gly 165 170 175 Gln Ser Pro Lys Leu Leu Ile Tyr Thr Val Ser Asn Arg Phe Ser Gly 180 185 190 Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 195 200 205 Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser 210 215 220 Gln Ser Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu 225 230 235 240 Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 245 250 255 Gly Ser Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 260 265 270 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 275 280 285 Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn 290 295 300 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 305 310 315 320 Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 325 330 335 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 340 345 350 Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys 355 360 365 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu 370 375 380 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 385 390 395 400 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 405 410 415 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 420 425 430 Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly 435 440 445 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 450 455 460 Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 465 470 475 <210> 728 <211> 487 <212> PRT <213> artificial sequence <220> <223> H398 scFv-GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nivolumab Fc fusion proteins <400> 728 Gln Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe 20 25 30 Tyr Ile Asn Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Tyr Pro Tyr Ser Gly His Ala Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Ala Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Phe 65 70 75 80 Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Val Arg Trp Asp Phe Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr 100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser 130 135 140 Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu 145 150 155 160 His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Val Gln Lys Pro Gly 165 170 175 Gln Ser Pro Lys Leu Leu Ile Tyr Thr Val Ser Asn Arg Phe Ser Gly 180 185 190 Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 195 200 205 Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser 210 215 220 Gln Ser Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu 225 230 235 240 Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 245 250 255 Gly Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 260 265 270 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 275 280 285 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 290 295 300 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 305 310 315 320 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 325 330 335 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 340 345 350 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 355 360 365 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 370 375 380 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 385 390 395 400 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 405 410 415 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 420 425 430 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 435 440 445 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 450 455 460 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 465 470 475 480 Leu Ser Leu Ser Leu Gly Lys 485 <210> 729 <211> 348 <212> PRT <213> artificial sequence <220> <223> DOMlh-574-16-ESKYGPPCPPCP-Nivolumab Fc fusion protein <400> 729 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Asp Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro 115 120 125 Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe 130 135 140 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 145 150 155 160 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 165 170 175 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 180 185 190 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 195 200 205 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 210 215 220 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 225 230 235 240 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 245 250 255 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 260 265 270 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 275 280 285 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 290 295 300 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 305 310 315 320 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 325 330 335 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 340 345 <210> 730 <211> 351 <212> PRT <213> artificial sequence <220> <223> DOMlh-574-16-GGGGSGGGGSGGGGS-Nivolumab Fc fusion protein <400> 730 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Asp Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 130 135 140 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 145 150 155 160 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 165 170 175 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 180 185 190 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 195 200 205 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 210 215 220 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 225 230 235 240 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 245 250 255 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 260 265 270 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 275 280 285 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 290 295 300 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 305 310 315 320 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 325 330 335 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 340 345 350 <210> 731 <211> 363 <212> PRT <213> artificial sequence <220> <223> DOMlh-574-16-GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nivoluma b Fc fusion protein <400> 731 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Gln Ile Ser Asn Thr Gly Asp Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Glu Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro 130 135 140 Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 145 150 155 160 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 165 170 175 Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn 180 185 190 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 195 200 205 Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 210 215 220 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 225 230 235 240 Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys 245 250 255 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu 260 265 270 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 275 280 285 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 290 295 300 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 305 310 315 320 Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly 325 330 335 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 340 345 350 Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 355 360 <210> 732 <211> 348 <212> PRT <213> artificial sequence <220> <223> DOMlh-549-ESKYGPPCPPCP-Nivolumab Fc fusion protein <400> 732 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Asp Tyr 20 25 30 Glu Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Glu Ser Gly Thr Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Arg Arg Phe Ser Ala Ser Thr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro 115 120 125 Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe 130 135 140 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 145 150 155 160 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 165 170 175 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 180 185 190 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 195 200 205 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 210 215 220 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 225 230 235 240 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 245 250 255 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 260 265 270 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 275 280 285 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 290 295 300 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 305 310 315 320 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 325 330 335 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 340 345 <210> 733 <211> 351 <212> PRT <213> artificial sequence <220> <223> DOMlh-549-GGGGSGGGGSGGGGS-Nivolumab Fc fusion protein <400> 733 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Asp Tyr 20 25 30 Glu Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Glu Ser Gly Thr Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Arg Arg Phe Ser Ala Ser Thr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 130 135 140 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 145 150 155 160 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 165 170 175 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 180 185 190 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 195 200 205 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 210 215 220 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 225 230 235 240 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 245 250 255 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 260 265 270 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 275 280 285 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 290 295 300 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 305 310 315 320 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 325 330 335 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 340 345 350 <210> 734 <211> 363 <212> PRT <213> artificial sequence <220> <223> DOMlh-549-GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nivolumab Fc fusion proteins <400> 734 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Asp Tyr 20 25 30 Glu Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Glu Ser Gly Thr Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Arg Arg Phe Ser Ala Ser Thr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro 130 135 140 Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 145 150 155 160 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 165 170 175 Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn 180 185 190 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 195 200 205 Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 210 215 220 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 225 230 235 240 Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys 245 250 255 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu 260 265 270 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 275 280 285 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 290 295 300 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 305 310 315 320 Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly 325 330 335 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 340 345 350 Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 355 360 <210> 735 <211> 348 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-208-ESKYGPPCPPCP-Nivolumab Fc fusion protein <400> 735 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro 115 120 125 Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe 130 135 140 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 145 150 155 160 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 165 170 175 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 180 185 190 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 195 200 205 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 210 215 220 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 225 230 235 240 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 245 250 255 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 260 265 270 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 275 280 285 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 290 295 300 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 305 310 315 320 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 325 330 335 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 340 345 <210> 736 <211> 351 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-208-GGGGSGGGGSGGGGS-Nivolumab Fc fusion protein <400> 736 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 130 135 140 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 145 150 155 160 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 165 170 175 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 180 185 190 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 195 200 205 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 210 215 220 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 225 230 235 240 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 245 250 255 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 260 265 270 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 275 280 285 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 290 295 300 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 305 310 315 320 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 325 330 335 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 340 345 350 <210> 737 <211> 363 <212> PRT <213> artificial sequence <220> <223> DOM1h-574-208-GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nivolum ab Fc fusion protein <400> 737 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Lys Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile Ser Asp Thr Ala Asp Arg Thr Tyr Tyr Ala His Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Tyr Thr Gly Arg Trp Val Pro Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro 130 135 140 Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 145 150 155 160 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 165 170 175 Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn 180 185 190 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 195 200 205 Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 210 215 220 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 225 230 235 240 Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys 245 250 255 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu 260 265 270 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 275 280 285 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 290 295 300 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 305 310 315 320 Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly 325 330 335 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 340 345 350 Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 355 360 <210> 738 <211> 348 <212> PRT <213> artificial sequence <220> <223> DOM1h-131-206-ESKYGPPCPPCP-Nivolumab Fc fusion protein <400> 738 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Asp Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr His Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro 115 120 125 Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe 130 135 140 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 145 150 155 160 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 165 170 175 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 180 185 190 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 195 200 205 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 210 215 220 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 225 230 235 240 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 245 250 255 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 260 265 270 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 275 280 285 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 290 295 300 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 305 310 315 320 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 325 330 335 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 340 345 <210> 739 <211> 351 <212> PRT <213> artificial sequence <220> <223> DOM1h-131-206-GGGGSGGGGSGGGGS-Nivolumab Fc fusion protein <400> 739 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Asp Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr His Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 130 135 140 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 145 150 155 160 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 165 170 175 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 180 185 190 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 195 200 205 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 210 215 220 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 225 230 235 240 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 245 250 255 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 260 265 270 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 275 280 285 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 290 295 300 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 305 310 315 320 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 325 330 335 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 340 345 350 <210> 740 <211> 363 <212> PRT <213> artificial sequence <220> <223> DOM1h-131-206-GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nivolum ab Fc fusion protein <400> 740 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30 Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Pro Pro Asp Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr His Cys 85 90 95 Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro 130 135 140 Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 145 150 155 160 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 165 170 175 Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn 180 185 190 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 195 200 205 Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 210 215 220 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 225 230 235 240 Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys 245 250 255 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu 260 265 270 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 275 280 285 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 290 295 300 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 305 310 315 320 Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly 325 330 335 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 340 345 350 Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 355 360 <210> 741 <211> 380 <212> PRT <213> artificial sequence <220> <223> TNF (R32W/S86T)-SCDKTH-Trastuzumab Fc fusion protein <400> 741 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Trp 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Thr Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu Ser Cys Asp 145 150 155 160 Lys Thr His Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <210> 742 <211> 389 <212> PRT <213> artificial sequence <220> <223> TNF (R32W/S86T)-GGGGSGGGGSGGGGS-Trastuzumab Fc fusion protein <400> 742 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Trp 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Thr Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu Gly Gly Gly 145 150 155 160 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Pro Glu Leu 165 170 175 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 180 185 190 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 195 200 205 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 210 215 220 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 225 230 235 240 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 245 250 255 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 260 265 270 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 275 280 285 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 290 295 300 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 305 310 315 320 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 325 330 335 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 340 345 350 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 355 360 365 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 370 375 380 Leu Ser Pro Gly Lys 385 <210> 743 <211> 395 <212> PRT <213> artificial sequence <220> <223> TNF (R32W/S86T)-GGGGSGGGGSGGGGS-SCDKTH-Trastuzumab Fc fusion proteins <400> 743 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Trp 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Thr Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu Gly Gly Gly 145 150 155 160 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Cys Asp Lys 165 170 175 Thr His Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 180 185 190 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 195 200 205 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 210 215 220 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 225 230 235 240 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 245 250 255 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 260 265 270 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 275 280 285 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 290 295 300 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 305 310 315 320 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 325 330 335 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 340 345 350 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 355 360 365 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 370 375 380 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 385 390 395 <210> 744 <211> 380 <212> PRT <213> artificial sequence <220> <223> TNF (V1M/R31C/C69V/Y87H/C101A/A145R)-SCDKTH-Trastuz umab Fc fusion protein <400> 744 Met Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Cys Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Val Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser His Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Ala Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Arg Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu Ser Cys Asp 145 150 155 160 Lys Thr His Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <210> 745 <211> 389 <212> PRT <213> artificial sequence <220> <223> TNF (V1M/R31C/C69V/Y87H/C101A/A145R)-GGGGSGGGGSGGGG S-Trastuzumab Fc fusion protein <400> 745 Met Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Cys Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Val Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser His Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Ala Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Arg Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu Gly Gly Gly 145 150 155 160 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Pro Glu Leu 165 170 175 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 180 185 190 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 195 200 205 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 210 215 220 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 225 230 235 240 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 245 250 255 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 260 265 270 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 275 280 285 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 290 295 300 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 305 310 315 320 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 325 330 335 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 340 345 350 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 355 360 365 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 370 375 380 Leu Ser Pro Gly Lys 385 <210> 746 <211> 395 <212> PRT <213> artificial sequence <220> <223> TNF (V1M/R31C/C69V/Y87H/C101A/A145R)-GGGGSGGGGSGGGG S-SCDKTH-Trastuzumab Fc fusion protein <400> 746 Met Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Cys Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Val Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser His Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Ala Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Arg Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu Gly Gly Gly 145 150 155 160 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Cys Asp Lys 165 170 175 Thr His Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 180 185 190 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 195 200 205 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 210 215 220 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 225 230 235 240 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 245 250 255 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 260 265 270 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 275 280 285 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 290 295 300 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 305 310 315 320 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 325 330 335 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 340 345 350 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 355 360 365 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 370 375 380 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 385 390 395 <210> 747 <211> 380 <212> PRT <213> artificial sequence <220> <223> TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q)-SCDKTH-Trastuzu mab Fc fusion protein <400> 747 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ser Thr Thr His Asn Gln Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu Ser Cys Asp 145 150 155 160 Lys Thr His Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <210> 748 <211> 389 <212> PRT <213> artificial sequence <220> <223> TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q)-GGGGSGGGGSGGGGS -Trastuzumab Fc fusion protein <400> 748 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ser Thr Thr His Asn Gln Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu Gly Gly Gly 145 150 155 160 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Pro Glu Leu 165 170 175 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 180 185 190 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 195 200 205 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 210 215 220 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 225 230 235 240 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 245 250 255 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 260 265 270 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 275 280 285 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 290 295 300 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 305 310 315 320 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 325 330 335 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 340 345 350 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 355 360 365 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 370 375 380 Leu Ser Pro Gly Lys 385 <210> 749 <211> 395 <212> PRT <213> artificial sequence <220> <223> TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q)-GGGGSGGGGSGGGGS -SCDKTH-Trastuzumab Fc fusion protein <400> 749 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ser Thr Thr His Asn Gln Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu Gly Gly Gly 145 150 155 160 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Cys Asp Lys 165 170 175 Thr His Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 180 185 190 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 195 200 205 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 210 215 220 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 225 230 235 240 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 245 250 255 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 260 265 270 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 275 280 285 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 290 295 300 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 305 310 315 320 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 325 330 335 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 340 345 350 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 355 360 365 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 370 375 380 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 385 390 395 <210> 750 <211> 380 <212> PRT <213> artificial sequence <220> <223> TNF (I97T/A145R)-SCDKTH-Trastuzumab Fc fusion protein <400> 750 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Thr Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Arg Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu Ser Cys Asp 145 150 155 160 Lys Thr His Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <210> 751 <211> 389 <212> PRT <213> artificial sequence <220> <223> TNF (I97T/A145R)-GGGGSGGGGSGGGGS-Trastuzumab Fc fusion protein <400> 751 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Thr Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Arg Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu Gly Gly Gly 145 150 155 160 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Pro Glu Leu 165 170 175 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 180 185 190 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 195 200 205 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 210 215 220 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 225 230 235 240 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 245 250 255 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 260 265 270 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 275 280 285 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 290 295 300 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 305 310 315 320 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 325 330 335 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 340 345 350 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 355 360 365 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 370 375 380 Leu Ser Pro Gly Lys 385 <210> 752 <211> 395 <212> PRT <213> artificial sequence <220> <223> TNF (I97T/A145R)-GGGGSGGGGSGGGGS-SCDKTH-Trastuzumab Fc fusion proteins <400> 752 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Thr Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Arg Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu Gly Gly Gly 145 150 155 160 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Cys Asp Lys 165 170 175 Thr His Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 180 185 190 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 195 200 205 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 210 215 220 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 225 230 235 240 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 245 250 255 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 260 265 270 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 275 280 285 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 290 295 300 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 305 310 315 320 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 325 330 335 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 340 345 350 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 355 360 365 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 370 375 380 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 385 390 395 <210> 753 <211> 386 <212> PRT <213> artificial sequence <220> <223> TNF (R32W/S86T)-ESKYGPPCPPCP-Nivolumab Fc fusion protein <400> 753 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Trp 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Thr Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu Glu Ser Lys 145 150 155 160 Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly 165 170 175 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 180 185 190 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 195 200 205 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 210 215 220 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg 225 230 235 240 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 245 250 255 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 260 265 270 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 275 280 285 Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu 290 295 300 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 305 310 315 320 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 325 330 335 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp 340 345 350 Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 355 360 365 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 370 375 380 Gly Lys 385 <210> 754 <211> 389 <212> PRT <213> artificial sequence <220> <223> TNF (R32W/S86T)-GGGGSGGGGSGGGGS-Nivolumab Fc fusion protein <400> 754 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Trp 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Thr Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu Gly Gly Gly 145 150 155 160 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Pro Glu Phe 165 170 175 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 180 185 190 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 195 200 205 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 210 215 220 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 225 230 235 240 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 245 250 255 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 260 265 270 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 275 280 285 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 290 295 300 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 305 310 315 320 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 325 330 335 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 340 345 350 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 355 360 365 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 370 375 380 Leu Ser Leu Gly Lys 385 <210> 755 <211> 401 <212> PRT <213> artificial sequence <220> <223> TNF(R32W/S86T)-GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nivolu mab Fc fusion protein <400> 755 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Trp 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Thr Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu Gly Gly Gly 145 150 155 160 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ser Lys Tyr 165 170 175 Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro 180 185 190 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 195 200 205 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 210 215 220 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 225 230 235 240 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 245 250 255 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 260 265 270 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 275 280 285 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 290 295 300 Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 305 310 315 320 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 325 330 335 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 340 345 350 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 355 360 365 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 370 375 380 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 385 390 395 400 Lys <210> 756 <211> 386 <212> PRT <213> artificial sequence <220> <223> TNF (V1M/R31C/C69V/Y87H/C101A/A145R)-ESKYGPPCPPCP-N ivolumab Fc fusion protein <400> 756 Met Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Cys Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Val Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser His Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Ala Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Arg Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu Glu Ser Lys 145 150 155 160 Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly 165 170 175 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 180 185 190 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 195 200 205 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 210 215 220 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg 225 230 235 240 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 245 250 255 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 260 265 270 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 275 280 285 Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu 290 295 300 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 305 310 315 320 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 325 330 335 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp 340 345 350 Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 355 360 365 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 370 375 380 Gly Lys 385 <210> 757 <211> 389 <212> PRT <213> artificial sequence <220> <223> TNF (V1M/R31C/C69V/Y87H/C101A/A145R)-GGGGSGGGGSGGGG S-Nivolumab Fc fusion protein <400> 757 Met Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Cys Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Val Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser His Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Ala Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Arg Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu Gly Gly Gly 145 150 155 160 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Pro Glu Phe 165 170 175 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 180 185 190 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 195 200 205 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 210 215 220 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 225 230 235 240 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 245 250 255 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 260 265 270 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 275 280 285 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 290 295 300 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 305 310 315 320 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 325 330 335 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 340 345 350 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 355 360 365 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 370 375 380 Leu Ser Leu Gly Lys 385 <210> 758 <211> 401 <212> PRT <213> artificial sequence <220> <223> TNF (V1M/R31C/C69V/Y87H/C101A/A145R)-GGGGSGGGGSGGGG S-ESKYGPPCPPCP-Nivolumab Fc fusion protein <400> 758 Met Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Cys Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Val Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser His Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Ala Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Arg Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu Gly Gly Gly 145 150 155 160 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ser Lys Tyr 165 170 175 Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro 180 185 190 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 195 200 205 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 210 215 220 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 225 230 235 240 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 245 250 255 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 260 265 270 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 275 280 285 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 290 295 300 Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 305 310 315 320 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 325 330 335 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 340 345 350 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 355 360 365 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 370 375 380 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 385 390 395 400 Lys <210> 759 <211> 386 <212> PRT <213> artificial sequence <220> <223> TNF(A84S/V85T/S86T/Y87H/Q88N/T89Q)-ESKYGPPCPPCP-Ni volumab Fc fusion protein <400> 759 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ser Thr Thr His Asn Gln Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu Glu Ser Lys 145 150 155 160 Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly 165 170 175 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 180 185 190 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 195 200 205 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 210 215 220 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg 225 230 235 240 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 245 250 255 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 260 265 270 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 275 280 285 Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu 290 295 300 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 305 310 315 320 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 325 330 335 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp 340 345 350 Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 355 360 365 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 370 375 380 Gly Lys 385 <210> 760 <211> 389 <212> PRT <213> artificial sequence <220> <223> TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q)-GGGGSGGGGSGGGGS -Nivolumab Fc fusion protein <400> 760 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ser Thr Thr His Asn Gln Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu Gly Gly Gly 145 150 155 160 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Pro Glu Phe 165 170 175 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 180 185 190 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 195 200 205 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 210 215 220 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 225 230 235 240 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 245 250 255 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 260 265 270 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 275 280 285 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 290 295 300 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 305 310 315 320 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 325 330 335 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 340 345 350 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 355 360 365 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 370 375 380 Leu Ser Leu Gly Lys 385 <210> 761 <211> 401 <212> PRT <213> artificial sequence <220> <223> TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q)-GGGGSGGGGSGGGGS -ESKYGPPCPPCP-Nivolumab Fc fusion protein <400> 761 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ser Thr Thr His Asn Gln Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu Gly Gly Gly 145 150 155 160 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ser Lys Tyr 165 170 175 Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro 180 185 190 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 195 200 205 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 210 215 220 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 225 230 235 240 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 245 250 255 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 260 265 270 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 275 280 285 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 290 295 300 Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 305 310 315 320 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 325 330 335 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 340 345 350 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 355 360 365 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 370 375 380 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 385 390 395 400 Lys <210> 762 <211> 386 <212> PRT <213> artificial sequence <220> <223> TNF (I97T/A145R)-ESKYGPPCPPCP-Nivolumab Fc fusion protein <400> 762 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Thr Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Arg Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu Glu Ser Lys 145 150 155 160 Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly 165 170 175 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 180 185 190 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 195 200 205 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 210 215 220 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg 225 230 235 240 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 245 250 255 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 260 265 270 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 275 280 285 Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu 290 295 300 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 305 310 315 320 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 325 330 335 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp 340 345 350 Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 355 360 365 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 370 375 380 Gly Lys 385 <210> 763 <211> 389 <212> PRT <213> artificial sequence <220> <223> TNF(I97T/A145R)-GGGGSGGGGSGGGGS-Nivolumab Fc fusion protein <400> 763 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Thr Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Arg Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu Gly Gly Gly 145 150 155 160 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Pro Glu Phe 165 170 175 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 180 185 190 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 195 200 205 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 210 215 220 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 225 230 235 240 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 245 250 255 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 260 265 270 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 275 280 285 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 290 295 300 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 305 310 315 320 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 325 330 335 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 340 345 350 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 355 360 365 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 370 375 380 Leu Ser Leu Gly Lys 385 <210> 764 <211> 401 <212> PRT <213> artificial sequence <220> <223> TNF(I97T/A145R)-GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nivol umab Fc fusion protein <400> 764 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Thr Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Arg Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu Gly Gly Gly 145 150 155 160 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ser Lys Tyr 165 170 175 Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro 180 185 190 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 195 200 205 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 210 215 220 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 225 230 235 240 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 245 250 255 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 260 265 270 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 275 280 285 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 290 295 300 Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 305 310 315 320 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 325 330 335 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 340 345 350 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 355 360 365 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 370 375 380 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 385 390 395 400 Lys <210> 765 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR2-selective agonist TNF mutein K65W <400> 765 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Trp Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 766 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR2-selective agonist TNF mutein D143Y <400> 766 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Tyr Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 767 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR2-selective agonist TNF mutein D143F <400> 767 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Phe Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 768 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR2-selective agonist TNF mutein D143N <400> 768 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asn Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 769 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR2-selective agonist TNF mutein D143E <400> 769 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Glu Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 770 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR2-selective agonist TNF mutein D143W <400> 770 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Trp Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 771 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR2-selective agonist TNF mutein D143V <400> 771 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Val Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 772 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR2-selective agonist TNF mutein A145R <400> 772 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Arg Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 773 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR2-selective agonist TNF mutein A145H <400> 773 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 His Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 774 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR2-selective agonist TNF mutein A145K <400> 774 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Lys Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 775 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR2-selective agonist TNF mutein A145F <400> 775 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Phe Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 776 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR2-selective agonist TNF mutein A145W <400> 776 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Trp Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 777 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR2-selective agonist TNF mutein E146Q <400> 777 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Gln Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 778 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR2-selective agonist TNF mutein E146H <400> 778 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala His Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 779 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR2-selective agonist TNF mutein E146K <400> 779 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Lys Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 780 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR2-selective agonist TNF mutein E146N <400> 780 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Asn Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 781 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR2-selective agonist TNF mutein D143N/A145R <400> 781 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asn Phe 130 135 140 Arg Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 782 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR2-selective agonist TNF mutein A145R/S147T <400> 782 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Arg Glu Thr Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 783 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR2-selective agonist TNF mutein Q88N/T89S/A145S/E146A/S147D <400> 783 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Asn Ser Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ser Ala Asp Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 784 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR2-selective agonist TNF mutein Q88N/A145I/E146G/S147D <400> 784 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Asn Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ile Gly Asp Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 785 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR2-selective agonist TNF mutein A145H/E146S/S147D <400> 785 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 His Ser Asp Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 786 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR2-selective agonist TNF mutein A145H/S147D <400> 786 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 His Glu Asp Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 787 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR2-selective agonist TNF mutein L29V/A145D/E146D/S147D <400> 787 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Val Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Asp Asp Asp Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 788 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR2-selective agonist TNF mutein A145N/E146D/S147D <400> 788 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Asn Asp Asp Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 789 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR2-selective agonist TNF mutein A145T/E146S/S147D <400> 789 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Thr Ser Asp Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 790 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR2-selective agonist TNF mutein A145Q/E146D/S147D <400> 790 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Gln Asp Asp Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 791 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR2-selective agonist TNF mutein A145T/E146D/S147D <400> 791 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Thr Asp Asp Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 792 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR2-selective agonist TNF mutein A145D/E146G/S147D <400> 792 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Asp Gly Asp Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 793 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR2-selective agonist TNF mutein A145D/S147D <400> 793 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Asp Glu Asp Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 794 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR2-selective agonist TNF mutein A145K/E146D/S147T <400> 794 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Lys Asp Thr Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 795 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR2-selective agonist TNF mutein A145R/E146T/S147D <400> 795 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Arg Thr Asp Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 796 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR2-selective agonist TNF mutein A145R/S147T <400> 796 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Arg Glu Thr Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 797 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR2-selective agonist TNF mutein E146D/S147D <400> 797 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Asp Asp Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 798 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR2-selective agonist TNF mutein D143V/F144L/A145S <400> 798 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Val Leu 130 135 140 Ser Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 799 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR2-selective agonist TNF mutein D143V/A145S <400> 799 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Val Phe 130 135 140 Ser Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 800 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNF07 (TNFR2-selective agonist TNF mutein S95C/G148C) <400> 800 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Cys Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Cys Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 801 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNFR2-selective TNF mutein A145T <400> 801 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Thr Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 802 <211> 157 <212> PRT <213> artificial sequence <220> <223> DN-TNF L57Y <400> 802 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Tyr Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 803 <211> 472 <212> PRT <213> artificial sequence <220> <223> scTNFR2 (human TNFR2-selective single chain agonist TNF mutein trimer D143N/A145R) <400> 803 Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn 1 5 10 15 Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala 20 25 30 Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro 35 40 45 Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln 50 55 60 Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile 65 70 75 80 Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser 85 90 95 Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr 100 105 110 Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg 115 120 125 Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asn Phe Arg Glu Ser 130 135 140 Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu Gly Gly Gly Gly Ser Ser 145 150 155 160 Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro 165 170 175 Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu 180 185 190 Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser 195 200 205 Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly 210 215 220 Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala 225 230 235 240 Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro 245 250 255 Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu 260 265 270 Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu 275 280 285 Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asn Phe Arg Glu Ser Gly 290 295 300 Gln Val Tyr Phe Gly Ile Ile Ala Leu Gly Gly Gly Gly Ser Ser Ser 305 310 315 320 Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro Gln 325 330 335 Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu Leu 340 345 350 Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser Glu 355 360 365 Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly Cys 370 375 380 Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala Val 385 390 395 400 Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro Cys 405 410 415 Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu Pro 420 425 430 Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu Ser 435 440 445 Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asn Phe Arg Glu Ser Gly Gln 450 455 460 Val Tyr Phe Gly Ile Ile Ala Leu 465 470 <210> 804 <211> 1808 <212> PRT 213 <br> <220> <223> chicken tenascin C (TNC) <220> <221> DOMAIN <222> (110)...(139) <223> trimerization domain <400> 804 Met Gly Leu Pro Ser Gln Val Leu Ala Cys Ala Ile Leu Gly Leu Leu 1 5 10 15 Tyr Gln His Ala Ser Gly Gly Leu Ile Lys Arg Ile Ile Arg Gln Lys 20 25 30 Arg Glu Thr Gly Leu Asn Val Thr Leu Pro Glu Asp Asn Gln Pro Val 35 40 45 Val Phe Asn His Val Tyr Asn Ile Lys Leu Pro Val Gly Ser Leu Cys 50 55 60 Ser Val Asp Leu Asp Thr Ala Ser Gly Asp Ala Asp Leu Lys Ala Glu 65 70 75 80 Ile Glu Pro Val Lys Asn Tyr Glu Glu His Thr Val Asn Glu Gly Asn 85 90 95 Gln Ile Val Phe Thr His Arg Ile Asn Ile Pro Arg Arg Ala Cys Gly 100 105 110 Cys Ala Ala Ala Pro Asp Ile Lys Asp Leu Leu Ser Arg Leu Glu Glu 115 120 125 Leu Glu Gly Leu Val Ser Ser Leu Arg Glu Gln Cys Ala Ser Gly Ala 130 135 140 Gly Cys Cys Pro Asn Ser Gln Thr Ala Glu Gly Arg Leu Asp Thr Ala 145 150 155 160 Pro Tyr Cys Ser Gly His Gly Asn Tyr Ser Thr Glu Ile Cys Gly Cys 165 170 175 Val Cys Glu Pro Gly Trp Lys Gly Pro Asn Cys Ser Glu Pro Ala Cys 180 185 190 Pro Arg Asn Cys Leu Asn Arg Gly Leu Cys Val Arg Gly Lys Cys Ile 195 200 205 Cys Glu Glu Gly Phe Thr Gly Glu Asp Cys Ser Gln Ala Ala Cys Pro 210 215 220 Ser Asp Cys Asn Asp Gln Gly Lys Cys Val Asp Gly Val Cys Val Cys 225 230 235 240 Phe Glu Gly Tyr Thr Gly Pro Asp Cys Gly Glu Glu Leu Cys Pro His 245 250 255 Gly Cys Gly Ile His Gly Arg Cys Val Gly Gly Arg Cys Val Cys His 260 265 270 Glu Gly Phe Thr Gly Glu Asp Cys Asn Glu Pro Leu Cys Pro Asn Asn 275 280 285 Cys His Asn Arg Gly Arg Cys Val Asp Asn Glu Cys Val Cys Asp Glu 290 295 300 Gly Tyr Thr Gly Glu Asp Cys Gly Glu Leu Ile Cys Pro Asn Asp Cys 305 310 315 320 Phe Asp Arg Gly Arg Cys Ile Asn Gly Thr Cys Phe Cys Glu Glu Gly 325 330 335 Tyr Thr Gly Glu Asp Cys Gly Glu Leu Thr Cys Pro Asn Asn Cys Asn 340 345 350 Gly Asn Gly Arg Cys Glu Asn Gly Leu Cys Val Cys His Glu Gly Phe 355 360 365 Val Gly Asp Asp Cys Ser Gln Lys Arg Cys Pro Lys Asp Cys Asn Asn 370 375 380 Arg Gly His Cys Val Asp Gly Arg Cys Val Cys His Glu Gly Tyr Leu 385 390 395 400 Gly Glu Asp Cys Gly Glu Leu Arg Cys Pro Asn Asp Cys His Asn Arg 405 410 415 Gly Arg Cys Ile Asn Gly Gln Cys Val Cys Asp Glu Gly Phe Ile Gly 420 425 430 Glu Asp Cys Gly Glu Leu Arg Cys Pro Asn Asp Cys His Asn Arg Gly 435 440 445 Arg Cys Val Asn Gly Gln Cys Glu Cys His Glu Gly Phe Ile Gly Glu 450 455 460 Asp Cys Gly Glu Leu Arg Cys Pro Asn Asp Cys Asn Ser His Gly Arg 465 470 475 480 Cys Val Asn Gly Gln Cys Val Cys Asp Glu Gly Tyr Thr Gly Glu Asp 485 490 495 Cys Gly Glu Leu Arg Cys Pro Asn Asp Cys His Asn Arg Gly Arg Cys 500 505 510 Val Glu Gly Arg Cys Val Cys Asp Asn Gly Phe Met Gly Glu Asp Cys 515 520 525 Gly Glu Leu Ser Cys Pro Asn Asp Cys His Gln His Gly Arg Cys Val 530 535 540 Asp Gly Arg Cys Val Cys His Glu Gly Phe Thr Gly Glu Asp Cys Arg 545 550 555 560 Glu Arg Ser Cys Pro Asn Asp Cys Asn Asn Val Gly Arg Cys Val Glu 565 570 575 Gly Arg Cys Val Cys Glu Glu Gly Tyr Met Gly Ile Asp Cys Ser Asp 580 585 590 Val Ser Pro Pro Thr Glu Leu Thr Val Thr Asn Val Thr Asp Lys Thr 595 600 605 Val Asn Leu Glu Trp Lys His Glu Asn Leu Val Asn Glu Tyr Leu Val 610 615 620 Thr Tyr Val Pro Thr Ser Ser Gly Gly Leu Asp Leu Gln Phe Thr Val 625 630 635 640 Pro Gly Asn Gln Thr Ser Ala Thr Ile His Glu Leu Glu Pro Gly Val 645 650 655 Glu Tyr Phe Ile Arg Val Phe Ala Ile Leu Lys Asn Lys Lys Ser Ile 660 665 670 Pro Val Ser Ala Arg Val Ala Thr Tyr Leu Pro Ala Pro Glu Gly Leu 675 680 685 Lys Phe Lys Ser Val Arg Glu Thr Ser Val Gln Val Glu Trp Asp Pro 690 695 700 Leu Ser Ile Ser Phe Asp Gly Trp Glu Leu Val Phe Arg Asn Met Gln 705 710 715 720 Lys Lys Asp Asp Asn Gly Asp Ile Thr Ser Ser Leu Lys Arg Pro Glu 725 730 735 Thr Ser Tyr Met Gln Pro Gly Leu Ala Pro Gly Gln Gln Tyr Asn Val 740 745 750 Ser Leu His Ile Val Lys Asn Asn Thr Arg Gly Pro Gly Leu Ser Arg 755 760 765 Val Ile Thr Thr Lys Leu Asp Ala Pro Ser Gln Ile Glu Ala Lys Asp 770 775 780 Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Ser Lys Pro Leu Ala Glu 785 790 795 800 Ile Glu Gly Ile Glu Leu Thr Tyr Gly Pro Lys Asp Val Pro Gly Asp 805 810 815 Arg Thr Thr Ile Asp Leu Ser Glu Asp Glu Asn Gln Tyr Ser Ile Gly 820 825 830 Asn Leu Arg Pro His Thr Glu Tyr Glu Val Thr Leu Ile Ser Arg Arg 835 840 845 Gly Asp Met Glu Ser Asp Pro Ala Lys Glu Val Phe Val Thr Asp Leu 850 855 860 Asp Ala Pro Arg Asn Leu Lys Arg Val Ser Gln Thr Asp Asn Ser Ile 865 870 875 880 Thr Leu Glu Trp Lys Asn Ser His Ala Asn Ile Asp Asn Tyr Arg Ile 885 890 895 Lys Phe Ala Pro Ile Ser Gly Gly Asp His Thr Glu Leu Thr Val Pro 900 905 910 Lys Gly Asn Gln Ala Thr Thr Arg Ala Thr Leu Thr Gly Leu Arg Pro 915 920 925 Gly Thr Glu Tyr Gly Ile Gly Val Thr Ala Val Arg Gln Asp Arg Glu 930 935 940 Ser Ala Pro Ala Thr Ile Asn Ala Gly Thr Asp Leu Asp Asn Pro Lys 945 950 955 960 Asp Leu Glu Val Ser Asp Pro Thr Glu Thr Thr Leu Ser Leu Arg Trp 965 970 975 Arg Arg Pro Val Ala Lys Phe Asp Arg Tyr Arg Leu Thr Tyr Val Ser 980 985 990 Pro Ser Gly Lys Lys Asn Glu Met Glu Ile Pro Val Asp Ser Thr Ser 995 1000 1005 Phe Ile Leu Arg Gly Leu Asp Ala Gly Thr Glu Tyr Thr Ile Ser Leu 1010 1015 1020 Val Ala Glu Lys Gly Arg His Lys Ser Lys Pro Thr Thr Ile Lys Gly 1025 1030 1035 1040 Ser Thr Glu Glu Glu Pro Glu Leu Gly Asn Leu Ser Val Ser Glu Thr 1045 1050 1055 Gly Trp Asp Gly Phe Gln Leu Thr Trp Thr Ala Ala Asp Gly Ala Tyr 1060 1065 1070 Glu Asn Phe Val Ile Gln Val Gln Gln Ser Asp Asn Pro Glu Glu Thr 1075 1080 1085 Trp Asn Ile Thr Val Pro Gly Gly Gln His Ser Val Asn Val Thr Gly 1090 1095 1100 Leu Lys Ala Asn Thr Pro Tyr Asn Val Thr Leu Tyr Gly Val Ile Arg 1105 1110 1115 1120 Gly Tyr Arg Thr Lys Pro Leu Tyr Val Glu Thr Thr Thr Thr Gly Ala His 1125 1130 1135 Pro Glu Val Gly Glu Leu Thr Val Ser Asp Ile Thr Pro Glu Ser Phe 1140 1145 1150 Asn Leu Ser Trp Thr Thr Thr Asn Gly Asp Phe Asp Ala Phe Thr Ile 1155 1160 1165 Glu Ile Ile Asp Ser Asn Arg Leu Leu Glu Pro Met Glu Phe Asn Ile 1170 1175 1180 Ser Gly Asn Ser Arg Thr Ala His Ile Ser Gly Leu Ser Pro Ser Thr 1185 1190 1195 1200 Asp Phe Ile Val Tyr Leu Tyr Gly Ile Ser His Gly Phe Arg Thr Gln 1205 1210 1215 Ala Ile Ser Ala Ala Ala Thr Thr Glu Ala Glu Pro Glu Val Asp Asn 1220 1225 1230 Leu Leu Val Ser Asp Ala Thr Pro Asp Gly Phe Arg Leu Ser Trp Thr 1235 1240 1245 Ala Asp Asp Gly Val Phe Asp Ser Phe Val Leu Lys Ile Arg Asp Thr 1250 1255 1260 Lys Arg Lys Ser Asp Pro Leu Glu Leu Ile Val Pro Gly His Glu Arg 1265 1270 1275 1280 Thr His Asp Ile Thr Gly Leu Lys Glu Gly Thr Glu Tyr Glu Ile Glu 1285 1290 1295 Leu Tyr Gly Val Ser Ser Gly Arg Arg Ser Gln Pro Ile Asn Ser Val 1300 1305 1310 Ala Thr Thr Val Val Gly Ser Pro Lys Gly Ile Ser Phe Ser Asp Ile 1315 1320 1325 Thr Glu Asn Ser Ala Thr Val Ser Trp Thr Pro Pro Arg Ser Arg Val 1330 1335 1340 Asp Ser Tyr Arg Val Ser Tyr Val Pro Ile Thr Gly Gly Thr Pro Asn 1345 1350 1355 1360 Val Val Thr Val Asp Gly Ser Lys Thr Arg Thr Lys Leu Val Lys Leu 1365 1370 1375 Val Pro Gly Val Asp Tyr Asn Val Asn Ile Ile Ser Val Lys Gly Phe 1380 1385 1390 Glu Glu Ser Glu Pro Ile Ser Gly Ile Leu Lys Thr Ala Leu Asp Ser 1395 1400 1405 Pro Ser Gly Leu Val Val Met Asn Ile Thr Asp Ser Glu Ala Leu Ala 1410 1415 1420 Thr Trp Gln Pro Ala Ile Ala Ala Val Asp Asn Tyr Ile Val Ser Tyr 1425 1430 1435 1440 Ser Ser Glu Asp Glu Pro Glu Val Thr Gln Met Val Ser Gly Asn Thr 1445 1450 1455 Val Glu Tyr Asp Leu Asn Gly Leu Arg Pro Ala Thr Glu Tyr Thr Leu 1460 1465 1470 Arg Val His Ala Val Lys Asp Ala Gln Lys Ser Glu Thr Leu Ser Thr 1475 1480 1485 Gln Phe Thr Thr Gly Leu Asp Ala Pro Lys Asp Leu Ser Ala Thr Glu 1490 1495 1500 Val Gln Ser Glu Thr Ala Val Ile Thr Trp Arg Pro Pro Arg Ala Pro 1505 1510 1515 1520 Val Thr Asp Tyr Leu Leu Thr Tyr Glu Ser Ile Asp Gly Arg Val Lys 1525 1530 1535 Glu Val Ile Leu Asp Pro Glu Thr Thr Ser Tyr Thr Leu Thr Glu Leu 1540 1545 1550 Ser Pro Ser Thr Gln Tyr Thr Val Lys Leu Gln Ala Leu Ser Arg Ser 1555 1560 1565 Met Arg Ser Lys Met Ile Gln Thr Val Phe Thr Thr Thr Gly Leu Leu 1570 1575 1580 Tyr Pro Tyr Pro Lys Asp Cys Ser Gln Ala Leu Leu Asn Gly Glu Val 1585 1590 1595 1600 Thr Ser Gly Leu Tyr Thr Ile Tyr Leu Asn Gly Asp Arg Thr Gln Pro 1605 1610 1615 Leu Gln Val Phe Cys Asp Met Ala Glu Asp Gly Gly Gly Trp Ile Val 1620 1625 1630 Phe Leu Arg Arg Gln Asn Gly Lys Glu Asp Phe Tyr Arg Asn Trp Lys 1635 1640 1645 Asn Tyr Val Ala Gly Phe Gly Asp Pro Lys Asp Glu Phe Trp Ile Gly 1650 1655 1660 Leu Glu Asn Leu His Lys Ile Ser Ser Gln Gly Gln Tyr Glu Leu Arg 1665 1670 1675 1680 Val Asp Leu Arg Asp Arg Gly Glu Thr Ala Tyr Ala Val Tyr Asp Lys 1685 1690 1695 Phe Ser Val Gly Asp Ala Lys Thr Arg Tyr Arg Leu Arg Val Asp Gly 1700 1705 1710 Tyr Ser Gly Thr Ala Gly Asp Ser Met Thr Tyr His Asn Gly Arg Ser 1715 1720 1725 Phe Ser Thr Phe Asp Lys Asp Asn Asp Ser Ala Ile Thr Asn Cys Ala 1730 1735 1740 Leu Ser Tyr Lys Gly Ala Phe Trp Tyr Lys Asn Cys His Arg Val Asn 1745 1750 1755 1760 Leu Met Gly Arg Tyr Gly Asp Asn Asn His Ser Gln Gly Val Asn Trp 1765 1770 1775 Phe His Trp Lys Gly His Glu Tyr Ser Ile Gln Phe Ala Glu Met Lys 1780 1785 1790 Leu Arg Pro Ser Ser Phe Arg Asn Leu Glu Gly Arg Arg Lys Arg Ala 1795 1800 1805 <210> 805 <211> 30 <212> PRT 213 <br> <220> <223> trimerization domain of Chicken Tenascin C (TNC) <400> 805 Ala Cys Gly Cys Ala Ala Ala Pro Asp Ile Lys Asp Leu Leu Ser Arg 1 5 10 15 Leu Glu Glu Leu Glu Gly Leu Val Ser Ser Leu Arg Glu Gln 20 25 30 <210> 806 <211> 2201 <212> PRT 213 <213> <220> <223> human tenascin C (TNC) <220> <221> DOMAIN <222> (110)...(139) <223> trimerization domain <400> 806 Met Gly Ala Met Thr Gln Leu Leu Ala Gly Val Phe Leu Ala Phe Leu 1 5 10 15 Ala Leu Ala Thr Glu Gly Gly Val Leu Lys Lys Val Ile Arg His Lys 20 25 30 Arg Gln Ser Gly Val Asn Ala Thr Leu Pro Glu Glu Asn Gln Pro Val 35 40 45 Val Phe Asn His Val Tyr Asn Ile Lys Leu Pro Val Gly Ser Gln Cys 50 55 60 Ser Val Asp Leu Glu Ser Ala Ser Gly Glu Lys Asp Leu Ala Pro Pro 65 70 75 80 Ser Glu Pro Ser Glu Ser Phe Gln Glu His Thr Val Asp Gly Glu Asn 85 90 95 Gln Ile Val Phe Thr His Arg Ile Asn Ile Pro Arg Arg Ala Cys Gly 100 105 110 Cys Ala Ala Ala Pro Asp Val Lys Glu Leu Leu Ser Arg Leu Glu Glu 115 120 125 Leu Glu Asn Leu Val Ser Ser Leu Arg Glu Gln Cys Thr Ala Gly Ala 130 135 140 Gly Cys Cys Leu Gln Pro Ala Thr Gly Arg Leu Asp Thr Arg Pro Phe 145 150 155 160 Cys Ser Gly Arg Gly Asn Phe Ser Thr Glu Gly Cys Gly Cys Val Cys 165 170 175 Glu Pro Gly Trp Lys Gly Pro Asn Cys Ser Glu Pro Glu Cys Pro Gly 180 185 190 Asn Cys His Leu Arg Gly Arg Cys Ile Asp Gly Gln Cys Ile Cys Asp 195 200 205 Asp Gly Phe Thr Gly Glu Asp Cys Ser Gln Leu Ala Cys Pro Ser Asp 210 215 220 Cys Asn Asp Gln Gly Lys Cys Val Asn Gly Val Cys Ile Cys Phe Glu 225 230 235 240 Gly Tyr Ala Gly Ala Asp Cys Ser Arg Glu Ile Cys Pro Val Pro Cys 245 250 255 Ser Glu Glu His Gly Thr Cys Val Asp Gly Leu Cys Val Cys His Asp 260 265 270 Gly Phe Ala Gly Asp Asp Cys Asn Lys Pro Leu Cys Leu Asn Asn Cys 275 280 285 Tyr Asn Arg Gly Arg Cys Val Glu Asn Glu Cys Val Cys Asp Glu Gly 290 295 300 Phe Thr Gly Glu Asp Cys Ser Glu Leu Ile Cys Pro Asn Asp Cys Phe 305 310 315 320 Asp Arg Gly Arg Cys Ile Asn Gly Thr Cys Tyr Cys Glu Glu Gly Phe 325 330 335 Thr Gly Glu Asp Cys Gly Lys Pro Thr Cys Pro His Ala Cys His Thr 340 345 350 Gln Gly Arg Cys Glu Glu Gly Gln Cys Val Cys Asp Glu Gly Phe Ala 355 360 365 Gly Val Asp Cys Ser Glu Lys Arg Cys Pro Ala Asp Cys His Asn Arg 370 375 380 Gly Arg Cys Val Asp Gly Arg Cys Glu Cys Asp Asp Gly Phe Thr Gly 385 390 395 400 Ala Asp Cys Gly Glu Leu Lys Cys Pro Asn Gly Cys Ser Gly His Gly 405 410 415 Arg Cys Val Asn Gly Gln Cys Val Cys Asp Glu Gly Tyr Thr Gly Glu 420 425 430 Asp Cys Ser Gln Leu Arg Cys Pro Asn Asp Cys His Ser Arg Gly Arg 435 440 445 Cys Val Glu Gly Lys Cys Val Cys Glu Gln Gly Phe Lys Gly Tyr Asp 450 455 460 Cys Ser Asp Met Ser Cys Pro Asn Asp Cys His Gln His Gly Arg Cys 465 470 475 480 Val Asn Gly Met Cys Val Cys Asp Asp Gly Tyr Thr Gly Glu Asp Cys 485 490 495 Arg Asp Arg Gln Cys Pro Arg Asp Cys Ser Asn Arg Gly Leu Cys Val 500 505 510 Asp Gly Gln Cys Val Cys Glu Asp Gly Phe Thr Gly Pro Asp Cys Ala 515 520 525 Glu Leu Ser Cys Pro Asn Asp Cys His Gly Gln Gly Arg Cys Val Asn 530 535 540 Gly Gln Cys Val Cys His Glu Gly Phe Met Gly Lys Asp Cys Lys Glu 545 550 555 560 Gln Arg Cys Pro Ser Asp Cys His Gly Gln Gly Arg Cys Val Asp Gly 565 570 575 Gln Cys Ile Cys His Glu Gly Phe Thr Gly Leu Asp Cys Gly Gln His 580 585 590 Ser Cys Pro Ser Asp Cys Asn Asn Leu Gly Gln Cys Val Ser Gly Arg 595 600 605 Cys Ile Cys Asn Glu Gly Tyr Ser Gly Glu Asp Cys Ser Glu Val Ser 610 615 620 Pro Pro Lys Asp Leu Val Val Thr Glu Val Thr Glu Glu Thr Val Asn 625 630 635 640 Leu Ala Trp Asp Asn Glu Met Arg Val Thr Glu Tyr Leu Val Val Tyr 645 650 655 Thr Pro Thr His Glu Gly Gly Leu Glu Met Gln Phe Arg Val Pro Gly 660 665 670 Asp Gln Thr Ser Thr Ile Ile Gln Glu Leu Glu Pro Gly Val Glu Tyr 675 680 685 Phe Ile Arg Val Phe Ala Ile Leu Glu Asn Lys Lys Ser Ile Pro Val 690 695 700 Ser Ala Arg Val Ala Thr Tyr Leu Pro Ala Pro Glu Gly Leu Lys Phe 705 710 715 720 Lys Ser Ile Lys Glu Thr Ser Val Glu Val Glu Trp Asp Pro Leu Asp 725 730 735 Ile Ala Phe Glu Thr Trp Glu Ile Ile Phe Arg Asn Met Asn Lys Glu 740 745 750 Asp Glu Gly Glu Ile Thr Lys Ser Leu Arg Arg Pro Glu Thr Ser Tyr 755 760 765 Arg Gln Thr Gly Leu Ala Pro Gly Gln Glu Tyr Glu Ile Ser Leu His 770 775 780 Ile Val Lys Asn Asn Thr Arg Gly Pro Gly Leu Lys Arg Val Thr Thr Thr 785 790 795 800 Thr Arg Leu Asp Ala Pro Ser Gln Ile Glu Val Lys Asp Val Thr Asp 805 810 815 Thr Thr Thr Ala Leu Ile Thr Trp Phe Lys Pro Leu Ala Glu Ile Asp Gly 820 825 830 Ile Glu Leu Thr Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr 835 840 845 Ile Asp Leu Thr Glu Asp Glu Asn Gln Tyr Ser Ile Gly Asn Leu Lys 850 855 860 Pro Asp Thr Glu Tyr Glu Val Ser Leu Ile Ser Arg Arg Gly Asp Met 865 870 875 880 Ser Ser Asn Pro Ala Lys Glu Thr Phe Thr Thr Gly Leu Asp Ala Pro 885 890 895 Arg Asn Leu Arg Arg Val Ser Gln Thr Asp Asn Ser Ile Thr Leu Glu 900 905 910 Trp Arg Asn Gly Lys Ala Ala Ile Asp Ser Tyr Arg Ile Lys Tyr Ala 915 920 925 Pro Ile Ser Gly Gly Asp His Ala Glu Val Asp Val Pro Lys Ser Gln 930 935 940 Gln Ala Thr Thr Lys Thr Thr Leu Thr Gly Leu Arg Pro Gly Thr Glu 945 950 955 960 Tyr Gly Ile Gly Val Ser Ala Val Lys Glu Asp Lys Glu Ser Asn Pro 965 970 975 Ala Thr Ile Asn Ala Ala Thr Glu Leu Asp Thr Pro Lys Asp Leu Gln 980 985 990 Val Ser Glu Thr Ala Glu Thr Ser Leu Thr Leu Leu Trp Lys Thr Pro 995 1000 1005 Leu Ala Lys Phe Asp Arg Tyr Arg Leu Asn Tyr Ser Leu Pro Thr Gly 1010 1015 1020 Gln Trp Val Gly Val Gln Leu Pro Arg Asn Thr Thr Ser Tyr Val Leu 1025 1030 1035 1040 Arg Gly Leu Glu Pro Gly Gln Glu Tyr Asn Val Leu Leu Thr Ala Glu 1045 1050 1055 Lys Gly Arg His Lys Ser Lys Pro Ala Arg Val Lys Ala Ser Thr Glu 1060 1065 1070 Gln Ala Pro Glu Leu Glu Asn Leu Thr Val Thr Glu Val Gly Trp Asp 1075 1080 1085 Gly Leu Arg Leu Asn Trp Thr Ala Ala Asp Gln Ala Tyr Glu His Phe 1090 1095 1100 Ile Ile Gln Val Gln Glu Ala Asn Lys Val Glu Ala Ala Arg Asn Leu 1105 1110 1115 1120 Thr Val Pro Gly Ser Leu Arg Ala Val Asp Ile Pro Gly Leu Lys Ala 1125 1130 1135 Ala Thr Pro Tyr Thr Val Ser Ile Tyr Gly Val Ile Gln Gly Tyr Arg 1140 1145 1150 Thr Pro Val Leu Ser Ala Glu Ala Ser Thr Gly Glu Thr Pro Asn Leu 1155 1160 1165 Gly Glu Val Val Val Val Ala Glu Val Gly Trp Asp Ala Leu Lys Leu Asn 1170 1175 1180 Trp Thr Ala Pro Glu Gly Ala Tyr Glu Tyr Phe Phe Ile Gln Val Gln 1185 1190 1195 1200 Glu Ala Asp Thr Val Glu Ala Ala Gln Asn Leu Thr Val Pro Gly Gly 1205 1210 1215 Leu Arg Ser Thr Asp Leu Pro Gly Leu Lys Ala Ala Thr His Tyr Thr 1220 1225 1230 Ile Thr Ile Arg Gly Val Thr Gln Asp Phe Ser Thr Thr Pro Leu Ser 1235 1240 1245 Val Glu Val Leu Thr Glu Glu Val Pro Asp Met Gly Asn Leu Thr Val 1250 1255 1260 Thr Glu Val Ser Trp Asp Ala Leu Arg Leu Asn Trp Thr Thr Pro Asp 1265 1270 1275 1280 Gly Thr Tyr Asp Gln Phe Thr Ile Gln Val Gln Glu Ala Asp Gln Val 1285 1290 1295 Glu Glu Ala His Asn Leu Thr Val Pro Gly Ser Leu Arg Ser Met Glu 1300 1305 1310 Ile Pro Gly Leu Arg Ala Gly Thr Pro Tyr Thr Val Thr Leu His Gly 1315 1320 1325 Glu Val Arg Gly His Ser Thr Arg Pro Leu Ala Val Glu Val Val Thr 1330 1335 1340 Glu Asp Leu Pro Gln Leu Gly Asp Leu Ala Val Ser Glu Val Gly Trp 1345 1350 1355 1360 Asp Gly Leu Arg Leu Asn Trp Thr Ala Ala Asp Asn Ala Tyr Glu His 1365 1370 1375 Phe Val Ile Gln Val Gln Glu Val Asn Lys Val Glu Ala Ala Gln Asn 1380 1385 1390 Leu Thr Leu Pro Gly Ser Leu Arg Ala Val Asp Ile Pro Gly Leu Glu 1395 1400 1405 Ala Ala Thr Pro Tyr Arg Val Ser Ile Tyr Gly Val Ile Arg Gly Tyr 1410 1415 1420 Arg Thr Pro Val Leu Ser Ala Glu Ala Ser Thr Ala Lys Glu Pro Glu 1425 1430 1435 1440 Ile Gly Asn Leu Asn Val Ser Asp Ile Thr Pro Glu Ser Phe Asn Leu 1445 1450 1455 Ser Trp Met Ala Thr Asp Gly Ile Phe Glu Thr Phe Thr Ile Glu Ile 1460 1465 1470 Ile Asp Ser Asn Arg Leu Leu Glu Thr Val Glu Tyr Asn Ile Ser Gly 1475 1480 1485 Ala Glu Arg Thr Ala His Ile Ser Gly Leu Pro Pro Ser Thr Asp Phe 1490 1495 1500 Ile Val Tyr Leu Ser Gly Leu Ala Pro Ser Ile Arg Thr Lys Thr Ile 1505 1510 1515 1520 Ser Ala Thr Ala Thr Thr Glu Ala Leu Pro Leu Leu Glu Asn Leu Thr 1525 1530 1535 Ile Ser Asp Ile Asn Pro Tyr Gly Phe Thr Val Ser Trp Met Ala Ser 1540 1545 1550 Glu Asn Ala Phe Asp Ser Phe Leu Val Thr Val Val Val Asp Ser Gly Lys 1555 1560 1565 Leu Leu Asp Pro Gln Glu Phe Thr Leu Ser Gly Thr Gln Arg Lys Leu 1570 1575 1580 Glu Leu Arg Gly Leu Ile Thr Gly Ile Gly Tyr Glu Val Met Val Ser 1585 1590 1595 1600 Gly Phe Thr Gln Gly His Gln Thr Lys Pro Leu Arg Ala Glu Ile Val 1605 1610 1615 Thr Glu Ala Glu Pro Glu Val Asp Asn Leu Leu Val Ser Asp Ala Thr 1620 1625 1630 Pro Asp Gly Phe Arg Leu Ser Trp Thr Ala Asp Glu Gly Val Phe Asp 1635 1640 1645 Asn Phe Val Leu Lys Ile Arg Asp Thr Lys Lys Gln Ser Glu Pro Leu 1650 1655 1660 Glu Ile Thr Leu Leu Ala Pro Glu Arg Thr Arg Asp Ile Thr Gly Leu 1665 1670 1675 1680 Arg Glu Ala Thr Glu Tyr Glu Ile Glu Leu Tyr Gly Ile Ser Lys Gly 1685 1690 1695 Arg Arg Ser Gln Thr Val Ser Ala Ile Ala Thr Thr Ala Met Gly Ser 1700 1705 1710 Pro Lys Glu Val Ile Phe Ser Asp Ile Thr Glu Asn Ser Ala Thr Val 1715 1720 1725 Ser Trp Arg Ala Pro Thr Ala Gln Val Glu Ser Phe Arg Ile Thr Tyr 1730 1735 1740 Val Pro Ile Thr Gly Gly Thr Pro Ser Met Val Thr Val Asp Gly Thr 1745 1750 1755 1760 Lys Thr Gln Thr Arg Leu Val Lys Leu Ile Pro Gly Val Glu Tyr Leu 1765 1770 1775 Val Ser Ile Ile Ala Met Lys Gly Phe Glu Glu Ser Glu Pro Val Ser 1780 1785 1790 Gly Ser Phe Thr Thr Ala Leu Asp Gly Pro Ser Gly Leu Val Thr Ala 1795 1800 1805 Asn Ile Thr Asp Ser Glu Ala Leu Ala Arg Trp Gln Pro Ala Ile Ala 1810 1815 1820 Thr Val Asp Ser Tyr Val Ile Ser Tyr Thr Gly Glu Lys Val Pro Glu 1825 1830 1835 1840 Ile Thr Arg Thr Val Ser Gly Asn Thr Val Glu Tyr Ala Leu Thr Asp 1845 1850 1855 Leu Glu Pro Ala Thr Glu Tyr Thr Leu Arg Ile Phe Ala Glu Lys Gly 1860 1865 1870 Pro Gln Lys Ser Ser Thr Ile Thr Ala Lys Phe Thr Thr Asp Leu Asp 1875 1880 1885 Ser Pro Arg Asp Leu Thr Ala Thr Glu Val Gln Ser Glu Thr Ala Leu 1890 1895 1900 Leu Thr Trp Arg Pro Pro Arg Ala Ser Val Thr Gly Tyr Leu Leu Val 1905 1910 1915 1920 Tyr Glu Ser Val Asp Gly Thr Val Lys Glu Val Ile Val Gly Pro Asp 1925 1930 1935 Thr Thr Ser Tyr Ser Leu Ala Asp Leu Ser Pro Ser Thr His Tyr Thr 1940 1945 1950 Ala Lys Ile Gln Ala Leu Asn Gly Pro Leu Arg Ser Asn Met Ile Gln 1955 1960 1965 Thr Ile Phe Thr Thr Ile Gly Leu Leu Tyr Pro Phe Pro Lys Asp Cys 1970 1975 1980 Ser Gln Ala Met Leu Asn Gly Asp Thr Thr Ser Gly Leu Tyr Thr Ile 1985 1990 1995 2000 Tyr Leu Asn Gly Asp Lys Ala Glu Ala Leu Glu Val Phe Cys Asp Met 2005 2010 2015 Thr Ser Asp Gly Gly Gly Trp Ile Val Phe Leu Arg Arg Lys Asn Gly 2020 2025 2030 Arg Glu Asn Phe Tyr Gln Asn Trp Lys Ala Tyr Ala Ala Gly Phe Gly 2035 2040 2045 Asp Arg Arg Glu Glu Phe Trp Leu Gly Leu Asp Asn Leu Asn Lys Ile 2050 2055 2060 Thr Ala Gln Gly Gln Tyr Glu Leu Arg Val Asp Leu Arg Asp His Gly 2065 2070 2075 2080 Glu Thr Ala Phe Ala Val Tyr Asp Lys Phe Ser Val Gly Asp Ala Lys 2085 2090 2095 Thr Arg Tyr Lys Leu Lys Val Glu Gly Tyr Ser Gly Thr Ala Gly Asp 2100 2105 2110 Ser Met Ala Tyr His Asn Gly Arg Ser Phe Ser Thr Phe Asp Lys Asp 2115 2120 2125 Thr Asp Ser Ala Ile Thr Asn Cys Ala Leu Ser Tyr Lys Gly Ala Phe 2130 2135 2140 Trp Tyr Arg Asn Cys His Arg Val Asn Leu Met Gly Arg Tyr Gly Asp 2145 2150 2155 2160 Asn Asn His Ser Gln Gly Val Asn Trp Phe His Trp Lys Gly His Glu 2165 2170 2175 His Ser Ile Gln Phe Ala Glu Met Lys Leu Arg Pro Ser Asn Phe Arg 2180 2185 2190 Asn Leu Glu Gly Arg Arg Lys Arg Ala 2195 2200 <210> 807 <211> 30 <212> PRT <213> Homo sapiens <220> <223> trimerization domain of human tenascin C (TNC) <400> 807 Ala Cys Gly Cys Ala Ala Ala Pro Asp Val Lys Glu Leu Leu Ser Arg 1 5 10 15 Leu Glu Glu Leu Glu Asn Leu Val Ser Ser Leu Arg Glu Gln 20 25 30 <210> 808 <211> 106 <212> PRT <213> artificial sequence <220> <223> EHD2 dimerization domain <400> 808 Asp Phe Thr Pro Pro Thr Val Lys Ile Leu Gln Ser Ser Cys Asp Gly 1 5 10 15 Gly Gly His Phe Pro Pro Thr Ile Gln Leu Leu Cys Leu Val Ser Gly 20 25 30 Tyr Thr Pro Gly Thr Ile Asn Ile Thr Trp Leu Glu Asp Gly Gln Val 35 40 45 Met Asp Val Asp Leu Ser Thr Ala Ser Thr Thr Gln Glu Gly Glu Leu 50 55 60 Ala Ser Thr Gln Ser Glu Leu Thr Leu Ser Gln Lys His Trp Leu Ser 65 70 75 80 Asp Arg Thr Tyr Thr Cys Gln Val Thr Tyr Gln Gly His Thr Phe Glu 85 90 95 Asp Ser Thr Lys Lys Cys Ala Asp Ser Asn 100 105 <210> 809 <211> 27 <212> PRT <213> artificial sequence <220> <223> Peptide linker between EHD2 and scTNFR2 in EHD2-scTNFR2 <400> 809 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 1 5 10 15 Gly Gly Gly Ser Gly Gly Ser Glu Phe Leu Ala 20 25 <210> 810 <211> 605 <212> PRT <213> artificial sequence <220> <223> EHD2-scTNFR2 <400> 810 Asp Phe Thr Pro Pro Thr Val Lys Ile Leu Gln Ser Ser Cys Asp Gly 1 5 10 15 Gly Gly His Phe Pro Pro Thr Ile Gln Leu Leu Cys Leu Val Ser Gly 20 25 30 Tyr Thr Pro Gly Thr Ile Asn Ile Thr Trp Leu Glu Asp Gly Gln Val 35 40 45 Met Asp Val Asp Leu Ser Thr Ala Ser Thr Thr Gln Glu Gly Glu Leu 50 55 60 Ala Ser Thr Gln Ser Glu Leu Thr Leu Ser Gln Lys His Trp Leu Ser 65 70 75 80 Asp Arg Thr Tyr Thr Cys Gln Val Thr Tyr Gln Gly His Thr Phe Glu 85 90 95 Asp Ser Thr Lys Lys Cys Ala Asp Ser Asn Gly Gly Gly Ser Gly Gly 100 105 110 Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly 115 120 125 Ser Glu Phe Leu Ala Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala 130 135 140 His Val Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn 145 150 155 160 Arg Arg Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn 165 170 175 Gln Leu Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val 180 185 190 Leu Phe Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His 195 200 205 Thr Ile Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu 210 215 220 Ser Ala Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu 225 230 235 240 Ala Lys Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu 245 250 255 Glu Lys Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu 260 265 270 Asn Phe Arg Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu Gly 275 280 285 Gly Gly Gly Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His 290 295 300 Val Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg 305 310 315 320 Arg Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln 325 330 335 Leu Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu 340 345 350 Phe Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr 355 360 365 Ile Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser 370 375 380 Ala Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala 385 390 395 400 Lys Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu 405 410 415 Lys Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asn 420 425 430 Phe Arg Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu Gly Gly 435 440 445 Gly Gly Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 450 455 460 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 465 470 475 480 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 485 490 495 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 500 505 510 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 515 520 525 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 530 535 540 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 545 550 555 560 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 565 570 575 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asn Phe 580 585 590 Arg Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 595 600 605 <210> 811 <211> 111 <212> PRT <213> artificial sequence <220> <223> MHD2 (IgM dimerization domain) <400> 811 Ala Glu Leu Pro Pro Lys Val Ser Val Phe Val Pro Pro Arg Asp Gly 1 5 10 15 Phe Phe Gly Asn Pro Arg Lys Ser Lys Leu Ile Cys Gln Ala Thr Gly 20 25 30 Phe Ser Pro Arg Gln Ile Gln Val Ser Trp Leu Arg Glu Gly Lys Gln 35 40 45 Val Gly Ser Gly Val Thr Thr Asp Gln Val Gln Ala Glu Ala Lys Glu 50 55 60 Ser Gly Pro Thr Thr Tyr Lys Val Thr Ser Thr Leu Thr Ile Lys Glu 65 70 75 80 Ser Asp Trp Leu Gly Gln Ser Met Phe Thr Cys Arg Val Asp His Arg 85 90 95 Gly Leu Thr Phe Gln Gln Asn Ala Ser Ser Met Cys Val Pro Asp 100 105 110 <210> 812 <211> 31 <212> PRT <213> artificial sequence <220> <223> stalk region of TNF that can be used as a linker in scTNFR2-Fc <400> 812 Gly Pro Gln Arg Glu Glu Phe Pro Arg Asp Leu Ser Leu Ile Ser Pro 1 5 10 15 Leu Ala Gln Ala Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys 20 25 30 <210> 813 <211> 6 <212> PRT <213> artificial sequence <220> <223> partial trastuzumab hinge sequence <400> 813 Ser Cys Asp Lys Thr His 1 5 <210> 814 <211> 15 <212> PRT <213> artificial sequence <220> <223> trastuzumab hinge sequence <400> 814 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 <210> 815 <211> 12 <212> PRT <213> artificial sequence <220> <223> nivolumab hinge sequence <400> 815 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 1 5 10 <210> 816 <211> 4 <212> PRT <213> artificial sequence <220> <223> gs linker <400> 816 Gly Ser Gly Ser One <210> 817 <211> 5 <212> PRT <213> artificial sequence <220> <223> gs linker <400> 817 Gly Gly Gly Gly Ser 1 5 <210> 818 <211> 15 <212> PRT <213> artificial sequence <220> <223> gs linker <400> 818 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 819 <211> 20 <212> PRT <213> artificial sequence <220> <223> gs linker <400> 819 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 820 <211> 8 <212> PRT <213> artificial sequence <220> <223> gs linker <400> 820 Gly Ser Gly Gly Ser Ser Gly Gly 1 5 <210> 821 <211> 12 <212> PRT <213> artificial sequence <220> <223> gs linker <400> 821 Gly Ser Ser Ser Gly Ser Gly Ser Gly Ser Ser Gly 1 5 10 <210> 822 <211> 13 <212> PRT <213> artificial sequence <220> <223> gs linker <400> 822 Gly Ser Ser Ser Gly Ser Gly Ser Gly Ser Ser Gly Gly 1 5 10 <210> 823 <211> 6 <212> PRT <213> artificial sequence <220> <223> gs linker <400> 823 Gly Gly Ser Ser Gly Gly 1 5 <210> 824 <211> 13 <212> PRT <213> artificial sequence <220> <223> gs linker <400> 824 Gly Gly Ser Ser Gly Gly Ser Gly Gly Ser Ser Ser Gly 1 5 10 <210> 825 <211> 18 <212> PRT <213> artificial sequence <220> <223> gs linker <400> 825 Gly Ser Ser Ser Gly Ser Gly Ser Gly Gly Ser Ser Ser Gly Ser Gly 1 5 10 15 Ser Gly <210> 826 <211> 18 <212> PRT <213> artificial sequence <220> <223> gs linker <400> 826 Gly Gly Ser Ser Gly Gly Ser Ser Gly Gly Gly Ser Ser Gly Gly Ser 1 5 10 15 Ser Gly <210> 827 <211> 6 <212> PRT <213> artificial sequence <220> <223> gs linker <400> 827 Gly Ser Ser Ser Gly Ser 1 5 <210> 828 <211> 12 <212> PRT <213> artificial sequence <220> <223> linker sequence <400> 828 Gly Ser Ala Gly Ser Ala Ala Gly Ser Gly Glu Phe 1 5 10 <210> 829 <211> 18 <212> PRT <213> artificial sequence <220> <223> linker sequences <400> 829 Lys Glu Ser Gly Ser Val Ser Ser Glu Gln Leu Ala Gln Phe Arg Ser 1 5 10 15 Leu Asp <210> 830 <211> 14 <212> PRT <213> artificial sequence <220> <223> linker sequences <400> 830 Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr 1 5 10 <210> 831 <211> 5 <212> PRT <213> artificial sequence <220> <223> Alpha helix-forming linker, (EAAAK)n <220> <221> REPEAT <222> (1)...(5) <223> occuring n times, where n= 1 to 10 <400> 831 Glu Ala Ala Ala Lys 1 5 <210> 832 <211> 7 <212> PRT <213> artificial sequence <220> <223> rigid linker, A(EAAAK)nA, where n = 2 to 5 <220> <221> REPEAT <222> (2)...(6) <223> occurring 2, 3, 4, or 5 times <400> 832 Ala Glu Ala Ala Ala Lys Ala 1 5 <210> 833 <211> 18 <212> PRT <213> artificial sequence <220> <223> C-terminal extension from human IgG linker <400> 833 Glu Leu Gln Leu Glu Glu Ser Ser Ala Glu Ala Gln Asp Gly Glu Leu 1 5 10 15 Asp Gly <210> 834 <211> 14 <212> PRT <213> artificial sequence <220> <223> C-terminal extension from human IgG variant linker <400> 834 Glu Leu Gln Leu Glu Glu Ser Ser Ala Glu Ala Gln Gly Gly 1 5 10 <210> 835 <211> 20 <212> PRT <213> artificial sequence <220> <223> human immunoglobulin light chain kappa leader signal peptide sequence; human Ig kappa chains V-III region signal peptide (IgGkSP) <400> 835 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly 20 <210> 836 <211> 6 <212> PRT <213> artificial sequence <220> <223> 6xHis tag <400> 836 His His His His His His 1 5 <210> 837 <211> 100 <212> PRT <213> artificial sequence <220> <223> SUMO sequence <400> 837 Gly Ser Leu Gln Asp Ser Glu Val Asn Gln Glu Ala Lys Pro Glu Val 1 5 10 15 Lys Pro Glu Val Lys Pro Glu Thr His Ile Asn Leu Lys Val Ser Asp 20 25 30 Gly Ser Ser Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr Pro Leu Arg 35 40 45 Arg Leu Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu Met Asp Ser 50 55 60 Leu Arg Phe Leu Tyr Asp Gly Ile Arg Ile Gln Ala Asp Gln Ala Pro 65 70 75 80 Glu Asp Leu Asp Met Glu Asp Asn Asp Ile Ile Glu Ala His Arg Glu 85 90 95 Gln Ile Gly Gly 100 <210> 838 <211> 108 <212> PRT <213> artificial sequence <220> <223> His tag-SUMO sequence <400> 838 Met Gly His His His His His Gly Ser Leu Gln Asp Ser Glu Val 1 5 10 15 Asn Gln Glu Ala Lys Pro Glu Val Lys Pro Glu Val Lys Pro Glu Thr 20 25 30 His Ile Asn Leu Lys Val Ser Asp Gly Ser Ser Glu Ile Phe Phe Lys 35 40 45 Ile Lys Lys Thr Thr Pro Leu Arg Arg Leu Met Glu Ala Phe Ala Lys 50 55 60 Arg Gln Gly Lys Glu Met Asp Ser Leu Arg Phe Leu Tyr Asp Gly Ile 65 70 75 80 Arg Ile Gln Ala Asp Gln Ala Pro Glu Asp Leu Asp Met Glu Asp Asn 85 90 95 Asp Ile Ile Glu Ala His Arg Glu Gln Ile Gly Gly 100 105 <210> 839 <211> 5 <212> PRT <213> artificial sequence <220> <223> human TNFR2 epitope <400> 839 Lys Cys Ser Pro Gly 1 5 <210> 840 <211> 5 <212> PRT <213> artificial sequence <220> <223> human TNFR2 epitope <400> 840 Lys Cys Arg Pro Gly 1 5 <210> 841 <211> 59 <212> PRT <213> artificial sequence <220> <223> human TNFR2 epitope <400> 841 Cys Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr 1 5 10 15 Arg Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala 20 25 30 Leu Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys 35 40 45 Arg Pro Gly Phe Gly Val Ala Arg Pro Gly Thr 50 55 <210> 842 <211> 40 <212> PRT <213> artificial sequence <220> <223> human TNFR2 epitope <400> 842 Thr Arg Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys 1 5 10 15 Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys 20 25 30 Cys Arg Pro Gly Phe Gly Val Ala 35 40 <210> 843 <211> 28 <212> PRT <213> artificial sequence <220> <223> human TNFR2 epitope <400> 843 Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys 1 5 10 15 Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys 20 25 <210> 844 <211> 15 <212> PRT <213> artificial sequence <220> <223> human TNFR2 epitope <400> 844 Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu 1 5 10 15 <210> 845 <211> 17 <212> PRT <213> artificial sequence <220> <223> human TNFR2 epitope <400> 845 Cys Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr 1 5 10 15 Arg <210> 846 <211> 7 <212> PRT <213> artificial sequence <220> <223> human TNFR2 epitope <400> 846 Ser Ser Asp Gln Val Glu Thr 1 5 <210> 847 <211> 20 <212> PRT <213> artificial sequence <220> <223> human TNFR2 epitope <400> 847 Gln Thr Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala 1 5 10 15 Lys Val Phe Cys 20 <210> 848 <211> 20 <212> PRT <213> artificial sequence <220> <223> human TNFR2 epitope <400> 848 Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro 1 5 10 15 Gly Phe Gly Val 20 <210> 849 <211> 38 <212> PRT <213> artificial sequence <220> <223> human TNFR2 epitope <400> 849 Cys Thr Arg Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr 1 5 10 15 Cys Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg 20 25 30 Lys Cys Arg Pro Gly Phe 35 <210> 850 <211> 50 <212> PRT <213> artificial sequence <220> <223> human TNFR2 epitope <400> 850 Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys Thr Cys Arg 1 5 10 15 Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu Cys 20 25 30 Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala Arg Pro Gly 35 40 45 Thr Glu 50 <210> 851 <211> 8 <212> PRT <213> artificial sequence <220> <223> human TNFR2 epitope <400> 851 Cys Ala Pro Leu Arg Lys Cys Arg 1 5 <210> 852 <211> 9 <212> PRT <213> artificial sequence <220> <223> human TNFR2 epitope <400> 852 Arg Lys Cys Arg Pro Gly Phe Gly Val 1 5 <210> 853 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 853 Cys Arg Pro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn 1 5 10 15 <210> 854 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 854 Cys Arg Pro His Gln Ile Cys Asn Val Val Gly Ala Pro Gly Asn 1 5 10 15 <210> 855 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 855 Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Ala Ala Cys Arg Pro 1 5 10 15 <210> 856 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 856 Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ala Ala Arg Pro His 1 5 10 15 <210> 857 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 857 Pro Ser Thr Ser Phe Leu Leu Pro Met Gly Pro Ser Pro Pro Ala 1 5 10 15 <210> 858 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 858 Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys 1 5 10 15 <210> 859 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 859 Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys 1 5 10 15 <210> 860 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 860 Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg 1 5 10 15 <210> 861 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 861 Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro 1 5 10 15 <210> 862 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 862 Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His 1 5 10 15 <210> 863 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 863 Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu 1 5 10 15 <210> 864 <211> 16 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 864 Cys Gln Leu Trp Asn Trp Val Pro Glu Ala Leu Ala Gly Gly Ser Arg 1 5 10 15 <210> 865 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 865 Leu Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gln Gly Gly Ser Gly Gly 1 5 10 15 Arg Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro 20 25 <210> 866 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 866 Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Ala Ala Arg Cys Ser 1 5 10 15 <210> 867 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 867 Leu Trp Asn Trp Val Pro Glu Xaa Leu Ser Cys Gly Gly Ser Gly Gly 1 5 10 15 Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Xaa 20 25 <210> 868 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 868 Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala Arg Pro Gly 1 5 10 15 <210> 869 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 869 Xaa Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Gly Gly Ser Gly Gly 1 5 10 15 Arg Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro 20 25 <210> 870 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 870 Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Gly Gly Ser Gly Gly 1 5 10 15 Arg Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro 20 25 <210> 871 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 871 Leu Trp Asn Trp Val Pro Glu Xaa Leu Ser Cys Gly Gly Ser Gly Gly 1 5 10 15 Ala Cys Thr Arg Glu Gln Asn Arg Ile Xaa Thr 20 25 <210> 872 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 872 Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ala Ala Gly Ser Arg 1 5 10 15 <210> 873 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 873 Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Xaa Gly Gly Ser Gly Gly 1 5 10 15 Arg Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro 20 25 <210> 874 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 874 Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Ala Ala Tyr Cys Ala 1 5 10 15 <210> 875 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 875 Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His Gln Ile Cys Asn 1 5 10 15 <210> 876 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 876 Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly 1 5 10 15 <210> 877 <211> 16 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 877 Cys Gln Leu Trp Asn Trp Val Pro Glu Ala Leu Ser Ala Gly Ser Arg 1 5 10 15 <210> 878 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 878 Arg Pro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala 1 5 10 15 <210> 879 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 879 Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Gly Gly Ser Gly Gly 1 5 10 15 Arg Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro 20 25 <210> 880 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 880 Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gly Gly Ser Gly Gly 1 5 10 15 Arg Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro 20 25 <210> 881 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (24)...(24) <223> Xaa is Cys residue protected with ACM protecting group <400> 881 Leu Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gln Gly Gly Ser Gly Gly 1 5 10 15 Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly 20 25 <210> 882 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <400> 882 Gln Glu Gly Cys Arg Leu Xaa Ala Pro Leu Arg Gly Gly Ser Gly Gly 1 5 10 15 Cys Arg Pro Gly Phe Gly Val Ala Arg Pro Gly 20 25 <210> 883 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 883 Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His Gln 1 5 10 15 <210> 884 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 884 Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Ala Ala Ser Arg Cys 1 5 10 15 <210> 885 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 885 Gln Thr Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His 1 5 10 15 <210> 886 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 886 Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Ser Gly Gly Ser Gly Gly 1 5 10 15 Arg Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro 20 25 <210> 887 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (9)...(9) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (15)...(15) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 887 Cys Glu Gln Asn Arg Ile Xaa Thr Xaa Arg Pro Gly Trp Tyr Xaa Ala 1 5 10 15 Cys Asn Ala Ser Met Asp Ala Val Xaa Thr Ser Thr Ser Pro Thr Arg 20 25 30 Cys <210> 888 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 888 Val Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Gly Gly Ser Gly Gly 1 5 10 15 Arg Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro 20 25 <210> 889 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 889 Trp Asn Trp Val Pro Glu Xaa Leu Ser Cys Gly Gly Gly Ser Gly Gly 1 5 10 15 Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Xaa 20 25 <210> 890 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Cys residue protected with ACM protecting group <400> 890 Leu Trp Asn Trp Val Pro Glu Xaa Leu Ser Cys Gly Gly Ser Gly Gly 1 5 10 15 Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile 20 25 <210> 891 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <400> 891 Val Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Gly Gly Ser Gly Gly 1 5 10 15 Gln Ala Xaa Thr Arg Glu Gln Asn Arg Ile Cys 20 25 <210> 892 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 892 Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gln Val Gly Gly Ser Gly Gly 1 5 10 15 Arg Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro 20 25 <210> 893 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (24)...(24) <223> Xaa is Cys residue protected with ACM protecting group <400> 893 Xaa Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Gly Gly Ser Gly Gly 1 5 10 15 Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly 20 25 <210> 894 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 894 Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys Ala Ala Arg Pro Gly 1 5 10 15 <210> 895 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (24)...(24) <223> Xaa is Cys residue protected with ACM protecting group <400> 895 Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gln Val Gly Gly Ser Gly Gly 1 5 10 15 Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly 20 25 <210> 896 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 896 Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Ala Gly Arg Pro Gly 1 5 10 15 <210> 897 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 897 Ile Cys Arg Pro His Gln Ile Cys Asn Val Ala Gly Ile Pro Gly 1 5 10 15 <210> 898 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 898 Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu Cys 1 5 10 15 <210> 899 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 899 Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Ala Ala Cys Arg Pro 1 5 10 15 <210> 900 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 900 Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser 1 5 10 15 <210> 901 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 901 Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gly Gly Ser Gly Gly 1 5 10 15 Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp 20 25 <210> 902 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 902 Val Pro Glu Xaa Leu Ser Cys Gly Ser Arg Xaa Gly Gly Ser Gly Gly 1 5 10 15 Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Xaa 20 25 <210> 903 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 903 Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gly Gly Ser Gly Gly 1 5 10 15 Arg Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro 20 25 <210> 904 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <400> 904 Glu Gly Cys Arg Leu Xaa Ala Pro Leu Arg Lys Gly Gly Ser Gly Gly 1 5 10 15 Cys Arg Pro Gly Phe Gly Val Ala Arg Pro Gly 20 25 <210> 905 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (22)...(22) <223> Xaa is Cys residue protected with ACM protecting group <400> 905 Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Gly Gly Ser Gly Gly 1 5 10 15 Met Cys Xaa Ser Lys Xaa Ser Pro Gly Gln His 20 25 <210> 906 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 906 Leu Trp Asn Trp Val Pro Glu Xaa Leu Ser Cys Gly Gly Ser Gly Gly 1 5 10 15 Cys Thr Arg Glu Gln Asn Arg Ile Xaa Thr Xaa 20 25 <210> 907 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Cys residue protected with ACM protecting group <400> 907 Lys Gln Glu Gly Cys Arg Leu Xaa Ala Pro Leu Gly Gly Ser Gly Gly 1 5 10 15 Cys Arg Pro Gly Phe Gly Val Ala Arg Pro Gly 20 25 <210> 908 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <400> 908 Xaa Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu Gly Gly Ser Gly Gly 1 5 10 15 Cys Arg Pro Gly Phe Gly Val Ala Arg Pro Gly 20 25 <210> 909 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <400> 909 Trp Tyr Xaa Ala Leu Ser Lys Gln Glu Gly Cys Gly Gly Ser Gly Gly 1 5 10 15 Cys Arg Pro Gly Phe Gly Val Ala Arg Pro Gly 20 25 <210> 910 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (10)...(10) <223> Xaa is Cys residue protected with ACM protecting group <400> 910 Leu Ser Lys Gln Glu Gly Cys Arg Leu Xaa Ala Gly Gly Ser Gly Gly 1 5 10 15 Cys Arg Pro Gly Phe Gly Val Ala Arg Pro Gly 20 25 <210> 911 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (24)...(24) <223> Xaa is Cys residue protected with ACM protecting group <400> 911 Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gly Gly Ser Gly Gly 1 5 10 15 Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly 20 25 <210> 912 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <400> 912 Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu Xaa Gly Gly Ser Gly Gly 1 5 10 15 Cys Arg Pro Gly Phe Gly Val Ala Arg Pro Gly 20 25 <210> 913 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 913 Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys Arg 1 5 10 15 <210> 914 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 914 Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu 1 5 10 15 <210> 915 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 915 Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Gly Gly Ser Gly Gly 1 5 10 15 Arg Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro 20 25 <210> 916 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 916 Trp Asn Trp Val Pro Glu Xaa Leu Ser Cys Gly Gly Gly Ser Gly Gly 1 5 10 15 Ala Cys Thr Arg Glu Gln Asn Arg Ile Xaa Thr 20 25 <210> 917 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <400> 917 Thr Ser Asp Val Val Xaa Lys Pro Cys Ala Pro Gly Gly Ser Gly Gly 1 5 10 15 Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His 20 25 <210> 918 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 918 Ile Cys Arg Pro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly 1 5 10 15 <210> 919 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (9)...(9) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <400> 919 Arg Leu Cys Ala Pro Leu Arg Lys Xaa Arg Pro Gly Gly Ser Gly Gly 1 5 10 15 Cys Lys Pro Xaa Ala Pro Gly Thr Phe Ser Asn 20 25 <210> 920 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 920 Pro Thr Arg Ser Met Ala Pro Gly Ala Val His Leu Pro Gln Pro 1 5 10 15 <210> 921 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (22)...(22) <223> Xaa is Cys residue protected with ACM protecting group <400> 921 Tyr Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu Gly Gly Ser Gly Gly 1 5 10 15 Met Cys Xaa Ser Lys Xaa Ser Pro Gly Gln His 20 25 <210> 922 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 922 Trp Val Pro Glu Xaa Leu Ser Cys Gly Ser Arg Gly Gly Ser Gly Gly 1 5 10 15 Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Xaa 20 25 <210> 923 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (18)...(18) <223> Xaa is Cys residue protected with ACM protecting group <400> 923 Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Gly Gly Ser Gly Gly 1 5 10 15 Ala Xaa Thr Arg Glu Gln Asn Arg Ile Cys Thr 20 25 <210> 924 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (24)...(24) <223> Xaa is Cys residue protected with ACM protecting group <400> 924 Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Ser Gly Gly Ser Gly Gly 1 5 10 15 Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly 20 25 <210> 925 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (10)...(10) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 925 Pro Glu Xaa Leu Ser Cys Gly Ser Arg Xaa Ser Gly Gly Ser Gly Gly 1 5 10 15 Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Xaa 20 25 <210> 926 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 926 Trp Val Pro Glu Xaa Leu Ser Cys Gly Ser Arg Gly Gly Ser Gly Gly 1 5 10 15 Ala Cys Thr Arg Glu Gln Asn Arg Ile Xaa Thr 20 25 <210> 927 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (22)...(22) <223> Xaa is Cys residue protected with ACM protecting group <400> 927 Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Gly Gly Ser Gly Gly 1 5 10 15 Met Cys Xaa Ser Lys Xaa Ser Pro Gly Gln His 20 25 <210> 928 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 928 Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly Pro 1 5 10 15 <210> 929 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 929 Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Gly Gly Ser Gly Gly 1 5 10 15 Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp 20 25 <210> 930 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 930 Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Gly Gly Ser Gly Gly 1 5 10 15 Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp 20 25 <210> 931 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (24)...(24) <223> Xaa is Cys residue protected with ACM protecting group <400> 931 Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Xaa Gly Gly Ser Gly Gly 1 5 10 15 Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly 20 25 <210> 932 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (22)...(22) <223> Xaa is Cys residue protected with ACM protecting group <400> 932 Pro Tyr Ala Pro Glu Pro Gly Ser Thr Cys Arg Gly Gly Ser Gly Gly 1 5 10 15 Met Cys Xaa Ser Lys Xaa Ser Pro Gly Gln His 20 25 <210> 933 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (18)...(18) <223> Xaa is Cys residue protected with ACM protecting group <400> 933 Val Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Gly Gly Ser Gly Gly 1 5 10 15 Ala Xaa Thr Arg Glu Gln Asn Arg Ile Cys Thr 20 25 <210> 934 <211> 22 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (21)...(21) <223> Xaa is Cys residue protected with ACM protecting group <400> 934 Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Xaa Thr Arg Glu Gln Asn 1 5 10 15 Arg Ile Cys Thr Xaa Arg 20 <210> 935 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <400> 935 Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Ser Gly Gly Ser Gly Gly 1 5 10 15 Gln Ala Xaa Thr Arg Glu Gln Asn Arg Ile Cys 20 25 <210> 936 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 936 Leu Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gln Gly Gly Ser Gly Gly 1 5 10 15 Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp 20 25 <210> 937 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (18)...(18) <223> Xaa is Cys residue protected with ACM protecting group <400> 937 Xaa Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu Gly Gly Ser Gly Gly 1 5 10 15 Leu Xaa Ala Pro Leu Arg Lys Cys Arg Pro Gly 20 25 <210> 938 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (22)...(22) <223> Xaa is Cys residue protected with ACM protecting group <400> 938 Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu Gly Gly Ser Gly Gly 1 5 10 15 Met Cys Xaa Ser Lys Xaa Ser Pro Gly Gln His 20 25 <210> 939 <211> 22 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <400> 939 Asn Arg Ile Xaa Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys 1 5 10 15 Gln Glu Gly Xaa Arg Leu 20 <210> 940 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (24)...(24) <223> Xaa is Cys residue protected with ACM protecting group <400> 940 Val Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Gly Gly Ser Gly Gly 1 5 10 15 Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly 20 25 <210> 941 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (22)...(22) <223> Xaa is Cys residue protected with ACM protecting group <400> 941 Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Gly Gly Ser Gly Gly 1 5 10 15 Met Cys Xaa Ser Lys Xaa Ser Pro Gly Gln His 20 25 <210> 942 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 942 Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Gly Gly Ser Gly Gly 1 5 10 15 Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His 20 25 <210> 943 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (24)...(24) <223> Xaa is Cys residue protected with ACM protecting group <400> 943 Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Gly Gly Ser Gly Gly 1 5 10 15 Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly 20 25 <210> 944 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <400> 944 Cys Ala Pro Leu Arg Lys Xaa Arg Pro Gly Phe Gly Gly Ser Gly Gly 1 5 10 15 Cys Lys Pro Xaa Ala Pro Gly Thr Phe Ser Asn 20 25 <210> 945 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 945 Val Pro Glu Xaa Leu Ser Cys Gly Ser Arg Xaa Gly Gly Ser Gly Gly 1 5 10 15 Ala Cys Thr Arg Glu Gln Asn Arg Ile Xaa Thr 20 25 <210> 946 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <400> 946 Trp Asn Trp Val Pro Glu Xaa Leu Ser Cys Gly Gly Gly Ser Gly Gly 1 5 10 15 Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile 20 25 <210> 947 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <400> 947 Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gly Gly Ser Gly Gly 1 5 10 15 Gln Ala Xaa Thr Arg Glu Gln Asn Arg Ile Cys 20 25 <210> 948 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (9)...(9) <223> Xaa is Cys residue protected with ACM protecting group <400> 948 Ser Lys Gln Glu Gly Cys Arg Leu Xaa Ala Pro Gly Gly Ser Gly Gly 1 5 10 15 Cys Arg Pro Gly Phe Gly Val Ala Arg Pro Gly 20 25 <210> 949 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 949 Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala Arg Pro Gly Thr 1 5 10 15 <210> 950 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 950 Trp Val Pro Glu Xaa Leu Ser Cys Gly Ser Arg Gly Gly Ser Gly Gly 1 5 10 15 Cys Thr Arg Glu Gln Asn Arg Ile Xaa Thr Xaa 20 25 <210> 951 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (18)...(18) <223> Xaa is Cys residue protected with ACM protecting group <400> 951 Trp Tyr Xaa Ala Leu Ser Lys Gln Glu Gly Cys Gly Gly Ser Gly Gly 1 5 10 15 Leu Xaa Ala Pro Leu Arg Lys Cys Arg Pro Gly 20 25 <210> 952 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (12)...(12) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 952 Cys Gly Xaa Arg Leu Xaa Ala Pro Leu Arg Lys Xaa Arg Pro Gly Phe 1 5 10 15 Cys Asn Ala Ser Met Asp Ala Val Xaa Thr Ser Thr Ser Pro Thr Arg 20 25 30 Cys <210> 953 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 953 Cys Gln Ile Xaa Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp 1 5 10 15 Cys Ala Pro Leu Arg Lys Xaa Arg Pro Gly Phe Gly Val Ala Arg Pro 20 25 30 Cys <210> 954 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 954 Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys Thr 1 5 10 15 <210> 955 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 955 Asn Trp Val Pro Glu Xaa Leu Ser Cys Gly Ser Gly Gly Ser Gly Gly 1 5 10 15 Ala Cys Thr Arg Glu Gln Asn Arg Ile Xaa Thr 20 25 <210> 956 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (22)...(22) <223> Xaa is Cys residue protected with ACM protecting group <400> 956 Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Gly Gly Ser Gly Gly 1 5 10 15 Met Cys Xaa Ser Lys Xaa Ser Pro Gly Gln His 20 25 <210> 957 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (24)...(24) <223> Xaa is Cys residue protected with ACM protecting group <400> 957 Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Gly Gly Ser Gly Gly 1 5 10 15 Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly 20 25 <210> 958 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 958 Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gly Gly Ser Gly Gly 1 5 10 15 Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp 20 25 <210> 959 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (10)...(10) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 959 Pro Glu Xaa Leu Ser Cys Gly Ser Arg Xaa Ser Gly Gly Ser Gly Gly 1 5 10 15 Ala Cys Thr Arg Glu Gln Asn Arg Ile Xaa Thr 20 25 <210> 960 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 960 Leu Ser Cys Gly Ser Arg Xaa Ser Ser Asp Gln Gly Gly Ser Gly Gly 1 5 10 15 Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Xaa 20 25 <210> 961 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 961 Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Gly Gly Ser Gly Gly 1 5 10 15 Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His 20 25 <210> 962 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (12)...(12) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (14)...(14) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 962 Cys Gln Ala Xaa Thr Arg Glu Gln Asn Arg Ile Xaa Thr Xaa Arg Pro 1 5 10 15 Cys Asn Ala Ser Met Asp Ala Val Xaa Thr Ser Thr Ser Pro Thr Arg 20 25 30 Cys <210> 963 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 963 Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Gly Gly Ser Gly Gly 1 5 10 15 Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp 20 25 <210> 964 <211> 17 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 964 Cys Gln Ala Ala Thr Arg Glu Gln Asn Arg Ile Gly Ala Ala Arg Pro 1 5 10 15 Cys <210> 965 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 965 Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Xaa Gly Gly Ser Gly Gly 1 5 10 15 Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp 20 25 <210> 966 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (14)...(14) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 966 Cys Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser Thr Xaa Arg Leu 1 5 10 15 Cys Asn Ala Ser Met Asp Ala Val Xaa Thr Ser Thr Ser Pro Thr Arg 20 25 30 Cys <210> 967 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (22)...(22) <223> Xaa is Cys residue protected with ACM protecting group <400> 967 Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gly Gly Ser Gly Gly 1 5 10 15 Met Cys Xaa Ser Lys Xaa Ser Pro Gly Gln His 20 25 <210> 968 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 968 Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly 1 5 10 15 <210> 969 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 969 Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ala Ala Cys Arg Leu 1 5 10 15 <210> 970 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (24)...(24) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 970 Cys Xaa Glu Asp Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu 1 5 10 15 Cys Gln Thr Ala Gln Met Xaa Xaa Ser Lys Xaa Ser Pro Gly Gln His 20 25 30 Cys <210> 971 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (9)...(9) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 971 Glu Xaa Leu Ser Cys Gly Ser Arg Xaa Ser Ser Gly Gly Ser Gly Gly 1 5 10 15 Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Xaa 20 25 <210> 972 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (17)...(17) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 972 Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Gly Gly Ser Gly Gly 1 5 10 15 Xaa Thr Arg Glu Gln Asn Arg Ile Cys Thr Xaa 20 25 <210> 973 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <400> 973 Leu Cys Ala Pro Leu Arg Lys Xaa Arg Pro Gly Gly Gly Ser Gly Gly 1 5 10 15 Cys Lys Pro Xaa Ala Pro Gly Thr Phe Ser Asn 20 25 <210> 974 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Cys residue protected with ACM protecting group <400> 974 Leu Trp Asn Trp Val Pro Glu Xaa Leu Ser Cys Gly Gly Ser Gly Gly 1 5 10 15 Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg 20 25 <210> 975 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 975 Asn Trp Val Pro Glu Xaa Leu Ser Cys Gly Ser Gly Gly Ser Gly Gly 1 5 10 15 Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Xaa 20 25 <210> 976 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (24)...(24) <223> Xaa is Cys residue protected with ACM protecting group <400> 976 Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Gly Gly Ser Gly Gly 1 5 10 15 Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly 20 25 <210> 977 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <400> 977 Tyr Xaa Ala Leu Ser Lys Gln Glu Gly Cys Arg Gly Gly Ser Gly Gly 1 5 10 15 Cys Arg Pro Gly Phe Gly Val Ala Arg Pro Gly 20 25 <210> 978 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (10)...(10) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (18)...(18) <223> Xaa is Cys residue protected with ACM protecting group <400> 978 Leu Ser Lys Gln Glu Gly Cys Arg Leu Xaa Ala Gly Gly Ser Gly Gly 1 5 10 15 Leu Xaa Ala Pro Leu Arg Lys Cys Arg Pro Gly 20 25 <210> 979 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <400> 979 Trp Tyr Xaa Ala Leu Ser Lys Gln Glu Gly Cys Gly Gly Ser Gly Gly 1 5 10 15 Lys Cys Arg Pro Gly Phe Gly Val Ala Arg Pro 20 25 <210> 980 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (12)...(12) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (30)...(30) <223> Xaa is Cys residue protected with ACM protecting group <400> 980 Cys Gly Xaa Arg Leu Xaa Ala Pro Leu Arg Lys Xaa Arg Pro Gly Phe 1 5 10 15 Cys Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser Thr Xaa Arg Leu 20 25 30 Cys <210> 981 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 981 Xaa Leu Ser Cys Gly Ser Arg Xaa Ser Ser Asp Gly Gly Ser Gly Gly 1 5 10 15 Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Xaa 20 25 <210> 982 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (9)...(9) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 982 Cys Asp Gln Val Glu Thr Gln Ala Xaa Thr Arg Glu Gln Asn Arg Ile 1 5 10 15 Cys Asn Ala Ser Met Asp Ala Val Xaa Thr Ser Thr Ser Pro Thr Arg 20 25 30 Cys <210> 983 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (18)...(18) <223> Xaa is Cys residue protected with ACM protecting group <400> 983 Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu Xaa Gly Gly Ser Gly Gly 1 5 10 15 Leu Xaa Ala Pro Leu Arg Lys Cys Arg Pro Gly 20 25 <210> 984 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 984 Trp Asn Trp Val Pro Glu Xaa Leu Ser Cys Gly Gly Gly Ser Gly Gly 1 5 10 15 Cys Thr Arg Glu Gln Asn Arg Ile Xaa Thr Xaa 20 25 <210> 985 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <400> 985 Leu Ser Lys Gln Glu Gly Xaa Arg Leu Cys Ala Gly Gly Ser Gly Gly 1 5 10 15 Cys Lys Pro Xaa Ala Pro Gly Thr Phe Ser Asn 20 25 <210> 986 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 986 Cys Thr Arg Glu Gln Asn Arg Ile Cys Thr Ala Ala Pro Gly Trp 1 5 10 15 <210> 987 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (18)...(18) <223> Xaa is Cys residue protected with ACM protecting group <400> 987 Tyr Xaa Ala Leu Ser Lys Gln Glu Gly Cys Arg Gly Gly Ser Gly Gly 1 5 10 15 Leu Xaa Ala Pro Leu Arg Lys Cys Arg Pro Gly 20 25 <210> 988 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (10)...(10) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <400> 988 Xaa Arg Leu Cys Ala Pro Leu Arg Lys Xaa Arg Gly Gly Ser Gly Gly 1 5 10 15 Cys Lys Pro Xaa Ala Pro Gly Thr Phe Ser Asn 20 25 <210> 989 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 989 Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Ala Ala Cys Ala Leu 1 5 10 15 <210> 990 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (12)...(12) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (28)...(28) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (30)...(30) <223> Xaa is Cys residue protected with ACM protecting group <400> 990 Cys Gly Xaa Arg Leu Xaa Ala Pro Leu Arg Lys Xaa Arg Pro Gly Phe 1 5 10 15 Cys Gln Ala Xaa Thr Arg Glu Gln Asn Arg Ile Xaa Thr Xaa Arg Pro 20 25 30 Cys <210> 991 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <400> 991 Asp Val Val Xaa Lys Pro Cys Ala Pro Gly Thr Gly Gly Ser Gly Gly 1 5 10 15 Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His 20 25 <210> 992 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 992 Gln Asn Arg Ile Xaa Thr Cys Arg Pro Gly Trp Gly Gly Ser Gly Gly 1 5 10 15 Tyr Cys Ala Leu Ser Lys Gln Glu Gly Xaa Arg 20 25 <210> 993 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 993 Pro Ser Thr Ser Phe Leu Leu Pro Met Gly Ala Ala Pro Pro Ala 1 5 10 15 <210> 994 <211> 22 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <400> 994 Glu Gly Cys Arg Leu Xaa Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe 1 5 10 15 Gly Val Ala Arg Pro Gly 20 <210> 995 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (18)...(18) <223> Xaa is Cys residue protected with ACM protecting group <400> 995 Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Ser Gly Gly Ser Gly Gly 1 5 10 15 Ala Xaa Thr Arg Glu Gln Asn Arg Ile Cys Thr 20 25 <210> 996 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 996 Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Ser Gly Gly Ser Gly Gly 1 5 10 15 Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp 20 25 <210> 997 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <400> 997 Leu Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gln Gly Gly Ser Gly Gly 1 5 10 15 Gln Ala Xaa Thr Arg Glu Gln Asn Arg Ile Cys 20 25 <210> 998 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (22)...(22) <223> Xaa is Cys residue protected with ACM protecting group <400> 998 Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gly Gly Ser Gly Gly 1 5 10 15 Met Cys Xaa Ser Lys Xaa Ser Pro Gly Gln His 20 25 <210> 999 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 999 Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly Pro Ser Pro Pro 1 5 10 15 <210> 1000 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <400> 1000 Asn Trp Val Pro Glu Xaa Leu Ser Cys Gly Ser Gly Gly Ser Gly Gly 1 5 10 15 Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile 20 25 <210> 1001 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 1001 Asn Arg Ile Xaa Thr Cys Arg Pro Gly Trp Tyr Gly Gly Ser Gly Gly 1 5 10 15 Tyr Cys Ala Leu Ser Lys Gln Glu Gly Xaa Arg 20 25 <210> 1002 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1002 Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Gly Gly Ser Gly Gly 1 5 10 15 Thr Ser Ser Thr Asp Ile Cys Arg Pro His Gln 20 25 <210> 1003 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (30)...(30) <223> Xaa is Cys residue protected with ACM protecting group <400> 1003 Cys Ala Pro Leu Arg Lys Xaa Arg Pro Gly Phe Gly Val Ala Arg Pro 1 5 10 15 Cys Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser Thr Xaa Arg Leu 20 25 30 Cys <210> 1004 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (22)...(22) <223> Xaa is Cys residue protected with ACM protecting group <400> 1004 Thr Pro Tyr Ala Pro Glu Pro Gly Ser Thr Cys Gly Gly Ser Gly Gly 1 5 10 15 Met Cys Xaa Ser Lys Xaa Ser Pro Gly Gln His 20 25 <210> 1005 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 1005 Leu Ser Cys Gly Ser Arg Xaa Ser Ser Asp Gln Gly Gly Ser Gly Gly 1 5 10 15 Cys Thr Arg Glu Gln Asn Arg Ile Xaa Thr Xaa 20 25 <210> 1006 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1006 Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly Pro Ser Pro 1 5 10 15 <210> 1007 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (9)...(9) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 1007 Glu Xaa Leu Ser Cys Gly Ser Arg Xaa Ser Ser Gly Gly Ser Gly Gly 1 5 10 15 Ala Cys Thr Arg Glu Gln Asn Arg Ile Xaa Thr 20 25 <210> 1008 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 1008 Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Xaa Gly Gly Ser Gly Gly 1 5 10 15 Thr Arg Glu Gln Asn Arg Ile Cys Thr Xaa Arg 20 25 <210> 1009 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <400> 1009 Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Gly Gly Ser Gly Gly 1 5 10 15 Gln Ala Xaa Thr Arg Glu Gln Asn Arg Ile Cys 20 25 <210> 1010 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1010 Xaa Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Gly Gly Ser Gly Gly 1 5 10 15 Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp 20 25 <210> 1011 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (9)...(9) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (18)...(18) <223> Xaa is Cys residue protected with ACM protecting group <400> 1011 Ser Lys Gln Glu Gly Cys Arg Leu Xaa Ala Pro Gly Gly Ser Gly Gly 1 5 10 15 Leu Xaa Ala Pro Leu Arg Lys Cys Arg Pro Gly 20 25 <210> 1012 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1012 Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Gly Gly Ser Gly Gly 1 5 10 15 Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His 20 25 <210> 1013 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 1013 Xaa Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Gly Gly Ser Gly Gly 1 5 10 15 Thr Arg Glu Gln Asn Arg Ile Cys Thr Xaa Arg 20 25 <210> 1014 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1014 Cys Ala Pro Leu Arg Lys Xaa Arg Pro Gly Phe Gly Val Ala Arg Pro 1 5 10 15 Cys Ala Pro Leu Arg Lys Xaa Arg Pro Gly Phe Gly Val Ala Arg Pro 20 25 30 Cys <210> 1015 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1015 Val Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Gly Gly Ser Gly Gly 1 5 10 15 Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp 20 25 <210> 1016 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <400> 1016 Val Xaa Lys Pro Cys Ala Pro Gly Thr Phe Ser Gly Gly Ser Gly Gly 1 5 10 15 Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His 20 25 <210> 1017 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <400> 1017 Trp Asn Trp Val Pro Glu Xaa Leu Ser Cys Gly Gly Gly Ser Gly Gly 1 5 10 15 Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg 20 25 <210> 1018 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1018 Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile 1 5 10 15 <210> 1019 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (28)...(28) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (30)...(30) <223> Xaa is Cys residue protected with ACM protecting group <400> 1019 Cys Ala Pro Leu Arg Lys Xaa Arg Pro Gly Phe Gly Val Ala Arg Pro 1 5 10 15 Cys Gln Ala Xaa Thr Arg Glu Gln Asn Arg Ile Xaa Thr Xaa Arg Pro 20 25 30 Cys <210> 1020 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1020 Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser 1 5 10 15 <210> 1021 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 1021 Leu Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gln Gly Gly Ser Gly Gly 1 5 10 15 Thr Arg Glu Gln Asn Arg Ile Cys Thr Xaa Arg 20 25 <210> 1022 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (12)...(12) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (28)...(28) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (30)...(30) <223> Xaa is Cys residue protected with ACM protecting group <400> 1022 Cys Gly Xaa Arg Leu Xaa Ala Pro Leu Arg Lys Xaa Arg Pro Gly Phe 1 5 10 15 Cys Gln Ala Xaa Thr Arg Glu Gln Asn Arg Ile Xaa Thr Xaa Arg Pro 20 25 30 Cys <210> 1023 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1023 Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His Gln Ile 1 5 10 15 <210> 1024 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1024 Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Ala Ala Arg Pro Gly 1 5 10 15 <210> 1025 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (9)...(9) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1025 Cys Ala Pro Glu Pro Gly Ser Thr Xaa Arg Leu Arg Glu Tyr Tyr Asp 1 5 10 15 Cys Asn Ala Ser Met Asp Ala Val Xaa Thr Ser Thr Ser Pro Thr Arg 20 25 30 Cys <210> 1026 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 1026 Arg Glu Gln Asn Arg Ile Xaa Thr Cys Arg Pro Gly Gly Ser Gly Gly 1 5 10 15 Tyr Cys Ala Leu Ser Lys Gln Glu Gly Xaa Arg 20 25 <210> 1027 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (14)...(14) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1027 Cys Ser Arg Xaa Ser Ser Asp Gln Val Glu Thr Gln Ala Xaa Thr Arg 1 5 10 15 Cys Ala Pro Leu Arg Lys Xaa Arg Pro Gly Phe Gly Val Ala Arg Pro 20 25 30 Cys <210> 1028 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <400> 1028 Val Val Xaa Lys Pro Cys Ala Pro Gly Thr Phe Gly Gly Ser Gly Gly 1 5 10 15 Thr Ser Ser Thr Asp Ile Cys Arg Pro His Gln 20 25 <210> 1029 <211> 22 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <400> 1029 Xaa Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr 1 5 10 15 Asp Ile Cys Arg Pro His 20 <210> 1030 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Cys residue protected with ACM protecting group <400> 1030 Thr Glu Thr Ser Asp Val Val Xaa Lys Pro Cys Gly Gly Ser Gly Gly 1 5 10 15 Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His 20 25 <210> 1031 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <400> 1031 Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Gly Gly Ser Gly Gly 1 5 10 15 Gln Ala Xaa Thr Arg Glu Gln Asn Arg Ile Cys 20 25 <210> 1032 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <400> 1032 Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Xaa Gly Gly Ser Gly Gly 1 5 10 15 Gln Ala Xaa Thr Arg Glu Gln Asn Arg Ile Cys 20 25 <210> 1033 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (17)...(17) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 1033 Val Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Gly Gly Ser Gly Gly 1 5 10 15 Xaa Thr Arg Glu Gln Asn Arg Ile Cys Thr Xaa 20 25 <210> 1034 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1034 Thr Arg Glu Gln Asn Arg Ile Xaa Thr Cys Arg Gly Gly Ser Gly Gly 1 5 10 15 Cys Ala Leu Ser Lys Gln Glu Gly Xaa Arg Leu 20 25 <210> 1035 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 1035 Thr Arg Glu Gln Asn Arg Ile Xaa Thr Cys Arg Gly Gly Ser Gly Gly 1 5 10 15 Tyr Cys Ala Leu Ser Lys Gln Glu Gly Xaa Arg 20 25 <210> 1036 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1036 Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Ala Gly Pro Gly Ala 1 5 10 15 <210> 1037 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (12)...(12) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (14)...(14) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1037 Cys Gln Ala Xaa Thr Arg Glu Gln Asn Arg Ile Xaa Thr Xaa Arg Pro 1 5 10 15 Cys Ala Pro Leu Arg Lys Xaa Arg Pro Gly Phe Gly Val Ala Arg Pro 20 25 30 Cys <210> 1038 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Cys residue protected with ACM protecting group <400> 1038 Ser Asp Val Val Xaa Lys Pro Cys Ala Pro Gly Gly Gly Ser Gly Gly 1 5 10 15 Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His 20 25 <210> 1039 <211> 17 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1039 Cys Ala Ala Thr Arg Glu Gln Asn Arg Ile Ala Ala Gly Arg Pro Gly 1 5 10 15 Cys <210> 1040 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (9)...(9) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa can be any naturally occurring amino acid <400> 1040 Cys Asp Gln Val Glu Thr Gln Ala Xaa Thr Arg Glu Gln Asn Arg Ile 1 5 10 15 Cys Ala Pro Leu Arg Lys Xaa Arg Pro Gly Phe Gly Val Ala Arg Pro 20 25 30 Cys <210> 1041 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 1041 Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Gly Gly Ser Gly Gly 1 5 10 15 Thr Arg Glu Gln Asn Arg Ile Cys Thr Xaa Arg 20 25 <210> 1042 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1042 Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly Pro Ser 1 5 10 15 <210> 1043 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (18)...(18) <223> Xaa is Cys residue protected with ACM protecting group <400> 1043 Leu Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gln Gly Gly Ser Gly Gly 1 5 10 15 Ala Xaa Thr Arg Glu Gln Asn Arg Ile Cys Thr 20 25 <210> 1044 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (30)...(30) <223> Xaa is Cys residue protected with ACM protecting group <400> 1044 Cys Leu Ser Lys Gln Glu Gly Xaa Arg Leu Xaa Ala Pro Leu Arg Lys 1 5 10 15 Cys Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser Thr Xaa Arg Leu 20 25 30 Cys <210> 1045 <211> 24 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1045 Cys Asn Trp Val Pro Glu Ala Leu Ser Ala Gly Pro Gly Ala Ser Ser 1 5 10 15 Asp Gln Val Glu Thr Gln Ala Cys 20 <210> 1046 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <400> 1046 Glu Thr Ser Asp Val Val Xaa Lys Pro Cys Ala Gly Gly Ser Gly Gly 1 5 10 15 Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His 20 25 <210> 1047 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <400> 1047 Val Xaa Lys Pro Cys Ala Pro Gly Thr Phe Ser Gly Gly Ser Gly Gly 1 5 10 15 Thr Ser Ser Thr Asp Ile Cys Arg Pro His Gln 20 25 <210> 1048 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (14)...(14) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1048 Cys Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala Val Xaa Thr Ser 1 5 10 15 Cys Ala Pro Leu Arg Lys Xaa Arg Pro Gly Phe Gly Val Ala Arg Pro 20 25 30 Cys <210> 1049 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Cys residue protected with ACM protecting group <400> 1049 Ser Asp Val Val Xaa Lys Pro Cys Ala Pro Gly Gly Gly Ser Gly Gly 1 5 10 15 Thr Ser Ser Thr Asp Ile Cys Arg Pro His Gln 20 25 <210> 1050 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1050 Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Gly Gly Ser Gly Gly 1 5 10 15 Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala 20 25 <210> 1051 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1051 Asp Ile Cys Arg Pro His Gln Ile Cys Asn Val Val Ala Ile Pro 1 5 10 15 <210> 1052 <211> 17 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1052 Cys Leu Trp Asn Trp Val Pro Glu Ala Leu Ser Ala Gly Ser Arg Ala 1 5 10 15 Cys <210> 1053 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (9)...(9) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1053 Cys Ala Pro Glu Pro Gly Ser Thr Xaa Arg Leu Arg Glu Tyr Tyr Asp 1 5 10 15 Cys Ala Pro Leu Arg Lys Xaa Arg Pro Gly Phe Gly Val Ala Arg Pro 20 25 30 Cys <210> 1054 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (22)...(22) <223> Xaa is Cys residue protected with ACM protecting group <400> 1054 Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Gly Gly Ser Gly Gly 1 5 10 15 Met Cys Xaa Ser Lys Xaa Ser Pro Gly Gln His 20 25 <210> 1055 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <400> 1055 Val Pro Glu Xaa Leu Ser Cys Gly Ser Arg Xaa Gly Gly Ser Gly Gly 1 5 10 15 Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile 20 25 <210> 1056 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 1056 Leu Ser Cys Gly Ser Arg Xaa Ser Ser Asp Gln Gly Gly Ser Gly Gly 1 5 10 15 Ala Cys Thr Arg Glu Gln Asn Arg Ile Xaa Thr 20 25 <210> 1057 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <400> 1057 Cys Xaa Glu Asp Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu 1 5 10 15 Cys Gln Ile Xaa Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp 20 25 30 Cys <210> 1058 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 1058 Ser Cys Gly Ser Arg Xaa Ser Ser Asp Gln Val Gly Gly Ser Gly Gly 1 5 10 15 Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Xaa 20 25 <210> 1059 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <400> 1059 Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Gly Gly Ser Gly Gly 1 5 10 15 Gln Ala Xaa Thr Arg Glu Gln Asn Arg Ile Cys 20 25 <210> 1060 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1060 Cys Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val His Leu Pro 1 5 10 15 Cys Ala Pro Leu Arg Lys Xaa Arg Pro Gly Phe Gly Val Ala Arg Pro 20 25 30 Cys <210> 1061 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <400> 1061 Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gln Val Gly Gly Ser Gly Gly 1 5 10 15 Gln Ala Xaa Thr Arg Glu Gln Asn Arg Ile Cys 20 25 <210> 1062 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (18)...(18) <223> Xaa is Cys residue protected with ACM protecting group <400> 1062 Lys Gln Glu Gly Cys Arg Leu Xaa Ala Pro Leu Gly Gly Ser Gly Gly 1 5 10 15 Leu Xaa Ala Pro Leu Arg Lys Cys Arg Pro Gly 20 25 <210> 1063 <211> 17 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1063 Cys Ala Ala Thr Arg Glu Gln Asn Arg Ile Ala Thr Ala Arg Pro Gly 1 5 10 15 Cys <210> 1064 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (16)...(16) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1064 Cys Xaa Xaa Ser Lys Xaa Ser Pro Gly Gln His Ala Lys Val Phe Xaa 1 5 10 15 Cys Ala Pro Leu Arg Lys Xaa Arg Pro Gly Phe Gly Val Ala Arg Pro 20 25 30 Cys <210> 1065 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1065 Cys Asp Val Val Xaa Lys Pro Xaa Ala Pro Gly Thr Phe Ser Asn Thr 1 5 10 15 Cys Asn Ala Ser Met Asp Ala Val Xaa Thr Ser Thr Ser Pro Thr Arg 20 25 30 Cys <210> 1066 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (18)...(18) <223> Xaa is Cys residue protected with ACM protecting group <400> 1066 Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gly Gly Ser Gly Gly 1 5 10 15 Ala Xaa Thr Arg Glu Gln Asn Arg Ile Cys Thr 20 25 <210> 1067 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (9)...(9) <223> Xaa is Cys residue protected with ACM protecting group <400> 1067 Glu Xaa Leu Ser Cys Gly Ser Arg Xaa Ser Ser Gly Gly Ser Gly Gly 1 5 10 15 Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile 20 25 <210> 1068 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (12)...(12) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (15)...(15) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1068 Cys Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Xaa Leu Ser Xaa Gly 1 5 10 15 Cys Asn Ala Ser Met Asp Ala Val Xaa Thr Ser Thr Ser Pro Thr Arg 20 25 30 Cys <210> 1069 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (17)...(17) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 1069 Leu Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gln Gly Gly Ser Gly Gly 1 5 10 15 Xaa Thr Arg Glu Gln Asn Arg Ile Cys Thr Xaa 20 25 <210> 1070 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (9)...(9) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1070 Cys Xaa Leu Ser Xaa Gly Ser Arg Xaa Ser Ser Asp Gln Val Glu Thr 1 5 10 15 Cys Ala Pro Leu Arg Lys Xaa Arg Pro Gly Phe Gly Val Ala Arg Pro 20 25 30 Cys <210> 1071 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Cys residue protected with ACM protecting group <400> 1071 Trp Val Pro Glu Xaa Leu Ser Cys Gly Ser Arg Gly Gly Ser Gly Gly 1 5 10 15 Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile 20 25 <210> 1072 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (9)...(9) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1072 Xaa Thr Arg Glu Gln Asn Arg Ile Xaa Thr Cys Gly Gly Ser Gly Gly 1 5 10 15 Cys Ala Leu Ser Lys Gln Glu Gly Xaa Arg Leu 20 25 <210> 1073 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1073 Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg 1 5 10 15 <210> 1074 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <400> 1074 Xaa Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Gly Gly Ser Gly Gly 1 5 10 15 Gln Ala Xaa Thr Arg Glu Gln Asn Arg Ile Cys 20 25 <210> 1075 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1075 Asn Arg Ile Xaa Thr Cys Arg Pro Gly Trp Tyr Gly Gly Ser Gly Gly 1 5 10 15 Cys Ala Leu Ser Lys Gln Glu Gly Xaa Arg Leu 20 25 <210> 1076 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (12)...(12) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (28)...(28) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (29)...(29) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (32)...(32) <223> Xaa is Cys residue protected with ACM protecting group <400> 1076 Cys Gly Xaa Arg Leu Xaa Ala Pro Leu Arg Lys Xaa Arg Pro Gly Phe 1 5 10 15 Cys Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Xaa Xaa Ser Lys Xaa 20 25 30 Cys <210> 1077 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1077 Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Ala Ala Cys Arg Pro 1 5 10 15 <210> 1078 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1078 Cys Thr Val Xaa Asp Ser Xaa Glu Asp Ser Thr Tyr Thr Gln Leu Trp 1 5 10 15 Cys Asn Ala Ser Met Asp Ala Val Xaa Thr Ser Thr Ser Pro Thr Arg 20 25 30 Cys <210> 1079 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1079 Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Gly Gly Ser Gly Gly 1 5 10 15 Gln Met Cys Xaa Ser Lys Xaa Ser Pro Gly Gln 20 25 <210> 1080 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Cys residue protected with ACM protecting group <400> 1080 Trp Val Pro Glu Xaa Leu Ser Cys Gly Ser Arg Gly Gly Ser Gly Gly 1 5 10 15 Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg 20 25 <210> 1081 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <400> 1081 Leu Ser Cys Gly Ser Arg Xaa Ser Ser Asp Gln Gly Gly Ser Gly Gly 1 5 10 15 Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile 20 25 <210> 1082 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (17)...(17) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 1082 Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Ser Gly Gly Ser Gly Gly 1 5 10 15 Xaa Thr Arg Glu Gln Asn Arg Ile Cys Thr Xaa 20 25 <210> 1083 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 1083 Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gly Gly Ser Gly Gly 1 5 10 15 Thr Arg Glu Gln Asn Arg Ile Cys Thr Xaa Arg 20 25 <210> 1084 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1084 Pro Tyr Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr 1 5 10 15 <210> 1085 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (16)...(16) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1085 Cys Xaa Xaa Ser Lys Xaa Ser Pro Gly Gln His Ala Lys Val Phe Xaa 1 5 10 15 Cys Asn Ala Ser Met Asp Ala Val Xaa Thr Ser Thr Ser Pro Thr Arg 20 25 30 Cys <210> 1086 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (14)...(14) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1086 Cys Thr Ser Ser Thr Asp Ile Xaa Arg Pro His Gln Ile Xaa Asn Val 1 5 10 15 Cys Asn Ala Ser Met Asp Ala Val Xaa Thr Ser Thr Ser Pro Thr Arg 20 25 30 Cys <210> 1087 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (16)...(16) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <400> 1087 Cys Xaa Xaa Ser Lys Xaa Ser Pro Gly Gln His Ala Lys Val Phe Xaa 1 5 10 15 Cys Ala Val Xaa Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly 20 25 30 Cys <210> 1088 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (10)...(10) <223> Xaa is Cys residue protected with ACM protecting group <400> 1088 Pro Glu Xaa Leu Ser Cys Gly Ser Arg Xaa Ser Gly Gly Ser Gly Gly 1 5 10 15 Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile 20 25 <210> 1089 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 1089 Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gly Gly Ser Gly Gly 1 5 10 15 Thr Arg Glu Gln Asn Arg Ile Cys Thr Xaa Arg 20 25 <210> 1090 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <400> 1090 Lys Gln Glu Gly Xaa Arg Leu Cys Ala Pro Leu Gly Gly Ser Gly Gly 1 5 10 15 Cys Lys Pro Xaa Ala Pro Gly Thr Phe Ser Asn 20 25 <210> 1091 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1091 Pro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly Gly Ser Gly Gly 1 5 10 15 Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala 20 25 <210> 1092 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1092 Cys Leu Pro Met Gly Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp 1 5 10 15 Cys Ala Pro Leu Arg Lys Xaa Arg Pro Gly Phe Gly Val Ala Arg Pro 20 25 30 Cys <210> 1093 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 1093 Xaa Leu Ser Cys Gly Ser Arg Xaa Ser Ser Asp Gly Gly Ser Gly Gly 1 5 10 15 Cys Thr Arg Glu Gln Asn Arg Ile Xaa Thr Xaa 20 25 <210> 1094 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <400> 1094 Xaa Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Gly Gly Ser Gly Gly 1 5 10 15 Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His 20 25 <210> 1095 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (30)...(30) <223> Xaa is Cys residue protected with ACM protecting group <400> 1095 Cys Gln Thr Ala Gln Met Xaa Xaa Ser Lys Xaa Ser Pro Gly Gln His 1 5 10 15 Cys Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser Thr Xaa Arg Leu 20 25 30 Cys <210> 1096 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1096 Thr Ser Ser Thr Asp Ile Cys Arg Pro His Gln Ile Cys Asn Val 1 5 10 15 <210> 1097 <211> 17 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1097 Cys Glu Gly Ala Arg Leu Ala Ala Pro Leu Arg Ala Gly Arg Pro Gly 1 5 10 15 Cys <210> 1098 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (12)...(12) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (14)...(14) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (30)...(30) <223> Xaa is Cys residue protected with ACM protecting group <400> 1098 Cys Gln Ala Xaa Thr Arg Glu Gln Asn Arg Ile Xaa Thr Xaa Arg Pro 1 5 10 15 Cys Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser Thr Xaa Arg Leu 20 25 30 Cys <210> 1099 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1099 Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gln Val Gly Gly Ser Gly Gly 1 5 10 15 Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp 20 25 <210> 1100 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (9)...(9) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (22)...(22) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa can be any naturally occurring amino acid <400> 1100 Cys Asp Gln Val Glu Thr Gln Ala Xaa Thr Arg Glu Gln Asn Arg Ile 1 5 10 15 Cys Ala Pro Leu Arg Xaa Xaa Arg Pro Gly Phe Gly Val Ala Arg Pro 20 25 30 Cys <210> 1101 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1101 Val Xaa Lys Pro Cys Ala Pro Gly Thr Phe Ser Gly Gly Ser Gly Gly 1 5 10 15 Asp Ile Cys Arg Pro His Gln Ile Xaa Asn Val 20 25 <210> 1102 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 1102 Xaa Leu Ser Cys Gly Ser Arg Xaa Ser Ser Asp Gly Gly Ser Gly Gly 1 5 10 15 Ala Cys Thr Arg Glu Gln Asn Arg Ile Xaa Thr 20 25 <210> 1103 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <400> 1103 Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gly Gly Ser Gly Gly 1 5 10 15 Gln Ala Xaa Thr Arg Glu Gln Asn Arg Ile Cys 20 25 <210> 1104 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (28)...(28) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (30)...(30) <223> Xaa is Cys residue protected with ACM protecting group <400> 1104 Cys Gln Thr Ala Gln Met Xaa Xaa Ser Lys Xaa Ser Pro Gly Gln His 1 5 10 15 Cys Gln Ala Xaa Thr Arg Glu Gln Asn Arg Ile Xaa Thr Xaa Arg Pro 20 25 30 Cys <210> 1105 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <400> 1105 Tyr Xaa Ala Leu Ser Lys Gln Glu Gly Cys Arg Gly Gly Ser Gly Gly 1 5 10 15 Lys Cys Arg Pro Gly Phe Gly Val Ala Arg Pro 20 25 <210> 1106 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 1106 Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Gly Gly Ser Gly Gly 1 5 10 15 Thr Asp Ile Cys Arg Pro His Gln Ile Xaa Asn 20 25 <210> 1107 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <400> 1107 Asp Ile Xaa Arg Pro His Gln Ile Cys Asn Val Gly Gly Ser Gly Gly 1 5 10 15 Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met 20 25 <210> 1108 <211> 17 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1108 Cys Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Gly Ala Ser Ala Gly 1 5 10 15 Cys <210> 1109 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1109 Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gly Gly Ser Gly Gly 1 5 10 15 Gln Met Cys Xaa Ser Lys Xaa Ser Pro Gly Gln 20 25 <210> 1110 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <400> 1110 Gln Glu Gly Cys Arg Leu Xaa Ala Pro Leu Arg Gly Gly Ser Gly Gly 1 5 10 15 Lys Cys Arg Pro Gly Phe Gly Val Ala Arg Pro 20 25 <210> 1111 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (9)...(9) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1111 Cys Asn Ala Ser Met Asp Ala Val Xaa Thr Ser Thr Ser Pro Thr Arg 1 5 10 15 Cys Ala Pro Leu Arg Lys Xaa Arg Pro Gly Phe Gly Val Ala Arg Pro 20 25 30 Cys <210> 1112 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (12)...(12) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (13)...(13) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (16)...(16) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1112 Cys Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Xaa Xaa Ser Lys Xaa 1 5 10 15 Cys Asn Ala Ser Met Asp Ala Val Xaa Thr Ser Thr Ser Pro Thr Arg 20 25 30 Cys <210> 1113 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 1113 Cys Gly Ser Arg Xaa Ser Ser Asp Gln Val Glu Gly Gly Ser Gly Gly 1 5 10 15 Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Xaa 20 25 <210> 1114 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1114 Cys Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly 1 5 10 15 Cys Asn Ala Ser Met Asp Ala Val Xaa Thr Ser Thr Ser Pro Thr Arg 20 25 30 Cys <210> 1115 <211> 24 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1115 Cys Leu Trp Asn Trp Val Pro Glu Ala Leu Ser Pro Gly Ser Arg Ala 1 5 10 15 Ser Ser Asp Gln Val Glu Thr Cys 20 <210> 1116 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (18)...(18) <223> Xaa is Cys residue protected with ACM protecting group <400> 1116 Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gln Val Gly Gly Ser Gly Gly 1 5 10 15 Ala Xaa Thr Arg Glu Gln Asn Arg Ile Cys Thr 20 25 <210> 1117 <211> 17 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1117 Cys Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly Pro Ser 1 5 10 15 Cys <210> 1118 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (12)...(12) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (14)...(14) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (30)...(30) <223> Xaa is Cys residue protected with ACM protecting group <400> 1118 Cys Gln Ala Xaa Thr Arg Glu Gln Asn Arg Ile Xaa Thr Xaa Arg Pro 1 5 10 15 Cys Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser Thr Xaa Arg Leu 20 25 30 Cys <210> 1119 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1119 Cys Ala Val Xaa Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly 1 5 10 15 Cys Ala Pro Leu Arg Lys Xaa Arg Pro Gly Phe Gly Val Ala Arg Pro 20 25 30 Cys <210> 1120 <211> 22 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (21)...(21) <223> Xaa is Cys residue protected with ACM protecting group <400> 1120 Gln Asn Arg Ile Xaa Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser 1 5 10 15 Lys Gln Glu Gly Xaa Arg 20 <210> 1121 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 1121 Val Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Gly Gly Ser Gly Gly 1 5 10 15 Thr Arg Glu Gln Asn Arg Ile Cys Thr Xaa Arg 20 25 <210> 1122 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <400> 1122 Xaa Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Gly Gly Ser Gly Gly 1 5 10 15 Thr Ser Ser Thr Asp Ile Cys Arg Pro His Gln 20 25 <210> 1123 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 1123 Asn Trp Val Pro Glu Xaa Leu Ser Cys Gly Ser Gly Gly Ser Gly Gly 1 5 10 15 Cys Thr Arg Glu Gln Asn Arg Ile Xaa Thr Xaa 20 25 <210> 1124 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (12)...(12) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (13)...(13) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (16)...(16) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1124 Cys Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Xaa Xaa Ser Lys Xaa 1 5 10 15 Cys Ala Pro Leu Arg Lys Xaa Arg Pro Gly Phe Gly Val Ala Arg Pro 20 25 30 Cys <210> 1125 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (17)...(17) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 1125 Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gly Gly Ser Gly Gly 1 5 10 15 Xaa Thr Arg Glu Gln Asn Arg Ile Cys Thr Xaa 20 25 <210> 1126 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <400> 1126 Gln Glu Gly Xaa Arg Leu Cys Ala Pro Leu Arg Gly Gly Ser Gly Gly 1 5 10 15 Cys Lys Pro Xaa Ala Pro Gly Thr Phe Ser Asn 20 25 <210> 1127 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1127 Cys Ala Pro Leu Arg Lys Xaa Arg Pro Gly Phe Gly Val Ala Arg Pro 1 5 10 15 Cys Asn Ala Ser Met Asp Ala Val Xaa Thr Ser Thr Ser Pro Thr Arg 20 25 30 Cys <210> 1128 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <400> 1128 Asp Ile Xaa Arg Pro His Gln Ile Cys Asn Val Gly Gly Ser Gly Gly 1 5 10 15 Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala 20 25 <210> 1129 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (12)...(12) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (14)...(14) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (28)...(28) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (30)...(30) <223> Xaa is Cys residue protected with ACM protecting group <400> 1129 Cys Gln Ala Xaa Thr Arg Glu Gln Asn Arg Ile Xaa Thr Xaa Arg Pro 1 5 10 15 Cys Gln Ala Xaa Thr Arg Glu Gln Asn Arg Ile Xaa Thr Xaa Arg Pro 20 25 30 Cys <210> 1130 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (14)...(14) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (28)...(28) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (30)...(30) <223> Xaa is Cys residue protected with ACM protecting group <400> 1130 Cys Thr Ser Ser Thr Asp Ile Xaa Arg Pro His Gln Ile Xaa Asn Val 1 5 10 15 Cys Gln Ala Xaa Thr Arg Glu Gln Asn Arg Ile Xaa Thr Xaa Arg Pro 20 25 30 Cys <210> 1131 <211> 32 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1131 Cys Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly 1 5 10 15 Cys Ala Pro Leu Arg Lys Xaa Arg Pro Gly Phe Gly Val Ala Arg Pro 20 25 30 <210> 1132 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <400> 1132 Val Pro Glu Xaa Leu Ser Cys Gly Ser Arg Xaa Gly Gly Ser Gly Gly 1 5 10 15 Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg 20 25 <210> 1133 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (14)...(14) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1133 Cys Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser Thr Xaa Arg Leu 1 5 10 15 Cys Ala Pro Leu Arg Lys Xaa Arg Pro Gly Phe Gly Val Ala Arg Pro 20 25 30 Cys <210> 1134 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (9)...(9) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1134 Cys Ile Xaa Arg Pro His Gln Ile Xaa Asn Val Val Ala Ile Pro Gly 1 5 10 15 Cys Ala Pro Leu Arg Lys Xaa Arg Pro Gly Phe Gly Val Ala Arg Pro 20 25 30 Cys <210> 1135 <211> 32 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1135 Cys Ser Thr Ser Phe Leu Leu Pro Met Gly Pro Ser Pro Pro Ala Glu 1 5 10 15 Cys Ala Pro Leu Arg Lys Xaa Arg Pro Gly Phe Gly Val Ala Arg Pro 20 25 30 <210> 1136 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (9)...(9) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 1136 Xaa Thr Arg Glu Gln Asn Arg Ile Xaa Thr Cys Gly Gly Ser Gly Gly 1 5 10 15 Tyr Cys Ala Leu Ser Lys Gln Glu Gly Xaa Arg 20 25 <210> 1137 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1137 Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Gly Gly Ser Gly Gly 1 5 10 15 Thr Ser Ser Thr Asp Ile Cys Arg Pro His Gln 20 25 <210> 1138 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1138 Cys Gln Thr Ala Gln Met Xaa Xaa Ser Lys Xaa Ser Pro Gly Gln His 1 5 10 15 Cys Asn Ala Ser Met Asp Ala Val Xaa Thr Ser Thr Ser Pro Thr Arg 20 25 30 Cys <210> 1139 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (9)...(9) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1139 Cys Asn Ala Ser Met Asp Ala Val Xaa Thr Ser Thr Ser Pro Thr Arg 1 5 10 15 Cys Asn Ala Ser Met Asp Ala Val Xaa Thr Ser Thr Ser Pro Thr Arg 20 25 30 Cys <210> 1140 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (12)...(12) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (15)...(15) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (24)...(24) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 1140 Cys Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Xaa Leu Ser Xaa Gly 1 5 10 15 Cys Leu Ser Lys Gln Glu Gly Xaa Arg Leu Xaa Ala Pro Leu Arg Lys 20 25 30 Cys <210> 1141 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1141 Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gly Gly Ser Gly Gly 1 5 10 15 Gln Met Cys Xaa Ser Lys Xaa Ser Pro Gly Gln 20 25 <210> 1142 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1142 Met Asp Ala Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala 1 5 10 15 <210> 1143 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (28)...(28) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (30)...(30) <223> Xaa is Cys residue protected with ACM protecting group <400> 1143 Cys Ala Val His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr 1 5 10 15 Cys Gln Ala Xaa Thr Arg Glu Gln Asn Arg Ile Xaa Thr Xaa Arg Pro 20 25 30 Cys <210> 1144 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 1144 Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Gly Gly Ser Gly Gly 1 5 10 15 Thr Arg Glu Gln Asn Arg Ile Cys Thr Xaa Arg 20 25 <210> 1145 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (24)...(24) <223> Xaa is Cys residue protected with ACM protecting group <400> 1145 Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gly Gly Ser Gly Gly 1 5 10 15 Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly 20 25 <210> 1146 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1146 Arg Glu Gln Asn Arg Ile Xaa Thr Cys Arg Pro Gly Gly Ser Gly Gly 1 5 10 15 Cys Ala Leu Ser Lys Gln Glu Gly Xaa Arg Leu 20 25 <210> 1147 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1147 Cys Gln Thr Ala Gln Met Xaa Xaa Ser Lys Xaa Ser Pro Gly Gln His 1 5 10 15 Cys Ala Pro Leu Arg Lys Xaa Arg Pro Gly Phe Gly Val Ala Arg Pro 20 25 30 Cys <210> 1148 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (10)...(10) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1148 Cys Xaa Thr Xaa Arg Pro Gly Trp Tyr Xaa Ala Leu Ser Lys Gln Glu 1 5 10 15 Cys Ala Pro Leu Arg Lys Xaa Arg Pro Gly Phe Gly Val Ala Arg Pro 20 25 30 Cys <210> 1149 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (10)...(10) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (14)...(14) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1149 Cys Asn Trp Val Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Xaa Ser Ser 1 5 10 15 Cys Asn Ala Ser Met Asp Ala Val Xaa Thr Ser Thr Ser Pro Thr Arg 20 25 30 Cys <210> 1150 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 1150 Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Gly Gly Ser Gly Gly 1 5 10 15 Thr Arg Glu Gln Asn Arg Ile Cys Thr Xaa Arg 20 25 <210> 1151 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1151 Tyr Asp Gln Thr Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly 1 5 10 15 <210> 1152 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1152 Cys Ser Pro Gly Gln His Ala Lys Val Phe Xaa Thr Lys Thr Ser Asp 1 5 10 15 Cys Ala Pro Leu Arg Lys Xaa Arg Pro Gly Phe Gly Val Ala Arg Pro 20 25 30 Cys <210> 1153 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1153 Cys Leu Ser Lys Gln Glu Gly Xaa Arg Leu Xaa Ala Pro Leu Arg Lys 1 5 10 15 Cys Asp Gln Val Glu Thr Gln Ala Xaa Thr Arg Glu Gln Asn Arg Ile 20 25 30 Cys <210> 1154 <211> 18 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1154 Cys Ser Asp Gln Val Glu Thr Gln Pro Gly Thr Arg Glu Gln Asn Arg 1 5 10 15 Ile Cys <210> 1155 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1155 Glu Gln Asn Arg Ile Xaa Thr Cys Arg Pro Gly Gly Gly Ser Gly Gly 1 5 10 15 Cys Ala Leu Ser Lys Gln Glu Gly Xaa Arg Leu 20 25 <210> 1156 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <400> 1156 Asp Val Val Xaa Lys Pro Cys Ala Pro Gly Thr Gly Gly Ser Gly Gly 1 5 10 15 Thr Ser Ser Thr Asp Ile Cys Arg Pro His Gln 20 25 <210> 1157 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Cys residue protected with ACM protecting group <400> 1157 Ser Thr Asp Ile Xaa Arg Pro His Gln Ile Cys Gly Gly Ser Gly Gly 1 5 10 15 Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala 20 25 <210> 1158 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1158 Cys Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly Pro 1 5 10 15 Cys Asn Ala Ser Met Asp Ala Val Xaa Thr Ser Thr Ser Pro Thr Arg 20 25 30 Cys <210> 1159 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1159 Gly Ala Val His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His 1 5 10 15 <210> 1160 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (12)...(12) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (13)...(13) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (16)...(16) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (28)...(28) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (30)...(30) <223> Xaa is Cys residue protected with ACM protecting group <400> 1160 Cys Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Xaa Xaa Ser Lys Xaa 1 5 10 15 Cys Gln Ala Xaa Thr Arg Glu Gln Asn Arg Ile Xaa Thr Xaa Arg Pro 20 25 30 Cys <210> 1161 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (16)...(16) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (28)...(28) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (30)...(30) <223> Xaa is Cys residue protected with ACM protecting group <400> 1161 Cys Xaa Xaa Ser Lys Xaa Ser Pro Gly Gln His Ala Lys Val Phe Xaa 1 5 10 15 Cys Gln Ala Xaa Thr Arg Glu Gln Asn Arg Ile Xaa Thr Xaa Arg Pro 20 25 30 Cys <210> 1162 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (13)...(13) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (16)...(16) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (28)...(28) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (30)...(30) <223> Xaa is Cys residue protected with ACM protecting group <400> 1162 Cys Gly Trp Tyr Xaa Ala Leu Ser Lys Gln Glu Gly Xaa Arg Leu Xaa 1 5 10 15 Cys Gln Ala Xaa Thr Arg Glu Gln Asn Arg Ile Xaa Thr Xaa Arg Pro 20 25 30 Cys <210> 1163 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (28)...(28) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (30)...(30) <223> Xaa is Cys residue protected with ACM protecting group <400> 1163 Cys Leu Ser Lys Gln Glu Gly Xaa Arg Leu Xaa Ala Pro Leu Arg Lys 1 5 10 15 Cys Gln Ala Xaa Thr Arg Glu Gln Asn Arg Ile Xaa Thr Xaa Arg Pro 20 25 30 Cys <210> 1164 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (14)...(14) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1164 Cys Thr Ser Ser Thr Asp Ile Xaa Arg Pro His Gln Ile Xaa Asn Val 1 5 10 15 Cys Ala Pro Leu Arg Lys Xaa Arg Pro Gly Phe Gly Val Ala Arg Pro 20 25 30 Cys <210> 1165 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Cys residue protected with ACM protecting group <400> 1165 Xaa Leu Ser Cys Gly Ser Arg Xaa Ser Ser Asp Gly Gly Ser Gly Gly 1 5 10 15 Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile 20 25 <210> 1166 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (9)...(9) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (30)...(30) <223> Xaa is Cys residue protected with ACM protecting group <400> 1166 Cys Asp Gln Val Glu Thr Gln Ala Xaa Thr Arg Glu Gln Asn Arg Ile 1 5 10 15 Cys Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser Thr Xaa Arg Leu 20 25 30 Cys <210> 1167 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (9)...(9) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (28)...(28) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (30)...(30) <223> Xaa is Cys residue protected with ACM protecting group <400> 1167 Cys Asn Ala Ser Met Asp Ala Val Xaa Thr Ser Thr Ser Pro Thr Arg 1 5 10 15 Cys Gln Ala Xaa Thr Arg Glu Gln Asn Arg Ile Xaa Thr Xaa Arg Pro 20 25 30 Cys <210> 1168 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1168 Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu Gly Gly Ser Gly Gly 1 5 10 15 Gln Met Cys Xaa Ser Lys Xaa Ser Pro Gly Gln 20 25 <210> 1169 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1169 Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Gly Gly Ser Gly Gly 1 5 10 15 Gln Met Cys Xaa Ser Lys Xaa Ser Pro Gly Gln 20 25 <210> 1170 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1170 Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Gly Ala Pro Gly Thr 1 5 10 15 <210> 1171 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (28)...(28) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (29)...(29) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (32)...(32) <223> Xaa is Cys residue protected with ACM protecting group <400> 1171 Cys Leu Ser Lys Gln Glu Gly Xaa Arg Leu Xaa Ala Pro Leu Arg Lys 1 5 10 15 Cys Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Xaa Xaa Ser Lys Xaa 20 25 30 Cys <210> 1172 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (13)...(13) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (16)...(16) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1172 Cys Gly Trp Tyr Xaa Ala Leu Ser Lys Gln Glu Gly Xaa Arg Leu Xaa 1 5 10 15 Cys Asn Ala Ser Met Asp Ala Val Xaa Thr Ser Thr Ser Pro Thr Arg 20 25 30 Cys <210> 1173 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (13)...(13) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1173 Cys Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Xaa Arg Pro His 1 5 10 15 Cys Asn Ala Ser Met Asp Ala Val Xaa Thr Ser Thr Ser Pro Thr Arg 20 25 30 Cys <210> 1174 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <400> 1174 Ser Cys Gly Ser Arg Xaa Ser Ser Asp Gln Val Gly Gly Ser Gly Gly 1 5 10 15 Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile 20 25 <210> 1175 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (18)...(18) <223> Xaa is Cys residue protected with ACM protecting group <400> 1175 Glu Gly Cys Arg Leu Xaa Ala Pro Leu Arg Lys Gly Gly Ser Gly Gly 1 5 10 15 Leu Xaa Ala Pro Leu Arg Lys Cys Arg Pro Gly 20 25 <210> 1176 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1176 Thr Ser Ser Thr Asp Ile Cys Arg Pro His Ala Ala Cys Asn Val 1 5 10 15 <210> 1177 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1177 Cys Arg Ser Gln His Thr Gln Pro Thr Pro Glu Pro Ser Thr Ala Pro 1 5 10 15 Cys Ala Pro Leu Arg Lys Xaa Arg Pro Gly Phe Gly Val Ala Arg Pro 20 25 30 Cys <210> 1178 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (9)...(9) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (28)...(28) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (30)...(30) <223> Xaa is Cys residue protected with ACM protecting group <400> 1178 Cys Asp Gln Val Glu Thr Gln Ala Xaa Thr Arg Glu Gln Asn Arg Ile 1 5 10 15 Cys Gln Ala Xaa Thr Arg Glu Gln Asn Arg Ile Xaa Thr Xaa Arg Pro 20 25 30 Cys <210> 1179 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1179 Cys Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val His Leu Pro 1 5 10 15 Cys Asn Ala Ser Met Asp Ala Val Xaa Thr Ser Thr Ser Pro Thr Arg 20 25 30 Cys <210> 1180 <211> 22 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Cys residue protected with ACM protecting group <400> 1180 Cys Gly Ser Arg Xaa Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr 1 5 10 15 Arg Glu Gln Asn Arg Ile 20 <210> 1181 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1181 Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Gly Gly Ser Gly Gly 1 5 10 15 Gln Met Cys Xaa Ser Lys Xaa Ser Pro Gly Gln 20 25 <210> 1182 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (9)...(9) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 1182 Glu Xaa Leu Ser Cys Gly Ser Arg Xaa Ser Ser Gly Gly Ser Gly Gly 1 5 10 15 Cys Thr Arg Glu Gln Asn Arg Ile Xaa Thr Xaa 20 25 <210> 1183 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1183 Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Gly Gly Ser Gly Gly 1 5 10 15 Asp Ala Val Cys Thr Ser Thr Ser Pro Thr Arg 20 25 <210> 1184 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1184 Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Ala Ala Ser Pro Pro 1 5 10 15 <210> 1185 <211> 17 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1185 Cys Glu Gln Asn Arg Ile Ala Thr Ala Arg Pro Ala Ala Tyr Ala Ala 1 5 10 15 Cys <210> 1186 <211> 18 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1186 Cys Gln Val Ala Phe Thr Pro Tyr Pro Gly Glu Pro Gly Ser Thr Ala 1 5 10 15 Arg Cys <210> 1187 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1187 Arg Pro His Gln Ile Cys Asn Val Val Ala Ile Gly Gly Ser Gly Gly 1 5 10 15 Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala 20 25 <210> 1188 <211> 15 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1188 Thr Arg Ser Met Ala Pro Gly Ala Val His Leu Pro Gln Pro Val 1 5 10 15 <210> 1189 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1189 Gln Asn Arg Ile Xaa Thr Cys Arg Pro Gly Trp Gly Gly Ser Gly Gly 1 5 10 15 Cys Ala Leu Ser Lys Gln Glu Gly Xaa Arg Leu 20 25 <210> 1190 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within TNFR2 that represents epitopes that TNFR2 agonist antibodies can bind to <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <400> 1190 Ala Leu Ser Lys Gln Glu Gly Xaa Arg Leu Cys Gly Gly Ser Gly Gly 1 5 10 15 Cys Lys Pro Xaa Ala Pro Gly Thr Phe Ser Asn 20 25 <210> 1191 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (15)...(15) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1191 Cys Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val Val Xaa Lys 1 5 10 15 Cys Ala Pro Leu Arg Lys Xaa Arg Pro Gly Phe Gly Val Ala Arg Pro 20 25 30 Cys <210> 1192 <211> 27 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <400> 1192 Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Gly Gly Ser Gly Gly 1 5 10 15 Thr Ser Ser Thr Asp Ile Cys Arg Pro His Gln 20 25 <210> 1193 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (14)...(14) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (28)...(28) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (30)...(30) <223> Xaa is Cys residue protected with ACM protecting group <400> 1193 Cys Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser Thr Xaa Arg Leu 1 5 10 15 Cys Gln Ala Xaa Thr Arg Glu Gln Asn Arg Ile Xaa Thr Xaa Arg Pro 20 25 30 Cys <210> 1194 <211> 33 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies or fragments thereof can bind to <220> <221> VARIANT <222> (10)...(10) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (13)...(13) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1194 Cys Gly Thr Glu Thr Ser Asp Val Val Xaa Lys Pro Xaa Ala Pro Gly 1 5 10 15 Cys Ala Pro Leu Arg Lys Xaa Arg Pro Gly Phe Gly Val Ala Arg Pro 20 25 30 Cys <210> 1195 <211> 185 <212> PRT <213> artificial sequence <220> <223> peptide derived from human TNFR2 with affinity for binding agonistic antibody MR2-1 <400> 1195 Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly 1 5 10 15 Gln His Ala Lys Val Phe Cys Thr Lys Thr Ser Asp Thr Val Cys Asp 20 25 30 Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu 35 40 45 Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln 50 55 60 Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp 65 70 75 80 Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu 85 90 95 Arg Lys Cys Arg Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr 100 105 110 Ser Asp Val Val Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr 115 120 125 Thr Ser Ser Thr Asp Ile Cys Arg Pro His Gln Ile Cys Asn Val Val 130 135 140 Ala Ile Pro Gly Asn Ala Ser Met Asp Ala Val Cys Thr Ser Thr Ser 145 150 155 160 Pro Thr Arg Ser Met Ala Pro Gly Ala Val His Leu Pro Gln Pro Val 165 170 175 Ser Thr Arg Ser Gln His Thr Gln Pro 180 185 <210> 1196 <211> 20 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies and fragments thereof can bind to <400> 1196 Gln Thr Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala 1 5 10 15 Lys Val Phe Cys 20 <210> 1197 <211> 20 <212> PRT <213> artificial sequence <220> <223> peptide derived from human TNFR2 with affinity for binding agonistic antibody MR2-1 <400> 1197 Arg Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala 1 5 10 15 Leu Ser Lys Gln 20 <210> 1198 <211> 20 <212> PRT <213> artificial sequence <220> <223> peptide derived from human TNFR2 with affinity for binding agonistic antibody MR2-1 <400> 1198 Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys Arg 1 5 10 15 Leu Cys Ala Pro 20 <210> 1199 <211> 20 <212> PRT <213> artificial sequence <220> <223> peptide derived from human TNFR2 with affinity for binding agonistic antibody MR2-1 <400> 1199 Leu Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys 1 5 10 15 Arg Pro Gly Phe 20 <210> 1200 <211> 20 <212> PRT <213> artificial sequence <220> <223> peptide derived from human TNFR2 with affinity for binding agonistic antibody MR2-1 <400> 1200 Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro 1 5 10 15 Gly Phe Gly Val 20 <210> 1201 <211> 5 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies and fragments thereof can bind to <400> 1201 Lys Cys Ser Pro Gly 1 5 <210> 1202 <211> 7 <212> PRT <213> artificial sequence <220> <223> human TNFR2 epitope to which agonist antibody MR2-1 binds <400> 1202 Ser Ser Asp Gln Val Glu Thr 1 5 <210> 1203 <211> 28 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies and fragments thereof can bind to <400> 1203 Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys 1 5 10 15 Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys 20 25 <210> 1204 <211> 40 <212> PRT <213> artificial sequence <220> <223> human TNFR2 epitope to which agonist antibody MR2-1 binds <400> 1204 Thr Arg Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys 1 5 10 15 Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys 20 25 30 Cys Arg Pro Gly Phe Gly Val Ala 35 40 <210> 1205 <211> 59 <212> PRT <213> artificial sequence <220> <223> peptide sequence within human TNFR2 that represents an epitope that TNFR2 agonist antibodies and fragments thereof can bind to <400> 1205 Cys Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr 1 5 10 15 Arg Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala 20 25 30 Leu Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys 35 40 45 Arg Pro Gly Phe Gly Val Ala Arg Pro Gly Thr 50 55 <210> 1206 <211> 17 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 that may bind agonistic TNFR2 antibodies and fragments thereof <400> 1206 Cys Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr 1 5 10 15 Arg <210> 1207 <211> 39 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 that may bind agonistic TNFR2 antibodies and fragments thereof <400> 1207 Cys Thr Arg Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr 1 5 10 15 Cys Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg 20 25 30 Lys Cys Arg Pro Ser Gly Phe 35 <210> 1208 <211> 50 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 that may bind agonistic TNFR2 antibodies and fragments thereof <400> 1208 Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys Thr Cys Arg 1 5 10 15 Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu Cys 20 25 30 Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala Arg Pro Gly 35 40 45 Thr Glu 50 <210> 1209 <211> 5 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 that may bind agonistic TNFR2 antibodies and fragments thereof <400> 1209 Lys Cys Arg Pro Gly 1 5 <210> 1210 <211> 8 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 that may bind agonistic TNFR2 antibodies and fragments thereof <400> 1210 Cys Ala Pro Leu Arg Lys Cys Arg 1 5 <210> 1211 <211> 8 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 that may bind agonistic TNFR2 antibodies and fragments thereof <400> 1211 Lys Cys Arg Pro Gly Phe Gly Val 1 5 <210> 1212 <211> 123 <212> PRT <213> artificial sequence <220> <223> TNFRAB1 (human TNFR2 antagonist antibody) heavy chain <220> <221> VARIANT <222> (26)...(32) <223> CDR-H1 = GFTFSSY <220> <221> VARIANT <222> (52)...(57) <223> CDR-H2 = SSGGSY <220> <221> VARIANT <222> (99)...(112) <223> CDR-H3 = QRVDGYSSYWYFDV <400> 1212 Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Gln Arg Val Asp Gly Tyr Ser Ser Tyr Trp Tyr Phe Asp Val 100 105 110 Trp Gly Ala Gly Thr Ala Val Thr Val Ser Ser 115 120 <210> 1213 <211> 108 <212> PRT <213> artificial sequence <220> <223> TNFRAB1 (human TNFR2 antagonist antibody) light chain <220> <221> VARIANT <222> (24)...(33) <223> CDR-L1 = SASSSVYYMY <220> <221> VARIANT <222> (49)...(55) <223> CDR-L2 = STSNLAS <220> <221> VARIANT <222> (88)...(96) <223> CDR-L3 = QQRRNYPYT <400> 1213 Asp Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Tyr Tyr Met 20 25 30 Tyr Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr 35 40 45 Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Arg Asn Tyr Pro Tyr Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala 100 105 <210> 1214 <211> 8 <212> PRT <213> artificial sequence <220> <223> TNFRAB2 (human TNFR2 antagonist antibody) heavy chain CDR-H1 variant 1 <400> 1214 Gly Tyr Thr Phe Thr Asp Tyr Leu 1 5 <210> 1215 <211> 8 <212> PRT <213> artificial sequence <220> <223> TNFRAB2 (human TNFR2 antagonist antibody) heavy chain CDR-H1 variant 2 <400> 1215 Gly Tyr Thr Phe Thr Asp Tyr Ile 1 5 <210> 1216 <211> 8 <212> PRT <213> artificial sequence <220> <223> TNFRAB2 (human TNFR2 antagonist antibody) heavy chain CDR-H2 <400> 1216 Val Asp Pro Glu Tyr Gly Ser Thr 1 5 <210> 1217 <211> 14 <212> PRT <213> artificial sequence <220> <223> TNFRAB2 (human TNFR2 antagonist antibody) heavy chain CDR-H3 <400> 1217 Ala Arg Asp Asp Gly Ser Tyr Ser Pro Phe Asp Tyr Trp Gly 1 5 10 <210> 1218 <211> 6 <212> PRT <213> artificial sequence <220> <223> TNFRAB2 (human TNFR2 antagonist antibody) light chain CDR-L1 <400> 1218 Gln Asn Ile Asn Lys Tyr 1 5 <210> 1219 <211> 3 <212> PRT <213> artificial sequence <220> <223> TNFRAB2 (human TNFR2 antagonist antibody) light chain CDR-L2 variant 1 <400> 1219 Thr Tyr Ser One <210> 1220 <211> 3 <212> PRT <213> artificial sequence <220> <223> TNFRAB2 (human TNFR2 antagonist antibody) light chain CDR-L2 variant 2 <400> 1220 Tyr Thr Ser One <210> 1221 <211> 9 <212> PRT <213> artificial sequence <220> <223> TNFRAB2 (human TNFR2 antagonist antibody) light chain CDR-L3 variant 1 <400> 1221 Cys Leu Gln Tyr Val Asn Leu Leu Thr 1 5 <210> 1222 <211> 9 <212> PRT <213> artificial sequence <220> <223> TNFRAB2 (human TNFR2 antagonist antibody) light chain CDR-L3 variant 2 <400> 1222 Cys Leu Gln Tyr Val Asn Leu Ile Thr 1 5 <210> 1223 <211> 14 <212> PRT <213> artificial sequence <220> <223> TNFR2A3 (human TNFR2 antagonist antibody) heavy chain CDR-H3 <400> 1223 Ala Arg Asp Asp Gly Ser Tyr Ser Pro Phe Asp Tyr Phe Gly 1 5 10 <210> 1224 <211> 8 <212> PRT <213> artificial sequence <220> <223> CDR-H2 of human TNFR2 antagonist antibody (or antigen-binding fragment thereof) <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Gly or Ala <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Asn, Gln, Ser, or Thr <400> 1224 Val Asp Pro Glu Tyr Xaa Xaa Thr 1 5 <210> 1225 <211> 14 <212> PRT <213> artificial sequence <220> <223> CDR-H3 of human TNFR2 antagonist antibody (or antigen-binding fragment thereof) <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Arg, Lys, or His <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Asp or Glu <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Gly or Ala <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Asn, Gln, Ser, or Thr <220> <221> VARIANT <222> (9)...(9) <223> Xaa is Ala, Val, Leu, Ile, Pro, Met, Trp, Phe, or Tyr <220> <221> VARIANT <222> (12)...(12) <223> Xaa is Ala, Val, Leu, Ile, Pro, Met, Trp, Phe, or Tyr <220> <221> VARIANT <222> (13)...(13) <223> Xaa is Asp or Glu <220> <221> VARIANT <222> (14)...(14) <223> Xaa is Ala, Val, Leu, Ile, Pro, Met, Trp, Phe, or Tyr <400> 1225 Gln Xaa Val Xaa Xaa Tyr Xaa Ser Xaa Trp Tyr Xaa Xaa Xaa 1 5 10 <210> 1226 <211> 14 <212> PRT <213> artificial sequence <220> <223> CDR-H3 of human TNFR2 antagonist antibody (or antigen-binding fragment thereof) <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Lys, Arg, or His <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Asp or Glu <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Gly or Ala <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Asn, Gln, Ser, or Thr <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Ala, Val, Leu, Ile, Pro, Met, Trp, Phe, or Tyr <220> <221> VARIANT <222> (10)...(10) <223> Xaa is Ala, Val, Leu, Ile, Pro, Met, Trp, Phe, or Tyr <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Asp or Glu <220> <221> VARIANT <222> (12)...(12) <223> Xaa is Ala, Val, Leu, Ile, Pro, Met, Trp, Phe, or Tyr <400> 1226 Ala Xaa Asp Xaa Xaa Xaa Xaa Ser Pro Xaa Xaa Xaa Trp Gly 1 5 10 <210> 1227 <211> 14 <212> PRT <213> artificial sequence <220> <223> CDR-H3 of human TNFR2 antagonist antibody (or antigen-binding fragment thereof) <220> <221> VARIANT <222> (1)...(1) <223> Xaa is any amino acid <220> <221> VARIANT <222> (3)...(3) <223> Xaa is any amino acid <220> <221> VARIANT <222> (6)...(6) <223> Xaa is any amino acid <220> <221> VARIANT <222> (8)...(8) <223> Xaa is any amino acid <220> <221> VARIANT <222> (10)...(10) <223> Xaa is any amino acid <220> <221> VARIANT <222> (11)...(11) <223> Xaa is any amino acid <220> <221> VARIANT <222> (14)...(14) <223> Xaa is any amino acid <400> 1227 Xaa Arg Xaa Asp Gly Xaa Ser Xaa Tyr Xaa Xaa Phe Asp Xaa 1 5 10 <210> 1228 <211> 14 <212> PRT <213> artificial sequence <220> <223> CDR-H3 of human TNFR2 antagonist antibody (or antigen-binding fragment thereof) <220> <221> VARIANT <222> (1)...(1) <223> Xaa is any amino acid <220> <221> VARIANT <222> (3)...(3) <223> Xaa is any amino acid <220> <221> VARIANT <222> (8)...(8) <223> Xaa is any amino acid <220> <221> VARIANT <222> (9)...(9) <223> Xaa is any amino acid <220> <221> VARIANT <222> (12)...(12) <223> Xaa is any amino acid <220> <221> VARIANT <222> (13)...(13) <223> Xaa is any amino acid <220> <221> VARIANT <222> (14)...(14) <223> Xaa is any amino acid <400> 1228 Xaa Arg Xaa Asp Gly Ser Tyr Xaa Xaa Phe Asp Xaa Xaa Xaa 1 5 10 <210> 1229 <211> 14 <212> PRT <213> artificial sequence <220> <223> CDR-H3 of human TNFR2 antagonist antibody (or antigen-binding fragment thereof) <400> 1229 Gln Arg Val Asp Gly Tyr Ser Ser Tyr Trp Tyr Phe Asp Val 1 5 10 <210> 1230 <211> 6 <212> PRT <213> artificial sequence <220> <223> CDR-H2 of human TNFR2 antagonist antibody (or antigen-binding fragment thereof) <400> 1230 Ser Ser Gly Gly Ser Tyr 1 5 <210> 1231 <211> 7 <212> PRT <213> artificial sequence <220> <223> CDR-H1 of human TNFR2 antagonist antibody (or antigen-binding fragment thereof) <400> 1231 Gly Phe Thr Phe Ser Ser Tyr 1 5 <210> 1232 <211> 8 <212> PRT <213> artificial sequence <220> <223> CDR-H1 of human TNFR2 antagonist antibody (or antigen-binding fragment thereof) <220> <221> VARIANT <222> (2)...(2) <223> Xaa is any amino acid <220> <221> VARIANT <222> (5)...(5) <223> Xaa is any amino acid <220> <221> VARIANT <222> (6)...(6) <223> Xaa is any amino acid <220> <221> VARIANT <222> (8)...(8) <223> Xaa is any amino acid <400> 1232 Gly Xaa Thr Phe Xaa Xaa Tyr Xaa 1 5 <210> 1233 <211> 8 <212> PRT <213> artificial sequence <220> <223> CDR-H1 of human TNFR2 antagonist antibody (or antigen-binding fragment thereof) <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Leu or Ile <400> 1233 Gly Tyr Thr Phe Thr Asp Tyr Xaa 1 5 <210> 1234 <211> 6 <212> PRT <213> artificial sequence <220> <223> CDR-L1 of human TNFR2 antagonist antibody (or antigen-binding fragment thereof) <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Ala, Val, Leu, Ile, Pro, Met, Trp, Phe, or Tyr <400> 1234 Gln Asn Ile Asn Lys Xaa 1 5 <210> 1235 <211> 10 <212> PRT <213> artificial sequence <220> <223> CDR-L1 of human TNFR2 antagonist antibody (or antigen-binding fragment thereof) <400> 1235 Ser Ala Ser Ser Ser Val Tyr Tyr Met Tyr 1 5 10 <210> 1236 <211> 15 <212> PRT <213> artificial sequence <220> <223> CDR-L1 of human TNFR2 antagonist antibody (or antigen-binding fragment thereof) <400> 1236 Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr Met His 1 5 10 15 <210> 1237 <211> 7 <212> PRT <213> artificial sequence <220> <223> CDR-L2 of human TNFR2 antagonist antibody (or antigen-binding fragment thereof) <400> 1237 Ser Thr Ser Asn Leu Ala Ser 1 5 <210> 1238 <211> 7 <212> PRT <213> artificial sequence <220> <223> CDR-L2 of human TNFR2 antagonist antibody (or antigen-binding fragment thereof) <400> 1238 Leu Ala Ser Asn Leu Glu Ser 1 5 <210> 1239 <211> 4 <212> PRT <213> artificial sequence <220> <223> framework region bound to the N-terminus of CDR-L2 of human TNFR2 antagonist antibody (or antigen-binding fragment thereof) <400> 1239 Leu Leu Ile Arg One <210> 1240 <211> 3 <212> PRT <213> artificial sequence <220> <223> framework region bound to the C-terminus of CDR-L2 of human TNFR2 antagonist antibody (or antigen-binding fragment thereof) <400> 1240 Thr Leu Glu One <210> 1241 <211> 9 <212> PRT <213> artificial sequence <220> <223> CDR-L3 of human TNFR2 antagonist antibody (or antigen-binding fragment thereof) <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Ala, Val, Leu, Ile, Pro, Met, Trp, Phe, or Tyr <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Leu or Ile <220> <221> VARIANT <222> (9)...(9) <223> Xaa is Asn, Gln, Ser, or Thr <400> 1241 Cys Leu Gln Xaa Val Asn Leu Xaa Xaa 1 5 <210> 1242 <211> 9 <212> PRT <213> artificial sequence <220> <223> CDR-L3 of human TNFR2 antagonist antibody (or antigen-binding fragment thereof) <400> 1242 Gln Gln Arg Arg Asn Tyr Pro Tyr Thr 1 5 <210> 1243 <211> 9 <212> PRT <213> artificial sequence <220> <223> CDR-L3 of human TNFR2 antagonist antibody (or antigen-binding fragment thereof) <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Leu or Ile <400> 1243 Cys Leu Gln Tyr Val Asn Leu Xaa Thr 1 5 <210> 1244 <211> 9 <212> PRT <213> artificial sequence <220> <223> CDR-L3 of human TNFR2 antagonist antibody (or antigen-binding fragment thereof) <400> 1244 Gln His Ser Arg Glu Leu Pro Arg Thr 1 5 <210> 1245 <211> 120 <212> PRT <213> artificial sequence <220> <223> excellent consensus sequence of a human antibody heavy chain variable domain <220> <221> VARIANT <222> (26)...(35) <223> CDR-H1 that can be replaced = GFTFSDYAMS <220> <221> VARIANT <222> (50)...(66) <223> CDR-H2 that can be replaced = VISENGSDTYYADSVKG <220> <221> VARIANT <222> (99)...(110) <223> CDR-H3 that can be replaced = DRGGAVSYFDVW <400> 1245 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Glu Asn Gly Ser Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Gly Gly Ala Val Ser Tyr Phe Asp Val Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 1246 <211> 109 <212> PRT <213> artificial sequence <220> <223> ideal consensus sequence of human antibody light chain variable domain <220> <221> VARIANT <222> (24)...(35) <223> CDR-L1 that can be replaced = RASQDVSSYLAW <220> <221> VARIANT <222> (50)...(56) <223> CDR-L2 that can be replaced = AASSLES <220> <221> VARIANT <222> (89)...(97) <223> CDR-L3 that can be replaced = QQYNSLPYT <400> 1246 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 <210> 1247 <211> 7 <212> PRT <213> artificial sequence <220> <223> Epitope within human TNFR2 that antagonistic antibodies or fragments that bind to <400> 1247 Asp Ser Thr Tyr Thr Gln Leu 1 5 <210> 1248 <211> 8 <212> PRT <213> artificial sequence <220> <223> Epitope within human TNFR2 that antagonistic antibodies or fragments that bind to <400> 1248 Pro Glu Cys Leu Ser Cys Gly Ser 1 5 <210> 1249 <211> 8 <212> PRT <213> artificial sequence <220> <223> Epitope within human TNFR2 that antagonistic antibodies or fragments that bind to <400> 1249 Arg Ile Cys Thr Cys Arg Pro Gly 1 5 <210> 1250 <211> 8 <212> PRT <213> artificial sequence <220> <223> Epitope within human TNFR2 that antagonistic antibodies or fragments that bind to <400> 1250 Cys Ala Pro Leu Arg Lys Cys Arg 1 5 <210> 1251 <211> 54 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic TNFR2 antibodies or fragments thereof can bind (at at least 5 continuous or discontinuous residues) <400> 1251 Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val 1 5 10 15 Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser Asp Gln Val Glu 20 25 30 Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro 35 40 45 Gly Trp Tyr Cys Ala Leu 50 <210> 1252 <211> 16 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic TNFR2 antibodies or fragments thereof can bind (at at least 5 continuous or discontinuous residues) <400> 1252 Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val 1 5 10 15 <210> 1253 <211> 16 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic TNFR2 antibodies or fragments thereof can bind (at at least 5 continuous or discontinuous residues) <400> 1253 Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser 1 5 10 15 <210> 1254 <211> 16 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic TNFR2 antibodies or fragments thereof can bind (at at least 5 continuous or discontinuous residues) <400> 1254 Thr Arg Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys 1 5 10 15 <210> 1255 <211> 16 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic TNFR2 antibodies or fragments thereof can bind (at at least 5 continuous or discontinuous residues) <400> 1255 Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro 1 5 10 15 <210> 1256 <211> 5 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic TNFR2 antibodies or fragments that bind <400> 1256 Lys Cys Arg Pro Gly 1 5 <210> 1257 <211> 8 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <400> 1257 Lys Cys Arg Pro Gly Phe Gly Val 1 5 <210> 1258 <211> 9 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies (such as TNFRAB1) or fragments thereof bind <400> 1258 Cys Lys Pro Cys Ala Pro Gly Thr Phe 1 5 <210> 1259 <211> 41 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic TNFR2 antibodies or fragments thereof can bind (at at least 5 continuous or discontinuous residues) <400> 1259 Ala Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala 1 5 10 15 Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro 20 25 30 His Gln Ile Cys Asn Val Val Ala Ile 35 40 <210> 1260 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (22)...(22) <223> Xaa is Cys residue protected with ACM protecting group <400> 1260 Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Gly Gly Ser Gly Gly 1 5 10 15 Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr 20 25 <210> 1261 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (22)...(22) <223> Xaa is Cys residue protected with ACM protecting group <400> 1261 Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gly Gly Ser Gly Gly 1 5 10 15 Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr 20 25 <210> 1262 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (22)...(22) <223> Xaa is Cys residue protected with ACM protecting group <400> 1262 Val Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Gly Gly Ser Gly Gly 1 5 10 15 Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr 20 25 <210> 1263 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (22)...(22) <223> Xaa is Cys residue protected with ACM protecting group <400> 1263 Leu Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gln Gly Gly Ser Gly Gly 1 5 10 15 Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr 20 25 <210> 1264 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1264 Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Gly Gly Ser Gly Gly 1 5 10 15 Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa Ala Leu 20 25 <210> 1265 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (22)...(22) <223> Xaa is Cys residue protected with ACM protecting group <400> 1265 Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gln Val Gly Gly Ser Gly Gly 1 5 10 15 Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr 20 25 <210> 1266 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (21)...(21) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 1266 Xaa Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Gly Gly Ser Gly Gly 1 5 10 15 Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa 20 25 <210> 1267 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1267 Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Ser Gly Gly Ser Gly Gly 1 5 10 15 Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa Ala Leu 20 25 <210> 1268 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (17)...(17) <223> Xaa is Cys residue protected with ACM protecting group <400> 1268 Glu Gly Cys Arg Leu Xaa Ala Pro Leu Arg Lys Gly Gly Ser Gly Gly 1 5 10 15 Xaa Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe 20 25 <210> 1269 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1269 Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gly Gly Ser Gly Gly 1 5 10 15 Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa Ala Leu 20 25 <210> 1270 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (21)...(21) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 1270 Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Xaa Gly Gly Ser Gly Gly 1 5 10 15 Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa 20 25 <210> 1271 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (21)...(21) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 1271 Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gly Gly Ser Gly Gly 1 5 10 15 Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa 20 25 <210> 1272 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (22)...(22) <223> Xaa is Cys residue protected with ACM protecting group <400> 1272 Xaa Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Gly Gly Ser Gly Gly 1 5 10 15 Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr 20 25 <210> 1273 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (22)...(22) <223> Xaa is Cys residue protected with ACM protecting group <400> 1273 Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gly Gly Ser Gly Gly 1 5 10 15 Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr 20 25 <210> 1274 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (21)...(21) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 1274 Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Gly Gly Ser Gly Gly 1 5 10 15 Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa 20 25 <210> 1275 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1275 Val Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Gly Gly Ser Gly Gly 1 5 10 15 Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp 20 25 <210> 1276 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (17)...(17) <223> Xaa is Cys residue protected with ACM protecting group <400> 1276 Gln Glu Gly Cys Arg Leu Xaa Ala Pro Leu Arg Gly Gly Ser Gly Gly 1 5 10 15 Xaa Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe 20 25 <210> 1277 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (21)...(21) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 1277 Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Gly Gly Ser Gly Gly 1 5 10 15 Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa 20 25 <210> 1278 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (17)...(17) <223> Xaa is Cys residue protected with ACM protecting group <400> 1278 Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu Xaa Gly Gly Ser Gly Gly 1 5 10 15 Xaa Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe 20 25 <210> 1279 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <400> 1279 Gln Glu Gly Cys Arg Leu Xaa Ala Pro Leu Arg Gly Gly Ser Gly Gly 1 5 10 15 Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val 20 25 <210> 1280 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <400> 1280 Tyr Xaa Ala Leu Ser Lys Gln Glu Gly Cys Arg Gly Gly Ser Gly Gly 1 5 10 15 Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly 20 25 <210> 1281 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (21)...(21) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 1281 Leu Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gln Gly Gly Ser Gly Gly 1 5 10 15 Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa 20 25 <210> 1282 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1282 Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Gly Gly Ser Gly Gly 1 5 10 15 Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp 20 25 <210> 1283 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (21)...(21) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 1283 Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Gly Gly Ser Gly Gly 1 5 10 15 Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa 20 25 <210> 1284 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 1284 Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Gly Gly Ser Gly Gly 1 5 10 15 Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa Ala 20 25 <210> 1285 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 1285 Val Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Gly Gly Ser Gly Gly 1 5 10 15 Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa Ala 20 25 <210> 1286 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1286 Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gln Val Gly Gly Ser Gly Gly 1 5 10 15 Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa Ala Leu 20 25 <210> 1287 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <400> 1287 Trp Tyr Xaa Ala Leu Ser Lys Gln Glu Gly Cys Gly Gly Ser Gly Gly 1 5 10 15 Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly 20 25 <210> 1288 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (10)...(10) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (17)...(17) <223> Xaa is Cys residue protected with ACM protecting group <400> 1288 Leu Ser Lys Gln Glu Gly Cys Arg Leu Xaa Ala Gly Gly Ser Gly Gly 1 5 10 15 Xaa Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe 20 25 <210> 1289 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (17)...(17) <223> Xaa is Cys residue protected with ACM protecting group <400> 1289 Lys Gln Glu Gly Cys Arg Leu Xaa Ala Pro Leu Gly Gly Ser Gly Gly 1 5 10 15 Xaa Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe 20 25 <210> 1290 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1290 Leu Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gln Gly Gly Ser Gly Gly 1 5 10 15 Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa Ala Leu 20 25 <210> 1291 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1291 Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Gly Gly Ser Gly Gly 1 5 10 15 Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp 20 25 <210> 1292 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (9)...(9) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (17)...(17) <223> Xaa is Cys residue protected with ACM protecting group <400> 1292 Ser Lys Gln Glu Gly Cys Arg Leu Xaa Ala Pro Gly Gly Ser Gly Gly 1 5 10 15 Xaa Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe 20 25 <210> 1293 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1293 Xaa Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Gly Gly Ser Gly Gly 1 5 10 15 Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp 20 25 <210> 1294 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1294 Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Xaa Gly Gly Ser Gly Gly 1 5 10 15 Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp 20 25 <210> 1295 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1295 Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Xaa Gly Gly Ser Gly Gly 1 5 10 15 Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp 20 25 <210> 1296 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1296 Val Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Gly Gly Ser Gly Gly 1 5 10 15 Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa Ala Leu 20 25 <210> 1297 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1297 Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Ser Gly Gly Ser Gly Gly 1 5 10 15 Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp 20 25 <210> 1298 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (22)...(22) <223> Xaa is Cys residue protected with ACM protecting group <400> 1298 Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Gly Gly Ser Gly Gly 1 5 10 15 Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr 20 25 <210> 1299 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1299 Xaa Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Gly Gly Ser Gly Gly 1 5 10 15 Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa Ala Leu 20 25 <210> 1300 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (21)...(21) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 1300 Val Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Gly Gly Ser Gly Gly 1 5 10 15 Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa 20 25 <210> 1301 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (21)...(21) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 1301 Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Ser Gly Gly Ser Gly Gly 1 5 10 15 Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa 20 25 <210> 1302 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (10)...(10) <223> Xaa is Cys residue protected with ACM protecting group <400> 1302 Leu Ser Lys Gln Glu Gly Cys Arg Leu Xaa Ala Gly Gly Ser Gly Gly 1 5 10 15 Arg Lys Cys Arg Pro Gly Phe Gly Val Ala Arg 20 25 <210> 1303 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1303 Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gly Gly Ser Gly Gly 1 5 10 15 Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp 20 25 <210> 1304 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (21)...(21) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 1304 Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gln Val Gly Gly Ser Gly Gly 1 5 10 15 Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa 20 25 <210> 1305 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1305 Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gly Gly Ser Gly Gly 1 5 10 15 Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa Ala Leu 20 25 <210> 1306 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <400> 1306 Tyr Xaa Ala Leu Ser Lys Gln Glu Gly Cys Arg Gly Gly Ser Gly Gly 1 5 10 15 Arg Lys Cys Arg Pro Gly Phe Gly Val Ala Arg 20 25 <210> 1307 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Cys residue protected with ACM protecting group <400> 1307 Lys Gln Glu Gly Cys Arg Leu Xaa Ala Pro Leu Gly Gly Ser Gly Gly 1 5 10 15 Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val 20 25 <210> 1308 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <400> 1308 Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu Xaa Gly Gly Ser Gly Gly 1 5 10 15 Arg Lys Cys Arg Pro Gly Phe Gly Val Ala Arg 20 25 <210> 1309 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 1309 Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Ser Gly Gly Ser Gly Gly 1 5 10 15 Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa Ala 20 25 <210> 1310 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 1310 Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Xaa Gly Gly Ser Gly Gly 1 5 10 15 Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa Ala 20 25 <210> 1311 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <400> 1311 Glu Gly Cys Arg Leu Xaa Ala Pro Leu Arg Lys Gly Gly Ser Gly Gly 1 5 10 15 Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val 20 25 <210> 1312 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <400> 1312 Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu Xaa Gly Gly Ser Gly Gly 1 5 10 15 Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val 20 25 <210> 1313 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <400> 1313 Xaa Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu Gly Gly Ser Gly Gly 1 5 10 15 Arg Lys Cys Arg Pro Gly Phe Gly Val Ala Arg 20 25 <210> 1314 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (17)...(17) <223> Xaa is Cys residue protected with ACM protecting group <400> 1314 Xaa Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu Gly Gly Ser Gly Gly 1 5 10 15 Xaa Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe 20 25 <210> 1315 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (21)...(21) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 1315 Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gly Gly Ser Gly Gly 1 5 10 15 Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa 20 25 <210> 1316 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (10)...(10) <223> Xaa is Cys residue protected with ACM protecting group <400> 1316 Leu Ser Lys Gln Glu Gly Cys Arg Leu Xaa Ala Gly Gly Ser Gly Gly 1 5 10 15 Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val 20 25 <210> 1317 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (9)...(9) <223> Xaa is Cys residue protected with ACM protecting group <400> 1317 Ser Lys Gln Glu Gly Cys Arg Leu Xaa Ala Pro Gly Gly Ser Gly Gly 1 5 10 15 Arg Lys Cys Arg Pro Gly Phe Gly Val Ala Arg 20 25 <210> 1318 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Cys residue protected with ACM protecting group <400> 1318 Lys Gln Glu Gly Cys Arg Leu Xaa Ala Pro Leu Gly Gly Ser Gly Gly 1 5 10 15 Arg Lys Cys Arg Pro Gly Phe Gly Val Ala Arg 20 25 <210> 1319 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <400> 1319 Gln Glu Gly Cys Arg Leu Xaa Ala Pro Leu Arg Gly Gly Ser Gly Gly 1 5 10 15 Arg Lys Cys Arg Pro Gly Phe Gly Val Ala Arg 20 25 <210> 1320 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <400> 1320 Xaa Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu Gly Gly Ser Gly Gly 1 5 10 15 Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly 20 25 <210> 1321 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <400> 1321 Xaa Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu Gly Gly Ser Gly Gly 1 5 10 15 Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val 20 25 <210> 1322 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 1322 Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gly Gly Ser Gly Gly 1 5 10 15 Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa Ala 20 25 <210> 1323 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1323 Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Gly Gly Ser Gly Gly 1 5 10 15 Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp 20 25 <210> 1324 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 1324 Xaa Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Gly Gly Ser Gly Gly 1 5 10 15 Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa Ala 20 25 <210> 1325 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (22)...(22) <223> Xaa is Cys residue protected with ACM protecting group <400> 1325 Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Ser Gly Gly Ser Gly Gly 1 5 10 15 Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr 20 25 <210> 1326 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 1326 Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Gly Gly Ser Gly Gly 1 5 10 15 Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa Ala 20 25 <210> 1327 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <400> 1327 Tyr Xaa Ala Leu Ser Lys Gln Glu Gly Cys Arg Gly Gly Ser Gly Gly 1 5 10 15 Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val 20 25 <210> 1328 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 1328 Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Gly Gly Ser Gly Gly 1 5 10 15 Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa Ala 20 25 <210> 1329 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (9)...(9) <223> Xaa is Cys residue protected with ACM protecting group <400> 1329 Ser Lys Gln Glu Gly Cys Arg Leu Xaa Ala Pro Gly Gly Ser Gly Gly 1 5 10 15 Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val 20 25 <210> 1330 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1330 Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gln Val Gly Gly Ser Gly Gly 1 5 10 15 Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp 20 25 <210> 1331 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <400> 1331 Glu Gly Cys Arg Leu Xaa Ala Pro Leu Arg Lys Gly Gly Ser Gly Gly 1 5 10 15 Arg Lys Cys Arg Pro Gly Phe Gly Val Ala Arg 20 25 <210> 1332 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1332 Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gly Gly Ser Gly Gly 1 5 10 15 Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp 20 25 <210> 1333 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <400> 1333 Trp Tyr Xaa Ala Leu Ser Lys Gln Glu Gly Cys Gly Gly Ser Gly Gly 1 5 10 15 Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val 20 25 <210> 1334 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <400> 1334 Gln Glu Gly Cys Arg Leu Xaa Ala Pro Leu Arg Gly Gly Ser Gly Gly 1 5 10 15 Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly 20 25 <210> 1335 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (17)...(17) <223> Xaa is Cys residue protected with ACM protecting group <400> 1335 Trp Tyr Xaa Ala Leu Ser Lys Gln Glu Gly Cys Gly Gly Ser Gly Gly 1 5 10 15 Xaa Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe 20 25 <210> 1336 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <400> 1336 Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu Xaa Gly Gly Ser Gly Gly 1 5 10 15 Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly 20 25 <210> 1337 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <400> 1337 Glu Gly Cys Arg Leu Xaa Ala Pro Leu Arg Lys Gly Gly Ser Gly Gly 1 5 10 15 Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly 20 25 <210> 1338 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (17)...(17) <223> Xaa is Cys residue protected with ACM protecting group <400> 1338 Tyr Xaa Ala Leu Ser Lys Gln Glu Gly Cys Arg Gly Gly Ser Gly Gly 1 5 10 15 Xaa Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe 20 25 <210> 1339 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1339 Leu Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gln Gly Gly Ser Gly Gly 1 5 10 15 Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp 20 25 <210> 1340 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (10)...(10) <223> Xaa is Cys residue protected with ACM protecting group <400> 1340 Leu Ser Lys Gln Glu Gly Cys Arg Leu Xaa Ala Gly Gly Ser Gly Gly 1 5 10 15 Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly 20 25 <210> 1341 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (22)...(22) <223> Xaa is Cys residue protected with ACM protecting group <400> 1341 Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Gly Gly Ser Gly Gly 1 5 10 15 Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr 20 25 <210> 1342 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Cys residue protected with ACM protecting group <400> 1342 Lys Gln Glu Gly Cys Arg Leu Xaa Ala Pro Leu Gly Gly Ser Gly Gly 1 5 10 15 Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly 20 25 <210> 1343 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (9)...(9) <223> Xaa is Cys residue protected with ACM protecting group <400> 1343 Ser Lys Gln Glu Gly Cys Arg Leu Xaa Ala Pro Gly Gly Ser Gly Gly 1 5 10 15 Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly 20 25 <210> 1344 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 1344 Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Gly Gly Ser Gly Gly 1 5 10 15 Thr Arg Glu Gln Asn Arg Ile Cys Thr Xaa Arg 20 25 <210> 1345 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 1345 Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Gly Gly Ser Gly Gly 1 5 10 15 Thr Arg Glu Gln Asn Arg Ile Cys Thr Xaa Arg 20 25 <210> 1346 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 1346 Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Xaa Gly Gly Ser Gly Gly 1 5 10 15 Thr Arg Glu Gln Asn Arg Ile Cys Thr Xaa Arg 20 25 <210> 1347 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1347 Val Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Gly Gly Ser Gly Gly 1 5 10 15 Arg Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro 20 25 <210> 1348 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1348 Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Gly Gly Ser Gly Gly 1 5 10 15 Arg Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro 20 25 <210> 1349 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1349 Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Ser Gly Gly Ser Gly Gly 1 5 10 15 Arg Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro 20 25 <210> 1350 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1350 Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gly Gly Ser Gly Gly 1 5 10 15 Arg Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro 20 25 <210> 1351 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1351 Leu Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gln Gly Gly Ser Gly Gly 1 5 10 15 Arg Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro 20 25 <210> 1352 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1352 Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gln Val Gly Gly Ser Gly Gly 1 5 10 15 Arg Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro 20 25 <210> 1353 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1353 Xaa Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Gly Gly Ser Gly Gly 1 5 10 15 Arg Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro 20 25 <210> 1354 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1354 Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gly Gly Ser Gly Gly 1 5 10 15 Arg Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro 20 25 <210> 1355 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1355 Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Gly Gly Ser Gly Gly 1 5 10 15 Arg Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro 20 25 <210> 1356 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1356 Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Gly Gly Ser Gly Gly 1 5 10 15 Arg Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro 20 25 <210> 1357 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1357 Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Xaa Gly Gly Ser Gly Gly 1 5 10 15 Arg Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro 20 25 <210> 1358 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (24)...(24) <223> Xaa is Cys residue protected with ACM protecting group <400> 1358 Val Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Gly Gly Ser Gly Gly 1 5 10 15 Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly 20 25 <210> 1359 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (24)...(24) <223> Xaa is Cys residue protected with ACM protecting group <400> 1359 Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Gly Gly Ser Gly Gly 1 5 10 15 Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly 20 25 <210> 1360 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (24)...(24) <223> Xaa is Cys residue protected with ACM protecting group <400> 1360 Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Ser Gly Gly Ser Gly Gly 1 5 10 15 Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly 20 25 <210> 1361 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (24)...(24) <223> Xaa is Cys residue protected with ACM protecting group <400> 1361 Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gly Gly Ser Gly Gly 1 5 10 15 Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly 20 25 <210> 1362 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (24)...(24) <223> Xaa is Cys residue protected with ACM protecting group <400> 1362 Leu Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gln Gly Gly Ser Gly Gly 1 5 10 15 Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly 20 25 <210> 1363 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (24)...(24) <223> Xaa is Cys residue protected with ACM protecting group <400> 1363 Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gln Val Gly Gly Ser Gly Gly 1 5 10 15 Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly 20 25 <210> 1364 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (24)...(24) <223> Xaa is Cys residue protected with ACM protecting group <400> 1364 Xaa Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Gly Gly Ser Gly Gly 1 5 10 15 Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly 20 25 <210> 1365 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (24)...(24) <223> Xaa is Cys residue protected with ACM protecting group <400> 1365 Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gly Gly Ser Gly Gly 1 5 10 15 Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly 20 25 <210> 1366 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (24)...(24) <223> Xaa is Cys residue protected with ACM protecting group <400> 1366 Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Gly Gly Ser Gly Gly 1 5 10 15 Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly 20 25 <210> 1367 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (24)...(24) <223> Xaa is Cys residue protected with ACM protecting group <400> 1367 Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Gly Gly Ser Gly Gly 1 5 10 15 Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly 20 25 <210> 1368 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (24)...(24) <223> Xaa is Cys residue protected with ACM protecting group <400> 1368 Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Xaa Gly Gly Ser Gly Gly 1 5 10 15 Glu Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly 20 25 <210> 1369 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1369 Val Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Gly Gly Ser Gly Gly 1 5 10 15 Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp 20 25 <210> 1370 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1370 Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Gly Gly Ser Gly Gly 1 5 10 15 Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp 20 25 <210> 1371 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1371 Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Ser Gly Gly Ser Gly Gly 1 5 10 15 Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp 20 25 <210> 1372 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1372 Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gly Gly Ser Gly Gly 1 5 10 15 Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp 20 25 <210> 1373 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1373 Leu Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gln Gly Gly Ser Gly Gly 1 5 10 15 Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp 20 25 <210> 1374 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1374 Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gln Val Gly Gly Ser Gly Gly 1 5 10 15 Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp 20 25 <210> 1375 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1375 Xaa Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Gly Gly Ser Gly Gly 1 5 10 15 Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp 20 25 <210> 1376 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1376 Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gly Gly Ser Gly Gly 1 5 10 15 Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp 20 25 <210> 1377 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1377 Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Gly Gly Ser Gly Gly 1 5 10 15 Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp 20 25 <210> 1378 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1378 Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Gly Gly Ser Gly Gly 1 5 10 15 Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp 20 25 <210> 1379 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (23)...(23) <223> Xaa is Cys residue protected with ACM protecting group <400> 1379 Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Xaa Gly Gly Ser Gly Gly 1 5 10 15 Gln Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp 20 25 <210> 1380 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (22)...(22) <223> Xaa is Cys residue protected with ACM protecting group <400> 1380 Val Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Gly Gly Ser Gly Gly 1 5 10 15 Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr 20 25 <210> 1381 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (22)...(22) <223> Xaa is Cys residue protected with ACM protecting group <400> 1381 Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Gly Gly Ser Gly Gly 1 5 10 15 Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr 20 25 <210> 1382 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (22)...(22) <223> Xaa is Cys residue protected with ACM protecting group <400> 1382 Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Ser Gly Gly Ser Gly Gly 1 5 10 15 Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr 20 25 <210> 1383 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (22)...(22) <223> Xaa is Cys residue protected with ACM protecting group <400> 1383 Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gly Gly Ser Gly Gly 1 5 10 15 Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr 20 25 <210> 1384 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (22)...(22) <223> Xaa is Cys residue protected with ACM protecting group <400> 1384 Leu Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gln Gly Gly Ser Gly Gly 1 5 10 15 Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr 20 25 <210> 1385 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (22)...(22) <223> Xaa is Cys residue protected with ACM protecting group <400> 1385 Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gln Val Gly Gly Ser Gly Gly 1 5 10 15 Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr 20 25 <210> 1386 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (22)...(22) <223> Xaa is Cys residue protected with ACM protecting group <400> 1386 Xaa Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Gly Gly Ser Gly Gly 1 5 10 15 Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr 20 25 <210> 1387 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (22)...(22) <223> Xaa is Cys residue protected with ACM protecting group <400> 1387 Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gly Gly Ser Gly Gly 1 5 10 15 Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr 20 25 <210> 1388 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (22)...(22) <223> Xaa is Cys residue protected with ACM protecting group <400> 1388 Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Gly Gly Ser Gly Gly 1 5 10 15 Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr 20 25 <210> 1389 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (22)...(22) <223> Xaa is Cys residue protected with ACM protecting group <400> 1389 Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Gly Gly Ser Gly Gly 1 5 10 15 Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr 20 25 <210> 1390 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (22)...(22) <223> Xaa is Cys residue protected with ACM protecting group <400> 1390 Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Xaa Gly Gly Ser Gly Gly 1 5 10 15 Asn Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr 20 25 <210> 1391 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (21)...(21) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 1391 Val Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Gly Gly Ser Gly Gly 1 5 10 15 Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa 20 25 <210> 1392 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (21)...(21) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 1392 Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Gly Gly Ser Gly Gly 1 5 10 15 Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa 20 25 <210> 1393 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (21)...(21) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 1393 Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Ser Gly Gly Ser Gly Gly 1 5 10 15 Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa 20 25 <210> 1394 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (21)...(21) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 1394 Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gly Gly Ser Gly Gly 1 5 10 15 Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa 20 25 <210> 1395 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (21)...(21) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 1395 Leu Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gln Gly Gly Ser Gly Gly 1 5 10 15 Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa 20 25 <210> 1396 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (21)...(21) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 1396 Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gln Val Gly Gly Ser Gly Gly 1 5 10 15 Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa 20 25 <210> 1397 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (21)...(21) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 1397 Xaa Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Gly Gly Ser Gly Gly 1 5 10 15 Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa 20 25 <210> 1398 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (21)...(21) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 1398 Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gly Gly Ser Gly Gly 1 5 10 15 Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa 20 25 <210> 1399 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (21)...(21) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 1399 Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Gly Gly Ser Gly Gly 1 5 10 15 Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa 20 25 <210> 1400 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (21)...(21) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 1400 Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Gly Gly Ser Gly Gly 1 5 10 15 Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa 20 25 <210> 1401 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (21)...(21) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (27)...(27) <223> Xaa is Cys residue protected with ACM protecting group <400> 1401 Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Xaa Gly Gly Ser Gly Gly 1 5 10 15 Arg Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa 20 25 <210> 1402 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 1402 Val Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Gly Gly Ser Gly Gly 1 5 10 15 Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa Ala 20 25 <210> 1403 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 1403 Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Gly Gly Ser Gly Gly 1 5 10 15 Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa Ala 20 25 <210> 1404 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 1404 Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Ser Gly Gly Ser Gly Gly 1 5 10 15 Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa Ala 20 25 <210> 1405 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 1405 Xaa Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Gly Gly Ser Gly Gly 1 5 10 15 Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa Ala 20 25 <210> 1406 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 1406 Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gly Gly Ser Gly Gly 1 5 10 15 Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa Ala 20 25 <210> 1407 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 1407 Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Gly Gly Ser Gly Gly 1 5 10 15 Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa Ala 20 25 <210> 1408 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 1408 Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Gly Gly Ser Gly Gly 1 5 10 15 Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa Ala 20 25 <210> 1409 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (20)...(20) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (26)...(26) <223> Xaa is Cys residue protected with ACM protecting group <400> 1409 Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Xaa Gly Gly Ser Gly Gly 1 5 10 15 Ile Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa Ala 20 25 <210> 1410 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1410 Val Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Gly Gly Ser Gly Gly 1 5 10 15 Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa Ala Leu 20 25 <210> 1411 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1411 Pro Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Gly Gly Ser Gly Gly 1 5 10 15 Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa Ala Leu 20 25 <210> 1412 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1412 Glu Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Ser Gly Gly Ser Gly Gly 1 5 10 15 Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa Ala Leu 20 25 <210> 1413 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (4)...(4) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1413 Xaa Leu Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gly Gly Ser Gly Gly 1 5 10 15 Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa Ala Leu 20 25 <210> 1414 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1414 Leu Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gln Gly Gly Ser Gly Gly 1 5 10 15 Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa Ala Leu 20 25 <210> 1415 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1415 Ser Xaa Gly Ser Arg Cys Ser Ser Asp Gln Val Gly Gly Ser Gly Gly 1 5 10 15 Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa Ala Leu 20 25 <210> 1416 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1416 Xaa Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Gly Gly Ser Gly Gly 1 5 10 15 Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa Ala Leu 20 25 <210> 1417 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1417 Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gly Gly Ser Gly Gly 1 5 10 15 Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa Ala Leu 20 25 <210> 1418 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1418 Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Gly Gly Ser Gly Gly 1 5 10 15 Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa Ala Leu 20 25 <210> 1419 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1419 Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Gly Gly Ser Gly Gly 1 5 10 15 Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa Ala Leu 20 25 <210> 1420 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (19)...(19) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (25)...(25) <223> Xaa is Cys residue protected with ACM protecting group <400> 1420 Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Xaa Gly Gly Ser Gly Gly 1 5 10 15 Cys Thr Xaa Arg Pro Gly Trp Tyr Xaa Ala Leu 20 25 <210> 1421 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (9)...(9) <223> Xaa is Cys residue protected with ACM protecting group <400> 1421 Xaa Thr Arg Glu Gln Asn Arg Ile Xaa Thr Cys Gly Gly Ser Gly Gly 1 5 10 15 Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln 20 25 <210> 1422 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Cys residue protected with ACM protecting group <400> 1422 Thr Arg Glu Gln Asn Arg Ile Xaa Thr Cys Arg Gly Gly Ser Gly Gly 1 5 10 15 Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln 20 25 <210> 1423 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <400> 1423 Arg Glu Gln Asn Arg Ile Xaa Thr Cys Arg Pro Gly Gly Ser Gly Gly 1 5 10 15 Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln 20 25 <210> 1424 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <400> 1424 Glu Gln Asn Arg Ile Xaa Thr Cys Arg Pro Gly Gly Gly Ser Gly Gly 1 5 10 15 Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln 20 25 <210> 1425 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (5)...(5) <223> Xaa is Cys residue protected with ACM protecting group <400> 1425 Gln Asn Arg Ile Xaa Thr Cys Arg Pro Gly Trp Gly Gly Ser Gly Gly 1 5 10 15 Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln 20 25 <210> 1426 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (4)...(4) <223> Xaa can be any naturally occurring amino acid <400> 1426 Asn Arg Ile Xaa Thr Cys Arg Pro Gly Trp Tyr Gly Gly Ser Gly Gly 1 5 10 15 Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln 20 25 <210> 1427 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (17)...(17) <223> Xaa is Cys residue protected with ACM protecting group <400> 1427 Trp Tyr Xaa Ala Leu Ser Lys Gln Glu Gly Cys Gly Gly Ser Gly Gly 1 5 10 15 Xaa Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe 20 25 <210> 1428 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (17)...(17) <223> Xaa is Cys residue protected with ACM protecting group <400> 1428 Tyr Xaa Ala Leu Ser Lys Gln Glu Gly Cys Arg Gly Gly Ser Gly Gly 1 5 10 15 Xaa Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe 20 25 <210> 1429 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (17)...(17) <223> Xaa is Cys residue protected with ACM protecting group <400> 1429 Xaa Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu Gly Gly Ser Gly Gly 1 5 10 15 Xaa Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe 20 25 <210> 1430 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (17)...(17) <223> Xaa is Cys residue protected with ACM protecting group <400> 1430 Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu Xaa Gly Gly Ser Gly Gly 1 5 10 15 Xaa Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe 20 25 <210> 1431 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (10)...(10) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (17)...(17) <223> Xaa is Cys residue protected with ACM protecting group <400> 1431 Leu Ser Lys Gln Glu Gly Cys Arg Leu Xaa Ala Gly Gly Ser Gly Gly 1 5 10 15 Xaa Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe 20 25 <210> 1432 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (9)...(9) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (17)...(17) <223> Xaa is Cys residue protected with ACM protecting group <400> 1432 Ser Lys Gln Glu Gly Cys Arg Leu Xaa Ala Pro Gly Gly Ser Gly Gly 1 5 10 15 Xaa Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe 20 25 <210> 1433 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (17)...(17) <223> Xaa is Cys residue protected with ACM protecting group <400> 1433 Lys Gln Glu Gly Cys Arg Leu Xaa Ala Pro Leu Gly Gly Ser Gly Gly 1 5 10 15 Xaa Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe 20 25 <210> 1434 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (17)...(17) <223> Xaa is Cys residue protected with ACM protecting group <400> 1434 Gln Glu Gly Cys Arg Leu Xaa Ala Pro Leu Arg Gly Gly Ser Gly Gly 1 5 10 15 Xaa Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe 20 25 <210> 1435 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <220> <221> VARIANT <222> (17)...(17) <223> Xaa is Cys residue protected with ACM protecting group <400> 1435 Glu Gly Cys Arg Leu Xaa Ala Pro Leu Arg Lys Gly Gly Ser Gly Gly 1 5 10 15 Xaa Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe 20 25 <210> 1436 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <400> 1436 Trp Tyr Xaa Ala Leu Ser Lys Gln Glu Gly Cys Gly Gly Ser Gly Gly 1 5 10 15 Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly 20 25 <210> 1437 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <400> 1437 Tyr Xaa Ala Leu Ser Lys Gln Glu Gly Cys Arg Gly Gly Ser Gly Gly 1 5 10 15 Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly 20 25 <210> 1438 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <400> 1438 Xaa Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu Gly Gly Ser Gly Gly 1 5 10 15 Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly 20 25 <210> 1439 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <400> 1439 Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu Xaa Gly Gly Ser Gly Gly 1 5 10 15 Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly 20 25 <210> 1440 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (10)...(10) <223> Xaa is Cys residue protected with ACM protecting group <400> 1440 Leu Ser Lys Gln Glu Gly Cys Arg Leu Xaa Ala Gly Gly Ser Gly Gly 1 5 10 15 Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly 20 25 <210> 1441 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (9)...(9) <223> Xaa is Cys residue protected with ACM protecting group <400> 1441 Ser Lys Gln Glu Gly Cys Arg Leu Xaa Ala Pro Gly Gly Ser Gly Gly 1 5 10 15 Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly 20 25 <210> 1442 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Cys residue protected with ACM protecting group <400> 1442 Lys Gln Glu Gly Cys Arg Leu Xaa Ala Pro Leu Gly Gly Ser Gly Gly 1 5 10 15 Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly 20 25 <210> 1443 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <400> 1443 Gln Glu Gly Cys Arg Leu Xaa Ala Pro Leu Arg Gly Gly Ser Gly Gly 1 5 10 15 Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly 20 25 <210> 1444 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <400> 1444 Glu Gly Cys Arg Leu Xaa Ala Pro Leu Arg Lys Gly Gly Ser Gly Gly 1 5 10 15 Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly 20 25 <210> 1445 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <400> 1445 Trp Tyr Xaa Ala Leu Ser Lys Gln Glu Gly Cys Gly Gly Ser Gly Gly 1 5 10 15 Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val 20 25 <210> 1446 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <400> 1446 Tyr Xaa Ala Leu Ser Lys Gln Glu Gly Cys Arg Gly Gly Ser Gly Gly 1 5 10 15 Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val 20 25 <210> 1447 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <400> 1447 Xaa Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu Gly Gly Ser Gly Gly 1 5 10 15 Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val 20 25 <210> 1448 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <400> 1448 Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu Xaa Gly Gly Ser Gly Gly 1 5 10 15 Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val 20 25 <210> 1449 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (10)...(10) <223> Xaa is Cys residue protected with ACM protecting group <400> 1449 Leu Ser Lys Gln Glu Gly Cys Arg Leu Xaa Ala Gly Gly Ser Gly Gly 1 5 10 15 Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val 20 25 <210> 1450 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (9)...(9) <223> Xaa is Cys residue protected with ACM protecting group <400> 1450 Ser Lys Gln Glu Gly Cys Arg Leu Xaa Ala Pro Gly Gly Ser Gly Gly 1 5 10 15 Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val 20 25 <210> 1451 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Cys residue protected with ACM protecting group <400> 1451 Lys Gln Glu Gly Cys Arg Leu Xaa Ala Pro Leu Gly Gly Ser Gly Gly 1 5 10 15 Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val 20 25 <210> 1452 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <400> 1452 Gln Glu Gly Cys Arg Leu Xaa Ala Pro Leu Arg Gly Gly Ser Gly Gly 1 5 10 15 Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val 20 25 <210> 1453 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <400> 1453 Glu Gly Cys Arg Leu Xaa Ala Pro Leu Arg Lys Gly Gly Ser Gly Gly 1 5 10 15 Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val 20 25 <210> 1454 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (3)...(3) <223> Xaa is Cys residue protected with ACM protecting group <400> 1454 Trp Tyr Xaa Ala Leu Ser Lys Gln Glu Gly Cys Gly Gly Ser Gly Gly 1 5 10 15 Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala 20 25 <210> 1455 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (2)...(2) <223> Xaa is Cys residue protected with ACM protecting group <400> 1455 Tyr Xaa Ala Leu Ser Lys Gln Glu Gly Cys Arg Gly Gly Ser Gly Gly 1 5 10 15 Arg Lys Cys Arg Pro Gly Phe Gly Val Ala Arg 20 25 <210> 1456 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (1)...(1) <223> Xaa is Cys residue protected with ACM protecting group <400> 1456 Xaa Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu Gly Gly Ser Gly Gly 1 5 10 15 Arg Lys Cys Arg Pro Gly Phe Gly Val Ala Arg 20 25 <210> 1457 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (11)...(11) <223> Xaa is Cys residue protected with ACM protecting group <400> 1457 Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu Xaa Gly Gly Ser Gly Gly 1 5 10 15 Arg Lys Cys Arg Pro Gly Phe Gly Val Ala Arg 20 25 <210> 1458 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (10)...(10) <223> Xaa is Cys residue protected with ACM protecting group <400> 1458 Leu Ser Lys Gln Glu Gly Cys Arg Leu Xaa Ala Gly Gly Ser Gly Gly 1 5 10 15 Arg Lys Cys Arg Pro Gly Phe Gly Val Ala Arg 20 25 <210> 1459 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (9)...(9) <223> Xaa is Cys residue protected with ACM protecting group <400> 1459 Ser Lys Gln Glu Gly Cys Arg Leu Xaa Ala Pro Gly Gly Ser Gly Gly 1 5 10 15 Arg Lys Cys Arg Pro Gly Phe Gly Val Ala Arg 20 25 <210> 1460 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (8)...(8) <223> Xaa is Cys residue protected with ACM protecting group <400> 1460 Lys Gln Glu Gly Cys Arg Leu Xaa Ala Pro Leu Gly Gly Ser Gly Gly 1 5 10 15 Arg Lys Cys Arg Pro Gly Phe Gly Val Ala Arg 20 25 <210> 1461 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (7)...(7) <223> Xaa is Cys residue protected with ACM protecting group <400> 1461 Gln Glu Gly Cys Arg Leu Xaa Ala Pro Leu Arg Gly Gly Ser Gly Gly 1 5 10 15 Arg Lys Cys Arg Pro Gly Phe Gly Val Ala Arg 20 25 <210> 1462 <211> 27 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <220> <221> VARIANT <222> (6)...(6) <223> Xaa is Cys residue protected with ACM protecting group <400> 1462 Glu Gly Cys Arg Leu Xaa Ala Pro Leu Arg Lys Gly Gly Ser Gly Gly 1 5 10 15 Arg Lys Cys Arg Pro Gly Phe Gly Val Ala Arg 20 25 <210> 1463 <211> 11 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <400> 1463 Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala 1 5 10 <210> 1464 <211> 13 <212> PRT <213> artificial sequence <220> <223> epitope within human TNFR2 to which antagonistic antibodies or fragments that bind <400> 1464 Val Val Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn 1 5 10 <210> 1465 <211> 645 <212> PRT <213> artificial sequence <220> <223> RB242 human EGFR ECD w/ modifications T15S and G564S (residue numbering does not include signal peptide) <220> <221> SIGNAL <222> (1)...(24) <223> signal peptide <220> <221> DOMAIN <222> (25)...(645) <223> EGFR/HER1 extracellular domain (ECD) <400> 1465 Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala 1 5 10 15 Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln 20 25 30 Gly Thr Ser Asn Lys Leu Ser Gln Leu Gly Thr Phe Glu Asp His Phe 35 40 45 Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn 50 55 60 Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys 65 70 75 80 Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val 85 90 95 Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr 100 105 110 Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn 115 120 125 Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu 130 135 140 His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu 145 150 155 160 Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met 165 170 175 Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro 180 185 190 Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln 195 200 205 Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg 210 215 220 Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys 225 230 235 240 Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp 245 250 255 Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro 260 265 270 Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly 275 280 285 Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His 290 295 300 Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu 305 310 315 320 Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val 325 330 335 Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn 340 345 350 Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp 355 360 365 Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr 370 375 380 Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu 385 390 395 400 Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp 405 410 415 Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln 420 425 430 His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu 435 440 445 Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser 450 455 460 Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu 465 470 475 480 Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu 485 490 495 Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro 500 505 510 Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn 515 520 525 Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Lys Leu Leu Glu Gly 530 535 540 Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro 545 550 555 560 Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro 565 570 575 Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Ser Pro His Cys Val 580 585 590 Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp 595 600 605 Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys 610 615 620 Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly 625 630 635 640 Pro Lys Ile Pro Ser 645 <210> 1466 <211> 621 <212> PRT <213> artificial sequence <220> <223> RB242 human EGFR ECD (without the signal peptide), with modifications T15S and G564S <400> 1466 Leu Glu Glu Lys Lys Val Cys Gln Gly Thr Ser Asn Lys Leu Ser Gln 1 5 10 15 Leu Gly Thr Phe Glu Asp His Phe Leu Ser Leu Gln Arg Met Phe Asn 20 25 30 Asn Cys Glu Val Val Leu Gly Asn Leu Glu Ile Thr Tyr Val Gln Arg 35 40 45 Asn Tyr Asp Leu Ser Phe Leu Lys Thr Ile Gln Glu Val Ala Gly Tyr 50 55 60 Val Leu Ile Ala Leu Asn Thr Val Glu Arg Ile Pro Leu Glu Asn Leu 65 70 75 80 Gln Ile Ile Arg Gly Asn Met Tyr Tyr Glu Asn Ser Tyr Ala Leu Ala 85 90 95 Val Leu Ser Asn Tyr Asp Ala Asn Lys Thr Gly Leu Lys Glu Leu Pro 100 105 110 Met Arg Asn Leu Gln Glu Ile Leu His Gly Ala Val Arg Phe Ser Asn 115 120 125 Asn Pro Ala Leu Cys Asn Val Glu Ser Ile Gln Trp Arg Asp Ile Val 130 135 140 Ser Ser Asp Phe Leu Ser Asn Met Ser Met Asp Phe Gln Asn His Leu 145 150 155 160 Gly Ser Cys Gln Lys Cys Asp Pro Ser Cys Pro Asn Gly Ser Cys Trp 165 170 175 Gly Ala Gly Glu Glu Asn Cys Gln Lys Leu Thr Lys Ile Ile Cys Ala 180 185 190 Gln Gln Cys Ser Gly Arg Cys Arg Gly Lys Ser Pro Ser Asp Cys Cys 195 200 205 His Asn Gln Cys Ala Ala Gly Cys Thr Gly Pro Arg Glu Ser Asp Cys 210 215 220 Leu Val Cys Arg Lys Phe Arg Asp Glu Ala Thr Cys Lys Asp Thr Cys 225 230 235 240 Pro Pro Leu Met Leu Tyr Asn Pro Thr Thr Tyr Gln Met Asp Val Asn 245 250 255 Pro Glu Gly Lys Tyr Ser Phe Gly Ala Thr Cys Val Lys Lys Cys Pro 260 265 270 Arg Asn Tyr Val Val Thr Asp His Gly Ser Cys Val Arg Ala Cys Gly 275 280 285 Ala Asp Ser Tyr Glu Met Glu Glu Asp Gly Val Arg Lys Cys Lys Lys 290 295 300 Cys Glu Gly Pro Cys Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu 305 310 315 320 Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys 325 330 335 Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe 340 345 350 Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu 355 360 365 Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln 370 375 380 Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu 385 390 395 400 Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val 405 410 415 Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile 420 425 430 Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala 435 440 445 Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr 450 455 460 Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln 465 470 475 480 Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro 485 490 495 Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val 500 505 510 Asp Lys Cys Lys Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn 515 520 525 Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn 530 535 540 Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His 545 550 555 560 Tyr Ile Asp Ser Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met 565 570 575 Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val 580 585 590 Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly 595 600 605 Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser 610 615 620 <210> 1467 <211> 643 <212> PRT <213> artificial sequence <220> <223> RB242 human HER3 ECD with modification Y246A (residue numbering does not include signal peptide) <220> <221> SIGNAL <222> (1)...(19) <223> signal peptide <220> <221> DOMAIN <222> (20)...(643) <223> HER3 extracellular domain (ECD) <400> 1467 Met Arg Ala Asn Asp Ala Leu Gln Val Leu Gly Leu Leu Phe Ser Leu 1 5 10 15 Ala Arg Gly Ser Glu Val Gly Asn Ser Gln Ala Val Cys Pro Gly Thr 20 25 30 Leu Asn Gly Leu Ser Val Thr Gly Asp Ala Glu Asn Gln Tyr Gln Thr 35 40 45 Leu Tyr Lys Leu Tyr Glu Arg Cys Glu Val Val Met Gly Asn Leu Glu 50 55 60 Ile Val Leu Thr Gly His Asn Ala Asp Leu Ser Phe Leu Gln Trp Ile 65 70 75 80 Arg Glu Val Thr Gly Tyr Val Leu Val Ala Met Asn Glu Phe Ser Thr 85 90 95 Leu Pro Leu Pro Asn Leu Arg Val Val Arg Gly Thr Gln Val Tyr Asp 100 105 110 Gly Lys Phe Ala Ile Phe Val Met Leu Asn Tyr Asn Thr Asn Ser Ser 115 120 125 His Ala Leu Arg Gln Leu Arg Leu Thr Gln Leu Thr Glu Ile Leu Ser 130 135 140 Gly Gly Val Tyr Ile Glu Lys Asn Asp Lys Leu Cys His Met Asp Thr 145 150 155 160 Ile Asp Trp Arg Asp Ile Val Arg Asp Arg Asp Ala Glu Ile Val Val 165 170 175 Lys Asp Asn Gly Arg Ser Cys Pro Pro Cys His Glu Val Cys Lys Gly 180 185 190 Arg Cys Trp Gly Pro Gly Ser Glu Asp Cys Gln Thr Leu Thr Lys Thr 195 200 205 Ile Cys Ala Pro Gln Cys Asn Gly His Cys Phe Gly Pro Asn Pro Asn 210 215 220 Gln Cys Cys His Asp Glu Cys Ala Gly Gly Cys Ser Gly Pro Gln Asp 225 230 235 240 Thr Asp Cys Phe Ala Cys Arg His Phe Asn Asp Ser Gly Ala Cys Val 245 250 255 Pro Arg Cys Pro Gln Pro Leu Val Ala Asn Lys Leu Thr Phe Gln Leu 260 265 270 Glu Pro Asn Pro His Thr Lys Tyr Gln Tyr Gly Gly Val Cys Val Ala 275 280 285 Ser Cys Pro His Asn Phe Val Val Asp Gln Thr Ser Cys Val Arg Ala 290 295 300 Cys Pro Pro Asp Lys Met Glu Val Asp Lys Asn Gly Leu Lys Met Cys 305 310 315 320 Glu Pro Cys Gly Gly Leu Cys Pro Lys Ala Cys Glu Gly Thr Gly Ser 325 330 335 Gly Ser Arg Phe Gln Thr Val Asp Ser Ser Asn Ile Asp Gly Phe Val 340 345 350 Asn Cys Thr Lys Ile Leu Gly Asn Leu Asp Phe Leu Ile Thr Gly Leu 355 360 365 Asn Gly Asp Pro Trp His Lys Ile Pro Ala Leu Asp Pro Glu Lys Leu 370 375 380 Asn Val Phe Arg Thr Val Arg Glu Ile Thr Gly Tyr Leu Asn Ile Gln 385 390 395 400 Ser Trp Pro Pro His Met His Asn Phe Ser Val Phe Ser Asn Leu Thr 405 410 415 Thr Ile Gly Gly Arg Ser Leu Tyr Asn Arg Gly Phe Ser Leu Leu Ile 420 425 430 Met Lys Asn Leu Asn Val Thr Ser Leu Gly Phe Arg Ser Leu Lys Glu 435 440 445 Ile Ser Ala Gly Arg Ile Tyr Ile Ser Ala Asn Arg Gln Leu Cys Tyr 450 455 460 His His Ser Leu Asn Trp Thr Lys Val Leu Arg Gly Pro Thr Glu Glu 465 470 475 480 Arg Leu Asp Ile Lys His Asn Arg Pro Arg Arg Asp Cys Val Ala Glu 485 490 495 Gly Lys Val Cys Asp Pro Leu Cys Ser Ser Gly Gly Cys Trp Gly Pro 500 505 510 Gly Pro Gly Gln Cys Leu Ser Cys Arg Asn Tyr Ser Arg Gly Gly Val 515 520 525 Cys Val Thr His Cys Asn Phe Leu Asn Gly Glu Pro Arg Glu Phe Ala 530 535 540 His Glu Ala Glu Cys Phe Ser Cys His Pro Glu Cys Gln Pro Met Glu 545 550 555 560 Gly Thr Ala Thr Cys Asn Gly Ser Gly Ser Asp Thr Cys Ala Gln Cys 565 570 575 Ala His Phe Arg Asp Gly Pro His Cys Val Ser Ser Cys Pro His Gly 580 585 590 Val Leu Gly Ala Lys Gly Pro Ile Tyr Lys Tyr Pro Asp Val Gln Asn 595 600 605 Glu Cys Arg Pro Cys His Glu Asn Cys Thr Gln Gly Cys Lys Gly Pro 610 615 620 Glu Leu Gln Asp Cys Leu Gly Gln Thr Leu Val Leu Ile Gly Lys Thr 625 630 635 640 His Leu Thr <210> 1468 <211> 624 <212> PRT <213> artificial sequence <220> <223> RB242 human HER3 ECD (without signal peptide), with modification Y246A <400> 1468 Ser Glu Val Gly Asn Ser Gln Ala Val Cys Pro Gly Thr Leu Asn Gly 1 5 10 15 Leu Ser Val Thr Gly Asp Ala Glu Asn Gln Tyr Gln Thr Leu Tyr Lys 20 25 30 Leu Tyr Glu Arg Cys Glu Val Val Met Gly Asn Leu Glu Ile Val Leu 35 40 45 Thr Gly His Asn Ala Asp Leu Ser Phe Leu Gln Trp Ile Arg Glu Val 50 55 60 Thr Gly Tyr Val Leu Val Ala Met Asn Glu Phe Ser Thr Leu Pro Leu 65 70 75 80 Pro Asn Leu Arg Val Val Arg Gly Thr Gln Val Tyr Asp Gly Lys Phe 85 90 95 Ala Ile Phe Val Met Leu Asn Tyr Asn Thr Asn Ser Ser His Ala Leu 100 105 110 Arg Gln Leu Arg Leu Thr Gln Leu Thr Glu Ile Leu Ser Gly Gly Val 115 120 125 Tyr Ile Glu Lys Asn Asp Lys Leu Cys His Met Asp Thr Ile Asp Trp 130 135 140 Arg Asp Ile Val Arg Asp Arg Asp Ala Glu Ile Val Val Lys Asp Asn 145 150 155 160 Gly Arg Ser Cys Pro Pro Cys His Glu Val Cys Lys Gly Arg Cys Trp 165 170 175 Gly Pro Gly Ser Glu Asp Cys Gln Thr Leu Thr Lys Thr Ile Cys Ala 180 185 190 Pro Gln Cys Asn Gly His Cys Phe Gly Pro Asn Pro Asn Gln Cys Cys 195 200 205 His Asp Glu Cys Ala Gly Gly Cys Ser Gly Pro Gln Asp Thr Asp Cys 210 215 220 Phe Ala Cys Arg His Phe Asn Asp Ser Gly Ala Cys Val Pro Arg Cys 225 230 235 240 Pro Gln Pro Leu Val Ala Asn Lys Leu Thr Phe Gln Leu Glu Pro Asn 245 250 255 Pro His Thr Lys Tyr Gln Tyr Gly Gly Val Cys Val Ala Ser Cys Pro 260 265 270 His Asn Phe Val Val Asp Gln Thr Ser Cys Val Arg Ala Cys Pro Pro 275 280 285 Asp Lys Met Glu Val Asp Lys Asn Gly Leu Lys Met Cys Glu Pro Cys 290 295 300 Gly Gly Leu Cys Pro Lys Ala Cys Glu Gly Thr Gly Ser Gly Ser Arg 305 310 315 320 Phe Gln Thr Val Asp Ser Ser Asn Ile Asp Gly Phe Val Asn Cys Thr 325 330 335 Lys Ile Leu Gly Asn Leu Asp Phe Leu Ile Thr Gly Leu Asn Gly Asp 340 345 350 Pro Trp His Lys Ile Pro Ala Leu Asp Pro Glu Lys Leu Asn Val Phe 355 360 365 Arg Thr Val Arg Glu Ile Thr Gly Tyr Leu Asn Ile Gln Ser Trp Pro 370 375 380 Pro His Met His Asn Phe Ser Val Phe Ser Asn Leu Thr Thr Ile Gly 385 390 395 400 Gly Arg Ser Leu Tyr Asn Arg Gly Phe Ser Leu Leu Ile Met Lys Asn 405 410 415 Leu Asn Val Thr Ser Leu Gly Phe Arg Ser Leu Lys Glu Ile Ser Ala 420 425 430 Gly Arg Ile Tyr Ile Ser Ala Asn Arg Gln Leu Cys Tyr His His Ser 435 440 445 Leu Asn Trp Thr Lys Val Leu Arg Gly Pro Thr Glu Glu Arg Leu Asp 450 455 460 Ile Lys His Asn Arg Pro Arg Arg Asp Cys Val Ala Glu Gly Lys Val 465 470 475 480 Cys Asp Pro Leu Cys Ser Ser Gly Gly Cys Trp Gly Pro Gly Pro Gly 485 490 495 Gln Cys Leu Ser Cys Arg Asn Tyr Ser Arg Gly Gly Val Cys Val Thr 500 505 510 His Cys Asn Phe Leu Asn Gly Glu Pro Arg Glu Phe Ala His Glu Ala 515 520 525 Glu Cys Phe Ser Cys His Pro Glu Cys Gln Pro Met Glu Gly Thr Ala 530 535 540 Thr Cys Asn Gly Ser Gly Ser Asp Thr Cys Ala Gln Cys Ala His Phe 545 550 555 560 Arg Asp Gly Pro His Cys Val Ser Ser Cys Pro His Gly Val Leu Gly 565 570 575 Ala Lys Gly Pro Ile Tyr Lys Tyr Pro Asp Val Gln Asn Glu Cys Arg 580 585 590 Pro Cys His Glu Asn Cys Thr Gln Gly Cys Lys Gly Pro Glu Leu Gln 595 600 605 Asp Cys Leu Gly Gln Thr Leu Val Leu Ile Gly Lys Thr His Leu Thr 610 615 620 <210> 1469 <211> 227 <212> PRT <213> artificial sequence <220> <223> Human IgG1 Fc with knob mutations S354C and T366W (by EU numbering) <220> <221> SITE <222> (1)...(10) <223> DKTHTCPPCP = portion of hinge region of human IgG1 <220> <221> DOMAIN <222> (11)...(227) <223> CH2 and CH3 domains of human IgG1 <400> 1469 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 1470 <211> 227 <212> PRT <213> artificial sequence <220> <223> Human IgG1 Fc with hole mutations Y349C, T366S, L368A and Y407V (by EU numbering) <220> <221> SITE <222> (1)...(10) <223> DKTHTCPPCP = portion of hinge region of human IgG1 <220> <221> DOMAIN <222> (11)...(227) <223> CH2 and CH3 domains of human IgG1 <400> 1470 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 1471 <211> 5 <212> PRT <213> artificial sequence <220> <223> gs linker <220> <221> REPEAT <222> (1)...(5) <223> occurring 1, 2, 3, 4 or 5 times <400> 1471 Gly Gly Gly Gly Ser 1 5 <210> 1472 <211> 5 <212> PRT <213> artificial sequence <220> <223> gs linker <220> <221> REPEAT <222> (1)...(5) <223> occurring 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times <400> 1472 Gly Gly Gly Gly Ser 1 5 <210> 1473 <211> 6 <212> PRT <213> artificial sequence <220> <223> Gly linker <400> 1473 Gly Gly Gly Gly Gly Gly 1 5 <210> 1474 <211> 8 <212> PRT <213> artificial sequence <220> <223> Gly linker <400> 1474 Gly Gly Gly Gly Gly Gly Gly Gly 1 5 <210> 1475 <211> 732 <212> PRT <213> artificial sequence <220> <223> Construct containing Vhh-4 <220> <221> SIGNAL <222> (1)...(19) <223> mouse immunoglobulin heavy-chain leader sequence <220> <221> DOMAIN <222> (20)...(138) <223> anti-TNFR1 dAb <220> <221> PEPTIDE <222> (139)... (147) <223> linker <220> <221> DOMAIN <222> (148)... (732) <223> Human Serum Albumin (HSA) <400> 1475 Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly 1 5 10 15 Val His Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ala His Glu Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ser His Ile Pro Pro Asp Gly Gln Asp Pro Phe Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr His Cys Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp 145 150 155 160 Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln 165 170 175 Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu 180 185 190 Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn 195 200 205 Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val 210 215 220 Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys 225 230 235 240 Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn 245 250 255 Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr 260 265 270 Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu 275 280 285 Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe 290 295 300 Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp 305 310 315 320 Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly 325 330 335 Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys 340 345 350 Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln 355 360 365 Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp 370 375 380 Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys 385 390 395 400 Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp 405 410 415 Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu 420 425 430 Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp 435 440 445 Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys 450 455 460 Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu 465 470 475 480 Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu 485 490 495 Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp 500 505 510 Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val 515 520 525 Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln 530 535 540 Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys 545 550 555 560 Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn 565 570 575 Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg 580 585 590 Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys 595 600 605 Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys 610 615 620 Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val 625 630 635 640 Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe 645 650 655 His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys 660 665 670 Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys 675 680 685 Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys 690 695 700 Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys 705 710 715 720 Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu 725 730 <210> 1476 <211> 19 <212> PRT 213 <213> <220> <223> mouse immunoglobulin heavy-chain leader sequence Uniprot: A0N1R4 <400> 1476 Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly 5 10 15 Val His Ser <210> 1477 <211> 7 <212> PRT <213> artificial sequence <220> <223> linker <400> 1477 Gly Gly Ser Gly Gly Gly Gly 1 5 <210> 1478 <211> 116 <212> PRT <213> artificial sequence <220> <223> Vhh-2 <400> 1478 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gln Tyr 20 25 30 Arg Met His Trp Val Arg Gln Ala Pro Gly Lys Ser Leu Glu Trp Val 35 40 45 Ser Ser Ile Asp Thr Arg Gly Ser Ser Thr Tyr Tyr Ala Asp Pro Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ala Val Thr Met Phe Ser Pro Phe Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val 115 <210> 1479 <211> 122 <212> PRT <213> artificial sequence <220> <223> <400> 1479 Glu Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Ala Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His 20 25 30 Glu Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser His Ile Pro Pro Asp Gly Gln Asp Pro Phe Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 His Cys Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Tyr Thr Val Ser Ser Ala 115 120

Claims (286)

A construct that is a tumor necrosis factor receptor 1 (TNFR1) antagonist construct of Formula 1:
(TNFR1 inhibitor) _n -linker _p - (active modifier) _q , where:
n and q are each an integer and are each independently 1, 2 or 3;
p is 0, 1, 2 or 3;
A TNFR1 inhibitor is a molecule that binds to and inhibits (antagonizes) the activity of TNFR1;
An active modifier is a moiety that modulates or alters the activity or pharmacological properties of a construct compared to a construct without the active modifier; and
The linker increases the flexibility of the construct, modulates or reduces the construct's steric effect or interaction with a receptor, and/or increases the construct's solubility in aqueous media. The construct of claim 1 , wherein the linker is selected from chemical linkers, polypeptide linkers, and combinations thereof. 3. The construct of claim 1 or 2, which is a fusion protein. 4. The construct of any preceding claim, wherein the linker contains a plurality of linker components. 5. The construct of any one of claims 1-4, wherein the TNFR1 inhibitor comprises a domain antibody (dAb). 6. The construct according to any one of claims 1 to 5, wherein when the TNFR1 inhibitor is a domain antibody (dAb), the active modifier is not an unmodified single Fc region or a human serum albumin antibody. 7. The construct of any one of claims 1-6, wherein the TNFR1 inhibitor inhibits TNFR1 signaling. 8. The construct of any one of claims 1-7, wherein the active modifier increases the serum half-life of the construct. 9. The construct of any one of claims 1-8, wherein the active modifier is albumin or Fc, which is modified with or without ADCC activity and/or with reduced or no CDC activity. 10. The construct of any preceding claim, wherein the TNFR1 inhibitor inhibits TNFR1 activity but does not antagonize tumor necrosis factor receptor 2 (TNFR2) activity. 11. The construct of claim 10, wherein the TNFR1 inhibitor inhibits TNFR1 signaling. A construct that is a multispecific construct comprising a TNFR1 inhibitor and a Treg expander, wherein the bispecific construct interacts with two different target receptors or antigens or epitopes on the receptor. 13. The construct of claim 12, which is bispecific for TFNR1 and a Treg expander. 14. The construct of claim 12 or 13, wherein the Treg expander is a TNFR2 agonist. 15. The construct of any one of claims 12-14, comprising a linker to provide flexibility, increase solubility, or mitigate or reduce steric hindrance or van der Waals interactions. 16. The construct of any one of claims 12-15, further comprising an activity modifier to alter the activity of the construct. The construct of any one of claims 12 to 16, having formula 2:
(TNFR1 inhibitor) _n - (active modifier) _r1 - (linker (L)) _p - (active modifier) _r2 - (TNFR2 agonist) _q , or
(TNFR1 inhibitor) _n - (active modifier) _r1 - (linker (L)) _p - (active modifier) _r2 - (Treg expander) _q ,
here:
n is 1, 2 or 3, p is 1, 2 or 3, q is 0, 1 or 2, r1 and r2 are each independently 0, 1 or 2; and
The components may be in the order specified or in any other order as long as the construct interacts with and antagonizes TNFR1 and TNFR2, agonizes TNFR2, or has Treg expander activity. 18. The construct of any one of claims 1-17, wherein the TNFR1 inhibitor moiety inhibits binding and/or signaling of TNFα to TNFR1. Constructs of Formula 3a or 3b:
(TNFR2 agonist or Treg expander) _n - linker _p - (active modifier) _q , formula 3a, or
(active modifier) _q - linker _p - (TNFR2 agonist or Treg expander) _n , formula 3b, where:
n and q are each an integer and are each independently 1, 2 or 3; p is 0, 1, 2 or 3;
Active modifiers are moieties that alter the pharmacological properties of a construct;
TNFR2 agonists interact with TNRFR2 to result in TNFR2 activity;
Treg expanders are molecules that include TNFR2 agonists and increase Treg cells; and
The linker increases flexibility and/or modulates or reduces the steric effect or interaction of the construct with a receptor; alter the solubility of the construct. 20. The method according to any one of claims 1 to 19, wherein the active modifier is an Fc region or a modified Fc region or short FcRnBP; and
wherein the linker is a linker comprising a hinge region or comprising G and S residues. 21. The construct of any preceding claim, wherein the linker has a sequence set forth in any one of SEQ ID NOs: 812-834 or is a PEG moiety linker. 22. The construct of any one of claims 1-21 comprising an active modifier which is a modified Fc region or peptide that increases the serum half-life of the construct. 23. The construct of any one of claims 1-22, comprising an Fc region or Fc dimer. 24. The construct of any one of claims 1 to 23, comprising an Fc region modified to have reduced ADCC and/or CDC activity. 25. The construct of claim 24, wherein the Fc is modified to have reduced or no ADCC activity. 26. The method of any one of claims 1 to 25,
TNFR1 inhibitors are any defined in the sequence listing listed below or known in the art;
The Treg expander is known in the art, is a TNFR2 agonist, or any Treg expander set forth in the sequence listing listed below, or is known in the art;
The linker is any listed in the Sequence Listing or below or known in the art; and
The construct, wherein the active modifier is any of those set forth in the Sequence Listing known in the art and/or set forth below. A construct that is a TNFR1 antagonist construct, comprising a TNFR1 inhibitor that is a single chain antibody or antigen binding portion thereof that specifically targets and inhibits TNFR1 but does not antagonize TNFR2, thereby preventing transient activation of TNFR1 through receptor clustering. . 28. The construct of claim 27, wherein the antibody or antigen-binding portion thereof comprises modifications that improve the structure and/or pharmacological properties of the construct. 29. The construct of any one of claims 1-28, which increases expression of regulatory T cells (Tregs) by agonizing TNFR2 signaling. 30. The construct of any one of claims 27-29, wherein the single chain antibody inhibits TNFR1 by inhibiting TNFR1 signaling. 31. The TNFR1 antagonist construct of any one of claims 27-30, wherein the antibody portion or antigen binding portion of the construct inhibits binding of TNFα to TNFR1. 31. The construct of any one of claims 27-30, wherein the antibody or antigen binding portion of the construct does not inhibit binding of TNFα to TNFR1 but inhibits TNFR1 signaling. 33. The construct of any one of claims 28-32, wherein the pharmacological property is increased serum half-life. 34. The construct of any one of claims 27-33, wherein the TNFR1 construct comprises an Fc modified to eliminate ADCC and/or CDC activity. 35. The construct of any one of claims 27-34, wherein the TNFR1 construct comprises an Fc dimer. 36. The construct of claim 35, wherein one Fc monomer comprises a hole and the other comprises a knob to form a heterodimer. The method of claim 35,
Knob mutation(s) are selected from among S354C, T366Y, T366W and T394W by EU numbering; and
the hole mutation(s) is selected from Y349C, T366S, L368A, F405A, Y407T, Y407A and Y407V by EU numbering;
wherein the Fc monomers form heterodimers. 38. The construct of any one of claims 1-37, comprising an Fc and being from Trastuzumab. 39. The construct of claim 38 comprising a linker that is a hinge region from an Fc region. 40. The construct of claim 39, wherein the hinge region is from Trastuzumab and is linked to the Fc region. 41. The construct of any one of claims 27-40 comprising a linker linked to an anti-TNFR1 antagonist antibody or antigen binding portion thereof. 42. The construct of any one of claims 27-41 comprising a linker connected to the Fc region either directly or through a hinge region to the anti-TNFR1 antagonist antibody or antigen binding portion thereof. 43. The Fc region according to any one of claims 1 to 42 comprising the sequence of amino acids set forth in any one of SEQ ID NOs: 10, 12, 14, 16, 27, 30, 1469 and 1470 or a modified Fc region. Containing, constructs. 44. The construct of any one of claims 1-43, wherein the construct comprises a hinge region connected to the Fc moiety. 45. The construct of any one of claims 1-44, wherein the construct binds to a neonatal Fc receptor (FcRn). The method of claim 45,
The TNFR1 construct contains a short FcRn binding peptide (FcRnBP); and
Short FcRn binding peptides (FcRnBPs) provide for interaction of the construct with FcRn and contain 6-25 or 10-20 amino acid residues. 47. The TNFR1 antagonist construct of claim 46, wherein the FcRnBP contains 12 to 20 residues or 15 residues or 16 residues. 48. The TNFR1 antagonist construct of claim 46 or 47, wherein the FcRn binding peptide (FcRnBP) comprises a peptide of any of SEQ ID NOs: 48-51. 49. The TNFR1 antagonist construct of any one of claims 46-48, wherein the FcRn-binding peptide (FcRnBP) consists of a peptide of any of SEQ ID NOs: 48-51. 50. The method of any one of claims 1-49, wherein the TNFR1 construct comprises an Fc heterodimer, wherein one Fc monomer comprises a hole and the other comprises a knob, and the resulting Fc dimer The construct is a heterodimer. 51. The construct of any one of claims 1-50, which is a TNFR1 antagonist construct comprising:
TNFR1 inhibitors;
Fc dimer; and
Treg expanders, wherein:
An Fc dimer comprises two complementary Fc monomers;
The TNFR1 inhibitor is linked to one Fc monomer and the Treg expander is linked to the other Fc monomer. 52. The construct of claim 51, wherein the Treg expander is a TNFR2 agonist. 53. The construct of claim 51 or 52, further comprising a second Treg expander linked to the same Fc monomer as the TNFR1 inhibitor, wherein the first and second Treg expanders are the same or different. 54. The construct of claim 53, wherein the second Treg expander is a TNFR2 agonist. 54. The construct of claim 53, wherein the Treg expander is the same. 56. The construct of any one of claims 51-55, wherein the TNFR1 inhibitor inhibits or blocks TNFR1 signaling. 57. The construct of any one of claims 51-56, wherein the TNFR1 inhibitor binds to TNFR1 and blocks or inhibits TNFα binding and TNFR1 signaling. 57. The construct of any one of claims 51-56, wherein the TNFR1 inhibitor binds TNFR1, does not or does not interfere with TNFa binding, and blocks or inhibits TNFR1 signaling. 59. The construct of any one of claims 51-58, wherein the Treg expander is a TNFR2 agonist. 60. The construct of claim 59, wherein the TNFR2 agonist stimulates or induces TNFR2 signaling. 61. The construct of any one of claims 51-60, wherein the Treg expander is a scFv, a VHH single domain antibody, or a TNFR2 agonist that is a Fab of a TNFR2 agonist monoclonal antibody. 62. The construct of any one of claims 1-61 comprising all or part of Trastuzumab and dimerized by N-terminal fusion with the C-terminus of Trastuzumab. 17. The construct of any one of claims 7-16, wherein the Treg expander is a TNFR2 agonist that is a small molecule, or a nucleic acid aptamer, or a peptide aptamer. 64. The construct of any one of claims 1-63, which is a TNFR2 agonist construct of Formula 3:
(Treg extender) _n - linker _p - (active modifier) _q , Formula 3a, or
(active modifier) _q - linker _p - (Treg extender) _n , formula 3b, where:
n and q are each an integer and are each independently 1, 2 or 3;
p is 0, 1, 2 or 3;
An active modifier is a moiety that modulates or alters the activity or pharmacological properties of a construct compared to a construct without the active modifier; and
The linker increases the flexibility of the construct, modulates or reduces the steric effect or interaction of the construct with a receptor, and/or increases the solubility of the construct in aqueous media. 65. The construct of any one of claims 51-64, wherein the Treg expander is a TNFR2 agonist. 66. The construct of claim 65, wherein the TNFR2 agonist stimulates or induces TNFR2 signaling. 67. The construct of any one of claims 51-66, wherein the Treg expander is a TNFR2 agonist that is a scFv, a VHH single domain antibody, or a Fab of a TNFR2 agonist monoclonal antibody. 68. The construct of claim 67, dimerized by C-terminal and N-terminal fusion of Trastuzumab. 69. The TNFR1 antagonist construct of any one of claims 51-68, wherein the Treg expander is a TNFR2 agonist that is a small molecule, or a nucleic acid or peptide aptamer. A construct comprising a TNFR1 inhibitor moiety linked to one or more Treg expanders via a central PEG linker, comprising at least two identical or different TNFR1 inhibitors, or comprising two identical or different Treg expanders. 71. The construct of claim 70, comprising a branched PEG moiety linking a TNFR1 inhibitor and one or more Treg expanders. The construct of claim 70 or 71 , having a structure selected from Formulas 4A-4D:
Equation 4A:

, or

n is 1 to 5;
R ¹ is H or CH ₃ , or CH ₂ CH ₃ or another C ₁ -C ₅ alkyl;

is a TNFR1 inhibitor (TNFR1 antagonist);

is a Treg expander; or
Equation 4B:

is a TNFR1 inhibitor (TNFR1 antagonist);

is a Treg expander;
n is 1 to 5; or
Equation 4C:

is a TNFR1 inhibitor (TNFR1 antagonist) or a Treg expander; and
n is 1 to 5; or
Equation 4D:

or
here
Each

are the same or different and are each independently selected from TNFR1 inhibitors (TNFR1 antagonists) and TNFR2 agonists;
Active modifiers are optional and may be linked to any suitable locus in the molecule; and
n is 1 to 5; 73. The construct of any one of claims 70-72, wherein the Treg expander is a TNFR2 agonist. 74. The method of any one of claims 1 to 73 comprising an active modifier, wherein:
The active modifier is an Fc region or an Fc region comprising a hinge region or other linker; and
wherein the Fc region or Fc region having a hinge region is an Fc that has been modified to reduce or eliminate ADCC and/or CDC activity. 75. The construct of claim 74, wherein the Fc or modified Fc is an IgG Fc or an IgG1 or IgG4 Fc. 76. The construct of any one of claims 1-75, which binds to the neonatal Fc receptor (FcRn). 77. The method of claim 76,
The construct contains a short FcRn binding peptide (FcRnBP); and
Short FcRn binding peptides (FcRnBPs) provide for interaction of the construct with FcRn and contain 6-25, such as 10-20, amino acid residues. 78. The construct of claim 77, wherein the FcRnBP contains 12 to 20 residues or 15 residues or 16 residues. 79. The TNFR1 antagonist construct of claim 78, wherein the FcRn binding peptide (FcRnBP) comprises a peptide of any of SEQ ID NOs: 48-51. 79. The TNFR1 antagonist construct of claim 78, wherein the FcRn binding peptide (FcRnBP) consists of a peptide of any of SEQ ID NOs: 48-51. 81. The construct of any one of claims 1-80, which is a TNFR1 antagonist construct comprising:
a) a TNFR1 selective TNFR1 inhibitor moiety;
b) optionally one or more linkers; and
c) optionally a half-life extending moiety, wherein
The antagonist construct includes at least one of b) and c). 82. The construct of claim 81, wherein the TNFR1 selective antagonist selectively binds to and inhibits TNFR1 signaling but not TNFR2 signaling. 83. The construct of claim 81 or 82, wherein the selective antagonist, TNFR1 inhibitor, comprises an antigen-binding fragment that selectively binds and inhibits TNFR1 signaling, but not TNFR2 signaling. 84. The construct of claim 83, wherein the antigen binding fragment that selectively binds to and inhibits TNFR1 signaling but not TNFR2 signaling comprises a domain antibody (dAb), scFv or Fab fragment. 85. The construct of any one of claims 1-84, wherein the TNFR1 inhibitor comprises an antigen-binding fragment of a human anti-TNFR1 antagonist monoclonal antibody. 86. The method of claim 85, wherein the human anti-TNFR1 antagonist monoclonal antibody is H398 comprising SEQ ID NO: 678, is ATROSAB, or an antigen binding portion thereof, or has at least 95% sequence identity to SEQ ID NO: 31 or 32 or 673 or 678 A construct that has a sequence with or is an antigen-binding portion thereof that binds to TNFR1. 87. The method of any one of claims 1 to 86, wherein the TNFR1 inhibitor comprises a domain antibody (dAb) or antigen binding portion thereof or a sequence of amino acids set forth in any one of SEQ ID NOs: 52-672, or TNFR1 inhibitor activity A construct comprising a sequence having at least 95% sequence identity that retains. 88. The method of any one of claims 1-87, wherein the TNFR1 inhibitor is a scFv set forth in any one of SEQ ID NOs: 673-678 or variants of these polypeptides having at least 90% or 95% sequence identity that retain TNFR1 inhibitor activity. Containing, constructs. 89. The method of any one of claims 1 to 88, wherein the TNFR1 inhibitor is a Fab or TNFR1 inhibitor set forth in any one of SEQ ID NOs: 679-682 or a sequence having at least 90% or 95% sequence identity that retains binding activity. Including, constructs. 90. The TNFR1 inhibitor according to any one of claims 1 to 89, wherein the TNFR1 inhibitor comprises a Nanobody whose sequence is set forth in SEQ ID NO: 683 or 684 or which has at least 90% or 95% sequence identity that retains the TNFR1 inhibitor or binding activity. Doing, construct. 91. The construct of any one of claims 1-90, wherein the TNFR1 inhibitor comprises dominant-negative tumor necrosis factor (DN-TNF) or a TNF mutein. 92. The method of claim 91, wherein the DN-TNF or TNF mutein is a soluble TNF molecule that confers selective inhibition of TNFR1 and comprises one or more amino acid substitutions selected from the following with respect to the sequence of soluble TNF set forth in SEQ ID NO: 2, Construct:
V1M, L29S, L29G, L29Y, R31C, R31E, R31N, R32Y, R32W, C69V, A84S, V85T, S86T, Y87H, Q88N, T89Q, I97T, C101A, A145R, E146R, L29S/R32W, L29S/S86T, R32W/ S86T, L29S/R32W/S86T, R31N/R32T, R31E/S86T, R31N/R32T/S86T, I97T/A145R, V1M/R31C/C69V/Y87H/C101A/A145R, and A84S/V85T/S86T/Y87H/Q88N /T89Q . 93. The method of claim 91 or 92, wherein the TNFR1 inhibitor TNF mutein is at least or A construct comprising a sequence having at least about 90% or 95% sequence identity or a fragment thereof that retains TNFR1 inhibitor activity. 94. The method of any one of claims 1 to 93, wherein the linker is all or part of the hinge sequence of Trastuzumab, SCDKTH corresponding to residues 222-227 of SEQ ID NO: 26 or the sequence EPKSCDKTHTCPPCP (residues of SEQ ID NO: 26) 219-233), or at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues thereof, or residues of SEQ ID NO:29 ESKYGPPCPPCP residues 212-223, or at least 98% thereof that are linkers or up to the entire sequence of the hinge region of trastuzumab that contains or has a sequence with 99% sequence identity. 95. The construct of any preceding claim, comprising a linker, wherein the linker comprises the sequence SCDKTH corresponding to residues 222-227 of SEQ ID NO:26. 96. The construct of any one of claims 1-95, comprising a linker, wherein the linker comprises a glycine-serine (GS) linker. 97. The method of claim 96, wherein the GS linker is (GlySer) _n (where n = 1-10); (GlySer ₂ ); (Gly ₄ Ser) _n (where n=1-10); (Gly ₃ Ser) _n (where n= 1-5); (SerGly ₄ ) _n (where n=1-5); (GlySerSerGly) _n (where n= 1-5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS. 98. The construct of any one of claims 1-97, wherein the linker comprises all or part of the hinge sequence of Trastuzumab corresponding to the GS linker and residues EPKSCDKTHTCPPCP (SEQ ID NO: 219-233 of 26). . 99. The construct of any preceding claim, wherein the linker comprises a GS linker and comprises the sequence SCDKTH corresponding to residues 217-222 of SEQ ID NO:31. 100. The construct of any one of claims 1-99, wherein the linker comprises a GS linker and all or part of the hinge sequence of nivolumab corresponding to residues 212-223 of SEQ ID NO:29. 101. The construct of any one of claims 1-100, comprising an active modifier, wherein the active modifier is an IgG Fc, a polyethylene glycol (PEG) molecule, or a half-life extending moiety that is human serum albumin (HSA). . 102. The construct of claim 101, wherein the IgG Fc is an IgG1 or IgG4 Fc. 103. The construct of claim 101 or 102, wherein the IgG1 Fc is the Fc of Trastuzumab set forth in SEQ ID NO: 27 or an amino acid sequence having at least 95% sequence identity thereto. 103. The construct of claim 101 or 102, wherein the IgG4 Fc is the Fc of nivolumab set forth in SEQ ID NO: 30 or an amino acid sequence having at least 95% sequence identity thereto. 105. The construct of any one of claims 101-104, wherein the IgG1 Fc is the Fc of human IgG1 set forth in SEQ ID NO:10. 105. The construct of any one of claims 101-104, wherein the IgG4 Fc is the Fc of human IgG4 set forth in SEQ ID NO: 16. 107. The construct of any one of claims 1-106, comprising a monovalent TNFR1 inhibitor. 108. The construct of any one of claims 81-107, comprising a linker comprising (Gly ₄ Ser) ₃ . 108. The construct of any one of claims 81-107, comprising a linker and comprising (Gly ₄ Ser) ₃ and SCDKTH (residues 217-222 of SEQ ID NO: 31). 110. The method of any one of claims 1 to 109, comprising a linker comprising (Gly ₄ Ser) ₃ and the hinge sequence of trastuzumab corresponding to residues 219-233 of SEQ ID NO: 26. sacrifice. 110. The construct of any one of claims 1-109, comprising a linker comprising (Gly ₄ Ser) ₃ and the hinge sequence of nivolumab corresponding to residues 212-223 of SEQ ID NO: 29. . 112. The method of any one of claims 1 to 111, wherein the sequence of residues set forth in any one of SEQ ID NOs: 704-764, or to the sequence of residues that inhibit TNFR1 and set forth in any one of SEQ ID NOs: 704-764 A construct comprising a construct having a sequence having at least or at least about 95% sequence identity. 113. The TNFR1 antagonist construct of any one of claims 1-112, wherein
The TNFR1 construct contains a short FcRn binding peptide (FcRnBP); and
Short FcRn binding peptides (FcRnBPs) provide for interaction of the construct with FcRn and contain 6-25, such as 10-20, amino acid residues. 114. The construct of claim 113, wherein the FcRnBP contains 12 to 20 residues or 15 residues or 16 residues. 114. The construct of claim 113, wherein the FcRn binding peptide (FcRnBP) comprises a peptide of any of SEQ ID NOs: 48-51. 114. The TNFR1 antagonist construct of claim 113, wherein the FcRn binding peptide (FcRnBP) consists of a peptide of any of SEQ ID NOs: 48-51. The construct of any one of claims 1 to 116 comprising:
a) domain antibodies that inhibit TNFR1;
b) increase flexibility; linkers that reduce steric effects or increase solubility; and
c) a half-life extending moiety. 118. The construct of claim 117, wherein the half-life extending moiety is not a human serum albumin antibody or an unmodified Fc. The construct of claim 117 or 118, which is a TNFR1 antagonist comprising:
a) a domain antibody (dAb) of any one of SEQ ID NOs: 52-672, or a scFv of any one of SEQ ID NOs: 673-678 or a Fab of any one of SEQ ID NOs: 679-682, or SEQ ID NOs: 683 or 684 or a TNF mutein of any one of SEQ ID NOs: 685-703;
b) (GlySer) _n (where n=1-10); (GlySer ₂ ); (Gly ₄ Ser) _n (where n=1-10); (Gly ₃ Ser) _n (where n= 1-5); (SerGly ₄ ) _n (where n=1-5); (GlySerSerGly) _n (where n= 1-5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and a GS linker selected from GSSSGS; and
c) a half-life extending moiety that is an IgG Fc. The method of any one of claims 117 to 119,
the GS linker is (GGGGS) ₃ ; and
The TNFR1 antagonist construct, wherein the IgG Fc is the Fc of Trastuzumab or the Fc of Nivolumab. 121. The construct of any one of claims 1-120, which is a TNFR1 antagonist construct comprising:
a) a domain antibody (dAb) of any one of SEQ ID NOs: 52-672, or a scFv of any one of SEQ ID NOs: 673-678 or a Fab of any one of SEQ ID NOs: 679-682, or SEQ ID NOs: 683 or 684 or a TNF mutein of any one of SEQ ID NOs: 685-703;
b) a linker selected from all or part of the hinge sequence of Trastuzumab and all or part of the hinge sequence of Nivolumab; and
c) a half-life extending moiety that is an IgG Fc. 121. The method of claim 121,
the linker comprises all or part of the hinge sequence of trastuzumab; and
wherein the IgG Fc is the Fc of Trastuzumab. 121. The method of claim 121,
the linker comprises all or part of the hinge sequence of nivolumab; and
wherein the IgG Fc is the Fc of nivolumab. 124. The construct of any one of claims 1-123, which is a TNFR1 antagonist construct comprising:
a) a domain antibody (dAb) of any one of SEQ ID NOs: 52-672, or a scFv of any one of SEQ ID NOs: 673-678 or a Fab of any one of SEQ ID NOs: 679-682, or SEQ ID NO: 683 or 684 or a TNF mutein of any one of SEQ ID NOs: 685-703;
b) (GlySer) _n (where n=1-10); (GlySer ₂ ); (Gly ₄ Ser) _n (where n=1-10); (Gly ₃ Ser) _n (where n= 1-5); (SerGly ₄ ) _n (where n=1-5); (GlySerSerGly) _n (where n= 1-5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and a GS linker selected from GSSSGS;
c) a second linker selected from all or part of the hinge sequence of Trastuzumab and all or part of the hinge sequence of Nivolumab; and
d) a half-life extending moiety that is an IgG Fc. 124. The method of claim 124,
the GS linker is (GGGGS) ₃ ;
the second linker comprises the sequence SCDKTH (residues 217-222 of SEQ ID NO: 31); and
wherein the IgG Fc is the Fc of Trastuzumab. 124. The method of claim 124,
the GS linker is (GGGGS) ₃ ;
the second linker comprises all or part of the hinge sequence of nivolumab; and
wherein the IgG Fc is the Fc of nivolumab. A construct, which is a TNFR1 agonist comprising:
a) a domain antibody (dAb) of any one of SEQ ID NOs: 52-672, or a scFv of any one of SEQ ID NOs: 673-678 or a Fab of any one of SEQ ID NOs: 679-682, or SEQ ID NOs: 683 or 684 or a TNF mutein of any one of SEQ ID NOs: 685-703;
b) (GlySer) _n (where n=1-10); (GlySer ₂ ); (Gly ₄ Ser) _n (where n=1-10); (Gly ₃ Ser) _n (where n= 1-5); (SerGly ₄ ) _n (where n=1-5); (GlySerSerGly) _n (where n= 1-5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and a GS linker selected from GSSSGS; and
c) a half-life extending moiety that is a PEG molecule. 128. The construct of claim 127, wherein the GS linker is (GGGGS) ₃ . 129. The construct of claim 127 or 128, wherein the PEG molecule has a molecular weight of 30 kDa or greater. The construct of any one of claims 1 - 129 , which is a TNFR1 agonist construct comprising:
a) a domain antibody (dAb) of any one of SEQ ID NOs: 52-672, or a scFv of any one of SEQ ID NOs: 673-678 or a Fab of any one of SEQ ID NOs: 679-682, or SEQ ID NOs: 683 or 684 or a TNF mutein of any one of SEQ ID NOs: 685-703;
b) (GlySer) _n (where n=1-10); (GlySer ₂ ); (Gly ₄ Ser) _n (where n=1-10); (Gly ₃ Ser) _n (where n= 1-5); (SerGly ₄ ) _n (where n=1-5); (GlySerSerGly) _n (where n= 1-5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and a GS linker selected from GSSSGS; and
c) a half-life extending moiety that is human serum albumin. 131. The construct of claim 130, wherein the GS linker is (GGGGS) ₃ . 132. The construct of any one of claims 1-131, which is a TNFR1 antagonist and is optimized to remove an immunogenic sequence or immunogenic epitope. 133. The construct of any one of claims 1-132, wherein the IgG Fc comprises one or more of the following modifications:
a) deformation(s) to introduce a knob-into-hole;
b) modification(s) to increase or enhance neonatal Fc receptor (FcRn) recycling; and
c) modification(s) to reduce or eliminate immune effector function. 133. The method of claim 133,
Knob mutations are selected from among S354C, T366Y, T366W and T394W by EU numbering; and
wherein the hole mutation is selected from Y349C, T366S, L368A, F405A, Y407T, Y407A and Y407V by EU numbering. 134. The TNFR1 antagonist construct of claim 133 or 134, wherein the modification(s) to increase or enhance FcRn recycling is selected from one or more of the following by EU numbering:
T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M2 52Y/T256Q, M252F/T256D, M252Y/ S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F, V259I/V308F/M428L, E294del/T307P/N4 34Y, and T256N/A378V/S383N/N434Y . 136. The method of any one of claims 133-135, wherein the immune effector function(s) is selected from among complement dependent cytotoxicity (CDC), antibody dependent cell mediated cytotoxicity (ADCC), and antibody dependent cell mediated phagocytosis (ADCP). A TNFR1 antagonist construct selected from one or more. 137. The TNFR1 antagonist construct of claim 136, wherein the modification(s) to reduce or eliminate immune effector function is selected from one or more of the following:
In IgG1: L235E, L234A/L235A, L234E/L235F/P331S, L234F/L235E/P331S, L234A/L235A/P329G, L234A/L235A/G237A/P238S/H268A/A330S/P331 by EU numbering S, G236R/L328R, G237A , E318A, D265A, E233P, N297A, N297Q, N297D, N297G, N297G/D265A, A330L, D270A, P329A, P331A, K322A, V264A, and F241A; and
In IgG4: L235E, F234A/L235A, S228P/L235E, and S228P/F234A/L235A by EU numbering. 138. The construct of any one of claims 1-137, which is a TNFR1 antagonist or multispecific or comprises a central PEG linker moiety and comprises a modified Fc region. 139. The construct of claim 138, wherein the Fc region is a modified IgG Fc and the modified IgG Fc comprises one or more of the following modifications:
a) deformation(s) to introduce a knob-into-hole; here
Knob mutations are selected from among S354C, T366Y, T366W and T394W by EU numbering; and
Hall mutations are variants selected from among Y349C, T366S, L368A, F405A, Y407T, Y407A and Y407V by EU numbering;
b) modification(s) to increase or enhance neonatal Fc receptor (FcRn) recycling, selected from one or more of the following:
T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M2 52Y/T256Q, M252F/T256D, M252Y/ S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F, V259I/V308F/M428L, E294del/T307P/N4 34Y, and T256N/A378V/S383N/N434Y ; and
c) modification(s) to increase or enhance one or more immune effector functions,
the immune effector function(s) is selected from one or more of CDC, ADCC and ADCP; and
Modification(s) selected from one or more of the following to increase or enhance immune effector function:
In IgG1: S239D, I332E, S239D/I332E, S239D/A330L/I332E, S298A/E333A/K334A in the first heavy chain; F243L/R292P/Y300L/V305I/P396L; L235V/F243L/R292P/Y300L/P396L; F243L/R292P/Y300L; L234Y/G236W/S298A and S239D/A330L/I332E in the second heavy chain; L234Y/L235Q/G236W/S239M/H268D/D270E/S298A in the first heavy chain and D270E/K326D/A330M/K334E in the second heavy chain; A327Q/P329A; D265A/S267A/H268A/D270A/K326A/S337A; T256A/K290A/S298A/E333A/K334A; G236A; G236A/I332E; G236A/S239D/I332E; G236A/S239D/A330L/I332E; introduction of a biantennary glycan at residue N297; residue N297; K326W; K326A; E333A; K326A/E333A; K326W/E333S; K326M/E333S; K222W/T223W; K222W/T223W/H224W; D221W/K222W; C220D/D221C; C220D/D221C/K222W/T223W; H268F/S324T; S267E; H268F; S324T; S267E/H268F/S324T; G236A/I332E/S267E/H268F/S324T; E345R; and introduction of afucosylated glycans at E345R/E430G/S440Y; by EU numbering. 140. The construct of any one of claims 1-139, comprising an Fc region comprising an IgG1 Fc comprising one or more modifications to increase binding to the inhibitory Fcγ receptor (FcγR) FcγRIIb. 141. The method of claim 140, wherein the modification that increases binding to FcγRIIb is selected from one or more of S267E, N297A, L328F, L351S, T366R, L368H, P395K, S267E / L328F and L351S / T366R / L368H / P395K by EU numbering , TNFR1 antagonist constructs. A construct that is a Treg expander construct, the construct comprising:
a) Treg expanders;
b) a linker that increases the flexibility of the construct and/or modulates or reduces the steric effect or interaction of the construct with a receptor and/or increases the solubility of the construct in aqueous media; and
c) an active modifier, which is a moiety that modulates or alters the activity or pharmacological properties of a construct compared to a construct without the active modifier. 142. The construct of any one of claims 142 or 1-141 comprising a Treg expander, wherein the Treg expander is a TNFR2 agonist. 144. The construct of claim 142 or 143, further comprising a TNFR1 inhibitor. 145. The construct of claim 143 or 144, wherein the TNFR2 agonist is a TNFR2 selective agonist. 146. The construct of any one of claims 1-145, which is a TNFR2 agonist construct comprising:
a) TNFR2 agonists;
b) a linker that increases the flexibility of the construct and/or modulates or reduces the steric effect or interaction of the construct with a receptor and/or increases the solubility of the construct in aqueous media; and
c) an active modifier, which is a moiety that modulates or alters the activity or pharmacological properties of a construct compared to a construct without the active modifier. 147. The construct of claim 146, wherein the TNFR2 agonist is a TNFR2 selective agonist. 148. The construct of any one of claims 142-147, comprising an active modifier, wherein the active modifier is a half-life extending moiety. 149. The construct of any one of claims 142-148, wherein the TNFR2 agonist selectively activates or antagonizes TNFR2 without activating or antagonizing TNFR1. 150. The construct of any one of claims 1-149, comprising a TNFR2 agonist, wherein the TNFR2 agonist binds one or more epitopes within TNFR2. 151. The construct of claim 150, wherein the TNFR2 is human TNFR2. 152. The construct of claim 151, wherein the epitope is selected from one or more epitopes comprising or consisting of the sequences of amino acids set forth in SEQ ID NOs: 839-865, 1202 and 1204. 153. The method of any one of claims 149-152, wherein the TNFR2 agonist comprises an agonist human anti-TNFR2 antibody or an antigen-binding fragment of a humanized anti-TNFR2 antibody, or an antigen-binding portion thereof, or a single chain form thereof. construct. 154. The construct of claim 153, wherein the agonist anti-TNFR2 antibody is selected from MR2-1 (also designated ab8161; US Pat. No. 9,821,010) or MAB2261 (US Pat. No. 9,821,010). 155. The construct of any one of claims 149-154, wherein the TNFR2 agonist is an antigen binding fragment selected from dAb, scFv or Fab fragments. 156. The construct of any one of claims 149-155, wherein the TNFR2 agonist is a TNFR2 selective agonist. 157. The construct of any one of claims 1-156, comprising a TNFR2 agonist, and the TNFR2 agonist comprising a TNFR2 agonist TNF mutein. 158. The method of claim 157, wherein the TNFR2 muteins are all K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, with respect to SEQ ID NO:2 D143N/A145R, A145R/S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/ S147D, A145N/E146D/S147D, A145T/E146S/S147D, A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A1 45R/S147T, E146D/S147D, D143V/ A construct that is a soluble TNF variant comprising one or more TNFR2 selective mutations selected from F144L/A145S, S95C/G148C, and D143V/A145S, and combinations of the foregoing. 158. The construct of claim 157, wherein the TNFR2 agonist is a TNF mutein comprising the mutations D143N/A145R. 160. The method of any one of claims 142-159, wherein the linker comprises all or part of the hinge sequence of Trastuzumab corresponding to residues 219-233 of SEQ ID NO: 26, or residues 212-23 of SEQ ID NO: 29 A construct comprising all or part of the hinge sequence of nivolumab corresponding to 223. 161. The construct of any one of claims 1-160, comprising a linker and comprising the sequence SCDKTH corresponding to residues 217-222 of SEQ ID NO:31. 162. The construct of claim 161 comprising a linker, wherein the linker comprises a glycine-serine (GS) linker. 163. The method of claim 162, wherein the GS linker is (GlySer) _n (where n = 1-10); (GlySer ₂ ); (Gly ₄ Ser) _n (where n=1-10); (Gly ₃ Ser) _n (where n= 1-5); (SerGly ₄ ) _n (where n=1-5); (GlySerSerGly) _n (where n= 1-5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS. 164. The construct of any one of claims 146-163, wherein the linker comprises a GS linker and all or part of the hinge sequence of trastuzumab corresponding to residues 219-233 of SEQ ID NO:26. 164. The construct of any one of claims 146-163, wherein the linker comprises a GS linker and the sequence SCDKTH corresponding to residues 217-222 of SEQ ID NO:31. 164. The construct of any one of claims 146-163, wherein the linker comprises a GS linker and all or part of the hinge sequence of nivolumab corresponding to residues 212-223 of SEQ ID NO:29. 167. The construct of any one of claims 1-166, comprising a half-life extending moiety, wherein the half-life extending moiety is an IgG Fc, a polyethylene glycol (PEG) molecule, or human serum albumin (HSA). 168. The construct of claim 167, wherein the IgG Fc is an IgG1 or IgG4 Fc. 169. The construct of claim 168, wherein the IgGl Fc is the Fc of Trastuzumab as set forth in SEQ ID NO:27. 169. The construct of claim 168, wherein the IgG4 Fc is the Fc of nivolumab as set forth in SEQ ID NO:30. 169. The construct of claim 168, wherein the IgG1 Fc is the Fc of human IgG1 set forth in SEQ ID NO:10. 169. The construct of claim 168, wherein the IgG4 Fc is the Fc of human IgG4 set forth in SEQ ID NO: 16. 173. The construct of any one of claims 146-172, wherein the TNFR2 agonist is monovalent. 174. The construct of any one of claims 1-173, which is a TNFR2 agonist construct that is bivalent. 174. The construct of any one of claims 1-173, which is a TNFR2 agonist construct that is trivalent. 176. The construct of any one of claims 1-175, wherein the construct is a TNFR2 agonist and the linker comprises (Gly ₄ Ser) ₃ . 177. The construct of any one of claims 1-176, which is the TNFR2 agonist construct TNFR2, wherein the linker comprises (Gly ₄ Ser) ₃ and SCDKTH (residues 217-222 of SEQ ID NO: 31). 118. The TNFR2 agonist construct according to any one of claims 1 to 117, wherein the linker comprises (Gly ₄ Ser) ₃ and the hinge sequence of trastuzumab corresponding to residues 219-233 of SEQ ID NO: 26 Doing, construct. 118. The TNFR2 agonist construct according to any one of claims 1 to 117, wherein the linker comprises (Gly ₄ Ser) ₃ and the hinge sequence of nivolumab corresponding to residues 212-223 of SEQ ID NO: 29 , construct. 180. The construct of any one of claims 1-179, comprising an active modifier that is a half-life extending moiety. 181. The construct of claim 180, wherein the half-life extending moiety is PEG. 181. The construct of claim 180, wherein the PEG has a molecular weight of at least or at least about 30 kDa. 181. The construct of claim 180, wherein the half-life extending moiety is human serum albumin (HSA). The construct of any one of claims 1 - 183 , which is a TNFR2 agonist construct comprising:
a) a TNFR2 agonist that binds to one or more epitopes in human TNFR2 selected from among the epitopes set forth in SEQ ID NOs: 839-865, 1202 and 1204;
b) (GlySer) _n (where n=1-10); (GlySer ₂ ); (Gly ₄ Ser) _n (where n=1-10); (Gly ₃ Ser) _n (where n= 1-5); (SerGly ₄ ) _n (where n=1-5); (GlySerSerGly) _n (where n= 1-5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and a GS linker selected from GSSSGS; and
c) an activity modifier that is a half-life extending moiety that is an IgG Fc. 184. The method of claim 184,
the GS linker is (GGGGS) ₃ ; and
wherein the IgG Fc is the Fc of Trastuzumab or the Fc of Nivolumab. The construct of any one of claims 1 - 185 , which is a TNFR2 agonist construct comprising:
a) a TNFR2 agonist that binds to one or more epitopes in human TNFR2 selected from among the epitopes set forth in SEQ ID NOs: 839-865, 1202 and 1204;
b) a linker selected from all or part of the hinge sequence of Trastuzumab and all or part of the hinge sequence of Nivolumab; and
c) an activity modifier that is a half-life extending moiety that is an IgG Fc. 186. The method of claim 186,
the linker comprises all or part of the hinge sequence of trastuzumab; and
wherein the IgG Fc is the Fc of Trastuzumab. 187. The method of claim 187,
the linker comprises all or part of the hinge sequence of nivolumab; and
wherein the IgG Fc is the Fc of nivolumab. The construct of any one of claims 1 - 183 , which is a TNFR2 construct comprising:
a) a TNFR2 agonist that binds to one or more epitopes in human TNFR2 selected from among the epitopes set forth in SEQ ID NOs: 839-865, 1202 and 1204;
b) (GlySer) _n (where n=1-10); (GlySer ₂ ); (Gly ₄ Ser) _n (where n=1-10); (Gly ₃ Ser) _n (where n= 1-5); (SerGly ₄ ) _n (where n=1-5); (GlySerSerGly) _n (where n= 1-5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and a GS linker selected from GSSSGS;
c) a second linker selected from all or part of the hinge sequence of Trastuzumab and all or part of the hinge sequence of Nivolumab; and
d) an activity modifier that is a half-life extending moiety that is an IgG Fc. 189. The method of claim 189,
the GS linker is (GGGGS) ₃ ;
the second linker comprises the sequence SCDKTH (residues 217-222 of SEQ ID NO: 31); and
wherein the IgG Fc is the Fc of Trastuzumab. 189. The method of claim 189,
the GS linker is (GGGGS) ₃ ;
the second linker comprises all or part of the hinge sequence of nivolumab; and
wherein the IgG Fc is the Fc of nivolumab. The construct of any one of claims 1 - 191 , which is a TNFR2 agonist construct comprising:
a) a TNFR2 agonist comprising an antigen-binding fragment of an agonist human anti-TNFR2 antibody selected from MR2-1 or MAB2261;
b) a linker comprising:
i) (GlySer) _n (where n = 1-10); (GlySer ₂ ); (Gly ₄ Ser) _n (where n=1-10); (Gly ₃ Ser) _n (where n= 1-5); (SerGly ₄ ) _n (where n=1-5); (GlySerSerGly) _n (where n= 1-5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and a GS linker selected from GSSSGS; and/or
ii) all or part of the hinge sequence of trastuzumab or all or part of the hinge sequence of nivolumab; and
c) an active modifier which is a half-life extending moiety selected from an IgG1 or IgG4 Fc, a PEG molecule and human serum albumin (HSA);
IgG1 Fc is the Fc of human IgG1 as shown in SEQ ID NO: 10 or the Fc of Trastuzumab as shown in SEQ ID NO: 27; and
wherein the PEG molecule has a molecular weight of at least or at least about 30 kDa. 193. The construct of any one of claims 1-192, which is or comprises a TNFR2 agonist construct comprising:
a) K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R with respect to SEQ ID NO: 2 /S147T, Q88N /T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145 T/E146S/S147D, A145Q/E146D /S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/S147D, D143V/F 144L/A145S, S95C/G148C, and a TNFR2 selective TNF mutein, which is a soluble TNF variant comprising one or more TNFR2 selective mutations selected from D143V/A145S;
b) a linker comprising:
i) (GlySer) _n (where n = 1-10); (GlySer ₂ ); (Gly ₄ Ser) _n (where n=1-10); (Gly ₃ Ser) _n (where n= 1-5); (SerGly ₄ ) _n (where n=1-5); (GlySerSerGly) _n (where n= 1-5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and a GS linker selected from GSSSGS; and/or
ii) all or part of the hinge sequence of trastuzumab or all or part of the hinge sequence of nivolumab; and
c) an active modifier which is a half-life extending moiety selected from an IgG1 or IgG4 Fc, a PEG molecule and human serum albumin (HSA);
IgG1 Fc is the Fc of human IgG1 as shown in SEQ ID NO: 10 or the Fc of Trastuzumab as shown in SEQ ID NO: 27; and
wherein the PEG molecule has a molecular weight of at least or at least about 30 kDa. 194. The construct of any one of claims 1-193, which is or comprises a TNFR2 agonist construct comprising:
a) a TNFR2 TNF mutein comprising the mutations D143N/A145R;
b) (GGGGS) ₃ linker; and
c) an active modifier that is a half-life extending moiety that is the Fc of Trastuzumab or the Fc of Nivolumab. 195. The construct of any one of claims 1-194, which is a TNFR2 agonist construct comprising:
a) a TNFR2 selective TNF mutein comprising the mutations D143N/A145R;
b) a second linker comprising the (GGGGS) ₃ linker and the sequence SCDKTH (residues 217-222 of SEQ ID NO: 31); and
c) an active modifier that is the Fc half-life extending moiety of Trastuzumab. A construct, which is a TNFR2 agonist construct comprising:
a) a TNFR2 selective TNF mutein comprising the mutations D143N/A145R;
b) a (GGGGS) ₃ linker and a second linker comprising all or part of the hinge sequence of nivolumab; and
c) an active modifier that is a half-life extending moiety that is the Fc of nivolumab. The construct of any one of claims 1 - 196 , which is a TNFR2 agonist construct comprising:
a) a TNFR2 selective TNF mutein comprising the mutations D143N/A145R;
b) a linker comprising all or part of the hinge sequence of Trastuzumab corresponding to residues 219-233 of SEQ ID NO:26; and
c) a half-life extending moiety that is the Fc of Trastuzumab. The construct of any one of claims 1 - 197 , which is or comprises a TNFR2 agonist construct comprising:
a) a TNFR2 selective TNF mutein comprising the mutations D143N/A145R;
b) a linker comprising all or part of the hinge sequence of nivolumab corresponding to residues 212-223 of SEQ ID NO:29; and
c) an active modifier that is a half-life extending moiety that is the Fc of nivolumab. 199. The construct of any one of claims 1-198, wherein the construct is a TNFR1 antagonist construct or a TNFR2 agonist construct or both, and the IgG Fc is a monomer or a dimer. 200. The construct of any one of claims 1-199 comprising a dAb. 201. The construct of claim 200, comprising a Vhh single or double chain (nanobody) containing dAb. 202. The construct of claim 201 comprising HSA linked directly or through a linker to the dAb. 203. The construct of claim 202, wherein the linker is a Gly-Ser(GS) linker and/or the HSA is linked to the C-terminus of the dAb directly or via a linker. 204. The dAb of SEQ ID NO: 59 linked via a linker to the HSA set forth in SEQ ID NO: 1475, comprising residues 20-732, Dom1h-131-206, according to any one of claims 1 to 203, or SEQ ID NO: 1475 A construct having at least 95%, 96%, 97%, 98%, 99% sequence identity to the construct of and having TNFR1 antagonist activity. The dAb of claim 200 or 203, as set forth in any of SEQ ID NOs: 52-83, 503-672, 1478 and 1479, and at least 95%, 96%, 97%, 98%, 99% sequences therewith A construct comprising variants thereof having the same identity, whereby the construct has TNFR1 antagonist activity. 206. The construct of claim 205, wherein the dAb has the sequence set forth in any one of SEQ ID NOs: 57-59, and the variant thereof has at least 95% sequence identity thereto, whereby the construct has TNFR1 antagonist activity. 207. The construct of claim 206, wherein the dAb is designated DOM1h-131-206 of SEQ ID NO: 59 and variants thereof having TNFR1 antagonist activity. 208. The construct of any one of claims 200-207, wherein the sequence of the dAb or required portion of the construct for administration to a human is humanized. 209. The construct of any one of claims 1-208, which comprises a TNFR2 agonist or is a TNFR2 agonist construct. 209. The method of any one of claims 1-208, which is or comprises a TNFR2 agonist construct, wherein the TNFR2 agonist has been modified to remove sequences of amino acids or epitopes that are immunogenic in the subject to be treated. , construct. 201. The construct of claim 200, wherein the subject is a human. 212. The construct of any one of claims 189-211, wherein the TNFR2 agonist is a TNFR2 selective agonist. 213. The construct of any one of claims 1-212, which is a TNFR2 agonist construct and comprises a modified IgG Fc, wherein the IgG Fc comprises one or more of the following modifications:
a) deformation(s) to introduce a knob-into-hole;
b) modification(s) to increase or enhance neonatal Fc receptor (FcRn) recycling; and
c) modification(s) to reduce or eliminate an immune effector function selected from one or more of complement dependent cytotoxicity (CDC), antibody dependent cell mediated cytotoxicity (ADCC) and antibody dependent cell mediated phagocytosis (ADCP). 213. The construct of claim 213, which is:
a) the modification(s) to introduce a knob-into-hole are selected from:
at least one knob mutation selected from among S354C, T366Y, T366W and T394W by EU numbering; and
at least one hole mutation selected from Y349C, T366S, L368A, F405A, Y407T, Y407A and Y407V by EU numbering, wherein Fc forms a dimer;
b) the variant(s) to increase or enhance FcRn recycling are, by EU numbering: N434A, N434W, N434S, N434Y, Y436H, M252Y/T256Q, M252F/T256D, M252Y/S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M4 28L, M428L/N434S, V259I/V308F, selected from one or more of V259I/V308F/M428L, E294del/T307P/N434Y, and T256N/A378V/S383N/N434Y; and
c) the modification(s) to reduce or eliminate immune effector function is selected from one or more of the following:
In IgG1: L235E, L234A/L235A, L234E/L235F/P331S, L234F/L235E/P331S, L234A/L235A/P329G, L234A/L235A/G237A/P238S/H268A/A330S/P331 by EU numbering S, G236R/L328R, G237A , E318A, D265A, E233P, N297A, N297Q, N297D, N297G, N297G/D265A, A330L, D270A, P329A, P331A, K322A, V264A, and F241A; and
In IgG4: L235E, F234A/L235A, S228P/L235E, and S228P/F234A/L235A by EU numbering. 214. The construct of any one of claims 1-213, wherein the TNFR2 agonist construct contains a modified IgG Fc, wherein the IgG Fc comprises one or more of the following modifications:
a) deformation(s) to introduce a knob-into-hole; here
Knob mutations are selected from among S354C, T366Y, T366W and T394W by EU numbering; and
Hall mutations are variants selected from among Y349C, T366S, L368A, F405A, Y407T, Y407A and Y407V by EU numbering;
b) modification(s) to increase or enhance neonatal Fc receptor (FcRn) recycling, selected from one or more of the following:
T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M2 52Y/T256Q, M252F/T256D, M252Y/ S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F, V259I/V308F/M428L, E294del/T307P/N4 34Y, and T256N/A378V/S383N/N434Y ; and
c) as modification(s) to increase or enhance immune effector function;
the immune effector function(s) is selected from one or more of CDC, ADCC and ADCP; and
The modification(s) to increase or enhance immune effector function are selected from one or more of the following:
In IgG1: S239D, I332E, S239D/I332E, S239D/A330L/I332E, S298A/E333A/K334A in the first heavy chain; F243L/R292P/Y300L/V305I/P396L; L235V/F243L/R292P/Y300L/P396L; F243L/R292P/Y300L; L234Y/G236W/S298A and S239D/A330L/I332E in the second heavy chain; L234Y/L235Q/G236W/S239M/H268D/D270E/S298A in the first heavy chain and D270E/K326D/A330M/K334E in the second heavy chain; A327Q/P329A; D265A/S267A/H268A/D270A/K326A/S337A; T256A/K290A/S298A/E333A/K334A; G236A; G236A/I332E; G236A/S239D/I332E; G236A/S239D/A330L/I332E; introduction of a biantennary glycan at residue N297; residue N297; K326W; K326A; E333A; K326A/E333A; K326W/E333S; K326M/E333S; K222W/T223W; K222W/T223W/H224W; D221W/K222W; C220D/D221C; C220D/D221C/K222W/T223W; H268F/S324T; S267E; H268F; S324T; S267E/H268F/S324T; G236A/I332E/S267E/H268F/S324T; E345R; and introduction of afucosylated glycans at E345R/E430G/S440Y; by EU numbering. 216. The method of any one of claims 1-215, which is or comprises a TNFR2 agonist construct comprising a modified IgG1 Fc, wherein the Fc is modified to increase binding to the inhibitory Fcγ receptor (FcγR) FcγRIIb. , construct. 216. The method of claim 216, wherein the modification that increases binding to FcγRIIb is selected from one or more of S267E, N297A, L328F, L351S, T366R, L368H, P395K, S267E / L328F and L351S / T366R / L368H / P395K by EU numbering , construct. 218. The method of any one of claims 1-217, which is or comprises a TNFR2 agonist construct, which selectively activates or agonizes TNFR2 without activating or antagonizing TNFR1, comprising:
a) TNFR2 agonists;
b) one or more linkers; and
c) an active modifier that is a half-life extending moiety,
The TNFR2 agonist construct is a fusion protein comprising a single chain TNFR2 selective TNF mutein trimer fused with a multimerization domain, comprising the formula:
MD-L1-TNFmut-L2-TNFmut-L3-TNFmut (formula II); or
TNFmut-L1-TNFmut-L2-TNFmut-L3-MD (formula III);
MDs are the same or different multimerization domains; TNFmut is a TNFR2 selective TNF mutein; and L1, L2 and L3 are linkers that may be the same or different. 219. The method of claim 218, wherein the TNF mutein is K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143 with respect to SEQ ID NO: 2 N /A145R, A145R/S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D , A145N/E146D/S147D, A145T /E146S/S147D, A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S 147T, E146D/S147D, D143V/F144L A construct comprising one or more TNFR2 selective mutations selected from /A145S, S95C/G148C, and D143V/A145S. 219. The construct of claim 218, wherein the TNF mutein comprises the TNFR2 selective mutations D143N/A145R. 221. The method of any one of claims 218-220, wherein the multimerization domain is EHD2 (SEQ ID NO: 808), MHD2 (SEQ ID NO: 811), the trimerization domain of chicken Tenascin C (TNC) (SEQ ID NO: 804; SEQ ID NO: 805), or the trimerization domain of human TNC (residues 110-139 of SEQ ID NO: 806, SEQ ID NO: 807). 222. The TNFR2 agonist construct of any one of claims 218-221, wherein the multimerization domain is an IgG1 Fc or IgG4 Fc, and wherein the IgG1 Fc or IgG4 Fc is also a half-life extending moiety. 223. The method of any one of claims 218-222, wherein the L1, L2 and/or L3 linker is (GGGGS) _n (where n = 1-5), or all or part of the stem region of TNF (SEQ ID NO: 812 ), a TNFR2 agonist construct independently selected from. 224. The TNFR2 agonist construct of any one of claims 218-223, wherein the linker between the TNFR2 agonist and the half-life extending moiety is:
(GlySer) _n (where n=1-10); (GlySer ₂ ); (Gly ₄ Ser) _n (where n=1-10); (Gly ₃ Ser) _n (where n= 1-5); (SerGly ₄ ) _n (where n=1-5); (GlySerSerGly) _n (where n= 1-5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and a GS linker selected from GSSSGS; or
a linker selected from all or part of the hinge sequence of Trastuzumab and all or part of the hinge sequence of Nivolumab; or
combination of them. 225. The TNFR2 agonist construct of any one of claims 218-224, wherein the half-life extending moiety is selected from:
an IgG1 Fc that is the Fc of human IgG1 shown in SEQ ID NO: 10 or the Fc of trastuzumab described in SEQ ID NO: 27;
an IgG4 Fc that is the Fc of human IgG4 as shown in SEQ ID NO: 16 or the Fc of nivolumab as shown in SEQ ID NO: 30;
PEG molecules of at least or at least about 30 kDa in size; and
Human Serum Albumin (HSA). 218. The construct of any one of claims 1-217, which is or comprises a TNFR2 agonist construct comprising:
a) a construct having the formula:
MD-L1-TNFmut-L2-TNFmut-L3-TNFmut (formula II); or
TNFmut-L1-TNFmut-L2-TNFmut-L3-MD (formula III);
MD is the multimerization domain; TNFmut is a TNFR2 selective TNF mutein; and L1, L2 and L3 are the same or different linkers, wherein
i) MD is EHD2 (SEQ ID NO: 808), MHD2 (SEQ ID NO: 811), the trimerization domain of chicken tenascin C (TNC) (residues 110-139 of SEQ ID NO: 804; SEQ ID NO: 805), or is selected from the trimerization domain of human TNC (residues 110-139 of SEQ ID NO: 806, SEQ ID NO: 807);
ii) L1, L2 and L3 are each (GGGGS) _n (where n = 1-5), or all or part of the stem region of TNF (SEQ ID NO: 812), or a mixture thereof; and
iii) the TNF mutein comprises the TNFR2 selective mutations D143N/A145R;
b) a half-life extending moiety selected from:
an IgG1 Fc that is the Fc of human IgG1 shown in SEQ ID NO: 10 or the Fc of trastuzumab described in SEQ ID NO: 27;
an IgG4 Fc that is the Fc of human IgG4 as shown in SEQ ID NO: 16 or the Fc of nivolumab as shown in SEQ ID NO: 30;
PEG molecules of at least or at least about 30 kDa in size; and
human serum albumin (HSA); and
c) a linker between the TNFR2 selective agonist and the half-life extending moiety comprising:
(GlySer) _n (where n=1-10); (GlySer ₂ ); (Gly ₄ Ser) _n (where n=1-10); (Gly ₃ Ser) _n (where n= 1-5); (SerGly ₄ ) _n (where n=1-5); (GlySerSerGly) _n (where n= 1-5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and a GS linker selected from GSSSGS; or
a linker selected from all or part of the hinge sequence of Trastuzumab and all or part of the hinge sequence of Nivolumab; or
or a combination thereof. 218. The construct of any one of claims 1-217, which is a TNFR2 agonist construct comprising:
MD-L1-TNFmut-L2-TNFmut-L3-TNFmut (formula II); or
TNFmut-L1-TNFmut-L2-TNFmut-L3-MD (formula III);
MD is the multimerization domain; TNFmut is a TNFR2 selective TNF mutein; and L1, L2 and L3 are the same or different linkers, wherein
i) MD is selected from IgG1 Fc or IgG4 Fc;
ii) L2 and L3 in formula II, and L1 and L2 in formula III are each independently (GGGGS) _n (where n = 1-5), or all or part of the stem region of TNF (SEQ ID NO: 812), or a combination thereof;
iii) L1 in Formula II and L3 in Formula III are each independently selected from:
(GlySer) _n (where n=1-10); (GlySer ₂ ); (Gly ₄ Ser) _n (where n=1-10); (Gly ₃ Ser) _n (where n= 1-5); (SerGly ₄ ) _n (where n=1-5); (GlySerSerGly) _n (where n= 1-5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and a GS linker selected from GSSSGS; or
a linker selected from all or part of the hinge sequence of Trastuzumab and all or part of the hinge sequence of Nivolumab; or
combinations thereof; and
iv) the TNF mutein comprises the TNFR2 selective mutations D143N/A145R. 228. The construct of claim 227, wherein MD is selected from:
an IgG1 Fc that is the Fc of human IgG1 shown in SEQ ID NO: 10 or the Fc of trastuzumab described in SEQ ID NO: 27; or
An IgG4 Fc that is the Fc of human IgG4 as shown in SEQ ID NO: 16 or the Fc of nivolumab as shown in SEQ ID NO: 30. 229. The method of claim 227 or 228, wherein MD is the IgG1 Fc of Trastuzumab, and the linker between MD and the adjacent TNF mutein is all of the hinge sequence of Trastuzumab corresponding to residues 219-233 of SEQ ID NO: 26 or a construct that is part of. 229. The construct of claim 227 or 228, wherein the MD is the IgG1 Fc of Trastuzumab and the linker between the MD and the adjacent TNF mutein comprises the sequence SCDKTH (residues 217-222 of SEQ ID NO:31). 231. The method of any one of claims 227-230, wherein MD is the IgG1 Fc of Trastuzumab, and the linker between MD and the adjacent TNF mutein is (Gly ₄ Ser) ₃ and residues 219-233 of SEQ ID NO: 26 A construct comprising the hinge sequence of trastuzumab corresponding to 229. The method of claim 227 or 228, wherein MD is the IgG1 Fc of Trastuzumab, and the linker between MD and the adjacent TNF mutein is (Gly ₄ Ser) ₃ and the sequence SCDKTH (residues 222-227 of SEQ ID NO:31) Containing, constructs. 229. The method of claim 227 or 228, wherein the MD is an IgG4 Fc of nivolumab, and the linker between the MD and the adjacent TNF mutein is all or part of the hinge sequence of nivolumab corresponding to residues 212-223 of SEQ ID NO: 29 Containing, constructs. 229. The method of claim 227 or 228, wherein MD is an IgG4 Fc of nivolumab, and the linker between MD and adjacent TNF mutein is nivolumab corresponding to (Gly ₄ Ser) ₃ and residues 212-223 of SEQ ID NO: 29 A construct comprising all or part of the hinge sequence of. 235. The construct of any one of claims 1-234, wherein the TNFR2 agonist is an agonist construct that has been modified to remove an immunogenic sequence or immunogenic epitope in a subject. 236. The construct of claim 235, wherein the subject is a human. 237. The construct of any one of claims 1-236, which is a TNFR2 agonist construct and comprises a modified IgG Fc, wherein the IgG Fc comprises one or more of the following modifications:
a) deformation(s) to introduce a knob-into-hole; here
the knob mutation is selected from one or more of S354C, T366Y, T366W and T394W by EU numbering; and
Hall mutations are variants selected from one or more of Y349C, T366S, L368A, F405A, Y407T, Y407A and Y407V by EU numbering;
b) modification(s) to increase or enhance neonatal Fc receptor (FcRn) recycling, selected from one or more of the following:
T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y4 by EU numbering 36H, M252Y/T256Q, M252F/ T256D, M252Y/S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F, V259I/V308F/M428L, E2 94del/T307P/N434Y, and T256N/A378V /S383N/N434Y; and
c) modification(s) to reduce or eliminate immune effector function, wherein
the immune effector function(s) is selected from one or more of CDC, ADCC and ADCP; and
The modification(s) to reduce or eliminate immune effector function is selected from one or more of the following:
In IgG1: L235E, L234A/L235A, L234E/L235F/P331S, L234F/L235E/P331S, L234A/L235A/P329G, L234A/L235A/G237A/P238S/H268A/A330S/P331 by EU numbering S, G236R/L328R, G237A , E318A, D265A, E233P, N297A, N297Q, N297D, N297G, N297G/D265A, A330L, D270A, P329A, P331A, K322A, V264A, and F241A; and
In IgG4: L235E, F234A/L235A, S228P/L235E, and S228P/F234A/L235A by EU numbering. 238. The construct of any one of claims 1-237, which is a TNFR2 agonist construct and comprises a modified IgG Fc, wherein the IgG Fc comprises one or more of the following modifications:
a) deformation(s) to introduce a knob-into-hole; here
the knob mutation is selected from one or more of S354C, T366Y, T366W and T394W by EU numbering; and
Hall mutations are variants selected from one or more of Y349C, T366S, L368A, F405A, Y407T, Y407A and Y407V by EU numbering;
b) modification(s) to increase or enhance neonatal Fc receptor (FcRn) recycling, selected from one or more of the following:
T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y4 by EU numbering 36H, M252Y/T256Q, M252F/ T256D, M252Y/S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F, V259I/V308F/M428L, E2 94del/T307P/N434Y, and T256N/A378V /S383N/N434Y; and
c) as modification(s) to increase or enhance immune effector function;
the immune effector function(s) is selected from one or more of CDC, ADCC and ADCP; and
The modification(s) to increase or enhance immune effector function are selected from one or more of the following:
In IgG1: S239D, I332E, S239D/I332E, S239D/A330L/I332E, S298A/E333A/K334A in the first heavy chain; F243L/R292P/Y300L/V305I/P396L; L235V/F243L/R292P/Y300L/P396L; F243L/R292P/Y300L; L234Y/G236W/S298A and S239D/A330L/I332E in the second heavy chain; L234Y/L235Q/G236W/S239M/H268D/D270E/S298A in the first heavy chain and D270E/K326D/A330M/K334E in the second heavy chain; A327Q/P329A; D265A/S267A/H268A/D270A/K326A/S337A; T256A/K290A/S298A/E333A/K334A; G236A; G236A/I332E; G236A/S239D/I332E; G236A/S239D/A330L/I332E; introduction of a biantennary glycan at residue N297; residue N297; K326W; K326A; E333A; K326A/E333A; K326W/E333S; K326M/E333S; K222W/T223W; K222W/T223W/H224W; D221W/K222W; C220D/D221C; C220D/D221C/K222W/T223W; H268F/S324T; S267E; H268F; S324T; S267E/H268F/S324T; G236A/I332E/S267E/H268F/S324T; E345R; and introduction of afucosylated glycans at E345R/E430G/S440Y; by EU numbering. 239. The TNFR2 agonist construct of any one of claims 1-238, which is a TNFR2 agonist construct comprising an IgG1 Fc that has been modified to increase binding to the inhibitory Fcγ receptor (FcγR) FcγRIIb. 240. The method of claim 239, wherein the modification that increases binding to FcγRIIb is selected from one or more of S267E, N297A, L328F, L351S, T366R, L368H, P395K, S267E / L328F and L351S / T366R / L368H / P395K by EU numbering , TNFR2 antagonist constructs. 241. The construct of any one of claims 1-240, which is a multispecific TNFR1 inhibitor/TNFR2 agonist construct and is of the formula:
(TNFR1 inhibitor) _n - linker (L) _p - (TNFR2 agonist) _q (Formula I), or
(TNFR1 inhibitor) _n - Linker (L) _p - (TNFR2 agonist) _q , or
(TNFR1 inhibitor) _n - (TNFR2 agonist) _q - linker (L) _p , or
(TNFR2 agonist) _q - (TNFR1 inhibitor) _n - Linker (L) _p , or
Any of the above, including a selective active modifier, wherein:
n = 1 or 2, p = 1, 2 or 3, q = 1 or 2;
TNFR1 inhibitors interact with TNFR1 to inhibit its activity;
An active modifier is a moiety that modulates or alters the activity or pharmacological properties of a construct compared to a construct without the active modifier; and
A linker increases the solubility of a construct, increases flexibility, or alters the steric effect of a structure. 242. The construct of any one of claims 1-241, which is a multispecific TNFR1 inhibitor/TNFR2 agonist construct:
TNFR1 inhibitors selectively inhibit or antagonize TNFR1 signaling without inhibiting or antagonizing TNFR2 signaling;
TNFR1 inhibitors do not interfere with the activation or action of TNFR2;
TNFR2 agonists selectively activate or agonize TNFR2 signaling without activating or agonizing TNFR1 signaling;
TNFR2 agonists do not interfere with inhibition or antagonism of TNFR1. The construct of claim 241 or 242, which is:
a) the TNFR1 inhibitor is selected from:
i) an antigen-binding fragment of a human anti-TNFR1 antagonist monoclonal antibody selected from H398 or ATROSAB or a polypeptide having a sequence having at least 95% sequence identity thereto; or
ii) a domain antibody (dAb) of any one of SEQ ID NOs: 52-672, or a scFv of any one of SEQ ID NOs: 673-678 or a Fab of any one of SEQ ID NOs: 679-682, or SEQ ID NOs: 683 or 684 or a TNF mutein of any one of SEQ ID NOs: 701-703, or a polypeptide having a sequence that has at least 95% sequence identity to any of the foregoing polypeptides and is a TNFR1 inhibitor; or
iii) a predominantly-negative tumor necrosis factor (DN-TNF) or TNF mutein comprising a soluble TNF molecule conferring selective inhibition of TNFR1 and having one or more amino acid substitutions selected from:
V1M, L29S, L29G, L29Y, R31C, R31E, R31N, R32Y, R32W, C69V, A84S, V85T, S86T, Y87H, Q88N, T89Q, I97T, C101A, A145R with respect to the sequence of soluble TNF set forth in SEQ ID NO: 2 , E146R, L29S/R32W, L29S/S86T, R32W/S86T, L29S/R32W/S86T, R31N/R32T, R31E/S86T, R31N/R32T/S86T, I97T/A145R, V1M/R31C/C69V/Y87H/C101 A/A145R , and A84S/V85T/S86T/Y87H/Q88N/T89Q;
b) the linker is selected from:
i) (GlySer) _n (where n = 1-10); (GlySer ₂ ); (Gly ₄ Ser) _n (where n=1-10); (Gly ₃ Ser) _n (where n= 1-5); (SerGly ₄ ) _n (where n=1-5); (GlySerSerGly) _n (where n= 1-5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and a GS linker selected from GSSSGS; and/or
ii) all or part of the hinge sequence of Trastuzumab corresponding to residues 219-233 of SEQ ID NO: 26, or all or part of the hinge sequence of Nivolumab corresponding to residues 212-223 of SEQ ID NO: 29; and
iii) IgG1 or IgG4 Fc, wherein:
the IgG1 Fc is selected from the IgG1 Fc of human IgG1 set forth in SEQ ID NO: 10, or the IgG1 Fc of Trastuzumab set forth in SEQ ID NO: 27;
the IgG4 Fc is selected from the IgG4 Fc of human IgG4 set forth in SEQ ID NO: 16 or the IgG4 Fc of nivolumab set forth in SEQ ID NO: 30; and
Optionally, the Fc comprises one or more modifications to introduce a knob-into-hole, increase or enhance neonatal Fc receptor (FcRn) recycling, and/or reduce or eliminate immune effector function; and
c) the TNFR2 agonist is selected from:
i) an antigen-binding fragment that binds to one or more epitopes in human TNFR2 selected from among the epitopes set forth in SEQ ID NOs: 839-865, 1202 and 1204; or
ii) an antigen-binding fragment of a potent human anti-TNFR2 antibody selected from MR2-1 or MAB2261; or
iii) K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145 with respect to SEQ ID NO: 2 R/S147T, Q88N /T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145 T/E146S/S147D, A145Q/E146D /S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/S147D, D143V/F 144L/A145S, S95C/G148C, and a TNFR2 selective TNF mutein, which is a soluble TNF variant comprising one or more TNFR2 selective mutations selected from D143V/A145S; or
iv) a single chain TNFR2 selective TNF mutein trimer comprising the mutations D143N/A145R, wherein the TNF mutein is (GGGGS) _n (where n = 1-5) or all or all of the stem region of TNF (SEQ ID NO: 812) single-chain TNFR2 selective TNF mutein trimers, linked by moieties; or
v) a TNFR2 selective agonist comprising the formula:
MD-L1-TNFmut-L2-TNFmut-L3-TNFmut (formula II); or
TNFmut-L1-TNFmut-L2-TNFmut-L3-MD (formula III);
MD is the multimerization domain; TNFmut is a TNFR2 selective TNF mutein; and L1, L2 and L3 are the same or different linkers, wherein
MD is EHD2 (SEQ ID NO: 808), MHD2 (SEQ ID NO: 811), the trimerization domain of chicken tenascin C (TNC) (residues 110-139 of SEQ ID NO: 804; SEQ ID NO: 805), or human TNC is selected from the trimerization domain of (residues 110-139 of SEQ ID NO: 806, SEQ ID NO: 807);
L1, L2 and L3 are each (GGGGS) _n (where n = 1-5), or all or part of the stem region of TNF (SEQ ID NO: 812), or a mixture thereof; and
TNF muteins include the TNFR2 selective mutations D143N/A145R. 243. The multispecific TNFR1 antagonist/TNFR2 agonist construct of claim 241 or 242:
a) the TNFR1 inhibitor is a domain antibody (dAb) of any one of SEQ ID NOs: 52-672, or a scFv of any one of SEQ ID NOs: 673-678 or a Fab of any one of SEQ ID NOs: 679-682, or SEQ ID NO: a Nanobody of 683 or 684, or a TNF mutein of any one of SEQ ID NOs: 701-703, or a sequence having at least or at least about 95% sequence identity therewith;
b) the linker comprises (GGGGS) ₃ , a polypeptide comprising the sequence SCDKTH (residues 222-227 of SEQ ID NO: 26), and the Fc of Trastuzumab; and
c) the TNFR2 agonist is K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R with respect to SEQ ID NO: 2 , A145R /S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D /S147D, A145T/E146S/S147D A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D S147D, D143V/F144L/A145S, S95C /G148C, and a TNFR2 selective TNF mutein, which is a soluble TNF variant comprising one or more TNFR2 selective mutations selected from D143V/A145S. The construct of claim 241 or 242, wherein:
a) the TNFR1 inhibitor is a domain antibody (dAb) of any one of SEQ ID NOs: 52-672, or a scFv of any one of SEQ ID NOs: 673-678 or a Fab of any one of SEQ ID NOs: 679-682, or SEQ ID NO: a Nanobody of 683 or 684, or a TNF mutein of any one of SEQ ID NOs: 701-703, or a sequence having at least or at least about 95% sequence identity therewith;
b) the linker comprises (GGGGS) ₃ , all or part of the hinge sequence of nivolumab, and the Fc of nivolumab; and
c) the TNFR2 agonist is K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R with respect to SEQ ID NO: 2 , A145R /S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D /S147D, A145T/E146S/S147D A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D S147D, D143V/F144L/A145S, S95C /G148C, and a TNFR2 selective TNF mutein, which is a soluble TNF variant comprising one or more TNFR2 selective mutations selected from D143V/A145S. The construct of claim 241 or 242, which is:
a) the TNFR1 inhibitor is a domain antibody (dAb) of any one of SEQ ID NOs: 52-672, or a scFv of any one of SEQ ID NOs: 673-678 or a Fab of any one of SEQ ID NOs: 679-682, or SEQ ID NO: a Nanobody of 683 or 684, or a TNF mutein of any one of SEQ ID NOs: 701-703, or a sequence having at least or at least about 95% sequence identity therewith;
b) the linker comprises (GGGGS) ₃ and the Fc of Trastuzumab; and
c) the TNFR2 agonist is K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R with respect to SEQ ID NO: 2 , A145R /S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D /S147D, A145T/E146S/S147D A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D S147D, D143V/F144L/A145S, S95C /G148C, and a TNFR2 selective TNF mutein, which is a soluble TNF variant comprising one or more TNFR2 selective mutations selected from D143V/A145S. The construct of claim 241 or 242, which is:
a) the TNFR1 inhibitor is a domain antibody (dAb) of any one of SEQ ID NOs: 52-672, or a scFv of any one of SEQ ID NOs: 673-678 or a Fab of any one of SEQ ID NOs: 679-682, or SEQ ID NO: a Nanobody of 683 or 684, or a TNF mutein of any one of SEQ ID NOs: 701-703, or a sequence having at least or at least about 95% sequence identity therewith;
b) the linker comprises (GGGGS) ₃ and the Fc of nivolumab; and
c) the TNFR2 agonist is K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R with respect to SEQ ID NO: 2 , A145R /S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D /S147D, A145T/E146S/S147D A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D S147D, D143V/F144L/A145S, S95C /G148C, and D143V/A145S, and a TNFR2 selective TNF mutein, which is a soluble TNF variant comprising one or more TNFR2 selective mutations selected from any combination of the foregoing mutations. The construct of any one of claims 241 - 247 comprising a modified Fc, wherein the IgG Fc comprises one or more of the following modifications:
a) deformation(s) to introduce a knob-into-hole;
b) modification(s) to increase or enhance neonatal Fc receptor (FcRn) recycling; and
c) modification(s) to reduce or eliminate immune effector function. 249. The construct of claim 248, wherein Fc comprises a knob-into-hole modification:
the knob mutation is selected from one or more of S354C, T366Y, T366W and T394W by EU numbering; and
The hole mutation is selected from one or more of Y349C, T366S, L368A, F405A, Y407T, Y407A and Y407V by EU numbering. 249. The construct of claim 248, wherein the Fc comprises a modification to increase or enhance FcRn recycling, and wherein the modification is selected from one or more of the following:
T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y4 by EU numbering 36H, M252Y/T256Q, M252F/ T256D, M252Y/S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F, V259I/V308F/M428L, E2 94del/T307P/N434Y, and T256N/A378V /S383N/N434Y. 249. The method of claim 248, wherein the Fc comprises a modification to an immune effector function selected from one or more of complement dependent cytotoxicity (CDC), antibody dependent cell mediated cytotoxicity (ADCC) and antibody dependent cell mediated phagocytosis (ADCP). , construct. 249. The construct of claim 248, comprising modification(s) to reduce or eliminate an immune effector function selected from one or more of the following:
In IgG1: L235E, L234A/L235A, L234E/L235F/P331S, L234F/L235E/P331S, L234A/L235A/P329G, L234A/L235A/G237A/P238S/H268A/A330S/P331 by EU numbering S, G236R/L328R, G237A , E318A, D265A, E233P, N297A, N297Q, N297D, N297G, N297G/D265A, A330L, D270A, P329A, P331A, K322A, V264A, and F241A; and
In IgG4: L235E, F234A/L235A, S228P/L235E, and S228P/F234A/L235A by EU numbering. The construct of any one of claims 241 - 247 , wherein the IgG Fc comprises one or more of the following modifications:
a) deformation(s) to introduce a knob-into-hole; here
the knob mutation is selected from one or more of S354C, T366Y, T366W and T394W by EU numbering; and
Hall mutations are variants selected from one or more of Y349C, T366S, L368A, F405A, Y407T, Y407A and Y407V by EU numbering;
b) modification(s) to increase or enhance neonatal Fc receptor (FcRn) recycling, selected from one or more of the following:
T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y4 by EU numbering 36H, M252Y/T256Q, M252F/ T256D, M252Y/S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F, V259I/V308F/M428L, E2 94del/T307P/N434Y, and T256N/A378V /S383N/N434Y; and
c) as modification(s) to increase or enhance immune effector function;
the immune effector function(s) is selected from one or more of CDC, ADCC and ADCP; and
The modification(s) to increase or enhance immune effector function are selected from one or more of the following:
In IgG1: S239D, I332E, S239D/I332E, S239D/A330L/I332E, S298A/E333A/K334A in the first heavy chain; F243L/R292P/Y300L/V305I/P396L; L235V/F243L/R292P/Y300L/P396L; F243L/R292P/Y300L; L234Y/G236W/S298A and S239D/A330L/I332E in the second heavy chain; L234Y/L235Q/G236W/S239M/H268D/D270E/S298A in the first heavy chain and D270E/K326D/A330M/K334E in the second heavy chain; A327Q/P329A; D265A/S267A/H268A/D270A/K326A/S337A; T256A/K290A/S298A/E333A/K334A; G236A; G236A/I332E; G236A/S239D/I332E; G236A/S239D/A330L/I332E; introduction of a biantennary glycan at residue N297; residue N297; K326W; K326A; E333A; K326A/E333A; K326W/E333S; K326M/E333S; K222W/T223W; K222W/T223W/H224W; D221W/K222W; C220D/D221C; C220D/D221C/K222W/T223W; H268F/S324T; S267E; H268F; S324T; S267E/H268F/S324T; G236A/I332E/S267E/H268F/S324T; E345R; and introduction of afucosylated glycans at E345R/E430G/S440Y; by EU numbering. 254. The construct of any one of claims 241-253, comprising an IgG1 Fc modified to increase binding to the inhibitory Fcγ receptor (FcγR) FcγRIIb. 255. The method of claim 254, wherein the modification that increases binding to FcγRIIb is selected from one or more of S267E, N297A, L328F, L351S, T366R, L368H, P395K, S267E / L328F and L351S / T366R / L368H / P395K by EU numbering , construct. 256. The construct of any one of claims 1-255, which is a multispecific TNFR1 antagonist/TNFR2 agonist:
TNFR1 antagonists are monovalent; and
TNFR2 agonists are monovalent. 256. The construct of any one of claims 1-255, which is a multispecific TNFR1 antagonist/TNFR2 agonist:
TNFR1 antagonists are monovalent; and
TNFR2 agonists are bivalent. The construct of any one of claims 241-257, which is a multispecific TNFR1 antagonist/TNFR2 agonist:
a) the TNFR1 antagonist is selected from:
i) an antigen-binding fragment of a human anti-TNFR1 antagonist monoclonal antibody selected from H398 or ATROSAB; or
ii) a domain antibody (dAb) of any one of SEQ ID NOs: 52-672, or a scFv of any one of SEQ ID NOs: 673-678 or a Fab of any one of SEQ ID NOs: 679-682, or SEQ ID NOs: 683 or 684 or a TNF mutein of any one of SEQ ID NOs: 701-703, or a sequence having at least or at least about 95% sequence identity therewith; or
iii) a predominantly-negative tumor necrosis factor (DN-TNF) or TNF mutein comprising a soluble TNF molecule conferring selective inhibition of TNFR1 and having one or more amino acid substitutions selected from:
V1M, L29S, L29G, L29Y, R31C, R31E, R31N, R32Y, R32W, C69V, A84S, V85T, S86T, Y87H, Q88N, T89Q, I97T, C101A, A145R with respect to the sequence of soluble TNF set forth in SEQ ID NO: 2 , E146R, L29S/R32W, L29S/S86T, R32W/S86T, L29S/R32W/S86T, R31N/R32T, R31E/S86T, R31N/R32T/S86T, I97T/A145R, V1M/R31C/C69V/Y87H/C101 A/A145R , and A84S/V85T/S86T/Y87H/Q88N/T89Q;
b) the linker is a branched chain PEG molecule of at least or at least about 30 kDa in size; and
c) the TNFR2 agonist is selected from:
i) an antigen-binding fragment that binds to one or more epitopes in human TNFR2 selected from among the epitopes set forth in SEQ ID NOs: 839-865, 1202 and 1204; or
ii) an antigen-binding fragment of a potent human anti-TNFR2 antibody selected from MR2-1 or MAB2261; or
iii) K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145 with respect to SEQ ID NO: 2 R/S147T, Q88N /T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145 T/E146S/S147D, A145Q/E146D /S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/S147D, D143V/F 144L/A145S, S95C/G148C, and a TNFR2 selective TNF mutein, which is a soluble TNF variant comprising one or more TNFR2 selective mutations selected from D143V/A145S; or
iv) a single chain TNFR2 selective TNF mutein trimer comprising the mutations D143N/A145R, wherein the TNF mutein is (GGGGS) _n (where n = 1-5) or all or all of the stem region of TNF (SEQ ID NO: 812) single-chain TNFR2 selective TNF mutein trimers, linked by moieties; or
v) a TNFR2 selective agonist comprising the formula:
MD-L1-TNFmut-L2-TNFmut-L3-TNFmut (formula II); or
TNFmut-L1-TNFmut-L2-TNFmut-L3-MD (formula III);
MD is the multimerization domain; TNFmut is a TNFR2 selective TNF mutein; and L1, L2 and L3 are the same or different linkers, wherein
MD is EHD2 (SEQ ID NO: 808), MHD2 (SEQ ID NO: 811), the trimerization domain of chicken tenascin C (TNC) (residues 110-139 of SEQ ID NO: 804; SEQ ID NO: 805), or human TNC is selected from the trimerization domain of (residues 110-139 of SEQ ID NO: 806, SEQ ID NO: 807);
L1, L2 and L3 are each (GGGGS) _n (where n = 1-5), or all or part of the stem region of TNF (SEQ ID NO: 812), or a mixture thereof; and
TNF muteins include the TNFR2 selective mutations D143N/A145R. 259. The construct of claim 258, wherein each of the TNFR1 antagonist and TNFR2 agonist is monovalent. 259. The construct of claim 258, wherein the TNFR1 antagonist is monovalent and the TNFR2 agonist is divalent. 261. The chronic inflammatory, autoimmune, neurodegenerative, demyelinating, or respiratory disease or disorder of any one of claims 1-260, or a disease characterized by overexpression of TNF or deregulated TNFR1 signaling in its pathogenesis. , a construct that is a multispecific TNFR1 antagonist/TNFR2 agonist for use in the treatment of a condition or disorder. Multispecific TNFR1 antagonists/ for the treatment of chronic inflammatory, autoimmune, neurodegenerative, demyelinating or respiratory diseases or disorders, or diseases, conditions or disorders characterized by overexpression of TNF or deregulated TNFR1 signaling in its etiology. Use of the construct of any one of claims 1 - 260 as a TNFR2 agonist. A composition comprising the construct of any one of claims 1 - 260 in a pharmaceutically acceptable carrier or vehicle. 263. Use according to claim 263, for the treatment of a chronic inflammatory, autoimmune, neurodegenerative, demyelinating or respiratory disease or disorder, or a disease, condition or disorder characterized by overexpression of TNF or deregulated TNFR1 signaling in its etiology. To do, the construct. 264. The chronic inflammatory, autoimmune, or chronic inflammatory disease of any one of claims 1 to 261, 262, or 263 or 264 characterized by overexpression of TNF or deregulated TNFR1 signaling in its pathogenesis. The neurodegenerative, demyelinating or respiratory disease or disorder, or the disease, condition or disorder is a construct, use, or composition selected from:
Rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, juvenile idiopathic arthritis (JIA), spondyloarthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis, inflammatory bowel disease (IBD), uveitis, fibrotic disease, endometriosis, lupus , multiple sclerosis (MS), congestive heart failure, cardiovascular disease, myocardial infarction (MI), atherosclerosis, metabolic disease, cytokine release syndrome, septic shock, sepsis, acute respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), SARS-CoV-2, influenza, acute and chronic neurodegenerative diseases, demyelinating diseases and disorders, stroke, Alzheimer's disease, Parkinson's disease, Behcet's disease, Dupuytren's disease, tumor necrosis factor receptor-associated periodic syndrome (TRAPS) , pancreatitis, type I diabetes, chronic obstructive pulmonary disease (COPD), chronic bronchitis, emphysema, transplant rejection, graft-versus-host disease (GvHD), lung inflammation, lung diseases and conditions, asthma, cystic fibrosis, idiopathic pulmonary fibrosis, acute fulminant viral or bacterial infections, pneumonia, hereditary genetic disorders with TNF/TNFR1 as a causative pathological vector, periodic fever syndrome, or cancer. The construct or use, or composition, of any one of claims 1-261, 262, or 263 or 264, for use in the treatment of rheumatoid arthritis. The construct of any one of claims 1 -261 or the use of claim 262 , or the composition of claim 263 or 264 for the treatment of rheumatoid arthritis. A construct comprising a TNFR2 antagonist and optionally a linker and optionally an active modifier. 269. The construct of claim 268, comprising Formula 5:
(TNFR2 antagonist) _n - linker _p - (active modifier) _q , or
linker _p - (active modifier) _q - (TNFR2 antagonist) _n , where:
n and q are each an integer and are each independently 1, 2 or 3;
p is 0, 1, 2 or 3;
A TNFR2 antagonist is a molecule capable of inhibiting (antagonizing) the activity of TNFR2 by interacting with TNFR2, thereby inhibiting the proliferation of Treg and/or inducing its death, and also inhibiting the proliferation and inducing the death of TNFR2-expressing tumor cells. ;
An active modifier is a moiety that modulates or alters the activity or pharmacological properties of a construct compared to a construct without the active modifier; and
The linker increases the flexibility of the construct, modulates or reduces the steric effect or interaction of the construct with a receptor, and/or increases the solubility of the construct in aqueous media. 269. The construct of claims 268 or 269, wherein each active modifier and linker are as defined and described for the construct of any one of claims 1-250. The construct of any one of claims 268 - 270 , wherein the TNFR2 antagonist is:
reduce and/or inhibit the proliferation of bone marrow derived suppressor cells (MDSCs);
Inducing apoptosis in MDSCs by binding TNFR2 expressed on the surface of MDSCs present in the tumor microenvironment;
Inhibition of Treg expansion and activity induces expansion of T effector cells, including cytotoxic CD8 ⁺ T cells. 272. The method of any one of claims 268-271, wherein the TNRF2 antagonist is an epitope in human TNFR2 containing one or more of residues KCRPG (corresponding to residues 142-146 of SEQ ID NO: 4), or for example residue 130 -149, 137-144 or 142-149, or a larger epitope containing at least 5 contiguous or discontinuous residues within these epitopes, and residues KCSPG (corresponding to residues 56-60 of SEQ ID NO: 4) does not bind to the epitope it contains; or the TNFR2 epitopes PECLSCGS (corresponding to residues 91-98 of SEQ ID NO: 4), RICTCRPG corresponding to residues 116-123 of SEQ ID NO: 4), CAPLRKCR (corresponding to residues 137-144 of SEQ ID NO: 4), LRKCRPGFGVA (corresponding to residues 140-150 of SEQ ID NO: 4), and/or VVCKPCAPGTFSN (corresponding to residues 159-171 of SEQ ID NO: 4), and/or residues 75-128, 86- of SEQ ID NO: 4 103, 111-128, or 150-190, which is an antibody, antigen-binding fragment thereof, or a single chain antibody that binds to an epitope containing at least 5 contiguous or discontinuous residues. 273. The method of any one of claims 268-272, wherein the antibody, fragment thereof, or single-chain form thereof has a greater affinity than the same antibody or antigen-binding fragment for a peptide containing the KCSPG sequence of human TNFR2 (SEQ ID NO: 839). A construct that binds an epitope containing one or more residues of the KCRPG sequence (SEQ ID NO: 840) with at least 10-fold greater affinity. The construct of any one of claims 268 - 273 , wherein the TNFR2 antagonist is an antibody or fragment or single chain form of an antibody selected from:
TNFRAB1 (see SEQ ID NOs: 1212 and 1213 for the heavy and light chain sequences of TNFRAB1, respectively), TNFRAB2 and TNFR2A3 (see, eg, US Patent Publication No. 2019/0144556 for a description of these antibodies);
TNFRAB1 (QRVDGYSSYWYFDV; corresponding to residues 99-112 of SEQ ID NO: 1212), TNFRAB2 (ARDDGSYSPFDYWG; SEQ ID NO: 1217) or TNFR2A3 (ARDDGSYSPFDYFG; SEQ ID NO: 1223), or a CDR with at least about 85% sequence identity there - a CDR-H3 sequence of TNFRAB1 that specifically binds to residues 130-149 containing residues KCRPG of TNFR2 with 40-fold higher affinity than H3 sequence, e.g., residues 48-67 containing residues KCSPG of TNFR2. Antibodies and antibody fragments and single chain forms containing The construct of any one of claims 268 - 274 , wherein the TNFR2 antagonist binds to one or more epitopes in TNFR2 selected from:
an epitope containing residues 137-144 (CAPLRKCR; SEQ ID NO: 851);
an epitope comprising one or more residues within positions 80-86 (DSTYTQL; SEQ ID NO: 1247), 91-98 (PECLSCGS; SEQ ID NO: 1248), and/or 116-123 (RICTCRPG; SEQ ID NO: 1249) of human TNFR2 ; and
TNFR2A3 selected from the first epitope is an epitope comprising residues 140-150 of human TNFR2 (LRKCRPGFGVA; SEQ ID NO: 1463) and containing a KCRPG motif, and/or residues 159-171 of human TNFR2 (VVCKPCAPGTFSN; SEQ ID NO: 1464) A second epitope containing 276. The method of any one of claims 268-275, wherein the TNFR2 antagonist is a CDR-H1 amino acid having the sequence set forth in any of SEQ ID NOs: 1214, 1215, and 1231-1233 to produce a humanized antagonistic TNFR2 antibody; The CDR-H2 sequence as set forth in any of 1216, 1224, and 1230, the CDR-H3 sequence as set forth in any of SEQ ID NOs: 1217, 1223, and 1225-1229, and/or at residues 99-112 of SEQ ID NO: 1212 corresponding CDR-H3 of TNFRAB1; the CDR-L1 sequence set forth in any of SEQ ID NOs: 1218 and 1234-1236, and/or the CDR-L1 sequence of TNFRAB1 corresponding to residues 24-33 of SEQ ID NO: 1213; the CDR-L2 sequence set forth in any of SEQ ID NOs: 1219, 1220, 1237 and 1238, or the CDR-L2 sequence of TNFRAB1 corresponding to residues 49-55 of SEQ ID NO: 1213; and/or a CDR-L3 sequence set forth in any of SEQ ID NOs: 1221, 1222, and 1241-1244, or a CDR-L3 sequence of TNFRAB1 corresponding to residues 88-96 of SEQ ID NO: 1213; and/or the CDR-H1 and CDR-H2 sequences of the consensus sequence of the human antibody heavy chain variable domain of SEQ ID NO: 1245 replaced with the corresponding CDR sequences of a phenotype neutral TNFR2 specific antibody, and/or of a phenotype neutral TNFR2 specific antibody. An antibody, fragment thereof, or short chain form thereof, which contains at least one of the CDR-L1, CDR-L2 and CDR-L3 sequences of the sequence of the human antibody light chain variable domain of SEQ ID NO: 1246 replaced with the corresponding CDR sequence. sacrifice. 276. The construct of any one of claims 268-275, wherein the TNFR2 antagonist specifically binds to an epitope in TNFR2 set forth in any one of SEQ ID NOs: 1247-1464. The construct of any one of claims 268 - 277 , wherein the TNFR2 antagonist specifically binds to an epitope(s) selected from:
(a) residues KCRPG corresponding to residues 142-146 of SEQ ID NO: 4, or residues 130-149, 137-144 or 142-149, or a larger epitope containing at least 5 contiguous or discontinuous residues within these epitopes; at least one epitope in human TNFR2 that contains at least one that does not bind to an epitope containing residues KCSPG corresponding to residues 56-60 of SEQ ID NO:4; and/or
(b) one or more TNFR2 epitopes comprising an amino acid sequence comprising:
PECLSCGS corresponding to residues 91-98 of SEQ ID NO: 4, and/or RICTCRPG corresponding to residues 116-123 of SEQ ID NO: 4, and/or
CAPLRKCR (corresponding to residues 137-144 of SEQ ID NO: 4), and/or LRKCRPGFGVA (corresponding to residues 140-150 of SEQ ID NO: 4), and/or VVCKPCAPGTFSN (corresponding to residues 159-171 of SEQ ID NO: 4) equivalent), and/or
An epitope containing at least 5 contiguous or discontinuous residues within residues 75-128, 86-103, 111-128, or 150-190 of SEQ ID NO:4. 279. The construct of any one of claims 268-278, comprising a small molecule TNFR2 antagonist. 280. The construct of claim 279, wherein the TNFR2 antagonist is thalidomide or an analog thereof. 281. The construct of claim 280, wherein the thalidomide analogs are lenalidomide and pomalidomide. 282. The construct of any one of claims 268-281 comprising a TNFR2 antagonist that reduces FoxP3 expression and inhibits the suppressive activity of Tregs. 283. The construct of claim 282, wherein the TNFR2 antagonist is a histone deacetylase inhibitor capable of reducing FoxP3 expression and inhibiting the suppressive activity of Tregs. 284. The construct of claim 282 or 283, wherein the inhibitor is panobinostat or cyclophosphamide or triptolide. 283. The construct of any one of claims 268-282 for use in the treatment of an infectious disease and a cancer expressing TNFR2. 286. The construct of claim 285, wherein the cancer is a cancer selected from: T cell lymphoma, such as Hodgkin's lymphoma and cutaneous non-Hodgkin's lymphoma, ovarian cancer, colon cancer, multiple myeloma, renal cell carcinoma, breast cancer, cervical cancer , endometrial cancer, glioma, head and neck cancer, liver cancer, and lung cancer.

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