RU2779489C2 - Antibodies binding cd3 - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, in particular to a multi-specific antibody specific to human CD3δε and a pathogen antigen, a composition containing it, as well as to a method for the production of the specified antibody. Polynucleotide encoding the above-mentioned antibody, as well as a vector and a cell, containing it, are also disclosed.

EFFECT: invention is effective for the treatment of infection.

31 cl, 6 ex, 12 dwg

Description

cross reference

This application claims priority of U.S. Provisional Application No. 62/352,698 filed June 21, 2016, U.S. Provisional Application No. 62/394,360 filed September 14, 2016, and U.S. Provisional Application No. 62/491,908 filed April 28, 2017 years, which are incorporated herein by reference in their entirety.

State of the art

The body's immune system serves as a defense against infection, injury, and cancer. Two separate but interconnected systems, the humoral and cellular immune systems, work together to protect the body. The humoral system is mediated by soluble factors called antibodies that neutralize products recognized as foreign by the body. Whereas, the cellular system includes cells such as T cells and macrophages that remove and neutralize foreign intruders.

T cell activation is critical to stimulate immune responses. T cells exhibit immunological specificity and direct most cellular immune responses. Although T cells do not secrete antibodies, they are required for B lymphocytes to secrete antibodies. T cell activation requires the participation of a number of cell surface molecules, such as the T cell receptor complex, and CD4 or CD8 molecules. The antigen-specific T-cell receptor (TCR) consists of a disulfide-linked heterodimer, a membrane glycoprotein with alpha and beta (α and β) or gamma and delta (γ and δ) chains. TCR is non-covalently bound to a complex of invariant proteins, designated CD3.

T cells are known to exert strong antitumor effects under numerous experimental conditions. Antibodies capable of effectively recruiting T cells against tumor cells have been available as bispecific antibodies, such as those directed against tumor-associated antigens (TAA) and agonistic T cell membrane proteins such as the TCR/CD3 complex. and CD28. These bispecific antibodies are capable of activating T cells regardless of their TCR specificity, resulting in specific lysis of cells bearing the respective TAAs.

However, although anti-CD3 bispecific antibodies can redirect T cell-mediated lysis to malignant cells, clinical trials with CD3-based bsAbs have shown high toxicity in patients. Non-specific activation of T cells by bsAbs can occur in an antigen-independent manner due to Fc/Fc receptor (FcR) interaction or in an antigen-dependent manner when the antigen is expressed on both normal and tumor cells. Both mechanisms may be responsible for the toxicity seen in previous clinical studies (See e.g. Link et al. (1998) Int. J. Cancer 77(2):251-6; Durben et al. Molecular Therapy (2015 ); 23 4, 648-655). Due to the resulting cytokine release syndrome, there have been significant limitations in the development of these antibodies for therapeutic purposes.

For example, Mack et al. (1995), PNAS 92:7021-7025 reported a bispecific single chain (bsoc) antibody with CD3 specificity referred to as bispecific T-cell engager (BiTE). However, as in earlier studies with various anti-CD3 bispecific antibodies, excessive T cell activation and cytokine release induced by in vivo use limit safe usable doses to less than 100 μg/day, resulting in serum concentrations below 1 ng. /ml

CD3-specific antibodies and bispecific antibodies derived from them are provided by this invention.

Publications

Antibodies to CD3 are described, for example, in US patents% 5585097; 5929212; 5968509; 6706.265; 6750325; 7381803; 7,728,114. Bispecific antibodies with CD3 binding specificity are described, for example, in US Pat. Nos. 7,262,276; 7635472; 7862813; and 8236308, each of which is specifically incorporated herein by reference.

The essence of the invention

Compositions and methods for their use are provided for a family of related antibodies that bind to and activate signaling through CD3, for example, activation of CD3 ⁺ T cells. The antibodies in the family contain a set of CDR sequences as defined herein. The antibody family provides a number of advantages that contribute to utility as a clinical drug(s). The antibodies in the family include members with a range of binding affinities allowing the selection of a particular sequence with the desired affinity. The ability to fine-tune affinity is of particular importance in order to control the level of CD3 activation in the individual being treated and thus to reduce toxicity. Members of the antibody family may have an affinity (KD) for CD3 in the range of about 10 ^-6 to about 10 ^-11 M. Some members of the antibody family of this invention cross-react with the cynomolgus CD3 protein and the necessary specific motif is identified for such a cross-reaction, such that allows selection of antibodies for preclinical or clinical trials on this basis.

Anti-CD3 antibodies that have an affinity (KD) of 50 nM or more, 100 nM or more, 500 nM or more, or 1 μM or more may be desirable to more closely mimic the TKR/MHC (major histocompatibility complex) interaction and minimize release toxic cytokines while maintaining effective lysis of tumor cells. In some embodiments, anti-CD3 antibodies are characterized or selected to reduce the propensity to induce cytokine release when bound to a competent T cell, for example, to release IL-2 and IFNγ. Antibodies can be selected for therapeutic applications that optimize tumor cell killing and reduced cytokine release, e.g., an antibody in the family of antibody sequences described herein induces cytokine release that is less than about half the maximum observed for a family member in a comparative analysis, and may be less, for example, less than and about 25% of the maximum observed for a family member in a comparative analysis.

In some embodiments, bispecific or multispecific antibodies are provided that comprise at least a heavy chain variable region from the antibody family of the invention and may comprise the heavy and light chain variable regions provided herein. Bispecific antibodies comprise at least the heavy chain variable region of an antibody specific for a protein other than CD3, and may comprise a heavy and light chain variable region. In some such embodiments, the second specificity of the antibody binds to a tumor associated antigen, a targeting antigen, eg, integrins and the like, a pathogenic antigen, a checkpoint protein, and the like. Various formats of bispecific antibodies are within the scope of the invention, including, without limitation, single chain polypeptides, double chain polypeptides, three chain polypeptides, four chain polypeptides and multiples thereof.

The family of CD3-specific antibodies of this invention contains a VH domain containing the sequences CDR1, CDR2 and CDR3 in a human VH framework. CDR sequences can be located, for example, in the region around amino acid residues 26-35; 53-59; and 98-117 for CDR1, CDR2 and CDR3, respectively, provided exemplary variable region sequences set forth in SEQ ID NOs: 1-68. One skilled in the art will appreciate that the CDR sequences may be in a different position if a different framework sequence is selected, although the order of the sequences will generally remain the same.

CDR sequences for an antibody of this invention may have the following sequence formulas. X is a variable amino acid, which may be specific amino acids as indicated below.

CDR1

_{G1 G2 S3} I _{4 x5 S6 X7 x8 x9} X ₁₀

wherein:

X ₅ can be any amino acid; in some embodiments of the invention, X ₅ is S or R;

X ₇ and X ₈ can be any amino acid; in some embodiments, X ₇ and X ₈ are independently S or G. In some embodiments, X ₇ X ₈ are SS or GG;

X ₉ can be any amino acid; in some embodiments of the invention, X ₉ is H or Y; in some embodiments of the invention, X ₉ is H;

X ₁₀ can be any amino acid; in some embodiments, X ₁₀ is Y or F; in some embodiments, X ₁₀ is Y.

In some embodiments, the CDR1 sequence has the formula: GGSIX ₅ SHHGY, with X ₅ as defined above. In some embodiments, the CDR1 sequence of an anti-CD3 antibody of the invention comprises the sequence set forth in any of SEQ ID NOs: 1-68, residues 26-35.

CDR2

I _1' X _2' X _3' S _4' G5 _' X _6' X _7'

wherein:

X _2' can be any amino acid; in some embodiments of the invention X _2' represents S, Y or H;

X _3' may be any amino acid; in some embodiments, X _3' is Y, H, or R;

X _6' can be any amino acid; in some embodiments, X _6' is S, N, or I, or R;

X _7' can be any amino acid; in some embodiments, X _7' is T or P.

In some embodiments, the CDR2 sequence has the formula: I X_2' X_3' S G S T; or I X_2' X_3' S G N P_2' and X_3' such as defined above. In some embodiments, the CDR2 sequence of an anti-CD3 antibody of the invention comprises the sequence set forth in any of SEQ ID NOs: 1-68, residues 53-59.

CDR3

X _1'' R _2'' W _3'' R _4'' H _5'' D _6'' I _7'' X _8'' X _9'' X _10'' Y _11'' P _12'' Y _{13 ''} Y _14'' Y _15'' Y _16'' G _17'' M _18'' D _19'' V _20''

wherein:

X _1'' can be any amino acid; in some embodiments, X _1'' is A or G.

X _8'' can be any amino acid; in some embodiments, X _8'' is L or F.

X _9'' can be any amino acid; in some embodiments, X _9'' is T or A

X _10'' can be any amino acid; in some embodiments, X _10'' is G, A, or R.

In some embodiments, X _8'' X _9'' X _10'' is FAA, the motif of which corresponds to antibodies that cross-react with cynomolgus CD3 protein. In other embodiments, X _8'' X _9'' X _10'' is LT A. In some embodiments, the CDR3 sequence of an anti-CD3 antibody of the invention comprises the sequence set forth in any of SEQ ID NOs: 1-68, residues 98-117.

In some embodiments, a VH CD3 binding domain is linked to a light chain variable region domain. In some such embodiments, the light chain is a fixed light chain. In some embodiments, the light chain comprises a VL domain with sequences of CDR1, CDR2, and CDR3 in a human VL framework. CDR sequences may be those contained in SEQ ID NO: 69. In some embodiments, the CDR sequence contains amino acid residues 27-32; 50-52; 89-97 for CDR1, CDR2, CDR3 respectively.

In some embodiments, the CDR sequences of an antibody of the invention are at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical to a CDR sequence or set of CDR sequences in any of SEQ ID NOs: 1-69. In some embodiments, the CDR sequence of this invention contains one, two, three or more amino acid substitutions with respect to the CDR sequence or set of CDR sequences in any of SEQ ID NOs: 1-69. In some embodiments, said amino acid substitution(s) is one or more at position 5 or 10 of CDR1, position 2, 6 or 7 of CDR2, position 1, 8, 9 or 10 of CDR3 relative to the formulas above.

In some embodiments, the bispecific antibody of the invention comprises the CD3 binding variable region described herein coupled to a light chain. In some embodiments, the light chain comprises the variable region sequence set forth in SEQ ID NO: 69 or a variable region containing the set of CDR sequences in SEQ ID NO: 69 and framework sequences. Various Fc sequences find use, including, without limitation, human IgG1, IgG2a, IgG2b, IgG3, IgG4, and the like. In some embodiments, the second arm of the bispecific antibody contains a variable region that specifically binds to a tumor-associated antigen. In some embodiments, the second arm of the bispecific antibody contains a variable region that specifically binds to BCMA. In some embodiments, the arm to BCMA is a single chain variable region, such as shown in FIG. 2B. In some embodiments, the BCMA arm comprises the variable region sequence set forth in SEQ ID NO: 70; or a tandem variable region sequence set forth in SEQ ID NO: 71. The anti-BCMA arm Fc sequence can be, without limitation, human IgG1, IgG2a, IgG2b, human IgG3, IgG4, and the like. CDR sequences may be those contained in SEQ ID NO: 70. In some embodiments, the CDR sequence contains amino acid residues 26-33; 51-58; 97-108 for CDR1, CDR2, CDR3 respectively.

In other embodiments, pharmaceutical compositions are provided comprising at least a VH CD3 binding domain of the invention, for example, a monospecific, bispecific, and the like antibody or antibody-like protein comprising at least a VH CD3 binding domain of the invention; and a pharmaceutically acceptable excipient. The composition may be lyophilized, suspended in solution, and the like. And it can be presented in the form of a standard dose.

In some embodiments, the invention provides a method of treating cancer, comprising administering to an individual having an indication for this, an effective dose of a monospecific, bispecific, or the like of an antibody of this invention. When the antibody is bispecific, the second antigen-binding site can specifically bind the tumor antigen, checkpoint protein, and the like. In various embodiments, the cancer is selected from the group consisting of ovarian cancer, breast cancer, gastrointestinal cancer, brain cancer, head and neck cancer, prostate cancer, colon cancer, lung cancer, leukemia, lymphoma, sarcoma, carcinomas, nerve cell tumors, squamous cell carcinomas, embryonic cell tumors, metastases, undifferentiated tumors, seminomas, melanomas, myelomas, neuroblastomas, mixed cell tumors, and neoplasias caused by infectious agents.

In some embodiments, the invention provides a method of treating an infectious disease, comprising administering to an individual having an indication for this, an effective dose of a monospecific, bispecific, and the like of an antibody of this invention. When the antibody is bispecific, the second antigen-binding site can specifically bind a pathogenic antigen, such as bacteria, viruses, or parasites.

In other embodiments, a method for producing a bispecific antibody of the invention is provided, comprising expressing antibody sequences, e.g., one or more light chain coding sequences, one or more heavy chain coding sequences, in a single host cell. In various embodiments, the host cell may be a prokaryotic or eukaryotic cell, such as a mammalian cell.

Brief description of graphic materials

The invention will be best understood from the following detailed description when read in conjunction with the accompanying drawings. The patent or application file contains at least one color graphic. Copies of this patent or patent application with color graphic material(s) will be made available by the Patent Office upon request and upon payment of the required fee. It should be emphasized that in accordance with established practice according to common practice, the various objects of the graphic materials are illustrated not to scale. On the contrary, the sizes of various objects are arbitrarily enlarged or reduced for clarity. The following figures are included in the graphics.

Fig. 1A-1B. (FAM1_aCD3_CDR_seqalign). In FIG. 1A illustrates the alignment of the CDR1, 2 and 3 regions of all members of the antibody family that recognizes human CD3. The CDR sequences correspond to amino acid residues 26-35; 53-59; and 98-117 for CDR1, CDR2 and CDR3, respectively, from the variable region sequences set forth in SEQ ID NOs: 1-68. In FIG. 1B illustrates fixed light chain CDR1, 2 and 3 regions (SEQ ID NO: 69); and type sequence to BCMA (SEQ ID NO: 70 and SEQ ID NO: 71).

In FIG. 2A-2E illustrate schematic models of human bispecific antibodies. In FIG. 2A illustrates an anti-CD3:tumor antigen bispecific antibody with a common light chain (total 3 unique chains). In FIG. 2B illustrates an anti-CD3:tumor antigen bispecific antibody with 2 unique light chains (4 unique chains in total). In FIG. 2C illustrates a CD3:tumor antigen bispecific antibody with a heavy chain of the tumor antigen-binding domain only (3 unique chains). In FIG. 2D illustrates an anti-CD3:tumor antigen bispecific antibody with an scFv tumor antigen-binding domain (3 unique chains in total). In FIG. 2E illustrates an anti-CD3:tumor antigen bispecific antibody with an scFv anti-CD3 binding domain (total 3 unique chains).

In FIG. 3 illustrates activation of human CD8+ T cells with the α-CD3/α-PD-L1 bispecific FlicAb. Purified human CD8+ T cells were co-cultured with bispecific at the indicated concentrations and tumor cells. Ramos (B cell lymphoma) is a PD-L1 negative cell line and HDLM2 (multiple myeloma) is a PD-L1 positive cell line. CD69 is a membrane molecule that is activated on activated T cells. The mean fluorescence intensity (MFI) of human CD8+ T cells stained with a fluorescently labeled anti-CD69 monoclonal antibody correlates with the degree of activation. Activation depended on the presence of both PD-L1-expressing and bispecific tumor cells.

In FIG. 4 illustrates tumor cell cytolysis with the α-CD3/α-PD-L1 bispecific FlicAb. Tumor cells (HDLM2) were incubated with purified human CD8+ T cells and bispecific antibodies. HDLM2 cells do not express CD20, and coculture with the α-CD3/α-CD20 bispecific FlicAb did not result in HDLM2 cell death. Only coculture of human CD8+ T cells and HDLM2 with the α-CD3/α-PD-L1 bispecific FlicAb results in significant killing. HDLM2 express PD-L1 on their surface.

In FIG. 5 illustrates a table summarizing the status of anti-CD3 antibodies in monospecific and bispecific format. Column 1 shows the sequence identifier for the VH sequence to CD3. Column 2 shows the SIF value for binding by Jurkat cells of the original monospecific (antibody) to CD3. Column 3 shows the SIF value for cynomolgus monkey T-cell binding of the original anti-CD3 monospecific (antibody). Column 4 shows the name of the aCD3:aBCMA bispecific antibody. Column 5 shows picograms of IL-2 released by pan T cells stimulated with a BCMA protein-binding bispecific antibody placed on plastic at the indicated dose. Column 6 shows picograms of IL-6 released by pan T cells stimulated with a BCMA protein-binding bispecific antibody placed on plastic at the indicated dose. Column 7 shows picograms of IL-10 released by pan T cells stimulated with a BCMA protein-binding bispecific antibody placed on plastic at the indicated dose. Column 8 shows picograms of IFN-γ released by pan-T cells stimulated with a BCMA protein-binding bispecific antibody placed on plastic at the indicated dose. Column 9 shows picograms of TNFα released by pan T cells stimulated with a BCMA protein-binding bispecific antibody placed on plastic at the indicated dose. Column 10 shows the EC50 of U266 tumor cell lysis mediated by bispecific antibodies in the presence of human pan T cells. Column 11 shows the percent lysis of U266 tumor cells in the presence of the bispecific antibody and human pan T cells at a dose of 333 ng/ml of the bispecific antibody. Column 12 shows the protein binding affinity of the arm for CD3 of the bispecific antibody as measured by Octet. Column 13 shows the SIF value for Jurkat cells to bind the bispecific antibody.

In FIG. 6 illustrates bispecific antibody-mediated tumor cell lysis. Four αCD3_fam1:aBCMA bispecific antibodies, each with a unique anti-CD3 arm and a common anti-BCMA arm, were tested for their ability to kill U266 BCMA+ tumor cells by redirecting activated primary T cells. In this experiment, U266 cells that express BCMA were mixed with activated pan T cells in an E:T ratio of 10:1 along with the addition of a bispecific antibody. The x-axis shows the concentration of the applied antibody, and the y-axis shows the % tumor cell lysis 6 hours after the addition of the antibody.

In FIG. 7 illustrates the bispecific killing activity of U266 correlated with IL-2 release. Comparison of the activity of tumor cell lysis mediated by bispecific antibodies with the release of the cytokine IL-2 is shown in the scatterplot. The correlation between IL-2 production and U266 tumor cell lysis is R ² = 0.37.

In FIG. 8 illustrates bispecific U266 killing activity correlated with IFN-γ release. Comparison of the activity of tumor cell lysis mediated by bispecific antibodies with the release of the cytokine IFN-γ is shown in the scatterplot. The correlation between IFN-γ production and U266 tumor cell lysis is R ² =0.53.

In FIG. 9 illustrates the bispecific killing activity of U266 correlated with binding affinity for CD3. Comparison of bispecific antibody-mediated U266 tumor cell lysis activity with CD3 binding affinity is depicted in a scatterplot. The correlation between EC50 killing U266 and protein binding affinity is R ² =0.93.

In FIG. 10A-10D illustrate bispecific antibody-mediated tumor cell lysis. The αCD3_F1F:αBCMA bispecific antibodies were analyzed for their ability to kill three different BCMA+ tumor cell lines and one BCMA-negative cell line by redirecting activated parental T cells. The antibodies consisted of the αCD3 arm (SEQ ID NO: 1 and SEQ ID NO: 69) and the αBCMA arm (SEQ ID NO: 70 or SEQ ID NO: 71). In this experiment, tumor cells were mixed with activated pan-T cells in an E:T ratio of 10:1 along with the addition of a bispecific antibody. In FIG. 10A illustrates the killing of RPMI-8226 cells. In FIG. 10B illustrates the killing of NCI-H929 cells, FIG. 10C illustrates the killing of U-266 cells and FIG. 10D illustrates killing of K562 cells, negative control. The x-axis shows the concentration of the applied antibody, and the y-axis shows the % tumor cell lysis 6 hours after the addition of the antibody.

In FIG. 11A-11D illustrate bispecific antibody mediated release of IL-2. The level of release of IL-2 cytokines was measured after culturing human T cells at rest with various tumor cell lines and increasing doses of the aCD3_F1F:aBCMA bispecific antibody (as illustrated in Fig. 10). In FIG. 11A illustrates IL-2 release stimulated by RPMI-8226 cells, FIG. 11B illustrates IL-2 release stimulated by NCI-H929 cells, FIG. 11C illustrates IL-2 release stimulated by U-266 cells and FIG. 11D illustrates IL-2 release stimulated by K562 cells, negative control.

In FIG. 12A-12D illustrate bispecific antibody mediated release of IFN-γ. The level of release of the cytokine IFN-γ was measured after culturing human T cells at rest with various tumor cell lines and increasing doses of the aCD3_F1F:aBCMA bispecific antibody (as illustrated in Fig. 10). In FIG. 12A illustrates IFN-γ release stimulated by RPMI-8226 cells, FIG. 12B illustrates IFN-γ release stimulated by NCI-H929 cells, FIG. 12C illustrates IFN-γ release stimulated by U-266 cells and FIG. 12D illustrates IFN-γ release stimulated by K562 cells, negative control.

Detailed Description of the Invention

To facilitate understanding of this invention, a number of terms are defined below.

Before describing these active agents and methods, it should be understood that this invention is not limited to the particular methodology, products, devices, and factors described, as such methods, devices, and formulations may, of course, be modified. It should also be understood that the terminology used herein is only intended to describe specific embodiments of the invention and is not intended to limit the scope of this invention, which will be limited only by the appended claims.

It should be noted that in this document and in the appended claims, the singular forms include the designated objects in the plural, unless otherwise directly follows from the context. Thus, for example, reference to "drug potential" refers to one or more such potential candidates, and reference to "method" includes references to equivalent steps and methods known to those skilled in the art, and so on.

Unless otherwise indicated, all technical and scientific terms used herein have the meanings normally implied by a person skilled in the art to which the invention belongs. All publications mentioned in this document are incorporated herein by reference for the purpose of describing and disclosing the devices, compositions and methods that are described in the publication and which can be used in connection with the invention described above.

Where a range of values is provided, it is to be understood that each intermediate value, up to the tenth unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limits of that range and any other specified or intermediate value within that specified range is covered by within the scope of the invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also included in this invention, subject to any specifically excluded limit in the specified range. In cases where the specified range includes one or both of these limits, the invention also includes ranges that exclude any or both of these included limits.

In the following description, numerous specific details are set forth in order to provide a more complete understanding of the present invention. However, for a person skilled in the art it will be obvious that this invention can be practiced without one or more of these specific details. In other cases, well-known features and procedures well known to those skilled in the art are not described in order to avoid obscuring the invention.

Generally, conventional methods of protein synthesis, recombinant cell culture and protein isolation, and recombinant DNA methods known to those skilled in the art are employed in the present invention. Such methods are described in detail in the literature, see, for example, Maniatis, Fritsch & Sambrook, Molecular Cloning: A Laboratory Manual (1982); Sambrook, Russell and Sambrook, Molecular Cloning: A Laboratory Manual (2001); Harlow, Lane and Harlow, Using Antibodies:A Laboratory Manual:Portable Protocol No. I, Cold Spring Harbor Laboratory (1998); and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory; (1988).

Definitions of terms

The term "comprising" means that the listed elements are required for the composition/method/kit, however, other elements may be included within the scope of the claims to form the composition/method/kit and the like.

The term "consisting essentially of" means limiting the scope of the described composition or method to the specified materials or step-by-step actions that do not significantly affect the main and new characteristic (characteristics) of the subject matter of the invention.

By "consisting of" is meant the exclusion of any element, step, or ingredient not listed in a claim from a composition, method, or kit.

The terms "drug", "treatment" and the like are used herein to generically refer to a desired pharmacological and/or physiological effect. The effect may be prophylactic in the sense of completely or partially preventing a disease or symptom thereof, and/or therapeutic in the sense of partially or completely curing a disease and/or an undesirable effect related to said disease. The term "treatment" as used herein encompasses any treatment of a disease in a mammal, and includes: (a) preventing the onset of the disease in a subject who may be predisposed to the disease but who has not been diagnosed with the disease; (b) inhibiting the disease, that is, stopping its development; or (c) alleviating the disease, ie regression of the disease. The therapeutic agent may be administered before, during, or after the onset of a disease or injury. The treatment of the current disease, in which the treatment stabilizes or alleviates the patient's undesirable clinical symptoms, is of particular interest. Such treatment is desirable to perform until the complete loss of function of the affected tissues. The drug in question can be administered during the symptomatic stage of the disease, and, in some cases, after the symptomatic stage of the disease.

"Therapeutically effective amount" means the amount of an active agent required to produce a therapeutically beneficial effect in a subject. For example, a "therapeutically effective amount" is an amount that induces, alleviates, or otherwise causes improvement in pathological symptoms, disease progression, or physiological conditions associated with the disease, or, or improves resistance to a disorder.

The terms “subject”, “individual” and “patient” are used interchangeably herein and refer to a mammal being evaluated for treatment and/or treatment. In one embodiment, the mammal is a human. The terms "subject", "individual" and "patient" include without limitation individuals with cancer, autoimmune diseases, pathogenic infections, and the like. Subjects may be humans, but include other mammals particularly useful as laboratory models of human diseases, such as mice, rats, and the like.

The terms "cancer", "neoplasm" and "tumor" are used interchangeably herein to refer to cells that exhibit autonomous unregulated growth such that they exhibit an aberrant growth phenotype characterized by a significant loss of control over cell proliferation. Cells of interest for detection, analysis, or treatment in this application include precancerous (eg, benign), malignant, pre-metastatic, metastatic, and non-metastatic cells. Cancer of almost every tissue is known. The phrase "cancer burden" refers to the number of cancer cells or the volume of cancer in a subject. Accordingly, a reduction in cancer burden means a reduction in the number of cancer cells or the volume of cancer in a subject. In this context, the term "cancer cell" refers to any cell that is a cancer cell or derived from a cancer cell, such as a clone of a cancer cell. Many types of cancers are known to those skilled in the art, including solid tumors such as carcinomas, sarcomas, glioblastomas, melanomas, lymphomas, myelomas, and the like, and circulating malignancies such as leukemias, including but not limited to B-cell leukemias. , T-cell leukemias and the like. Examples of cancer include, but are not limited to, ovarian cancer, breast cancer, colon cancer, lung cancer, prostate cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer urinary tract, thyroid cancer, kidney cancer, carcinoma, melanoma, head and neck cancer, and brain cancer.

The terms "antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated response in which non-specific cytotoxic cells that express Fc receptors, such as natural killer cells, neutrophils, and macrophages, recognize the bound antibody on the target cell and cause lysis of the target cell. . ADCC activity can be assessed using methods such as those described in US Pat. No. 5,821,337. ADCC refers to antibody-dependent cell-mediated phagocytosis.

"Effector cells" are leukocytes that express one or more constant region receptors and perform effector functions.

A "cytokine" is a protein released by one cell to act on another cell as an intercellular mediator.

The term "non-immunogenic" refers to a material that does not initiate, provoke, or enhance an immune response, which immune response includes an adaptive and/or innate immune response.

The term "isolated" means that the material is removed from its original environment (eg, the natural environment, if it occurs naturally). For example, a natural polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide separated from materials co-existing with it in the natural system is isolated. Such nucleotides may be included in a vector, and/or such polynucleotides or polypeptides may be included in a composition and still be isolated in the sense that such vector or composition is not part of its natural environment.

By "pharmaceutically acceptable substance" is meant an excipient which can be used in the preparation of a pharmaceutical composition and which is generally safe, non-toxic and desirable; this term includes excipients acceptable for both veterinary use and pharmaceutical use in the treatment of humans. Such excipients may be solid, liquid, semi-liquid or, in the case of an aerosol composition, gaseous.

By "pharmaceutically acceptable salts and esters" is meant salts and esters that are pharmaceutically acceptable and have the desired pharmacological properties. Such salts include salts which may be formed by the reaction of acidic protons present in the compounds with inorganic or organic bases. Suitable inorganic salts include alkali metal salts such as sodium and potassium, magnesium, calcium and aluminum. Suitable organic salts include those derived from organic bases, eg amine bases, eg ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine and the like. Such salts also include acid addition salts formed with inorganic acids (eg, hydrochloric and hydrobromic acid) and organic acids (eg, acetic acid, citric acid, maleic acid, and alkane and arenesulfonic acids, such as methanesulfonic acid and benzenesulfonic acid). Pharmaceutically acceptable esters include esters derived from carboxy, sulfonyloxy and phosphonoxy groups present in the compounds, eg C _1-6 alkyl esters. When two acidic groups are present, the pharmaceutically acceptable salt or ester may be a monoacid-monosalt or ester, or a double salt or ester, and similarly, when more than two acidic groups are present, some or all of such groups may be in salt form or esterified form. The compounds whose names are mentioned in this invention may be present outside the salt or ester form, or in the form of a salt and/or ester, and the name of such compounds is intended to include both the parent compound (not a salt and not an ester) and its pharmaceutically acceptable salts and esters. In addition, some compounds named in this invention may be present in more than one stereoisomeric form, and the names of such compounds are intended to include all single stereoisomers and all mixtures (racemic or otherwise) of such stereoisomers.

The terms "pharmaceutically acceptable", "physiologically tolerable" and their grammatical forms in relation to compositions, carriers, diluents and reagents are used on an equal footing and mean that these materials can be administered or applied to the human body without undesirable physiological effects that could prevent composition introduction.

"Homology" between two sequences is defined by sequence identity. If the two sequences to be compared to each other differ in length, sequence identity preferably refers to the percentage of nucleotide residues of the shorter sequence that are identical to the nucleotide residues of the longer sequence. Typically, sequence identity can be determined using computer programs such as Bestfit (Wisconsin Sequence Analysis Package, version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive Madison, Wis. 53711). The Bestfit program uses the local Smith-Waterman homology algorithm (Smith and Waterman), Advances in Applied Mathematics 2 (1981), 482-489, to find the segment with the highest sequence identity between two sequences. When using Bestfit or another sequence alignment program to determine if a particular sequence is, for example, 95% identical to the reference sequence of the present invention, the parameters are preferably adjusted so that the percent identity is calculated over the entire length of the reference sequence, and when this to allow gaps in homology up to 5% of the total number of nucleotides in the reference sequence. In the case of Bestfit, the so-called optional parameters are preferably left at their preset ("default") values. Deviations that appear when comparing this sequence and the sequences described above, proposed in this invention, may be caused by, for example, addition, deletion, substitution, insertion or recombination. Such sequence comparisons can preferably be performed using the program "fasta20u66" (Version 2.0u66, September 1998, by William R. Pearson and the University of Virginia; see also W.R. Pearson (1990), Methods in Enzymology 183, 63-98 , attached examples, and http://workbench.sdsc.edu/). The "default" parameter settings may be used for this purpose.

The term "variant" refers to polypeptides having amino acid sequences that differ to some extent from the native sequence polypeptide. Typically, amino acid sequence variants will have at least about 80% sequence identity, more preferably at least about 90% sequence homology. Amino acid sequence variants may have substitutions, deletions and/or insertions at certain positions in the reference amino acid sequence.

The term "vector" in the context of this document means a nucleic acid molecule capable of carrying another nucleic acid connected to it. One type of vector is a "plasmid", which refers to a cyclic double-stranded loop of DNA into which additional DNA segments can be ligated. Another type of vector is a viral vector in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). Other vectors (eg, non-episomal mammalian vectors) can be integrated into the genome of a host cell when introduced into the host cell and thus replicate along with the host genome. Moreover, some vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "recombinant vectors"). In general, expression vectors used in recombinant DNA technologies are often in the form of plasmids. In this specification, "plasmid" and "vector" may be used interchangeably, as the plasmid is the most commonly used form of the vector.

As used herein, the term "host cell" (or "recombinant host cell") is intended to refer to a cell that has been genetically altered or is capable of being genetically altered by the introduction of an exogenous polynucleotide, such as a recombinant plasmid or vector. It should be understood that such terms are intended to refer not only to the specific cell under study, but also to cells derived from it. Since some modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may not be essentially identical to the parent cell, but are still included in the term "host cell" as used herein.

"Binding affinity" generally refers to the strength of the total number of non-covalent interactions between one binding site of a molecule (eg, an antibody or other binding molecule) and its binding partner (eg, an antigen or receptor). The affinity of a molecule X for its partner Y can generally be represented as a dissociation constant (Kd). Affinity can be measured using conventional methods known in the art, including those described herein. Low affinity antibodies bind the antigen (or receptor) weakly and tend to dissociate easily, while high affinity antibodies bind the antigen (or receptor) more tightly and tend to stay bound longer.

Unless specifically stated otherwise, the term "conjugate", as described and stated herein, is defined as a heterogeneous molecule formed by the covalent attachment of one or more antibody fragments to one or more polymer molecule(s), wherein the heterogeneous molecule is soluble in water, that is, soluble in bodily fluids such as blood, yet the heterogeneous molecule is free of any structured aggregate. The conjugate of interest is PEG. In the context of the above definition, the term "structured aggregate" refers to (1) any aggregate of molecules in aqueous solution having a spheroid or spheroid shell structure such that the heterogeneous molecule is not in a micelle or other emulsion structure, and is not fixed in a lipid bilayer, vesicle or liposome; and (2) any aggregate of molecules in solid or insoluble form, such as an array of chromatographic beads, that does not release the heterogeneous molecule into solution upon contact with the aqueous phase. Accordingly, the term "conjugate" as defined herein encompasses the aforementioned heterogeneous molecule as a precipitate, precipitate, biodegradable matrix, or other solid capable of releasing the heterogeneous molecule into aqueous solution upon hydration of the solid.

The word "label" as used herein refers to a detectable compound or composition that is directly or indirectly conjugated to an antibody. The label itself may be detectable per se (eg, radioisotope or fluorescent labels) or, in the case of an enzymatic label, may catalyze such chemical changes in the substrate compound or composition as are detectable.

By "solid phase" is meant an anhydrous matrix to which an antibody of the invention can adhere. Examples of solid phases covered by this document include those formed partially or wholly from glass (eg, controlled pore glass), polysaccharides (eg, agarose), polyacrylamides, polystyrene, polyvinyl alcohol, and silicones. In certain embodiments of the invention, depending on the context, the solid phase may contain the well of the analytical tablet; in others it is a purification column (for example, an affinity chromatography column). The term also includes a discontinuous solid phase of discrete particles, such as described in US patent No. 4,275,149.

Antibodies, also called immunoglobulins, generally contain at least one heavy chain and one light chain, wherein the amino-terminal domain of the heavy and light chains is variable in sequence and is therefore commonly referred to as a variable region domain, or a heavy chain variable domain (VH) and a variable domain light chain (VH). These two domains typically bind to each other to form a specific binding region, although it is also discussed here that specific binding can also be obtained using only heavy chain variable sequences, and a large number of non-natural antibody configurations are known and used in the art.

A "functional" or "biologically active" antibody or antigen-binding molecule (including heavy chain-only antibodies and bispecific three-chain antibody-like molecules (TCAs) herein) is an antibody or molecule capable of exhibiting one or more of their natural activities in structural, regulatory, biochemical or biophysical phenomena. For example, a functional antibody or other binding molecule, such as TCA, may have the ability to specifically bind an antigen, and this binding, in turn, may cause or alter cellular or molecular events, such as signal transduction or enzymatic activity. A functional antibody or other binding molecule, such as TCA, can also block ligand or receptor activation or act as an agonist or antagonist. The ability of an antibody or other binding molecule, such as TCA, to exhibit one or more of its natural activities depends on several factors, including the correct folding and assembly of polypeptide chains.

The term "antibody" in this document is used in its broadest sense; it specifically encompasses monoclonal antibodies, polyclonal antibodies, monomers, dimers, multimers, polyspecific antibodies (e.g., bispecific antibodies), heavy chain-only antibodies, three-chain antibodies, single-chain Fv fragments, nanobodies, and the like, and also includes antibody fragments, provided they have the desired biological activity (Miller et al. (2003) Jour. of Immunology 170:4854-4861). The antibodies may be murine, human, humanized, chimeric, or may be derived from other animal species.

The term antibody may refer to a full length heavy chain, a full length light chain, an intact immunoglobulin molecule; or an immunologically active fragment of any of these polypeptides, that is, a polypeptide containing an antigen-binding site that immunospecifically binds an antigen or target of interest, or a fragment thereof, and such targets include a cancer cell or cells producing autoimmune antibodies associated with an autoimmune disease, but are not limited to them. An immunoglobulin described herein may comprise any suitable Fc region, including, without limitation, IgG, IgE, IgM, IgD, IgA, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2 of a human or other mammalian, e. immunoglobulin molecules, including engineered subclasses with altered Fc fragments that reduce or enhance the activity of effector cells. Immunoglobulins can be derived from any animal species. In one aspect, the immunoglobulin is primarily of human origin.

The term “variable” refers to the fact that the sequences of certain portions of the variable domains vary greatly between antibodies and contribute to the binding and specificity of each particular antibody for its particular antigen. At the same time, variability is not evenly distributed across the variable domains of antibodies. It is concentrated in three segments of both the light and heavy chain variable domains called hypervariable regions. More conserved fragments of variable domains are called framework regions (FRs). Each of the native heavy and light chain variable domains contains four FRs, predominantly taking on a beta-sheet configuration, connected by three hypervariable regions that form loops connecting the beta-sheet structure, and in some cases forming part of the beta-sheet structure. The hypervariable regions of each chain are associated with each other in close proximity to the FR and, together with the hypervariable regions of the other chain, are involved in the formation of the antigen-binding site of antibodies (see Kabat et al. (1991), Sequences of Proteins of Immunological Interest, 5th Ed. . Public Health Service, National Institutes of Health, Bethesda, MD). The constant domains are not directly involved in antibody-antigen binding, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity (ADCC).

The term "hypervariable region", as used herein, refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region may contain amino acid residues from the "complementarity determining region" or "CDR" and/or these residues from the "hypervariable loop". "Framework region" or "FR" residues are variable domain residues other than hypervariable region residues as defined herein.

Variable regions of interest include at least one CDR sequence from the variable regions provided herein, typically at least 2 CDR sequences, and more typically 3 CDR sequences. Generic CDR designations are shown herein, however, one skilled in the art will appreciate that a number of CDR definitions are commonly used, including the Kabat definition (see "Zhao et al., A germline knowledge based computational approach for determining antibody complementarity determining regions". Mol Immunol 2010; 47:694-700), which is based on sequence variability and is the most commonly used. Chothia's definition is based on the location of structural loop regions (Chothia et al. "Conformations of immunoglobulin hypervariable regions." Nature. 1989; 342:877-883). Alternative definitions of CDR of interest include, without limitation, those disclosed in Honegger, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool." J Mol Biol. year 2001; 309:657-670; Ofran et al. "Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B cell epitopes." J Immunol. 2008; 181:6230-6235; Almagro "Identification of differences in the specificity-determining residues of antibodies that recognize antigens of different size: implications for the rational design of antibody repertoires." J Mol Recognit. 2004; 17:132-143; and Padlanet et al. "Identification of specificity-determining residues in antibodies." Faceb J. 1995; 9:133-139., each of which is specifically incorporated herein by reference.

The term "monoclonal antibody" as used herein refers to an antibody derived from a population of substantially homogeneous antibodies, i.e., individual antibodies within the population are identical except for naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific because they are directed against a single antigenic site. In addition, compared to polyclonal antibody preparations that contain different antibodies against different determinants (epitopes), each monoclonal antibody is directed against a single antigen determinant. In addition to specificity, monoclonal antibodies have the advantage that they can be synthesized in a form that is not contaminated with other antibodies. The modifier "monoclonal" indicates that the antibody is derived from a substantially homogeneous population of antibodies; it should not be interpreted as requiring the production of an antibody by any particular method.

Antibodies herein include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequences of antibodies of a particular animal species or antibodies belonging to a particular class or subclass, while the remainder of the chain(s) is identical or homologous to the corresponding sequences of antibodies from another animal species or antibodies belonging to another class or subclass, as well as fragments of such antibodies, provided that they demonstrate the desired biological activity (US patent No. 4,816,567; and Morrison et al. (1984) Proc. Natl Acad. Sci. USA, 81:6851-6855). Chimeric antibodies of interest include "primatized" antibodies containing variable domain antigen-binding sequences derived from a non-human primate (eg, Old World marmosets, monkeys, and the like) and human constant region sequences.

An "intact antibody chain" as used herein is a chain containing a full length variable region and a full length constant region. An intact "standard" antibody contains an intact light chain and an intact heavy chain, as well as a light chain constant domain (CL) and heavy chain constant domains - CH1, hinge, CH2 and CH3 (for secreted IgG). Other isotypes, such as IgM or IgA, may contain other CH domains. The constant domains can be native sequence constant domains (eg, human native sequence constant domains) or amino acid sequence variants thereof. An intact antibody may have one or more "effector functions" that refer to its biological activity attributable to the Fc constant region (native sequence Fc region or amino acid sequence of a variant Fc region) of the antibody. Examples of antibody effector functions include C1q binding; complement-dependent cytotoxicity; binding to the Fc receptor; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and downregulation of cell surface receptors. Variants of the constant region include options that change the effector profile, binding to Fc receptors, and the like.

Depending on the amino acid sequences of the heavy chain constant domains, intact antibodies can be assigned to different "classes". There are five major classes of intact immunoglobulin antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further subdivided into "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structure and three-dimensional configuration of various classes of immunoglobulins are well known. Ig forms include hinge modifications or hingeless forms (Roux et al. (1998) J. Immunol. 161:4083-4090; Lund et al. (2000) Eur. J. Biochem. 267:7246-7256; USA 2005 /0048572; USA 2004/0229310). The light chains of antibodies from any vertebrate species can be assigned to one of two distinct types, called κ and λ chains, based on the amino acid sequence of their constant domains.

A "functional Fc region" has an "effector function" of a native sequence Fc region. Exemplary effector functions include C1q binding; CPC (complement dependent cytotoxicity); Fc receptor binding; ADCC; ADCP (antibody-dependent cellular phagocytosis); downregulation of cell surface receptors (eg B-cell receptor) and the like. Such effector functions typically require interaction of the Fc region with a receptor, such as FcγRI receptors; FcγRIIA; FcγRIIB1; FcγRIIB2; FcγRIIIA; FcγRIIIB, and a low affinity FcRn receptor; their evaluation can be performed using various analyzes described, for example, in the definitions in this document. A "dead" Fc region is an Fc region mutated to remain active in relation to, for example, increased serum half-life, but does not activate a high affinity Fc receptor.

The "native sequence of the Fc region" contains an amino acid sequence identical to the amino acid sequence of the naturally occurring Fc region. Native human sequence Fc regions include the native sequence of the human IgG1 Fc region (other than the A and A allotypes); native sequence of the human IgG2 Fc region; native sequence of the human IgG3 Fc region; and the native sequence of the human IgG4 Fc region, as well as naturally occurring variants thereof.

A "variant Fc region" contains an amino acid sequence that differs from the native sequence of the Fc region by modifying at least one amino acid, preferably by substituting one or more amino acids. Preferably, the variant Fc region contains at least one amino acid substitution compared to the native sequence Fc region or Fc region of the parent polypeptide, for example, from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in native sequence Fc-region or Fc-region of the original polypeptide. The variant Fc region of the present invention preferably shares at least about 80% homology with the native sequence of the Fc region and/or with the Fc region of the parent polypeptide, and most preferably at least about 90% homology with them, more preferably at least about 95% homology to them.

Variant Fc sequences may contain three amino acid substitutions in the CH2 region to reduce FcγRI binding at positions 234, 235 and 237 according to the EC index (see Duncan et al. (1988) Nature 332:563). Two amino acid substitutions at complement binding site C1q at positions 330 and 331, according to the EC index, impair complement fixation (see, Tao et al., J. Exp. Med. 178:661 (1993) and Canfield and Morrison, J. Exp Med. 173:1483 (1991)). Replacing residues at positions 233-236 of human IgG1 or IgG2 and at positions 327, 330 and 331 of IgG4 significantly attenuates ADCC and CDC (see, for example, Armor KL. et al., 1999 Eur J Immunol. 29(8):2613 -24 and Shields RL et al 2001 J Biol Chem 276(9):6591-604). Other variants of the Fc region are possible, including, without limitation, a variant in which the region capable of forming a disulfide bond is removed, or a variant in which some amino acid residues at the N-terminus of the native Fc form are removed or a methionine residue is added to them . Thus, in one embodiment of the present invention, one or more of the Fc fragments of the scFc molecule may contain one or more mutations in the hinge region to eliminate the possibility of formation of disulfide bonds. In yet another embodiment, the Fc hinge region may be completely removed. In yet another embodiment, the molecule may contain a variant Fc region.

In addition, a variant Fc region can be engineered to remove or greatly attenuate effector functions through substitution, deletion, or addition of amino acid residues to affect complement or Fc receptor binding. As a non-limiting example, a deletion at a complement binding site, such as the C1q binding site, is possible. Methods for obtaining such derived immunoglobulin Fc fragment sequences are described in International Patent Publication Nos. WO 97/34631 and WO 96/32478. In addition, the Fc domain can be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, and the like.

The Fc region may be in a form containing native carbohydrate chains, an increased number of carbohydrate chains compared to the native form, or a reduced number of carbohydrate chains compared to the native form, or may be in an aglycosylated or deglycosylated form. The increase, decrease, removal or other modifications of carbohydrate chains can be carried out using methods often used in the art, for example, a chemical method, an enzymatic method, or through expression in a genetically engineered producer cell line. Such cell lines may include microorganisms, eg Pichia pastoris and a mammalian cell line, eg CHO cells, which naturally express glycosylation enzymes. In addition, microorganisms or cells can be engineered to express glycosylation enzymes or rendered incapable of expressing glycosylation enzymes (see, for example, Hamilton et al., Science, 313:1441 (2006); Kanda, et al., J. Biotechnology, 130:300 (2007) Kitagawa et al J Biol Chem 269(27):17872 (1994) Ujita-Lee et al J Biol Chem 264(23): 13848 (1989); Imai-Nishiya et al., BMC Biotechnology 7:84 (2007); and WO 07/055916). As an example of engineered cells with modified sialylation activity, the alpha-2,6-sialyltransferase 1 gene was recombinantly introduced into Chinese hamster ovary cells and sf9 cells. Antibodies expressed by these recombinant cells are sialylated with the exogenous gene product. An additional method for obtaining Fc molecules with a modified number of carbohydrate residues compared to a plurality of native molecules involves separating said plurality of molecules into glycosylated and non-glycosylated fractions, for example, using lectin affinity chromatography (see, for example, WO 07/117505). It has been shown that the presence of specific glycosylation functional groups leads to a change in the function of immunoglobulins. For example, removal of carbohydrate chains from the Fc molecule results in a dramatic decrease in binding affinity for the C1q fragment of the first complement component C1 and a reduction or loss of antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CCC) and, therefore, no induction of unnecessary immune responses in vivo. Additional important modifications include sialylation and fucosylation: the presence of sialic acid in IgG correlated with anti-inflammatory activity (see, for example, Kaneko, et al., Science 313:760 (2006)), while the removal of fucose from IgG leads to increased ADCC activity (see, for example, Shoj-Hosaka, et al., J. Biochem., 140:777 (2006)).

In alternative embodiments, the antibodies of this invention may contain an Fc sequence with enhanced effector functions, such as increased FcγRIIIA binding and increased ADCC activity. For example, attachment of fucose to an N-linked glycan at the Asn-297 region of Fc sterically hinders Fc interaction with FcγRIIIA, and removal of fucose by glycoengineering can enhance binding to FcγRIIIA, resulting in a >50-fold increase in ADCC activity compared to control wild-type IgG1. . Protein engineering makes it possible to obtain various variants with increased Fc binding affinity for FcγRIIIA due to amino acid mutations in the IgG1 Fc region. Notably, the alanine triple mutant S298A/E333A/K334A shows a 2-fold increase in FcγRIIIA binding and ADCC function. Variants S239D/I332E (2X) and S239D/I332E/A330L (3X) are characterized by a significant increase in binding affinity for FcγRIIIA and increased ADCC in vitro and in vivo. Other Fc region variants detected by phage display also showed improved FcγRIIIA binding and increased tumor cell killing in mouse xenograft models. See, for example, Liu et al. (2014) JBC 289(6):3571-90, specifically incorporated herein by reference.

The term "antibody containing an Fc region" refers to an antibody containing an Fc region. The C-terminal lysine residue (residue 447 according to the EC numbering system) in the Fc region can be removed, for example, by purification of the antibody or by recombinant engineering of the nucleic acid encoding the antibody. Accordingly, an antibody containing an Fc region of the invention may include an antibody with or without K447.

"Fv" is the minimum antibody fragment containing a complete antigen recognition and antigen binding site. The CD3 binding antibodies of the invention comprise a dimer of one heavy chain variable region domain and one light chain variable region domain joined by a rigid non-covalent bond; however, additional antibodies, for example, for use in a multispecific configuration, may contain a VH in the absence of a VL sequence. Even a single variable domain (or half of an Fv containing only three antigen-specific hypervariable regions) has the ability to recognize and bind an antigen, although the affinity may be lower than that of the binding site of the two domains.

The Fab fragment also contains a light chain constant domain and a heavy chain first constant domain (CH1). Fab' fragments differ from Fab fragments in an additional few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. In this description, Fab'-SH is the designation Fab', in which the cysteine(s) residue(s) of the constant domains bear at least one free thiol group. F(ab') ₂ fragments of the antibody were originally generated as pairs of Fab' fragments with hinged cysteine residues between them. Other variants of chemical binding of antibody fragments are also known.

"Humanized" forms of non-human (eg, rodent) antibodies, including single chain antibodies, are chimeric antibodies (including single chain antibodies) containing minimal sequences derived from a non-human immunoglobulin. See, for example, Jones et al. (1986) Nature 321:522-525; Chothia et al. (1989) Nature 342:877; Riechmann et al., (1992) J. Mol. Biol. 224, 487-499; Foote and Winter, (1992) J. Mol. Biol. 224:487-499; Presta et al., (1993) J. Immunol. 151, 2623-2632; Werther et al., (1996) J. Immunol. Methods 157:4986-4995; and Presta et al., (2001) Thromb. haemost. 85:379-389. See US Pat. Nos. 5,225,539 for additional information; 6,548,640; 6,982,321; 5,585,089; 5,693,761; 6,407,213; Jones et al. (1986) Nature, 321:522-525; and Riechmann et al. (1988) Nature 332:323-329.

As used herein, the term "single chain antibody" means a single polypeptide chain containing one or more antigen-binding domains that bind an antigen epitope, such domains being derived from or having sequence identity with an antibody heavy or light chain variable region. Portions of such a variable region may be encoded by V _H or V _L gene segments, D and J _H gene segments, or J _L gene segments. The variable region may be encoded by rearranged V _H DJ _H , V _L DJ _H , V _H J _L or V _L J _L gene segments. The V-, D-, and J- gene segments can be derived from humans and various animals, including birds, fish, sharks, mammals, rodents, non-human primates, camels, llamas, rabbits, and the like.

The CD3 binding antibodies of this invention find particular use in multispecific configurations, which include, without limitation, bispecific antibodies, trifunctional antibodies, and the like. A wide variety of protein methods and configurations are known and used in bispecific monoclonal antibodies (BsAbs), trispecific antibodies, and the like.

The first generation BsAbs consisted of two heavy and two light chains, one each from two different antibodies. Two Fab regions are directed against two antigens. The Fc region consists of two heavy chains and forms a third binding site for the Fc receptor on immune cells (see, for example, Lindhofer et al., The Journal of Immunology, Vol. 155, pp. 219-225, 1995). Antibodies can be from the same or different species. For example, cell lines expressing rat and mouse antibodies secrete a functional bispecific Ab due to a preferential species-limited pairing of heavy and light chains. In other embodiments of the invention, the Fc regions are designed such that they only fit together in certain ways.

Other types of bispecific antibodies include chemically linked Fabs consisting of only Fab regions. Two chemically linked Fab or Fab2 fragments form an artificial antibody that binds to two different antigens, making it a type of bispecific antibody. Antigen-binding fragments (Fab or Fab2) of two different monoclonal antibodies are produced, linked by chemical means such as a thioether (see, Glennie, M J et al., Journal of immunology 139, pp. 2367-75, 1987; Peter Borchmann et al., Blood, Vol. 100, No. 9, pp. 3101-3107, 2002).

Various other methods for producing polyvalent artificial antibodies have been developed by recombinant fusion of the variable domains of two antibodies. A single chain variable fragment (scFv) is a fusion protein of immunoglobulin heavy (VH) and light chain (VL) variable regions linked to a short linker peptide of 10-25 amino acids. Typically, the linker is saturated with glycine for flexibility and serine or threonine for solubility, and may connect the N-terminus of the VH to the C-terminus of the VL, or vice versa. Bispecific single chain variable fragments (di-scFvs, bi-scFvs) can be constructed by linking two scFvs with different specificities. One peptide chain with two VH and two VL regions is produced, resulting in divalent scFvs.

Bispecific tandem scFvs are also known as T-cell-attracting bispecific activators (BiTEs). Bispecific scFvs can be created with linker peptides that are too short for two variable regions to be folded together (about five amino acids long), causing the scFvs to dimerize. This type is known as a diabody (Adams et al., British journal of cancer 77, pp. 1405-12, 1998). Dual-Affinity Re-Targeting (DART) Platform Technology (Macrogenics, Rockville, MD). This protein fusion technology uses two single chain variable fragments (scFv) of different antibodies on a single peptide chain of about 55 kilodaltons. SCORPION Therapeutics (Emergent Biosolutions, Inc., Seattle, WA) combines two antigen-binding domains into a single-stranded protein. One binding domain is at the C-terminus and the second binding domain is at the N-terminus of an effector domain based on immunoglobulin Fc regions.

Tetravalent and bispecific antibody-like proteins also include DVD-Ig, which are constructed from two monoclonal antibodies (Wu, C. et al., Nature Biotechnology, 25, pp. 1290-1297, 2007). To construct the DVD-Ig molecule, the V domains of the two mAbs are fused in tandem using a short linker (TVAAP) with the light chain variable domain (VL) of the first antibody at the N-terminus followed by the VL and Ck of the other antibodies to form the light chain of the DVD- Ig. Similarly, the heavy chain variable regions (VH) of two mAbs are fused in tandem using a short linker (ASTKGP) with the first antibody at the N-terminus followed by the other antibody and the heavy chain constant domains to form the heavy chain (VH1/VL1) of the protein. DVD-Ig. All light chain and heavy chain constant domains are retained in the DVD-Ig construct as they are critical for the formation of a disulfide-linked complete IgG-like molecule. Co-transfection of mammalian cells with expression vectors encoding the DVD-Ig light chain and heavy chain results in the secretion of a single species IgG-like molecule with a molecular weight of approximately 200 kDa. This molecule now has four binding sites, 2 from each mAb.

The term "bispecific three-chain antibody-like molecule" or "TCA" is used herein to refer to antibody-like molecules comprising, consisting essentially of, or consisting of three polypeptide subunits, two of which include, consist essentially of, or consist of one heavy and one light chain of a monoclonal antibody, or functional antigen-binding fragments of such antibody chains, comprising an antigen-binding region and at least one CH domain. This heavy chain/light chain pair has binding specificity for the first antigen. The third polypeptide subunit contains, consists essentially of, or consists of a heavy chain-only antibody comprising an Fc region containing CH2 and/or CH3 and/or CH4 domains, in the absence of a CH1 domain, and an antigen-binding domain that binds an epitope of a second antigen or other an epitope of the first antigen, wherein such binding domain is derived from or has sequence identity with the heavy or light chain variable region of the antibody. Portions of such a variable region may be encoded by V _H or V _L gene segments, D and J _H gene segments, or J _L gene segments. The variable region may be encoded by rearranged V _H DJ _H , V _L DJ _H , V _H J _L or V _L J _L gene segments.

A TCA protein using "heavy chain-only antibody" or "heavy chain antibody" or "heavy chain polypeptide", as used herein, means a single chain antibody containing CH2 and/or CH3 and/or CH4 heavy chains, but lacking a CH1 domain . In one embodiment, the heavy chain antibody consists of an antigen-binding domain, at least a portion of the hinge region, and CH2 and CH3 domains. In another embodiment, the heavy chain antibody consists of an antigen-binding domain, at least a portion of the hinge region, and a CH2 domain. In a further embodiment, the heavy chain antibody consists of an antigen-binding domain, at least a portion of the hinge region, and a CH3 domain. Heavy chain antibodies in which the CH2 and/or CH3 domain is truncated are also included in this document. In a further embodiment, the heavy chain consists of an antigen-binding domain and at least one CH domain (CH1, CH2, CH3, or CH4), but without the hinge region. The heavy chain-only antibody may be in the form of a dimer in which two heavy chains are disulfide-linked to each other or covalently or non-covalently linked to each other. The heavy chain antibody may belong to the IgG subclass, but antibodies belonging to other subclasses such as the IgM, IgA, IgD and IgE subclass are also included in this document. In a particular embodiment, the heavy chain antibody is of the IgG1, IgG2, IgG3, or IgG4 subtype, in particular the IgG1 subtype.

Heavy chain antibodies make up about one-fourth of the IgG antibodies produced by camelids, such as camelids and llamas (Hamers-Casterman C., et al. Nature. 363, 446-448 (1993)). These antibodies are formed by two heavy chains but lack light chains. As a consequence, the variable antigen-binding portion is referred to as the VHH domain and is the smallest naturally occurring intact antigen-binding site only about 120 amino acids long (Desmyter A., et al. J. Biol. Chem. 276, 26285-26290 (2001)). Heavy chain antibodies with high specificity and affinity can be produced against various antigens by immunization (van der Linden, R. H., et al. Biochim. Biophys. Acta. 1431, 37-46 (1999)), and the VHH part can be easily cloned and expressed in yeast (Frenken, L.G.J., et al. J. Biotechnol. 78, 11-21 (2000)). Their levels of expression, solubility and stability are significantly higher than those of classical F(ab) or Fv fragments (Ghahroudi, M. A. et al. FEBS Lett. 414, 521-526 (1997)). Sharks have also been shown to have a single VH-like domain in their antibodies, called VNAR. (Nuttall et al. Eur. J. Biochem. 270, 3543-3554 (2003); Nuttall et al., Function and Bioinformatics 55, 187-197 (2004); Dooley et al., Molecular Immunology 40, 25- 33 (2003)).

An antibody or antigen-binding molecule, including heavy chain-only antibodies and bispecific three-chain antibody-like molecules (TCAs) herein, "which binds" an antigen of interest, is a molecule that binds the antigen with sufficient affinity such that the antibody or binding molecule useful as a diagnostic and/or therapeutic agent when targeting an antigen, and has no significant cross interaction with other proteins. In such embodiments, the degree of binding of the antibody or other binding molecule to the non-target antigen will be no more than 10%, as determined by the fluorescence activated cell sorting (FACS) or radioimmunoprecipitation (RIA) assay.

Proteins of the Invention

The present invention provides a family of related antibodies that bind to and activate signaling through CD3, for example by activating CD3 ⁺ T cells. The antibodies in the family comprise a set of CDR sequences as defined herein, exemplified by the provided VH sequences of SEQ ID NOs: 1-68 and the exemplary VL sequence of SEQ ID NOs: 69. The antibody family provides a number of advantages that contribute to utility as a clinical drug(s). The antibodies in the family include members with a range of binding affinities allowing the selection of a particular sequence with the desired affinity. The ability to fine-tune affinity is of particular importance in order to control the level of CD3 activation in the individual being treated and thus to reduce toxicity. For example, if low levels of tumor antigens (less than 10,000 molecules per cell) are targeted, high affinity CD3 binders (<30 nM) are expected to be used. If tumor antigens present in high abundance (greater than 50,000 molecules per cell) are the target, low affinity CD3 binders (>50 nM) are preferred.

A suitable antibody may be selected from a library for development and use, including, without limitation, use as a bispecific antibody. Determination of affinity for a candidate protein can be performed using methods known in the art, for example, Biacore measurements and the like. Members of the antibody family may have an affinity for CD3 with Kd from about 10 ^-6 to about 10 ^-11 , including without limitation: from about 10 ^-6 to about 10 ^-10 ; from about 10 ^-6 to about 10 ^-9 ; from about 10 ^-6 to about 10 ^-8 ; from about 10 ^-8 to about 10 ^-11 ; about 10 ^-8 to about 10 ^-10 ; from about 10 ^-8 to about 10 ^-9 ; from about 10 ^-9 to about 10 ^-11 ; from about 10 ^-9 to about 10 ^-10 ; or any value in those ranges. The choice of affinity can be confirmed by biological assessment of T cell activation, for example, in vitro or in a preclinical model, and an assessment of potential toxicity.

Certain members of the antibody family of this invention cross-react with the cynomolgus CD3 protein and the required specific motif is identified for such cross-reactivity, allowing antibodies to be selected for preclinical or clinical trials on that basis. Antibodies having the FAA amino acid motif in the heavy chain CDR3 have been found to be particularly effective in cross-reacting with non-human primate CD3 proteins.

Engagement of the T cell receptor (TCR) by binding of MH-peptide complexes or anti-TCR/CD3 antibodies initiates T cell activation. Examples of anti-TCR/CD3 antibodies that activate T cells are OKT3 and UCHT1. These anti-CD3 antibodies cross-compete for binding to CD3 on T cells and are commonly used in T cell activation assays. The anti-CD3 antibodies of this invention cross-compete with OKT3 for binding to human CD3. Depending on the binding affinity of CD3 and the epitope on CD3, anti-CD3 antibodies activated T cells with different functional outcomes. In vitro incubation of human T cells with low affinity anti-CD3 antibodies resulted in incomplete T cell activation, low production of IL-2 and IL-10. In contrast, high affinity CD3 binders activated T cells to produce significantly more IL-2 and other cytokines. Low affinity anti-CD3 antibodies are considered partial agonists that selectively induce some effector functions, strong tumor killing, and CD69 activation, but do not induce others, such as IL-2 and IL-10 production. The high affinity binders of this invention are full agonists that activate many of the immune effector functions of T cells. The strength of the interaction with CD3 and the recognizable epitope resulted in qualitatively different T cell activation. The maximum production of cytokines by T cells activated with low-affinity anti-CD3 antibodies was lower than the maximum activation by high-affinity anti-CD3 antibodies. In some embodiments, an antibody of the invention results in lower release of one or both of IL-2 and IL-10 when combined with T cells in an activation assay compared to an anti-CD3 reference antibody in the same assay, wherein the reference antibody may represent is ID 304703 (or an antibody of equivalent affinity). The maximum release of IL-2 and/or IL-10 may be less than about 75% release with the reference antibody, less than about 50% release with the reference antibody, less than about 25% release with the reference antibody, and may be less than than about 10% release with a reference antibody.

In some embodiments of the invention, bispecific or multispecific antibodies are provided, which may have any of the configurations discussed herein, including, without limitation, the bispecific three-chain configuration. Bispecific antibodies comprise at least the heavy chain variable region of an antibody specific for a protein other than CD3, and may comprise a heavy and light chain variable region. In some such embodiments, the second specificity of the antibody is associated with a tumor associated antigen, a targeting antigen, eg, integrins and the like, a pathogenic antigen, a checkpoint protein, and the like. Various formats of bispecific antibodies are within the scope of the invention, including, without limitation, single chain polypeptides, double chain polypeptides, three chain polypeptides, four chain polypeptides and multiples thereof.

The family of CD3-specific antibodies of this invention contains a VH domain containing the sequences CDR1, CDR2 and CDR3 in a human VH framework. CDR sequences can be located, for example, in the region of amino acid residues 26-35; 53-59; and 98-117 for CDR1, CDR2 and CDR3, respectively, provided exemplary variable region sequences set forth in SEQ ID NOs: 1-68. One skilled in the art will appreciate that the CDR sequences may be in a different position if a different framework sequence is selected, although the order of the sequences will generally remain the same.

CDR sequences for an antibody of this invention may have the following sequence formulas. X is a variable amino acid, which may be specific amino acids as indicated below.

CDR1

_{G1 G2 S3} I _{4 x5 S6 X7 x8 x9} X ₁₀

wherein:

X ₅ can be any amino acid; in some embodiments of the invention, X ₅ is S or R;

X ₇ and X ₈ can be any amino acid; in some embodiments, X ₇ and X ₈ are independently S or G. In some embodiments, X ₇ X ₈ are SS or GG;

X ₉ can be any amino acid; in some embodiments of the invention, X ₉ is H or Y; in some embodiments of the invention, X ₉ is H;

X ₁₀ can be any amino acid; in some embodiments, X ₁₀ is Y or F; in some embodiments, X ₁₀ is Y.

In some embodiments, the CDR1 sequence has the formula: GGSIX _5' SHHGY, with X ₅ as defined above. In some embodiments, the CDR1 sequence of an anti-CD3 antibody of the invention comprises the sequence set forth in any of SEQ ID NOs: 1-68, residues 26-35.

CDR2

I _1' X _2' X _3' S _4' G5 _' X _6' X _7'

wherein:

X _2' can be any amino acid; in some embodiments of the invention X _2' represents S, Y or H;

X _3' may be any amino acid; in some embodiments, X _3' is Y, H, or R;

X _6' can be any amino acid; in some embodiments, X _6' is S, N, or I, or R;

X _7' can be any amino acid; in some embodiments, X _7' is T or P.

In some embodiments, the CDR2 sequence has the formula: I X_2' X_3' S G S T; or I X_2' X_3' S G N P_2' and X_3' such as defined above. In some embodiments, the CDR2 sequence of an anti-CD3 antibody of the invention comprises the sequence set forth in any of SEQ ID NOs: 1-68, residues 53-59.

CDR3

X _1'' R _2'' W _3'' R _4'' H _5'' D _6'' I _7'' X _8'' X _9'' X _10'' Y _11'' P _12'' Y _{13 ''} Y _14'' Y _15'' Y _16'' G _17'' M _18'' D _19'' V _20''

wherein:

X _1'' can be any amino acid; in some embodiments, X _1' is A or G.

X _8'' can be any amino acid; in some embodiments, X _8'' is L or F.

X _9'' can be any amino acid; in some embodiments, X _9'' is T or A

X _10'' can be any amino acid; in some embodiments, X _10'' is G, A, or R.

In some embodiments, X _8'' X _9'' X _10'' is FAA, the motif of which corresponds to antibodies that cross-react with cynomolgus CD3 protein. In other embodiments, X _8'' X _9'' X _10'' is LT A. In some embodiments, the CDR3 sequence of an anti-CD3 antibody of the invention comprises the sequence set forth in any of SEQ ID NOs: 1-68, residues 98-117.

In some embodiments, a VH CD3 binding domain is linked to a light chain variable region domain. In some such embodiments, the light chain is a fixed light chain. In some embodiments, the light chain comprises a VL domain with sequences of CDR1, CDR2, and CDR3 in a human VL framework. CDR sequences may be sequences from SEQ ID NO: 69. In some embodiments, the CDR1 sequence contains amino acid residues 27-32; 50-52; 89-97 for CDR1, CDR2, CDR3 respectively.

In some embodiments, the CDR sequences of an antibody of the invention are at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical to a CDR sequence or set of CDR sequences in any of SEQ ID NOs: 1-68. In some embodiments, the CDR sequence of this invention contains one, two, three or more amino acid substitutions with respect to the CDR sequence or set of CDR sequences in any of SEQ ID NOs: 1-68. In some embodiments, said amino acid substitution(s) is one or more at position 5 or 10 of CDR1, position 2, 6 or 7 of CDR2, position 1, 8, 9 or 10 of CDR3 relative to the formulas above.

When the protein of the invention is a bispecific antibody, one binding fragment, i.e. a combination of VH/VL or VH alone, is specific for human CD3 while the other arm can be specific for target cells, including cancer cells such as ovarian cells. , breast, gastrointestinal tract, brain, head and neck, prostate, colon and lung cancer and the like, as well as hematological tumors such as B-cell tumors, including leukemias, lymphomas, sarcomas, carcinomas, tumors cell tumors, squamous cell carcinomas, embryonic cell tumors, metastases, undifferentiated tumors, seminomas, melanomas, myelomas, neuroblastomas, mixed cell tumors, neoplasms caused by infectious agents and other malignancies, cells infected by a pathogen, autoreactive cells causing inflammation and /or an autoimmune reaction. The non-CD3 portion may also be specific for an immunoregulatory protein, as will be described herein.

Tumor-associated antigens (TAA) are relatively limited to tumor cells, while tumor-specific antigens (TSA) are unique to tumor cells. TSA and TAA are usually parts of intracellular molecules expressed on the cell surface as part of the major histocompatibility complex.

Tissue-specific differentiation antigens are molecules present on tumor cells and their normal cellular counterparts. Tumor-associated antigens known to be recognized by therapeutic mAbs fall into several different categories. Hematopoietic differentiation antigens are glycoproteins that are commonly associated with cluster of differentiation (CD) groups and include CD20, CD30, CD33 and CD52. Cell surface differentiation antigens are a diverse group of glycoproteins and carbohydrates that are found on the surface of both normal and tumor cells. Antigens that are involved in growth and differentiation signaling are often growth factors and growth factor receptors. Growth factors that are targets for antibodies in cancer patients include CEA, epidermal growth factor receptor (EGFR; also known as ERBB1), ERBB2 (also known as HER2), ERBB3, MET (also known as HGFR) , insulin-like growth factor 1 receptor (IGF1R - insulin-like growth factor 1 receptor), ephrin A3 receptor (EPHA3), TNF-dependent apoptosis-inducing ligand 1 receptor (TRAILR1 (tumor necrosis factor (TNF)-related apoptosis-inducing ligand receptor 1); also known as TNFRSF10A), TRAILR2 (also known as TNFRSF10B), and nuclear factor κB activator receptor ligand (RANKL; also known as TNFSF11). Antigens involved in angiogenesis are usually proteins or growth factors that support the formation of a new microvasculature, including vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR), integrin αVβ3 and integrin α5β1. The tumor stroma and extracellular matrix are essential supporting structures for a tumor. Stromal and extracellular matrix antigens that are therapeutic targets include fibroblast activation protein (FAP) and tenascin.

Examples of monoclonal antibody drugs useful in bispecific configurations include, without limitation, rituximab; Ibritumomab; Tiuxetan; Tositumomab; brentuximab; vedotin; Gemtuzumab; ozogamicin; Alemtuzumab; IGN101; adecatumumab; Labetuzumab; huA33; Pemtumomab; oregovomab; CC49 (minretumomab); cG250; J591; MOv18; MORAb-003 (farletuzumab); 3F8, ch14. eighteen; KW-2871; hu3S193; IgN311; Bevacizumab; IM-2C6; CDP791; Etaracizumab; Volociximab; cetuximab, panitumumab, nimotuzumab; 806; Trastuzumab; pertuzumab; MM-121; AMG 102, METMAB; SCH 900105; AVE1642, IMC-A12, MK-0646, R1507; CP 751871; KB004; IIIA4; Mapatumumab (HGS-ETR1); HGS-ETR2; CS-1008; Denosumab; Sibrotuzumab; F19; and 81C6.

The immune checkpoint receptors that have been most extensively studied in the context of clinical cancer immunotherapy are cytotoxic T-lymphocyte-associated antigen 4 (CTLA4; also known as CD152) and programmed cell death protein 1 (PD1). cell death protein 1; also known as CD279), both are inhibitory receptors. The clinical activity of antibodies that block any of these receptors implies that antitumor immunity can be enhanced at several levels and that combinatorial strategies can be reasonably designed based on mechanistic considerations and preclinical models.

The two ligands for PD1 are PD1 ligand 1 (PDL1; also known as B7-H1 and CD274) and PDL2 (also known as B7-DC and CD273). PDL1 is expressed on cancer cells and, by binding to its PD1 receptor on T cells, inhibits T cell activation/function.

Lymphocyte activation gene 3 (LAG3; also known as CD223), 2B4 (also known as CD244), B and T lymphocyte attenuator (BTLA; also known as CD272), T cell membrane protein 3 (TIM3; also known as like HAVcr2), the adenosine A2a receptor (A2aR) and the cytotoxicity suppression receptor family have been associated with inhibition of lymphocyte activity and in some cases with induction of lymphocyte anergy. Targeting antibodies to these receptors can be used in the methods of this invention.

Agents that agonize the immune costimulatory molecule are also useful in the methods of this invention. Such agents include either CD40 and OX40 agonists. CD40 is a co-stimulatory protein found in antigen presenting cells (APCs) and required for their activation. These APCs include phagocytes (macrophages and dendritic cells) and B cells. CD40 is part of the TNF receptor family. The primary activating signaling molecules for CD40 are IFNγ and the CD40 ligand (CD40L). Stimulation via CD40 activates macrophages.

Anti-CCR4 (CD194) antibodies of interest include humanized monoclonal antibodies directed against C-C chemokine receptor 4 (CCR4) with potential anti-inflammatory and anti-tumor activities. CCR2 is expressed on free macrophages, which can be found in various inflammatory conditions such as rheumatoid arthritis; and have also been identified as being expressed on tumor-stimulating macrophages. CCR2 is also expressed on regulatory T cells, and the CCR2 ligand, CCL2, mediates the recruitment of regulatory T cells in tumors. Regulatory T cells suppress the response to antitumor T cells, and therefore their inhibition or depletion of their population is desirable.

Obtaining proteins according to this invention

Although antibodies can be made by chemical synthesis, they are usually made by recombinant DNA techniques, such as co-expression of all the chains that make up a protein in a single recombinant host cell, or co-expression of a heavy chain polypeptide and an antibody, such as a human antibody. In addition, the heavy and light chains of an antibody can also be expressed using a single polycistronic expression vector. Purification of individual polypeptides is achieved using standard protein purification methods such as affinity (protein A) chromatography, size exclusion chromatography and/or hydrophobic interaction chromatography. The bispecifics differ sufficiently in size and hydrophobicity that purification can be carried out using standard procedures.

The amount of antibody and heavy chain polypeptide produced in a single host cell can be minimized by designing antibody and heavy chain constant regions such that homodimerization is favored over heterodimerization, e.g. , such as "bulge-to-cavity" strategies (see WO 96/27011)). Therefore, another aspect of the present invention is to provide a method for producing a recombinant host bispecific method comprising the step of: expressing in a recombinant host cell a nucleic acid sequence encoding at least two heavy chain polypeptides, wherein said heavy chain polypeptides differ in their constant regions in sufficient to reduce or prevent homodimer formation but increase bispecific formation.

When a protein contains three chains, such as FlicAb, they can be produced by co-expressing the three chains (2 heavy chains and one light chain) that make up the molecule in a single recombinant host cell.

For recombinant production of proteins herein, one or more nucleic acids encoding all strands, eg 2, 3, 4 and the like, are isolated and inserted into a replicable vector for further cloning (DNA amplification) or for expression. Many vectors are available. Vector components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes and an enhancer element, a promoter, and a transcription termination sequence.

In a preferred embodiment, the host cell according to the method of the invention is capable of expressing human immunoglobulin at a high level, i.e. at least 1 pg/cell/day, preferably at least 10 pg/cell/day and even more preferably at least 20 pg/cell/day or more without the need for amplification of nucleic acid molecules encoding single strands in said host cell.

pharmaceutical composition

Another aspect of this invention is the provision of pharmaceutical compositions containing one or more proteins according to this invention in admixture with a suitable pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers used herein are, but are not limited to, adjuvants, solid carriers, water, buffers or other carriers used in the art for storage of therapeutic components or combinations thereof.

Therapeutic protein formulations used in accordance with this invention are prepared for storage by mixing proteins having the desired purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (see, for example, Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), for example, in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations employed and include buffering agents such as phosphate, citrate and other organic salts; antioxidants including ascorbic acid and methionine; preservatives (eg octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl or benzyl alcohol; alkyl parabens, eg methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (containing less than 10 residues) polypeptides; proteins, eg serum albumin, gelatin or immunoglobulins; hydrophilic polymers, for example polyvinylpyrrolidone; amino acids, for example glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars, for example sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes) and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

Anti-CD3 antibody formulations are described, for example, in US Patent Publication No. 20070065437, the full disclosure of which is incorporated herein by reference. Similar formulations can be used for the proteins of this invention. The main components of such formulations are a pH buffering agent effective in the range of 3.0 to 6.2, a salt, a surfactant, and an effective amount of a CD3 specific bispecific.

Application methods

Methods are provided for treating or reducing morbidity, including, without limitation, infection, autoimmune disease, primary or metastatic cancer, and the like, in a drug regimen comprising contacting target cells with an antigen binding composition of the present invention, especially when the antigen binding composition is a multispecific antibody. suitable for the condition being treated, for example, where one binding fragment specifically binds to a tumor associated antigen to treat the respective cancer cells; a binding fragment specific for the pathogen of interest to treat the corresponding infection, and the like. Such methods include administering to a subject in need thereof a therapeutically effective amount or effective dose of the agents of this invention, including, without limitation, combinations of a reagent with a chemotherapeutic drug, radiation therapy, or surgery.

Effective dosages of the compositions of this invention for treating a disease will vary depending on many different factors, including the route of administration, the target site, the physiological state of the patient, whether the patient is a human or an animal, other medications administered, and whether preventive or therapeutic treatment. Usually, the patient is a human, but non-human mammals can also be treated, for example, domestic animals such as dogs, cats, horses and the like, laboratory mammals such as rabbits, mice, rats and the like, and the like. . Dosages for treatment can be adjusted to optimize safety and efficacy.

Dosage levels can be easily determined by the skilled physician and can be changed as needed, for example, if the subject's response to therapy needs to be changed. The amount of active ingredient that can be combined with carrier materials to form a single dosage form will vary depending on the individual being treated and the particular route of administration. Unit dosage forms typically contain from about 1 mg to about 500 mg of the active ingredient.

In some embodiments of the invention, the therapeutic dosage of the agent may be from about 0.0001 to 100 mg/kg or more, usually from 0.01 to 5 mg/kg, based on the body weight of the host. For example, dosages may be 1 mg/kg body weight or 10 mg/kg body weight, or in the range of 1-10 mg/kg. A typical treatment regimen includes administration once every two weeks or once a month or once every 3-6 months. Therapeutic formulations of this invention are typically administered multiple times. The intervals between administration of each dose may be weekly, monthly or yearly. The intervals can also be uneven, as indicated by measuring the level of the therapeutic drug in the patient's blood. Alternatively, the therapeutics of this invention may be administered as a sustained release formulation, in which case less frequent administration is necessary. The dosage and frequency will vary depending on the half-life of the polypeptide in the patient.

For prophylactic purposes, a relatively small dose may be administered at relatively long intervals over an extended period of time. Some patients continue to receive treatment throughout their lives. In other therapeutic applications, a relatively high dosage is sometimes required at relatively short intervals until the progression of the disease is reduced or stopped, and preferably until the patient shows a partial or complete improvement in the symptoms of the disease. After that, the patient can be assigned a prophylactic regimen.

In yet other embodiments, the methods of this invention include treating, reducing, or preventing tumor growth, tumor metastasis, or tumor invasion of cancers, including carcinomas, hematologic malignancies such as leukemias and lymphomas, melanomas, sarcomas, gliomas, and the like. For prophylactic use, pharmaceutical compositions or medicaments are administered to a patient at risk for, or otherwise susceptible to, a disease in an amount sufficient to eliminate or reduce the risk, reduce the severity, or delay the onset of a disease, including the biochemical, histological and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes present during the development of the disease.

Compositions for treating a disease may be administered by parenteral, topical, intravenous, intratumoral, oral, subcutaneous, intra-arterial, intracranial, intraperitoneal, intranasal, or intramuscular routes. A typical route of administration is intravenous or intratumoral, although other routes may be equally effective.

Typically, the compositions are prepared as injectable solutions, either as liquid solutions or suspensions; solid forms suitable for dissolution or suspension in liquid vehicles prior to injection may also be prepared. The drug may also be emulsified or encapsulated in liposomes or microparticles such as polylactide, polyglycolide or copolymer to enhance the adjuvant effect as discussed above. Langer, Science 249:1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28:97-119, 1997. The agents of this invention may be administered in the form of an injection of depot agents or an implant formulation, which may be formulated to provide sustained or pulsatile release of the active ingredient. Pharmaceutical compositions are typically formulated to be sterile, substantially isotonic, and fully compliant with all US Good Manufacturing Practice (GMP) regulations. US Food and Drug Administration.

The toxicity of the proteins described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining LD ₅₀ (dose lethal to 50% of the population) or LD ₁₀₀ (dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used in determining a dosage range that is not toxic for human use. The dosage of the proteins described herein is preferably in the range of circulating concentrations that include an effective dose with little or no toxicity. The dosage may vary within this range depending on the dosage form used and the route of administration used. The precise composition, route of administration, and dosage may be selected by the individual physician based on the condition of the patient.

Pharmaceutical compositions can be administered in various unit dosage forms depending on the route of administration. For example, unit dosage forms suitable for oral administration include, but are not limited to, powder, tablets, pills, capsules, and lozenges. It is recognized that the compositions of this invention when administered orally must be protected from the effects of digestion. This is usually achieved either by complexing the molecules with the composition to render them resistant to acid and enzymatic hydrolysis, or by packaging the molecules in a suitably stable carrier such as a liposome or protective barrier. Means of protecting agents from the digestive system are well known in the art.

Compositions for administration typically contain an antibody or other ablative agent dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. Various aqueous vehicles may be used, for example buffered saline and the like. These solutions are sterile and generally free of unwanted substances. These compositions can be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable excipients which are necessary to suit physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. similar. The concentration of the active agent in these formulations can vary widely and will be selected primarily based on fluid volumes, viscosity, body weight, and the like, in accordance with the particular route of administration chosen and the needs of the patient (e.g., Remington's Pharmaceutical Science (15th ed.). ., 1980) and Goodman & Gillman, The Pharmacological Basis of Therapeutics (Hardman et al., eds., 1996)).

Also included within the scope of this invention are kits containing the active agents and their formulations of the invention and instructions for use. The kit may further contain at least one additional reagent, for example, a chemotherapeutic drug and the like. Kits usually include a label indicating the intended use of the contents of the kit. The term "label" includes any written or recorded materials supplied with or with the kit, or otherwise accompanying the kit.

The compositions may be administered for therapeutic treatment. The compositions are administered to the patient in an amount sufficient to substantially remove target cells as described above. An amount sufficient to achieve this is defined as the "therapeutically effective dose" which may provide an improvement in overall survival. The compositions can be administered once or multiple times, depending on the dosage and frequency desired and tolerated by the patient. The specific dose required for treatment will depend on the medical diagnosis and medical history of the mammal, as well as other factors such as age, weight, sex, route of administration, efficacy, and the like.

Now that the invention has been fully described, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit or scope of the invention.

Examples

Example 1

Genetically engineered rats expressing heavy chain-only antibodies

The human IgH locus was constructed and assembled from several parts that included modification and fusion of the rat C-region genes, which were then fused downstream of the human V _H 6-DJ _H region. The two BACs with separate clusters of human V _H genes were then co-introduced with the BAC encoding the assembled (human V _H 6-DJ _H -rat C) fragment.

Were obtained transgenic rats carrying artificial heavy chain immunoglobulin loci in a disordered configuration. The included constant region genes encode IgM, IgD, IgG2b, IgE, IgA and a 3' enhancer. RT-PCR and serum analysis (ELISA) of transgenic rats revealed productive rearrangement of transgenic immunoglobulin loci and serum expression of various isotypes of heavy chain-only antibodies. Transgenic rats were cross-bred with mutated endogenous heavy chain and light chain loci previously described in US Patent Publication 2009/0098134 A1. Analysis of these animals demonstrated inactivation of rat immunoglobulin heavy and light chain expression and high expression of heavy chain antibodies with variable regions encoded by the human V, D and J genes. Immunization of transgenic rats resulted in serum responses with high titers of antigen-specific heavy chain antibodies. These transgenic rats expressing heavy chain antibodies with the human VDJ region were named UniRats.

Example 2

Genetically engineered rats expressing fixed light chain antibodies

Sets of transgenic human antibodies were generated from H chains with a diverse (V _H -DJ _H ) _n rearrangement in combination with a unique L chain. To do this, the rearranged L chain, human Vk-Jk1-Ck, was integrated into the rat germline via DNA microinjection, and the resulting transgenic animals were crossed with a previously described rat line that naturally expresses the human H chain set (Osborn et al. , year 2013). This new line of rats has been named OmniFlic.

Immunization of OmniFlic rats with a variety of different antigens produced high levels of antigen-specific IgG, similar to other transgenic rats carrying the same IgH locus. Analysis of the set by RT-PCR revealed highly variable rearrangements of the V _H gene at high transcript and protein levels. In addition, only one L chain product was identified, also highly expressed.

Antigen-specific binders from OmniFlic were generated by next generation sequencing (NGS) and selected from cDNA libraries (yeast, E. coli, phage) that, after sequencing, identified a variety of H chain transcripts. For expression in mammalian cells, hypermutated H chain constructs were transfected in combination with the original Igκ transgenic sequence. Mutational changes were not allowed in this rearranged Vk-Jk1-Ck and always the same L chain was expressed with different H chain products to generate monoclonal human IgG.

Example 3

Obtaining antigen-specific antibodies in transgenic rats.

To obtain antigen-specific heavy chain antibodies, rats expressing genetically engineered rats were immunized in two ways.

Immunization with recombinant extracellular domains of PD-L1 and BCMA. Recombinant extracellular domains of PD-L1 and BCMA were purchased from R&D Systems, diluted with sterile saline and combined with adjuvant. The immunogens were either combined with complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA), or with Titermax and Ribi adjuvants. The first immunization (priming) with the immunogen in CFA or Titermax was administered to the left and right legs. After the first immunization with immunogens in CFA, two more immunizations in IFA (booster injections) or 4 more immunizations in Ribi and another immunization in Titermax were administered to each leg. This sequence of immunizations results in the development of B cells producing high affinity antibodies. Immunogen concentrations were 10 μg per leg. Serum was collected from rats at the last bloodletting to determine serum titers.

To obtain antibodies to human CD3δε, genetically engineered rats were immunized using DNA-based immunization protocols.

OmniFlic rats were immunized with human and cynomolgus macaque CD3 epsilon/delta constructs at Aldevron, Inc. (Fargo, ND) using GENOVAC antibody technology. Draining lymph nodes were harvested after final immunization and RNA was isolated. After cDNA synthesis, the IgH heavy chain antibody set was characterized using next generation sequencing and our own in-house software. Candidate antigen-specific VH sequences were selected showing evidence of antigen-specific positive selection. Several hundred VH sequences encoding FlicAb were selected for gene assembly and cloned into an expression vector. Subsequently, fully human FlicAb IgG1 antibodies were expressed in HEK cells for analysis by Flow and ELISA. Human FlicAbs were tested for binding to primary human T cells and Jurkat cells using flow. In addition, human FlicAbs were tested using recombinant CD3δε proteins in ELISA. All FlicAbs with positive binding to human T cells are listed in FIG. 1. Selected sequences were further characterized in T cell activation assays.

Example 4 Characterization of OmniFlic Anti-CD3 Antibodies

The antibodies generated from the campaign as described above were further characterized for their ability to activate T cells to produce cytokines and to activate CD69 on their surface. For the quantitative determination of IL-2 produced by Jurkat cells or peripheral blood lymphocytes, the max BioLegend ELISA kit was used. The results of the experiment using human peripheral blood T cells and various concentrations of OmniFlic antibodies are illustrated in FIG. 3.

Example 5 Characterization of Bispecific Antibodies

FlicAb to CD3 ID 304703 (SEQ ID NO: 39) was selected for further bispecific development. This FlicAb cross-reacts with cynomolgus macaque CD3 and significantly stimulates human T cells. Two types of bispecifics were obtained (see Fig. 2 for a schematic representation). To create bispecific FlicAbs, the "knob-in-trough" technology was used (Protein Engineering vol. 9, no. 7, pp. 617-621, 1996, 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization. John B. B. Ridgway, Leonard G. Presta, and Paul Carter). The C-terminus of the heavy chain was labeled with a C-tag with a ridge, and heterodimeric antibodies were purified using CaptureSelect C-Tag Affinity Matrix (Thermo Fischer Scientific). A bispecific FlicAb with one arm reactive with CD3 (ID 304703) and the other with human PD-L1 was produced and shown to activate human CD8+ T cells only in the presence of PD-L1 positive tumor cells (Fig. 3). HDLM2 is a multiple myeloma cell line that expresses PD-L1 on the surface. Ramos is a Burkitt's lymphoma cell line that is PD-L1 negative. CD69 expression was used as a recordable indicator. The bispecific antibody was used at the indicated concentrations.

As illustrated in FIG. 4, tumor cells (HDLM2 that express PD-L1 on the cell surface) were incubated with purified human CD8+ T cells and bispecific antibodies. HDLM2 cells do not express CD20, and coculture with the α-CD3/α-CD20 bispecific FlicAb did not result in HDLM2 cell death. Only coculture of human CD8+ T cells and HDLM2 with the α-CD3/α-PD-L1 bispecific FlicAb resulted in significant kill.

In FIG. 5 summarizes the data for antibodies in monospecific and bispecific format. Column 1 shows the sequence identifier for VH to CD3 sequence ID NO: 304703 (SEQ ID NO: 39), 314171 (SEQ ID NO: 13), 313306 (SEQ ID NO: 1), 313329 (SEQ ID NO: 6) and 313283 (SEQ ID NO: 18). Column 2 shows the SIF value for binding by Jurkat cells of the original monospecific (antibody) to CD3. Column 3 shows the SIF value for cynomolgus monkey T-cell binding of the original anti-CD3 monospecific (antibody). Column 5 shows picograms of IL-2 released by pan T cells stimulated with a BCMA protein-binding bispecific antibody placed on plastic at the indicated dose. Column 6 shows picograms of IL-6 released by pan T cells stimulated with a BCMA protein-binding bispecific antibody placed on plastic at the indicated dose. Column 7 shows picograms of IL-10 released by pan T cells stimulated with a BCMA protein-binding bispecific antibody placed on plastic at the indicated dose. Column 8 shows picograms of IFN-γ released by pan-T cells stimulated with a BCMA protein-binding bispecific antibody placed on plastic at the indicated dose. Column 9 shows picograms of TNFα released by pan T cells stimulated with a BCMA protein-binding bispecific antibody placed on plastic at the indicated dose. Column 10 shows the EC50 of U266 tumor cell lysis mediated by bispecific antibodies in the presence of human pan T cells. Column 11 shows the percent lysis of U266 tumor cells in the presence of the bispecific antibody and human pan T cells at a dose of 333 ng/ml of the bispecific antibody. Column 12 shows the protein binding affinity of the arm for CD3 of the bispecific antibody as measured by Octet. Column 13 shows the SIF value for Jurkat cells to bind the bispecific antibody.

Example 6

Correlation of bispecific killing activity and cytokine release.

As illustrated in FIG. 6, four αCD3_fam1:aBCMA bispecific antibodies, each with a unique anti-CD3 arm (as shown) and a common anti-BCMA arm, were tested for the ability to kill U266 BCMA+ tumor cells by redirecting activated primary T cells. In this experiment, U266 cells that express BCMA were mixed with activated pan T cells in an E:T ratio of 10:1 along with the addition of a bispecific antibody. The x-axis shows the concentration of the applied antibody, and the y-axis shows the % tumor cell lysis 6 hours after the addition of the antibody. Killing activity correlated with IL-2 release (FIG. 7); with IFN-γ release (FIG. 8) and with CD3 binding affinity (FIG. 9). The correlation between IL-2 production and lysis of U266 tumor cells is R ² =0.37. The correlation between IFN-γ production and U266 tumor cell lysis is R ² =0.53. The correlation between EC50 killing U266 and protein binding affinity is R ² =0.93.

The αCD3_F1F:αBCMA bispecific antibodies were analyzed for their ability to kill three different BCMA+ tumor cell lines and one BCMA-negative cell line by redirecting activated parental T cells. The antibodies consisted of the αCD3 arm (SEQ ID NO: 1 and SEQ ID NO: 69) and the αBCMA arm (SEQ ID NO: 70 or SEQ ID NO: 71) (illustrated in FIGS. 10-12). In this experiment, tumor cells were mixed with activated pan-T cells in an E:T ratio of 10:1 along with the addition of a bispecific antibody. In FIG. 10A illustrates killing of RPMI-8226 cells, FIG. 10B illustrates the killing of NCI-H929 cells, panel C illustrates the killing of U-266 cells, and FIG. 10D illustrates killing of K562 cells, negative control. The x-axis shows the concentration of the applied antibody, and the y-axis shows the % lysis of tumor cells 6 hours after the addition of the antibody.

In FIG. 11 illustrates the level of IL-2 cytokine release measured after culturing human T cells at rest with various tumor cell lines and increasing doses of the aCD3_F1F:aBCMA bispecific antibody (as illustrated in FIG. 10). In FIG. 11A illustrates IL-2 release stimulated by RPMI-8226 cells, FIG. 11B illustrates IL-2 release stimulated by NCI-H929 cells, FIG. 11C illustrates IL-2 release stimulated by U-266 cells and FIG. 11D illustrates IL-2 release stimulated by K562 cells, negative control.

The level of release of the cytokine IFN-γ was measured after culturing human T cells at rest with various tumor cell lines and increasing doses of the aCD3_F1F:aBCMA bispecific antibody (as illustrated in Fig. 10). In FIG. 12A illustrates IFN-γ release stimulated by RPMI-8226 cells, FIG. 12B illustrates IFN-γ release stimulated by NCI-H929 cells, FIG. 12C illustrates IFN-γ release stimulated by U-266 cells and FIG. 12D illustrates IFN-γ release stimulated by K562 cells, negative control.

The examples are provided in such a way as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use this invention, and are not intended to limit the scope of what the inventors consider their invention, nor are they intended to represent what the experiments below are all the experiments that were performed. Care has been taken to ensure accuracy with respect to the numbers used (eg amounts, temperatures, and the like), but some experimental errors and deviations must be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

While the present invention has been described with reference to specific embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the present invention. In addition, many modifications can be made in order to adapt a particular situation, material, composition of matter, process, process steps or step to the object, spirit and scope of this invention. All such modifications are intended to be within the scope of the appended claims.

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Sequence listing

<110> TENEOBIO, INC.

<120> CD3 BINDING ANTIBODIES

<130> 60792.00027US01 (TNO-0010-US1)

<140> 16/312.743

<141> 2019-07-22

<150> PCT/US2017/038373

<151> 2017-06-20

<150> 62/491.908

<151> 2017-04-28

<150> 62/394.360

<151> 2016-09-14

<150> 62/352.698

<151> 2016-06-21

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Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asp Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Thr Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Ala Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 22

<211> 128

<212> PRT

<213> Homo sapiens

<400> 22

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Val Ser Gly Gly Ser Ile Arg Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Leu

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Arg Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 23

<211> 128

<212> PRT

<213> Homo sapiens

<400> 23

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Val Ser Gly Gly Ser Ile Arg Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln Asn Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Ala Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 24

<211> 128

<212> PRT

<213> Homo sapiens

<400> 24

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Ile Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Thr Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Gly Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 25

<211> 128

<212> PRT

<213> Homo sapiens

<400> 25

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Ile Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Ala Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 26

<211> 128

<212> PRT

<213> Homo sapiens

<400> 26

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Arg Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Ile Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Gly Arg Trp Arg His Asp Ile Leu Thr Ala Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 27

<211> 128

<212> PRT

<213> Homo sapiens

<400> 27

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Arg Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Val Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 28

<211> 128

<212> PRT

<213> Homo sapiens

<400> 28

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Arg Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln Tyr Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile His Ser Gly Ser Thr Asn Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Ile Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Leu

65 70 75 80

Ser Leu Lys Val Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Phe Ala Ala Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 29

<211> 128

<212> PRT

<213> Homo sapiens

<400> 29

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Arg Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln Tyr Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile His Arg Ser Gly Asn Pro Asn Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Leu Thr Ile Leu Leu Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Met Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Ala Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 30

<211> 128

<212> PRT

<213> Homo sapiens

<400> 30

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Arg Ser Gly

20 25 30

Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Ala Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 31

<211> 128

<212> PRT

<213> Homo sapiens

<400> 31

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly His Phe Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile His His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Ile Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Gly Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 32

<211> 128

<212> PRT

<213> Homo sapiens

<400> 32

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly His Phe Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Ile Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Gly Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 33

<211> 128

<212> PRT

<213> Homo sapiens

<400> 33

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly His Phe Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Ala Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 34

<211> 128

<212> PRT

<213> Homo sapiens

<400> 34

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Asp

35 40 45

Trp Ile Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Ala Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 35

<211> 128

<212> PRT

<213> Homo sapiens

<400> 35

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile His Ser Gly Ser Thr Asn Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Ser Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Ala Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 36

<211> 128

<212> PRT

<213> Homo sapiens

<400> 36

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile His Ser Gly Ser Thr Asn Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Phe Ala Ala Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 37

<211> 128

<212> PRT

<213> Homo sapiens

<400> 37

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile His Arg Ser Gly Asn Pro Asn Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Leu Thr Ile Leu Leu Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Ser Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Ala Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 38

<211> 128

<212> PRT

<213> Homo sapiens

<400> 38

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile His Arg Ser Gly Asn Pro Asn Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Leu Thr Ile Leu Leu Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Ala Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 39

<211> 128

<212> PRT

<213> Homo sapiens

<400> 39

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile His Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Ile Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Phe Ala Ala Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 40

<211> 128

<212> PRT

<213> Homo sapiens

<400> 40

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Ser Tyr Ser Gly Ser Thr His Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Met Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Gly Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 41

<211> 128

<212> PRT

<213> Homo sapiens

<400> 41

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Ala Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 42

<211> 128

<212> PRT

<213> Homo sapiens

<400> 42

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr His Ser Gly Ile Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Ala Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 43

<211> 128

<212> PRT

<213> Homo sapiens

<400> 43

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr His Ser Gly Asn Thr Tyr Tyr Lys Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Met Asn Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Ala Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 44

<211> 128

<212> PRT

<213> Homo sapiens

<400> 44

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Ile Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Ala Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 45

<211> 128

<212> PRT

<213> Homo sapiens

<400> 45

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Ile Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Ala Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 46

<211> 128

<212> PRT

<213> Homo sapiens

<400> 46

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Ile Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Gly Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 47

<211> 128

<212> PRT

<213> Homo sapiens

<400> 47

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Ile Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Ser Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Ala Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 48

<211> 128

<212> PRT

<213> Homo sapiens

<400> 48

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Ile Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Gly Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 49

<211> 128

<212> PRT

<213> Homo sapiens

<400> 49

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Ile Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Gly Arg Trp Arg His Asp Ile Leu Thr Gly Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 50

<211> 128

<212> PRT

<213> Homo sapiens

<400> 50

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Ser Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Gly Arg Trp Arg His Asp Ile Leu Thr Gly Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 51

<211> 128

<212> PRT

<213> Homo sapiens

<400> 51

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Ala Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 52

<211> 128

<212> PRT

<213> Homo sapiens

<400> 52

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Gly Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 53

<211> 128

<212> PRT

<213> Homo sapiens

<400> 53

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Gln Ser Arg Val Phe Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Asn Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Ala Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 54

<211> 128

<212> PRT

<213> Homo sapiens

<400> 54

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Ile Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Ala Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 55

<211> 128

<212> PRT

<213> Homo sapiens

<400> 55

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Arg Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Ile Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Ala Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 56

<211> 128

<212> PRT

<213> Homo sapiens

<400> 56

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Met Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Arg Leu Asn Ser Val Thr Ala Val Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Ala Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 57

<211> 128

<212> PRT

<213> Homo sapiens

<400> 57

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln Tyr Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile His His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Ile Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Ala Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 58

<211> 128

<212> PRT

<213> Homo sapiens

<400> 58

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Val Ser Gly Gly Ser Ile Arg Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Gly Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 59

<211> 128

<212> PRT

<213> Homo sapiens

<400> 59

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Arg Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln Tyr Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Val Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Ala Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 60

<211> 128

<212> PRT

<213> Homo sapiens

<400> 60

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Asn Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Ala Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 61

<211> 128

<212> PRT

<213> Homo sapiens

<400> 61

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Ile Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Ala Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 62

<211> 128

<212> PRT

<213> Homo sapiens

<400> 62

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Ile Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Gly Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 63

<211> 128

<212> PRT

<213> Homo sapiens

<400> 63

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Ile Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Gly Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 64

<211> 128

<212> PRT

<213> Homo sapiens

<400> 64

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Gly Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 65

<211> 128

<212> PRT

<213> Homo sapiens

<400> 65

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Ala Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 66

<211> 128

<212> PRT

<213> Homo sapiens

<400> 66

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln Tyr Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile His Arg Ser Gly Asn Pro Asn Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Leu Thr Ile Leu Leu Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Gly Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 67

<211> 128

<212> PRT

<213> Homo sapiens

<400> 67

Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Val Ser Gly Gly Ser Ile Arg Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Tyr Ser Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Ser Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Ala Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 68

<211> 128

<212> PRT

<213> Homo sapiens

<400> 68

Arg Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Trp Arg His Asp Ile Leu Thr Gly Tyr Pro Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 69

<211> 107

<212> PRT

<213> Homo sapiens

<400> 69

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 70

<211> 243

<212> PRT

<213> Homo sapiens

<400> 70

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val

35 40 45

Ser Gly Ile Arg Gly Ser Asp 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 Gly Glu Asn Asp Gly Pro Phe Asp His Arg Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Glu Val Gln Leu

115 120 125

Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu

130 135 140

Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Tyr Gly Met Ser Trp

145 150 155 160

Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val Ser Gly Ile Arg

165 170 175

Gly Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe

180 185 190

Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn

195 200 205

Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Gln Gly

210 215 220

Glu Asn Asp Gly Pro Phe Asp His Arg Gly Gln Gly Thr Leu Val Thr

225 230 235 240

Val Ser Ser

<210> 71

<211> 119

<212> PRT

<213> Homo sapiens

<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 Val Ser Ser Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val

35 40 45

Ser Gly Ile Arg Gly Ser Asp 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 Gly Glu Asn Asp Gly Pro Phe Asp His Arg Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 72

<211> 10

<212> PRT

<213> Homo sapiens

<400> 72

Gly Gly Ser Ile Arg Ser Gly Gly His Tyr

1 5 10

<210> 73

<211> 7

<212> PRT

<213> Homo sapiens

<400> 73

Ile Ser Tyr Ser Gly Ser Thr

fifteen

<210> 74

<211> 20

<212> PRT

<213> Homo sapiens

<400> 74

Ala Arg Trp Arg His Asp Ile Leu Thr Ala Tyr Pro Tyr Tyr Tyr Tyr

1 5 10 15

Gly Met Asp Val

twenty

<210> 75

<211> 7

<212> PRT

<213> Homo sapiens

<400> 75

Ile His His Ser Gly Ser Thr

fifteen

<210> 76

<211> 20

<212> PRT

<213> Homo sapiens

<400> 76

Ala Arg Trp Arg His Asp Ile Phe Thr Thr Tyr Pro Tyr Tyr Tyr Tyr

1 5 10 15

Gly Met Asp Val

twenty

<210> 77

<211> 10

<212> PRT

<213> Homo sapiens

<400> 77

Gly Gly Ser Ile Ser Ser Gly Gly His Tyr

1 5 10

<210> 78

<211> 7

<212> PRT

<213> Homo sapiens

<400> 78

Ile His Tyr Ser Gly Ser Thr

fifteen

<210> 79

<211> 20

<212> PRT

<213> Homo sapiens

<400> 79

Ala Arg Trp Arg His Asp Ile Phe Ala Ala Tyr Pro Tyr Tyr Tyr Tyr

1 5 10 15

Gly Met Asp Val

twenty

<210> 80

<211> 7

<212> PRT

<213> Homo sapiens

<400> 80

Ile His Arg Ser Gly Asn Pro

fifteen

<210> 81

<211> 6

<212> PRT

<213> Homo sapiens

<400> 81

Gln Ser Val Ser Ser Asn

fifteen

<210> 82

<211> 9

<212> PRT

<213> Homo sapiens

<400> 82

Gln Gln Tyr Asn Asn Trp Pro Trp Thr

fifteen

<210> 83

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<220>

<221> MOD_RES

<222> (5)..(5)

<223> Any amino acids

<220>

<221> MOD_RES

<222> (7)..(10)

<223> Any amino acids

<220>

<223> See specification as filed for detailed description of

substitutions and preferred embodiments

<400> 83

Gly Gly Ser Ile Xaa Ser Xaa Xaa Xaa Xaa

1 5 10

<210> 84

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<220>

<221> MOD_RES

<222> (1)..(1)

<223> Any amino acids

<220>

<221> MOD_RES

<222> (8)..(10)

<223> Any amino acids

<220>

<223> See specification as filed for detailed description of

substitutions and preferred embodiments

<400> 84

Xaa Arg Trp Arg His Asp Ile Xaa Xaa Xaa Tyr Pro Tyr Tyr Tyr Tyr

1 5 10 15

Gly Met Asp Val

twenty

<210> 85

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<400> 85

Thr Val Ala Ala Pro

fifteen

<210> 86

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<400> 86

Ala Ser Thr Lys Gly Pro

fifteen

<210> 87

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<220>

<221> MOD_RES

<222> (5)..(5)

<223> Any amino acids

<220>

<223> See specification as filed for detailed description of

substitutions and preferred embodiments

<400> 87

Gly Gly Ser Ile Xaa Ser His His Gly Tyr

1 5 10

<210> 88

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<220>

<221> MOD_RES

<222> (2)..(3)

<223> Any amino acids

<220>

<223> See specification as filed for detailed description of

substitutions and preferred embodiments

<400> 88

Ile Xaa Xaa Ser Gly Ser Thr

fifteen

<210> 89

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<220>

<221> MOD_RES

<222> (2)..(3)

<223> Any amino acids

<220>

<223> See specification as filed for detailed description of

substitutions and preferred embodiments

<400> 89

Ile Xaa Xaa Ser Gly Asn Pro

fifteen

<210> 90

<211> 10

<212> PRT

<213> Homo sapiens

<400> 90

Gly Gly Ser Ile Ser Ser Ser Ser His Tyr

1 5 10

<210> 91

<211> 10

<212> PRT

<213> Homo sapiens

<400> 91

Gly Gly Ser Ile Arg Ser Gly Gly Tyr Tyr

1 5 10

<210> 92

<211> 10

<212> PRT

<213> Homo sapiens

<400> 92

Gly Gly Ser Ile Ser Ser Gly Gly His Phe

1 5 10

<210> 93

<211> 7

<212> PRT

<213> Homo sapiens

<400> 93

Ile Tyr His Ser Gly Ser Thr

fifteen

<210> 94

<211> 7

<212> PRT

<213> Homo sapiens

<400> 94

Ile Tyr Tyr Ser Gly Ser Thr

fifteen

<210> 95

<211> 7

<212> PRT

<213> Homo sapiens

<400> 95

Ile Ser Tyr Ser Gly Arg Thr

fifteen

<210> 96

<211> 7

<212> PRT

<213> Homo sapiens

<400> 96

Ile Tyr His Ser Gly Ile Thr

fifteen

<210> 97

<211> 7

<212> PRT

<213> Homo sapiens

<400> 97

Ile Tyr His Ser Gly Asn Thr

fifteen

<210> 98

<211> 20

<212> PRT

<213> Homo sapiens

<400> 98

Gly Arg Trp Arg His Asp Ile Leu Thr Ala Tyr Pro Tyr Tyr Tyr Tyr

1 5 10 15

Gly Met Asp Val

twenty

<210> 99

<211> 20

<212> PRT

<213> Homo sapiens

<400> 99

Ala Arg Trp Arg His Asp Ile Leu Thr Gly Tyr Pro Tyr Tyr Tyr Tyr

1 5 10 15

Gly Met Asp Val

twenty

<210> 100

<211> 20

<212> PRT

<213> Homo sapiens

<400> 100

Gly Arg Trp Arg His Asp Ile Leu Thr Gly Tyr Pro Tyr Tyr Tyr Tyr

1 5 10 15

Gly Met Asp Val

twenty

<210> 101

<211> 20

<212> PRT

<213> Homo sapiens

<400> 101

Ala Arg Trp Arg His Asp Ile Leu Thr Arg Tyr Pro Tyr Tyr Tyr Tyr

1 5 10 15

Gly Met Asp Val

twenty

<210> 102

<211> 20

<212> PRT

<213> Homo sapiens

<400> 102

Ala Arg Trp Arg His Asp Ile Leu Thr Val Tyr Pro Tyr Tyr Tyr Tyr

1 5 10 15

Gly Met Asp Val

twenty

<210> 103

<211> 8

<212> PRT

<213> Homo sapiens

<400> 103

Gly Phe Thr Val Ser Ser Tyr Gly

fifteen

<210> 104

<211> 8

<212> PRT

<213> Homo sapiens

<400> 104

Ile Arg Gly Ser Asp Gly Ser Thr

fifteen

<210> 105

<211> 12

<212> PRT

<213> Homo sapiens

<400> 105

Ala Lys Gln Gly Glu Asn Asp Gly Pro Phe Asp His

1 5 10

<---

Claims (48)

1. A multispecific antibody specific for human CD3δε and a pathogen antigen, comprising a first binding fragment that specifically binds to human CD3δε and a second binding fragment that specifically binds to a pathogen antigen, characterized in that this first binding fragment contains: the first heavy chain variable domain containing: (a) CDR1 sequence GGSIRSGGHY, CDR2 sequence ISYSGST and CDR3 sequence ARWRHDILTAYPYYYYGMDV; or (b) CDR1 sequence GGSIRSGGHY, CDR2 sequence IHHSGST and CDR3 sequence ARWRHDIFTTYPYYYYGMDV; or (c) CDR1 sequence GGSISSGGHY, CDR2 sequence IHYSGST and CDR3 sequence ARWRHDIFAAYPYYYYGMDV; (d) CDR1 sequence GGSIRSGGHY, CDR2 sequence IHRSGNP and CDR3 sequence ARWRHDIFAAYPYYYYGMDV; or (e) CDR1 sequence GGSISSGGHY, CDR2 sequence IHHSGST and CDR3 sequence ARWRHDIFAAYPYYYYGMDV; and a light chain variable domain containing a CDR1 sequence containing QSVSSN, a CDR2 sequence containing GAS, and a CDR3 sequence containing QQYNNWPWT in a human VL framework. 2. The multispecific antibody of claim 1, wherein the CDR1, CDR2, and CDR3 sequences of said first heavy chain variable domain are presented in a human VH framework. 3. Multispecific antibody according to any one of paragraphs. 1, 2, characterized in that the heavy chain variable domain contains the amino acid sequence set forth in any of SEQ ID NOs: 18, 1, 39, 6 and 13. 4. Multispecific antibody according to any one of paragraphs. 1-3, characterized in that the light chain variable domain contains the amino acid sequence of SEQ ID NO: 69. 5. Multispecific antibody according to any one of paragraphs. 1-4, characterized in that upon contact with T cells in activation assays, the multispecific antibody induces the release of reduced levels of one or both of IL-2 and IL-10 relative to the reference anti-CD3 antibody. 6. Multispecific antibody according to any one of paragraphs. 1-5 containing the Fc region. 7. Multispecific antibody according to claim 6, characterized in that the Fc region was designed to reduce effector functions. 8. Multispecific antibody according to any one of paragraphs. 1-7, characterized in that the affinity (KD) of the protein for human CD3 delta epsilon is from about 10 ^-6 M to about 10 ^-11 M. 9. Multispecific antibody according to any one of paragraphs. 1-7, characterized in that it cross-reacts with human and cynomolgus monkey CD3 protein. 10. Multispecific antibody according to any one of paragraphs. 1-9, characterized in that it contains a single chain. 11. Multispecific antibody according to any one of paragraphs. 1-9, characterized in that it contains two chains or a plurality of chains. 12. Multispecific antibody according to any one of paragraphs. 1-9, characterized in that it contains three chains. 13. Multispecific antibody according to any one of paragraphs. 1-9, characterized in that it contains three chains and two antigen-binding arms, wherein the first antigen-binding arm contains an antibody heavy chain and an antibody light chain, and the second antigen-binding arm contains an antibody heavy chain that does not contain a CH1 domain. 14. Multispecific antibody according to any one of paragraphs. 1-13, characterized in that upon contact with T cells in activation assays, said multispecific antibody induces the release of reduced levels of one or both of IL-2 and IL-10 relative to a reference anti-CD3 antibody; and induces over 30% tumor cytotoxicity in standard in vitro assays using tumor cells and human T cells. 15. The multispecific antibody of claim 14, wherein the second binding fragment, which specifically binds to the pathogen antigen, is a heavy chain domain only. 16. A multispecific antibody according to claim 15, characterized in that the second binding fragment, which specifically binds to the pathogen antigen, contains a light chain variable domain. 17. The multispecific antibody of claim 15 wherein the light chain variable domain is the same as the light chain variable domain of the CD3 binding region. 18. Directed against CD3 pharmaceutical composition containing a therapeutically effective amount of multispecific antibodies according to any one of paragraphs. 1-17. 19. Pharmaceutical composition according to claim 18 in a single dose formulation. 20. A polynucleotide encoding a multispecific antibody according to any one of paragraphs. 1-17. 21. An expression vector containing a polynucleotide according to claim 20. 22. Recombinant host cell for the production of multispecific antibodies according to any one of paragraphs. 1-17, containing the vector according to item 21. 23. The method of obtaining multispecific antibodies according to any one of paragraphs. 1-17, which includes growing a recombinant host cell according to claim 22 under conditions that allow the expression of a multispecific antibody, and isolating the multispecific antibody from the cells. 24. A method of treating an infection, comprising the introduction of an individual effective dose of a multispecific antibody according to any one of paragraphs. 1-17 or a pharmaceutical composition according to claim 18. 25. The use of multispecific antibodies according to any one of paragraphs. 1-17 for preparing a medicament for treating an infection. 26. The method according to any one of paragraphs. 23 and 24 or the application of claim 25, where the individual is a human. 27. Bispecific antibody specific for human CD3δε and pathogen antigen, containing: (i) a CD3-binding heavy chain variable domain in the human VH framework; (ii) a CD3-binding light chain variable domain containing a CDR1 sequence containing QSVSSN, a CDR2 sequence containing GAS, and a CDR3 sequence containing QQYNNWPWT in a human VL framework region; as well as (iii) a single chain variable domain in a single chain or tandem configuration that specifically binds to said pathogen antigen, wherein the bispecific antibody is characterized in that the CD3-binding heavy chain variable domain comprises: (a) CDR1 sequence GGSIRSGGHY, CDR2 sequence ISYSGST and CDR3 sequence ARWRHDILTAYPYYYYGMDV; or (b) CDR1 sequence GGSIRSGGHY, CDR2 sequence IHHSGST and CDR3 sequence ARWRHDIFTTYPYYYYGMDV; or (c) the CDR1 sequence GGSISSGGHY, the CDR2 sequence IHYSGST and the CDR3 sequence ARWRHDIFAAYPYYYYGMDV; (d) the CDR1 sequence GGSIRSGGHY, the CDR2 sequence IHRSGNP and the CDR3 sequence ARWRHDIFAAYPYYYYGMDV; or (e) CDR1 sequence GGSISSGGHY, CDR2 sequence IHHSGST and CDR3 sequence ARWRHDIFAAYPYYYYGMDV. 28. A bispecific antibody according to claim 27 containing an Fc region. 29. The bispecific antibody of claim 28, wherein the heavy chain CD3-binding variable domain contains the sequence of SEQ ID NO: 18, and the light chain variable domain contains the sequence of SEQ ID NO: 69. 30. The bispecific antibody of claim 27 comprising a heavy chain CD3-binding variable domain comprising the sequences CDR1, CDR2 and CDR3 of SEQ ID NO: 18 in a human VH framework region. 31. The bispecific antibody of claim 27 comprising a light chain variable domain comprising the sequences CDR1, CDR2 and CDR3 of SEQ ID NO: 69 in the human VL framework region.

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