WO2018136725A1

WO2018136725A1 - Innate immune cell inducible binding proteins and methods of use

Info

Publication number: WO2018136725A1
Application number: PCT/US2018/014396
Authority: WO
Inventors: Patrick Baeuerle; Robert B. Dubridge; Holger Wesche; Luke Evnin; Jeanmarie Guenot; Richard J. Austin; Bryan D. LEMON
Original assignee: Harpoon Therapeutics, Inc.
Priority date: 2017-01-19
Filing date: 2018-01-19
Publication date: 2018-07-26

Abstract

Provided herein are conditionally activated antigen-binding proteins comprising a protease-activated domain binding to an innate immune cell, at least one half-life extension domain, and two or more domains binding to one or more target antigens. Also provided are pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells for making such antigen-binding proteins. Also disclosed are methods of using the disclosed antigen-binding proteins in the prevention, and/or treatment diseases, conditions and disorders.

Description

INNATE IMMUNE CELL INDUCIBLE BINDING PROTEINS AND METHODS OF

USE

CROSS-REFERENCE

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 62/448,342, filed January 19, 2017, which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] The selective destruction of an individual cell or a specific cell type is often desirable in a variety of clinical settings. For example, it is a primary goal of cancer therapy to specifically destroy tumor cells, while leaving healthy cells and tissues intact and undamaged. One such method is by inducing an immune response against the tumor, to make immune effector cells such as natural killer (NK) cells or cytotoxic T lymphocytes (CTLs) attack and destroy tumor cells.

SUMMARY OF THE INVENTION

[0003] Provided herein are conditional binding protein, pharmaceutical compositions thereof, as nucleic acids, recombinant expression vectors and host cells for making such antigen binding proteins, and methods of use for the treatment of diseases, disorders, or conditions.

[0004] In some aspects, there are provided antigen-binding proteins, comprising a single polypeptide chain comprising two or more inactive innate immune cell binding domains, two or more target antigen binding domains, one or more half-life extension domains, and one or more protease cleavage domains; wherein upon protease cleavage of the protease cleavage domain and binding the target antigens by the target antigen binding domains, the innate immune cell binding domain becomes active and binds to an innate immune cell. In some embodiments, the innate immune cell binding domains bind to a cell surface antigen selected from CDlc, CD83, CD141, CD209, MHC II, CD123, CD303, CD304, CD16, CD56, CDld, CD160, PLZF, NKG2d, CD94-NKG2A/C/E , CD14, CD64, CD15, 2D7 antigen, CD203c, FcsRIa, CDl lb, CD193, EMR1, and Siglec-8 and activates an innate immune cell selected from dendritic cells, plasmacytoid dendritic cells, natural killer cells, natural killer T cells, monocytes, neutrophils, basophils, and eosinophils, wherein the innate immune cell is not a T cell. In some

embodiments, the target antigen binding domains bind to the same target antigen. In some embodiments, the target antigen binding domains bind to different target antigens. In some embodiments, the protease cleavage domain is cleaved by at least one of a serine protease, a cysteine protease, an aspartate protease, a threonine protease, a glutamic acid protease, a metalloproteinase, a gelatinase, and an asparagine peptide lyase. In some embodiments, the protease cleavage domain is cleaved by at least one of a Cathepsin B, a Cathepsin C, a Cathepsin D, a Cathepsin E, a Cathepsin K, a Cathepsin L, a kallikrein, a hKl, a hK10, a hK15, a plasmin, a collagenase, a Type IV collagenase, a stromelysin, a Factor Xa, a chymotryp sin-like protease, a trypsin-like protease, a elastase-like protease, a subtili sin-like protease, an actinidain, a bromelain, a calpain, a caspase, a caspase-3, a Mirl-CP, a papain, a HIV-1 protease, a HSV protease, a CMV protease, a chymosin, a renin, a pepsin, a matriptase, a legumain, a plasmepsin, a nepenthesin, a metalloexopeptidase, a metalloendopeptidase, a matrix metalloprotease (MMP), a MMPl, a MMP2, a MMP3, a MMP8, a MMP9, a MMPlO, a MMPl l, a MMP12, a MMP13, a MMP14, an ADAM10, an ADAM12, an urokinase plasminogen activator (uPA), an

enterokinase, a prostate-specific antigen (PSA, hK3), an interleukin-ΐβ converting enzyme, a thrombin, a FAP (FAP-a), a dipeptidyl peptidase, and a dipeptidyl peptidase IV (DPPIV/CD26). In some embodiments, the protease cleavage domain is cleaved at the site of a tumor. In some embodiments, the protease is expressed by a cell in a microenvironment of the tumor. In some embodiments, the protease cleavage domain is cleaved in the blood of a subject. In some embodiments, the protein further comprises two or more protease cleavage domains. In some embodiments, one or more innate immune cell binding domains comprise a polypeptide derived from a single-chain variable fragment (scFv) specific to a cell surface marker on an innate immune cell, wherein the innate immune cell is not a T cell. In some embodiments, one or more innate immune cell binding domains are specific for a dendritic cell. In some embodiments, one or more innate immune cell binding domains are specific for a plasmacytoid dendritic cell. In some embodiments, one or more innate immune cell binding domains are specific for a natural killer cell. In some embodiments, one or more innate immune cell binding domains are specific for a natural killer T cell. In some embodiments, one or more innate immune cell binding domains are specific for a monocyte. In some embodiments, one or more innate immune cell binding domains are specific for a neutrophil. In some embodiments, one or more innate immune cell binding domains are specific for a basophil. In some embodiments, one or more innate immune cell binding domains are specific for an eosinophil. In some embodiments, one or more innate immune cell binding domains comprise complementary determining regions (CDRs) selected from the group consisting of Lorvotuzumab, 3C12C, CSL362, 3G8, rMil2, E4, NNC141-0100. In some embodiments, one or more innate immune cell binding domains are humanized. In some embodiments, one or more activated innate immune cell binding domains have a KD binding 1000 nM or less to innate immune cells. In some embodiments, one or more activated innate immune cell binding domains have a KD binding 100 nM or less to innate immune cell innate immune cells. In some embodiments, one or more activated innate immune cell binding domains have a KD binding 10 nM or less to innate immune cells. In some embodiments, one or more innate immune cell binding domains have crossreactivity with cynomolgus innate immune cells. In some embodiments, one or more innate immune cell binding domains comprise an amino acid sequence provided herein. In some embodiments, one or more half-life extension domains comprise a binding domain to human serum albumin. In some embodiments, one or more half-life extension domains comprise a scFv, a variable heavy domain (VH), a variable light domain (VL), a nanobody, a peptide, a ligand, or a small molecule. In some embodiments, one or more half-life extension domains comprise a scFv. In some embodiments, one or more half-life extension domains comprise a VH domain.

[0005] The antigen-binding protein of any one of claims 1 to 28, wherein one or more half-life extension domains comprise a VL domain. In some embodiments, one or more half-life extension domains comprise a nanobody. In some embodiments, one or more half-life extension domains comprise a peptide. In some embodiments, one or more half-life extension domains comprise a ligand. In some embodiments, one or more half-life extension domains comprise an Fc domain. In some embodiments, at least one half-life extension domain is at the N-terminus of the protein prior to protease cleavage. In some embodiments, at least one half-life extension domain is at the C-terminus of the protein prior to protease cleavage. In some embodiments, at least one half-life extension domain is not at the C-terminus or the N-terminus of the protein prior to protease cleavage. In some embodiments, the protease cleavage domain is in the half- life extension domain or the innate immune cell binding domain. In some embodiments, the protease cleavage domain is not in the half-life extension domain or the innate immune cell binding domain. In some embodiments, the target antigen binding domains independently comprise a scFv, a VH domain, a VL domain, a non-Ig domain, or a ligand that specifically binds to the target antigen. In some embodiments, at least one target antigen binding domains specifically bind to a cell surface molecule. In some embodiments, at least one target antigen binding domains specifically bind to a tumor antigen. In some embodiments, the target antigen binding domains specifically and independently bind to an antigen selected from at least one of EpCAM, EGFR, HER-2, HER-3, cMet, CEA, and FoIR. In some embodiments, the target antigen binding domains specifically and independently bind to two different antigens, wherein at least one of the antigens is selected from one of EpCAM, EGFR, HER-2, HER-3, cMet, CEA, and FoIR. In some embodiments, the protein prior to cleavage of the protease cleavage domain is less than about 100 kDa. In some embodiments, the protein after cleavage of the protease cleavage domain is about 25 to about 75 kDa. In some embodiments, the protein prior to protease cleavage has a size that is above the renal threshold for first-pass clearance. In some embodiments, the protein prior to protease cleavage has an elimination half-time of at least about 50 hours. In some embodiments, the protein prior to protease cleavage has an elimination half-time of at least about 100 hours. In some embodiments, the protein has increased tissue penetration or tissue distribution as compared to an IgG to the same target antigen. In some embodiments, the protein has improved pharmacokinetics as compared to an IgG to the same target antigen. In some embodiments, the protein has reduced or eliminated target mediated drug disposition through innate immune cell binding as compared to an IgG to an innate immune cell. In some embodiments, the protein has a shallower alpha phase and higher exposure in the beta phase as compared to an IgG to the same target antigen. In some embodiments, the protein has a larger therapeutic window with smaller peak/trough differences in exposure as compared to an IgG to the same target antigen.

[0006] In other aspects, there are provided, polynucleotide encoding an antigen-binding protein according to any one of above embodiments. In additional aspects, there are provided vectors comprising the above polynucleotides. In further aspects, there are provided, host cells transformed with the above vectors.

[0007] In additional aspects, there are provided pharmaceutical compositions comprising (i) the antigen-binding protein according to any one of the above embodiments, the polynucleotide according to the above embodiments, the vector according to the above embodiments, or the host cell according to the above embodiments, and (ii) a pharmaceutically acceptable carrier.

[0008] In further aspects, there are provided, processes for the production of an antigen-binding protein of any one of the above embodiments, said process comprising culturing a host transformed or transfected with a vector comprising a nucleic acid sequence encoding an antigen-binding protein of any one of the above embodiments under conditions allowing the expression of the protein and recovering and purifying the produced protein from the culture.

[0009] In additional aspects, there are provided, methods for the treatment or amelioration of a proliferative disease, a tumorous disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, a graft-versus-host disease or a host-versus-graft disease comprising the administration of an antigen-binding protein of any one of the above embodiments to a subject in need of such a treatment or amelioration. In some embodiments, the subject is a human. In some

embodiments, the method further comprises administration of an agent in combination with the antigen-binding protein of any one of the above embodiments.

[0010] In additional aspects, there are provided, antigen-binding proteins, wherein the protein comprises a single polypeptide chain comprising a protease cleavage domain (P) separating the chain into a first and second region; wherein the first region comprises an anti- innate immune cell VH binding domain (IVH) and a target antigen binding domain (Tl) and the second region comprises an anti- innate immune cell VL binding domain (IVL) and a target antigen binding domain (T2); wherein the protein optionally comprises a half-life extension domain (H) in the first region, second region, or both regions, and wherein upon activation by protease cleavage of P and binding the target antigen by Tl and T2, the first and second regions associate to form a complete anti- innate immune cell VL/VH binding domain that binds innate immune cells.

[0011] In further aspects, there are provided, antigen-binding proteins, wherein the protein comprises a single polypeptide chain comprising a protease cleavage domain (P) separating the chain into a first and second region; wherein the first region comprises an anti- innate immune cell VL binding domain (IVL) and a target antigen binding domain (Tl) and the second region comprises an anti- innate immune cell VH binding domain (IVH) and a target antigen binding domain (T2); wherein the protein optionally comprises a half-life extension domain (H) in the first region, second region, or both regions, and wherein upon activation by protease cleavage of P and binding of the target antigen by Tl and T2, the first and second regions associate to form a complete anti- innate immune cell VL/VH binding domain that binds innate immune cells.

[0012] In additional aspects, there are provided, antigen-binding proteins, wherein the protein comprises a single polypeptide chain comprises a first and second region; wherein the first region comprises an anti- innate immune cell VH binding domain (IVH), an inactive anti- innate immune cell VL binding domain (IVLi) which associates with IVH and a target antigen binding domain (Tl); wherein the second region comprises a an anti- innate immune cell VL binding domain (IVL); an inactive anti- innate immune cell VH binding domain (IVHi) which associates with IVH and a target antigen binding domain (T2); wherein the protein optionally comprises a half-life extension domain (H) in the first region, second region, or both regions, and wherein IVLi and IVHi each comprise at least one protease cleavage domains; and wherein upon activation by protease cleavage the protease cleavage domains and binding the target antigen by Tl and T2, the first and second regions associate to form a complete anti- innate immune cell VL/VH binding domain that binds innate immune cells.

[0013] In further aspects, there are provided, antigen-binding proteins, wherein the protein comprises a single polypeptide chain comprises a first and second region; wherein the first region comprises an anti- innate immune cell VL binding domain (IVL), an inactive anti- innate immune cell VH binding domain (IVHi) which associates with IVL and a target antigen binding domain (Tl); wherein the second region comprises a an anti- innate immune cell VH binding domain (IVH); an inactive anti- innate immune cell VL binding domain (IVLi) which associates with IVL and a target antigen binding domain (T2); wherein the protein optionally comprises a half-life extension domain (H) in the first region, second region, or both regions, and wherein IVLi and IVHi each comprise at least one protease cleavage domains; and wherein upon activation by protease cleavage the protease cleavage domains and binding the target antigen by Tl and T2, the first and second regions associate to form a complete anti- innate immune cell VL/VH binding domain that binds innate immune cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative

embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0015] Figure 1 shows an exemplary antigen binding protein. In this example, the anti-CD16 VH and VL domains are separated by a protease cleavage site which keeps the anti -CD 16 VH and VL domains from folding properly and binding to CD 16 on an innate immune cell. Figure 1 also shows the cleaved antigen binding protein, where the VH and VL domains are folded such that they are able to bind to CD 16 and the anti -target domains are bound to the target antigen on the surface of the target cell. This example also has a half-life extension domain.

[0016] Figure 2 shows an exemplary dual-target antigen binding protein. In this example, the anti-CD 16VH and VL domains are separated by a protease cleavage site which keeps the anti- CD16VH and VL domains from folding properly and binding to CD 16 on an innate immune cell. Figure 2 also shows the cleaved antigen binding protein, where the VH and VL domains are folded such that they are able to bind CD16and each anti -target domain is bound to its target antigen on the surface of the target cell. This example also has a half-life extension domain.

[0017] Figure 3 shows an exemplary antigen binding protein. In this example, the anti- CD 16VH and VL domains are bound to protease cleavable VL and VH domains that together do not bind to CD 16. Once these domains are cleaved, the anti-CD 16VH and VL domains are able to fold and associate with CD16 on an innate immune cell. This example also has two anti- target domains that in some cases bind to two different antigens and in some cases bind to the same antigen. This example also has a half-life extension domain.

DETAILED DESCRIPTION OF THE INVENTION

[0018] Described herein are antigen-binding proteins, such as tri-specific, quad-specific antigen, and multi-specific binding proteins, pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells for making such antigen-binding proteins. Also provided are methods of using the disclosed antigen-binding proteins in the prevention, and/or treatment of diseases, conditions and disorders. The antigen-binding proteins are capable of specifically binding to one or more target antigen as well as at least one protein on an innate immune cell, and optionally a half-life extension domain, such as an HSA binding domain. Binding to an innate immune cell is only possible once activated by a protease and binding to the target antigen(s). It is to be understood that in some embodiments, protease cleavage of the protease cleavage domain occurs before target antigen binding domain binding to the target antigen. It is also to be understood that in some embodiments, protease cleavage of the protease cleavage domain occurs after target antigen binding domain binding to the target antigen.

Figures 1, 2 and 3 depict three non-limiting examples of an antigen-binding protein.

[0019] The antigen-binding proteins described herein are designed to allow specific targeting of cells expressing a target antigen by recruiting innate immune cells. This improves specificity compared to therapeutics that bind to innate immune cells and a target antigen which may or may not be expressed by a target cell, such as a tumor or cancer cell. In contrast, by activating innate immune cell binding specifically in the microenvironment of the target cell, where the target antigen and proteases are highly expressed, the antigen-binding proteins can crosslink innate immune cells with cells expressing a target antigen in a highly specific fashion, thereby directing the therapeutic potential of the innate immune cell towards the target cell. The antigen-binding proteins described herein engage innate immune cells via protease-activated binding to the surface-expressed surface antigens, which are uniquely found on the surface of innate immune cells. Simultaneous binding of several antigen-binding proteins to an innate immune cell and to a target antigen expressed on the surface of particular cells causes innate immune cell activation and mediates the subsequent lysis of the particular target antigen expressing cell. Thus, antigen-binding proteins are contemplated to display strong, specific and efficient target cell killing. In some embodiments, the antigen-binding proteins described herein stimulate target cell killing by innate immune cells to eliminate pathogenic cells in protease-rich microenvironments (e.g., tumor cells, virally or bacterially infected cells, autoreactive T cells, etc). In some of such embodiments, cells are eliminated selectively, thereby reducing the potential for toxic side effects. In other embodiments, the same polypeptides could be used to enhance the elimination of endogenous cells for therapeutic effect, such as B or T lymphocytes in autoimmune disease, or hematopoietic stem cells (HSCs) for stem cell transplantation.

Proteases known to be associated with diseased cells or tissues include but are not limited to serine proteases, cysteine proteases, aspartate proteases, threonine proteases, glutamic acid proteases, metalloproteases, asparagine peptide lyases, serum proteases, cathepsins, Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin E, Cathepsin K, Cathepsin L, kallikreins, hKl, hK10, hK15, plasmin, collagenase, Type IV collagenase, stromelysin, Factor Xa, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtili sin-like protease, actinidain, bromelain, calpain, caspases, caspase-3, Mirl-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matriptase, legumain, plasmepsin, nepenthesin, metalloexopeptidases, metalloendopeptidases, matrix metalloproteases (MMP), MMP1, MMP2, MMP3, MMP8, MMP9, MMP 13, MMP11, MMP 14, urokinase plasminogen activator (uPA), enterokinase, prostate-specific antigen (PSA, hK3), interleukin-ΐβ converting enzyme, thrombin, FAP (FAP-a), dipeptidyl peptidase, and dipeptidyl peptidase IV (DPPIV/CD26).

[0020] The antigen-binding proteins described herein confer further therapeutic advantages over traditional monoclonal antibodies and other smaller bispecific molecules. Bi-specific molecules are designed to bind to a target cell via a cell-specific marker associated with a pathogenic cell. Toxicities are possible when, in some cases, healthy cells or tissues express the same marker as the pathogenic cell. One benefit to an antigen binding protein is that binding to an innate immune cell is dependent upon activation by a protease expressed by the target cell, such as a tumor cell, and binding of the antigen binding domains to one or more target antigens, for example a tumor antigen. The antigen-binding proteins comprise an inactive innate immune cell binding domain comprising VH and VL domains separated by one or more protease cleavage sites. In the protease-rich environment of the target cell, the protease cleavage sites are cleaved allowing the VH and VL domains to fold properly and bind to an innate immune cell when one or more target antigens are bound. In the absence of protease cleavage, the innate immune cell binding domain is inactive and cannot bind to an innate immune cell.

[0021] The antigen-binding proteins described herein comprise at least one protease cleavage site comprising an amino acid sequence that is cleaved by at least one protease. In some cases, the antigen-binding proteins described herein comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more protease cleavage sites that are cleaved by at least one protease. In some cases, the protease cleavage site comprises an amino acid sequence recognized by a protease is a MMP9 cleavage site comprising a polypeptide having an amino acid sequence LEATA (SEQ ID NO: 4).

[0022] The antigen-binding proteins described herein confer additional therapeutic advantages over traditional monoclonal antibodies and other smaller bispecific molecules. Generally, the effectiveness of recombinant protein pharmaceuticals depends heavily on the intrinsic pharmacokinetics of the protein itself. One such additional benefit here is that the antigen- binding proteins described herein have extended pharmacokinetic elimination half-time due to having a half-life extension domain, for example a binding domain specific to HSA. In this respect, the antigen-binding proteins described herein have an extended serum elimination half- time of about two, three, about five, about seven, about 10, about 12, or about 14 days in some embodiments. This contrasts to other binding proteins such as BiTE or DART molecules which have relatively much shorter elimination half-times. For example, the BiTE CD19xCD3 bispecific scFv-scFv fusion molecule requires continuous intravenous infusion (i.v.) drug delivery due to its short elimination half-time. The longer intrinsic half-times of the antigen- binding proteins solve this issue thereby allowing for increased therapeutic potential such as low-dose pharmaceutical formulations, decreased periodic administration and/or novel pharmaceutical compositions.

[0023] The antigen-binding proteins described herein also have an optimal size for enhanced tissue penetration and distribution and enhanced reduced first pass renal clearance. Because the kidney generally filters out molecules below 50 kDa, efforts to reduce clearance in the design of protein therapeutics have focused on increasing molecular size through protein fusions, glycosylation, or the addition of polyethylene glycol polymers (i.e., PEG). However, while increasing the size of a protein therapeutic may prevent renal clearance, the downside is that the larger size also prevents penetration of the molecule into the target tissues. The antigen-binding proteins described herein avoid this by associating with albumin which will prevent renal clearance while also having a small size that allows enhanced tissue penetration and distribution and optimal efficacy. Accordingly, the antigen-binding proteins described herein, in some embodiments have a size of about 50 kD to about 80 kD, about 50 kD to about 75 kD, about 50 kD to about 70 kD, or about 50 kD to about 65 kD. Thus, the size of the antigen-binding proteins is advantageous over IgG antibodies which are about 150 kD and the BiTE and DART diabody molecules which are about 55 kD but are not half-life extended and therefore are cleared quickly through the kidney.

[0024] Another feature of the antigen-binding proteins described herein is that they are of a single-polypeptide design with flexible linkage of their domains. This allows for facile production and manufacturing of the antigen-binding proteins as they can be encoded by single cDNA molecule to be easily incorporated into a vector. Further, because the antigen-binding proteins described herein are a monomeric single polypeptide chain, there are no chain pairing issues or a requirement for dimerization. It is contemplated that the antigen-binding proteins described herein have a reduced tendency to aggregate unlike other reported molecules such as bispecific BiTE proteins.

[0025] In one aspect, the antigen binding proteins, in pre-activated form, comprise a single polypeptide chain comprising a first region and a second region separated by at least one protease cleavage domain (P). In an embodiment, the first region comprises an anti-innate immune cell V_H binding domain (IV_H) and a target antigen binding domain (Ti). In an embodiment, the second region comprises an anti-innate immune cell V_L binding domain (IV_L) and a target antigen binding domain (T₂). In an embodiment, the antigen-binding domain optionally comprises a half-life extension domain (H) in the first region. In an embodiment, the antigen-binding domain optionally comprises a half-life extension domain (H) in the second region. Once, activated by a protease cleaving the protease cleavage domain (P) and target antigen binding domains T₁ and T₂ binding the target antigens, the anti- innate immune cell binding domains IV_H and IV_L are activated to bind to an innate immune cell. The domains in an antigen binding protein are contemplated to be arranged in any order within each region, with a protease cleavage domain (P) in the center of the pre-activated polypeptide. Further, each region may be in any order within the pre-activated polypeptide. Thus, by way of example only, it is contemplated that exemplary domain order of the antigen-binding proteins include but are not limited to:

c) IV_H-T_!-P-IV_L-T,,

d) T_!-IV_H-P-IV_L-T₂,

g) IV_H-T H-P-T.-IV_L,

h) IV_H-T P-H-T.-IV_L,

k) H-T_!-IV_H-P-T V_L,

1) T H-IV_H-P-T.-IV_L,

o) T_i-rv_H-p-T₂-H-iv_L,

P) T_i-rv_H-p-T₂-iv_L-H,

q) H-IV_H-T_!-P-IV_L-T,,

IV_H-H-T_!-P-IV_L-T,,

s) IV_H-T_!-H-P-IV_L-T,,

t) IV_H-T_!-P-H-IV_L-T,,

u) IV_H-T_!-P-IV_L-H-T,,

v) IV_H-T_!-P -IV_L-T₂-H,

w) H-T_!-IV_H-P-IV_L-T,,

x) T_!-H-IV_H-P-IV_L-T,,

y) T_!-TV_H-H-P-IV_L-T,,

z) T_!-IV_H-P-H-IV_L-T₂,

aa) T_I-TV_H-P-IV_L-H-T₂, and bb) Ti-rV_H-P-IV_L-T₂-H.

[0026] In one aspect, the antigen-binding proteins, in pre-activated form, comprise a single polypeptide chain comprising a first region and a second region. In an embodiment, the first region comprises an anti- innate immune cell V_H binding domain (IV_H), an inactive anti- innate immune cell V_L binding domain (IV_L;) which associates with IV_H, and a target antigen binding domain (Ti), wherein IV_L; comprises at least one protease cleavage domain (P). In an embodiment, the second region comprises an anti- innate immune cell V_L binding domain (IV_L), an inactive anti- innate immune cell V_H binding domain (IV_H,) which associates with IV_L, and a target antigen binding domain (T₂), wherein IV comprises at least one protease cleavage domain (P). In an embodiment, the antigen-binding domain optionally comprises a half-life extension domain (H) in the first region. In an embodiment, the antigen-binding domain optionally comprises a half-life extension domain (H) in the second region. Once, activated by a protease cleaving the protease cleavage domains (P) and target antigen binding domains Ti and T₂ binding the target antigens, the anti- innate immune cell binding domains IV_H and IV_L are activated to bind to an innate immune cell. An example of this type of antigen binding protein is described in Figure 3.

[0027] In the antigen-binding proteins described herein, the domains are linked by internal linkers LI, L2, L3, and L4 where LI links the first and second domain of the antigen-binding proteins, L2 links the second and third domains of the antigen-binding proteins, L3 links the third and fourth domains of the antigen-binding proteins, and L4 links the fourth and fifth domains of the protease activated antigen -binding proteins. Linkers LI, L2, L3, and L4 have an optimized length and/or amino acid composition. In some embodiments, linkers LI, L2, L3, and L4 are the same length and amino acid composition. In other embodiments, LI, L2, L3, and L4 are different. In certain embodiments, internal linkers LI, L2, L3, and/or L4 are "short", i.e., consist of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues. Thus, in certain instances, the internal linkers consist of about 12 or less amino acid residues. In the case of 0 amino acid residues, the internal linker is a peptide bond. In certain embodiments, internal linkers LI, L2, L3, and/or L4 are "long", i.e., consist of 15, 20 or 25 amino acid residues. In some

embodiments, these internal linkers consist of about 3 to about 15, for example 8, 9 or 10 contiguous amino acid residues. Regarding the amino acid composition of the internal linkers LI, L2, L3, and L4, peptides are selected with properties that confer flexibility to the antigen- binding proteins, do not interfere with the binding domains as well as resist cleavage from proteases. For example, glycine and serine residues generally provide protease resistance.

Examples of internal linkers suitable for linking the domains in the antigen-binding proteins include but are not limited to (GS)„, (GGS)„, (GGGS)„, (GGSG)„, (GGSGG)„, or (GGGGS)„, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, internal linker LI, L2, and/or L3 is (GGGGS)₄ or (GGGGS)₃.

Innate Immune Cell Binding Domain

[0028] Innate immune cells are a part of the innate immune system. Innate immune cells recognize and respond to pathogens via recognition of pathogens. They play a variety of roles in immunity including but not limited to, recruitment of other immune cells via cytokine secretion, activation of the complement cascade, identification and removal of foreign substances in the body, and presenting antigen to other immune cells. Innate immune cells do not include T cells, such as CD4+ T cells or CD8+ T cells. Innate immune cells include multiple cell types such as dendritic cells, plasmacytoid dendritic cells, natural killer cells, natural killer T cells, monocytes, neutrophils, basophils, and eosinophils. Recruitment and activation of innate immune cells to the site of a target antigen, for example at a diseased cell or tissue leads to activation of the immune system at that site. Each innate immune cell, is recruited by at least one cell surface marker expressed by the cell, which is specific for the innate immune cell type. Non-limiting examples of innate immune cell surface markers include but are not limited to in dendritic cells: CDlc, CD83, CD141, CD209, and MHC II; plasmacytoid dendritic cells: CD123, CD303, and CD304; natural killer cells: CD16, CD56; natural killer T cells: CDld, CD160, PLZF, NKG2d, and CD94-NKG2A/C/E; monocytes: CD14, CD16, CD47, and CD64; neutrophils: CD15, CD16, and CD89; basophils: 2D7 antigen, CD123, CD203c, and FcsRIa; eosinophils: CDl lb, CD193, EMR1, and Siglec-8.

[0029] In one aspect, the antigen-binding proteins described herein comprise a domain which specifically binds to an innate immune cell when activated by a protease. In one aspect, the antigen-binding proteins described herein comprise two or more domains which when activated by a protease specifically bind to a human innate immune cell, wherein the innate immune cell is not a T cell. In some embodiments, the antigen-binding proteins described herein comprise two or more domains which when activated by a protease which specifically binds to a dendritic cell. In some embodiments, the antigen-binding proteins described herein comprise two or more domains which when activated by a protease specifically bind to a plasmacytoid dendritic cell. In some embodiments, the antigen-binding proteins described herein comprise two or more domains which when activated by a protease specifically bind to a natural killer cell. In some embodiments, the antigen-binding proteins described herein comprise two or more domains which when activated by a protease specifically bind to a natural killer T cell. In some embodiments, the antigen-binding proteins described herein comprise two or more domains which when activated by a protease specifically bind to a monocyte. In some embodiments, the antigen-binding proteins described herein comprise two or more domains which when activated by a protease specifically bind to a neutrophil. In some embodiments, the antigen-binding proteins described herein comprise two or more domains which when activated by a protease specifically bind to an eosinophil. In some embodiments, the antigen -binding proteins described herein comprise two or more domains which when activated by a protease specifically bind to a basophil.

[0030] In some embodiments, the protease cleavage site is between the anti- innate immune cell VH and VL domains and keeps them from folding and binding to an innate immune cell. Once the protease cleavage site is cleaved by a protease present at the target cell, the anti- innate immune cell VH and VL domains are able to fold and bind to an innate immune cell. In an alternate embodiment, the protease cleavage site is designed into a non- innate immune cell binding VL and VH domain that binds to the anti- innate immune cell VH and VL domains. Cleavage of the protease cleavage site by a protease present at the target cell removes the non- innate immune cell binding VL and VH domain and allows the anti- innate immune cell VH and VL domain to fold and to bind an innate immune cell.

[0031] The antigen binding proteins described herein comprise a domain which specifically binds to an innate immune cell when activated by a protease. In one embodiment, the domain which specifically binds to an innate immune cell comprises a VH domain and a VL domain separated by at least one protease cleavage site. When the protease cleavage site is cleaved, the VH domain and the VL domain are able to fold and therefore bind to an innate immune cell. In some embodiments, the protease cleavage site is in a loop region. In some embodiments, the protease cleavage site is within the VH and/or the VL domains and the protease cleavage sites are cleaved revealing the VH and/or the VL domains allowing them to fold and therefore bind to an innate immune cell.

[0032] In further embodiments, the antigen-binding proteins described herein comprise two or more domains which when activated by a protease specifically bind to an innate immune cell surface marker including but are not limited to CDlc, CD83, CD 141, CD209, MHC II, CD 123, CD303, CD304, CD16, CD56, CDld, CD160, PLZF, KG2d, CD94- KG2A/C/E , CD14, CD16, CD47, CD64, CD15, CD16, CD89, 2D7 antigen, CD123, CD203c, FcsRIa, CDl lb, CD193, EMR1, and Siglec-8.. In certain instances, the antigen-binding proteins described herein comprise two or more domains which when activated by a protease specifically bind innate immune cell surface marker.

[0033] In certain embodiments, the innate immune cell binding domain of the antigen-binding proteins described herein exhibit not only potent innate immune cell binding affinities with human innate immune cells, but show also excellent cross reactivity with the respective cynomolgus monkey innate immune cell proteins. In some instances, the innate immune cell binding domain of the antigen-binding proteins is cross-reactive with innate immune cells from cynomolgus monkey. In certain instances, human xynomolgous K_D ratios for innate immune cell binding are between 5 and 0.2.

[0034] In some embodiments, the innate immune cell binding domain of the antigen binding protein can be any domain that binds to innate immune cell including but not limited to domains from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody. In some instances, it is beneficial for the innate immune cell binding domain to be derived from the same species in which the antigen binding protein will ultimately be used in. For example, for use in humans, it may be beneficial for the innate immune cell binding domain of the antigen binding protein to comprise human or humanized residues from the antigen binding domain of an antibody or antibody fragment.

[0035] Thus, in one aspect, the antigen-binding domain comprises a humanized or human antibody or an antibody fragment, or a murine antibody or antibody fragment. In one

embodiment, the humanized or human anti-innate immune cell binding domain comprises one or more (e.g., all three) light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) of a humanized or human anti- innate immune cell binding domain described herein, and/or one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a humanized or human anti -innate immune cell binding domain described herein, e.g., a humanized or human anti-innate immune cell binding domain comprising one or more, e.g., all three, LC CDRs and one or more, e.g., all three, HC CDRs.

[0036] In some embodiments, the humanized or human anti-innate immune cell binding domain comprises a humanized or human light chain variable region specific to innate immune cell where the light chain variable region specific to innate immune cell comprises human or non- human light chain CDRs in a human light chain framework region. In certain instances, the light chain framework region is a λ (lambda) light chain framework. In other instances, the light chain framework region is a κ (kappa) light chain framework.

[0037] In some embodiments, the humanized or human anti-innate immune cell binding domain comprises a humanized or human heavy chain variable region specific to innate immune cell where the heavy chain variable region specific to innate immune cell comprises human or non- human heavy chain CDRs in a human heavy chain framework region. [0038] In certain instances, the complementary determining regions of the heavy chain and/or the light chain are derived from known anti-innate immune cell antibodies, such as, for example, Lorvotuzumab, 3C12C, CSL362, 3G8, rMil2, E4, NNC141-0100.

[0039] In one embodiment, the anti-innate immune cell binding domain is a single chain variable fragment (scFv) comprising a light chain and a heavy chain of an amino acid sequence provided herein. As used herein, "single chain variable fragment" or "scFv" refers to a antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker, and capable of being expressed as a single polypeptide chain, and wherein the scFv retains the specificity of the intact antibody from which it is derived. In an embodiment, the anti-innate immune cell binding domain comprises: a light chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 30, 20 or 10 modifications (e.g., substitutions) of an amino acid sequence of a light chain variable region provided herein, or a sequence with 95-99% identity with an amino acid sequence provided herein; and/or a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 30, 20 or 10 modifications (e.g., substitutions) of an amino acid sequence of a heavy chain variable region provided herein, or a sequence with 95-99% identity to an amino acid sequence provided herein. In one embodiment, the humanized or human anti-innate immune cell binding domain is a scFv, and a light chain variable region comprising an amino acid sequence described herein, is attached to a heavy chain variable region comprising an amino acid sequence described herein, via a scFv linker. The light chain variable region and heavy chain variable region of a scFv can be, e.g., in any of the following orientations: light chain variable region- scFv linker-heavy chain variable region or heavy chain variable region- scFv linker-light chain variable region.

[0040] In some instances, scFvs which bind to innate immune cell are prepared according to known methods. For example, scFv molecules can be produced by linking VH and VL regions together using flexible polypeptide linkers. The scFv molecules comprise a scFv linker (e.g., a Ser-Gly linker) with an optimized length and/or amino acid composition. Accordingly, in some embodiments, the length of the scFv linker is such that the VH or VL domain can associate intermolecularly with the other variable domain to form the innate immune cell binding site. In certain embodiments, such scFv linkers are "short", i.e. consist of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues. Thus, in certain instances, the scFv linkers consist of about 12 or less amino acid residues. In the case of 0 amino acid residues, the scFv linker is a peptide bond. In some embodiments, these scFv linkers consist of about 3 to about 15, for example 8, 9 or 10 contiguous amino acid residues. Regarding the amino acid composition of the scFv linkers, peptides are selected that confer flexibility, do not interfere with the variable domains as well as allow inter-chain folding to bring the two variable domains together to form a functional innate immune cell binding site. For example, scFv linkers comprising glycine and serine residues generally provide protease resistance. In some embodiments, linkers in a scFv comprise glycine and serine residues. The amino acid sequence of the scFv linkers can be optimized, for example, by phage-display methods to improve the innate immune cell binding and production yield of the scFv. Examples of peptide scFv linkers suitable for linking a variable light chain domain and a variable heavy chain domain in a scFv include but are not limited to (GS)_n, (GGS)_n, (GGGS)„, (GGSG)„, (GGSGG)„, or (GGGGS)„, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the scFv linker can be (GGGGS)₄ or (GGGGS)₃. Variation in the linker length may retain or enhance activity, giving rise to superior efficacy in activity studies.

[0041] In some embodiments, innate immune cell binding domain of an antigen binding protein has an affinity to innate immune cells with a K_D of 1000 nM or less, 100 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 1 nM or less, or 0.5 nM or less. In some embodiments, the innate immune cell binding domain of an antigen binding protein has an affinity to CDlc, CD83, CD141, CD209, MHC II, CD123, CD303, CD304, CD16, CD56, CD Id, CD 160, PLZF, KG2d, CD94- KG2A/C/E , CD 14, CD 16, CD64, CD 15 CD 16, 2D7 antigen, CD123, CD203c, FcsRIa, CDl lb, CD193, EMRl, or Siglec-8with a K_D of 1000 nM or less, 100 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 1 nM or less, or 0.5 nM or less. In further embodiments, innate immune cell binding domain of an antigen binding protein has low affinity to an innate immune cell, i.e., about 100 nM or greater.

[0042] The affinity to bind to innate immune cell can be determined, for example, by the ability of the antigen binding protein itself or its innate immune cell binding domain to bind to an innate immune cell surface marker coated on an assay plate; displayed on a microbial cell surface; in solution; etc. The binding activity of the antigen binding protein itself or its innate immune cell binding domain of the present disclosure to innate immune cell can be assayed by immobilizing the cell surface marker (e.g., CDlc, CD83, CD141, CD209, MHC II, CD123, CD303, CD304, CD16, CD56, CDld, CD160, PLZF, KG2d, CD94- KG2A/C/E , CD14, CD16, CD64, CD15 CD16, 2D7 antigen, CD123, CD203c, FcsRIa, CDl lb, CD193, EMRl, or Siglec-8) or the antigen binding protein itself or its innate immune cell binding domain, to a bead, substrate, cell, etc. Agents can be added in an appropriate buffer and the binding partners incubated for a period of time at a given temperature. After washes to remove unbound material, the bound protein can be released with, for example, SDS, buffers with a high pH, and the like and analyzed, for example, by Surface Plasmon Resonance (SPR). Protease Cleavage Domains

[0043] Protease cleavage domains are polypeptides having a sequence recognized and cleaved in a sequence-specific manner. Antigen binding proteins contemplated herein, in some cases, comprise a protease cleavage domain recognized in a sequence-specific manner by a matrix metalloprotease (MMP), for example a MMP9. In some cases, the protease cleavage domain recognized by a MMP9 comprises a polypeptide having an amino acid sequence

PR(S/T)(L/I)(S/T) (SEQ ID NO: 3). In some cases, the protease cleavage domain recognized by a MMP9 comprises a polypeptide having an amino acid sequence LEATA (SEQ ID NO: 4). In some cases, the protease cleavage domain is recognized in a sequence-specific manner by a MMP11. In some cases, the protease cleavage domain recognized by a MMP11 comprises a polypeptide having an amino acid sequence GGAANLVRGG (SEQ IN NO: 3). In some cases, the protease cleavage domain is recognized by a protease disclosed in Table 1. In some cases, the protease cleavage domain recognized by a protease disclosed in Table 1 comprises a polypeptide having an amino acid sequence selected from a sequence disclosed in Table 1 (SEQ ID NOS: 1-42).

[0044] Proteases are proteins that cleave proteins, in some cases, in a sequence-specific manner. Proteases include but are not limited to serine proteases, cysteine proteases, aspartate proteases, threonine proteases, glutamic acid proteases, metalloproteases, asparagine peptide lyases, serum proteases, cathepsins, Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin E, Cathepsin K, Cathepsin L, kallikreins, hKl, hK10, hK15, plasmin, collagenase, Type IV collagenase, stromelysin, Factor Xa, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtilisin-like protease, actinidain, bromelain, calpain, caspases, caspase-3, Mirl-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matriptase, legumain, plasmepsin, nepenthesin, metalloexopeptidases, metalloendopeptidases, matrix metalloproteases (MMP), MMP1, MMP2, MMP3, MMP8, MMP9, MMP 13, MMP11, MMP 14, urokinase plasminogen activator (uPA), enterokinase, prostate-specific antigen (PSA, hK3), interleukin-ΐβ converting enzyme, thrombin, FAP (FAP-a), dipeptidyl peptidase, and dipeptidyl peptidase IV (DPPTV/CD26).

[0045] Table 1: Exemplary Proteases and Protease Cleavage Domain Sequences

MMP11 GGAANLVRGG 5

MMP14 SGRIGFLRTA 6

MMP PLGLAG 7

MMP PLGLAX 8

MMP PLGC(me)AG 9

MMP ESPAYYTA 10

MMP RLQLKL 11

MMP RLQLKAC 12

MMP2, MMP9, MMP 14 EP(Cit)G(Hof)YL 13

Urokinase plasminogen activator (uPA) SGRSA 14

Urokinase plasminogen activator (uPA) DAFK 15

Urokinase plasminogen activator (uPA) GGGRR 16

Lysosomal Enzyme GFLG 17

Lysosomal Enzyme ALAL 18

Lysosomal Enzyme FK 19

Cathepsin B NLL 20

Cathepsin D PIC(Et)FF 21

Cathepsin K GGPRGLPG 22

Prostate Specific Antigen HSSKLQ 23

Prostate Specific Antigen HSSKLQL 24

Prostate Specific Antigen HSSKLQEDA 25

Herpes Simplex Virus Protease LVLASSSFGY 26

HIV Protease GVSQNYPIVG 27

CMV Protease GVVQASCRLA 28

Thrombin F(Pip)RS 29

Thrombin DPRSFL 30

Thrombin PPRSFL 31

Caspase-3 DEVD 32

Caspase-3 DEVDP 33

Caspase-3 KGSGDVEG 34

Interleukin 1β converting enzyme GWEHDG 35

Enterokinase EDDDDKA 36

FAP KQEQ PGST 37

Kallikrein 2 GKAFRR 38 Plasmin DAFK 39

Plasmin DVLK 40

Plasmin DAFK 41

TOP ALLLALL 42

[0046] Proteases are known to be secreted by some diseased cells and tissues, for example tumor or cancer cells, creating a microenvironment that is rich in proteases or a protease-rich microenvironment. In some case, the blood of a subject is rich in proteases. In some cases, cells surrounding the tumor secrete proteases into the tumor microenvironment. Cells surrounding the tumor secreting proteases include but are not limited to the tumor stromal cells,

myofibroblasts, blood cells, mast cells, B cells, NK cells, regulatory T cells, macrophages, cytotoxic T lymphocytes, dendritic cells, mesenchymal stem cells, polymorphonuclear cells, and other cells. In some cases, proteases are present in the blood of a subject, for example proteases that target amino acid sequences found in microbial peptides. This feature allows for targeted therapeutics such as antigen-binding proteins to have additional specificity because T cells will not be bound by the antigen binding protein except in the protease rich microenvironment of the targeted cells or tissue.

Half-Life Extension Domain

[0047] Contemplated herein are domains which extend the half-life of an antigen-binding domain. Such domains are contemplated to include but are not limited to HSA binding domains, Fc domains, small molecules, and other half-life extension domains known in the art. In some embodiments, Fc and albumin binding domains extend half-lives by increasing the size of the peptide drug. In some embodiments, Fc and albumin binding domains bind to the neonatal Fc receptor, FcRn, which, at least in some cases prevents degradation of the fusion protein in the endosome. In some embodiments, Fc domains, or HSA binding domains improve solubility and stability of the antigen binding domain.

[0048] Human serum albumin (HSA) (molecular mass ~67 kDa) is the most abundant protein in plasma, present at about 50 mg/ml (600 μΜ), and has a half-life of around 20 days in humans. HSA serves to maintain plasma pH, contributes to colloidal blood pressure, functions as carrier of many metabolites and fatty acids, and serves as a major drug transport protein in plasma.

[0049] Noncovalent association with albumin extends the elimination half-time of short lived proteins. For example, a recombinant fusion of an albumin binding domain to a Fab fragment resulted in an in vivo clearance of 25- and 58-fold and a half-life extension of 26- and 37-fold when administered intravenously to mice and rabbits respectively as compared to the

administration of the Fab fragment alone. . In another example, when insulin is acylated with fatty acids to promote association with albumin, a protracted effect was observed when injected subcutaneously in rabbits or pigs. Together, these studies demonstrate a linkage between albumin binding and prolonged action.

[0050] In one aspect, the antigen-binding proteins described herein comprise a half-life extension domain, for example a domain which specifically binds to HSA. In some embodiments, the HSA binding domain of an antigen binding protein can be any domain that binds to HSA including but not limited to domains from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody. In some embodiments, the HSA binding domain is a single chain variable fragments (scFv), single- domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) of camelid derived nanobody, peptide, ligand or small molecule specific for HSA. In certain embodiments, the HSA binding domain is a single- domain antibody. In other embodiments, the HSA binding domain is a peptide. In further embodiments, the HSA binding domain is a small molecule. It is contemplated that the HSA binding domain of an antigen binding protein is fairly small and no more than 25 kD, no more than 20 kD, no more than 15 kD, or no more than 10 kD in some embodiments. In certain instances, the HSA binding is 5 kD or less if it is a peptide or small molecule.

[0051] The half-life extension domain of an antigen binding protein provides for altered pharmacodynamics and pharmacokinetics of the antigen binding protein itself. As above, the half-life extension domain extends the elimination half-time. The half-life extension domain also alters pharmacodynamic properties including alteration of tissue distribution, penetration, and diffusion of the antigen-binding protein. In some embodiments, the half-life extension domain provides for improved tissue (including tumor) targeting, tissue penetration, tissue distribution, diffusion within the tissue, and enhanced efficacy as compared with a protein without a half-life extension binding domain. In one embodiment, therapeutic methods effectively and efficiently utilize a reduced amount of the antigen-binding protein, resulting in reduced side effects, such as reduced non-tumor cell cytotoxicity.

[0052] Further, characteristics of the half-life extension domain, for example a HSA binding domain, include the binding affinity of the HSA binding domain for HSA. Affinity of said HSA binding domain can be selected so as to target a specific elimination half-time in a particular antigen-binding protein. Thus, in some embodiments, the HSA binding domain has a high binding affinity. In other embodiments, the HSA binding domain has a medium binding affinity. In yet other embodiments, the HSA binding domain has a low or marginal binding affinity. Exemplary binding affinities include K_D concentrations at 10 nM or less (high), between 10 nM and 100 nM (medium), and greater than 100 nM (low). As above, binding affinities to HSA are determined by known methods such as Surface Plasmon Resonance (SPR).

[0053] The half-life extension domain of an antigen binding protein extends the half-life of an antigen binding protein to at least about two days, about three days, about four days, about five days, about six days, about seven days, about eight days, about nine days, about ten days, or more. In some embodiments, the half-life extension domain extends the half-life of an antigen binding protein to at least 2-3 days, 3-4 days, 4-5, days, 5-6 days, 6-7 days, 7-8 days, 2-5 days, 5-8 days, 2-10 days, or more. In some embodiments, the half-life extension domain extends the half-life of an antigen binding domain to at least about five days. In some embodiments, the half-life extension domain extends the half-life of an antigen binding protein to five or more days.

Target Antigen Binding Domain

[0054] In addition to the described innate immune cell binding and half-life extension domains, the antigen-binding proteins described herein also comprise at least two more domains that bind to one or more target antigens. It is contemplated herein that an antigen binding protein is cleaved, for example, in a disease-specific microenvironment or in the blood of a subject at the protease cleavage domain and that each target antigen binding domain will bind to a target antigen on a target cell, thereby activating the innate immune cell binding domain to bind an innate immune cell. At least one target antigen is involved in and/or associated with a disease, disorder or condition. In particular, a target antigen associated with a proliferative disease, a tumorous disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, a graft-versus-host disease or a host-versus-graft disease. In some embodiments, a target antigen is a tumor antigen expressed on a tumor cell. Alternatively in some embodiments, a target antigen is associated with a pathogen such as a virus or bacterium. At least one target antigen may also be directed against healthy tissue.

[0055] In some embodiments, a target antigen is a cell surface molecule such as a protein, lipid or polysaccharide. In some embodiments, a target antigen is a on a tumor cell, virally infected cell, bacterially infected cell, damaged red blood cell, arterial plaque cell, or fibrotic tissue cell. It is contemplated herein that upon binding more than one target antigen, two inactive innate immune cell binding domains are co-localized and form an active innate immune cell binding domain on the surface of the target cell. In some embodiments, the antigen binding protein comprises more than one target antigen binding domain to activate an inactive innate immune cell binding domain in the antigen binding protein. In some embodiments the antigen binding protein comprises more than one target antigen binding domain to enhance the strength of binding to the target cell. In some embodiments the antigen binding protein comprises more than one target antigen binding domain to enhance the strength of binding to the target cell. In some embodiments, more than one antigen binding domain comprises the same antigen binding domain. In some embodiments, more than one antigen binding domain comprises different antigen binding domains. For example, two different antigen binding domains known to be dually expressed in a diseased cell or tissue, for example a tumor or cancer cell, can enhance binding or selectivity of an antigen binding protein for a target.

[0056] Antigen-binding proteins contemplated herein include at least one antigen binding domain, wherein the antigen binding domain binds to at least one target antigen. Target antigens, in some cases, are expressed on the surface of a diseased cell or tissue, for example a tumor or a cancer cell. Target antigens include but are not limited to EpCAM, EGFR, HER-2, HER-3, c-Met, FoIR, and CEA. Antigen-binding proteins disclosed herein, also include proteins comprising two antigen binding domains that bind to two different target antigens known to be expressed on a diseased cell or tissue. Exemplary pairs of antigen binding domains include but are not limited to EGFR/CEA, EpCAM/CEA, and HER-2/HER-3.

[0057] The design of the antigen-binding proteins described herein allows the binding domain to one or more target antigens to be flexible in that the binding domain to a target antigen can be any type of binding domain, including but not limited to, domains from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody. In some embodiments, the binding domain to a target antigen is a single chain variable fragment (scFv), single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) of camelid derived nanobody. In other embodiments, the binding domain to a target antigen is a non-Ig binding domain, i.e., antibody mimetic, such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, and monobodies. In further embodiments, the binding domain to one or more target antigens is a ligand or peptide that binds to or associates with one or more target antigens.

Antigen binding protein Pharmacokinetics

[0058] The antigen-binding proteins described herein have certain advantages that would be recognized by one of skill in the art. For example, antigen-binding proteins described herein have improved pharmacokinetics over traditional antibody therapeutics. Improved

pharmacokinetics of antigen-binding proteins herein are attributed to at least the half-life extension domain and the innate immune cell binding domain. Half-life extension domains, as disclosed herein, include various polypeptides including but not limited to Fc domains and polypeptides binding to HSA. Innate immune cell binding domains herein have unique properties which give superior pharmacokinetics. The innate immune cell binding domains herein do not bind to an innate immune cell until they are activated by at least cleavage of at least one protease cleavage domain and binding of the antigen binding domains to target antigens. Therefore, enhanced pharmacokinetics of antigen binding proteins herein is attributed at least in part to reduced or eliminated target mediated drug disposition through innate immune cell binding in the circulation of a person. Improved pharmacokinetics comprises at least one of a shallower alpha phase and higher exposure in the beta phase. Antigen binding proteins described herein, thus have a larger therapeutic window with smaller peak/trough differences in exposure when compared to traditional antibody therapeutics.

Antigen binding protein Modifications

[0059] The antigen-binding proteins described herein encompass derivatives or analogs in which (i) an amino acid is substituted with an amino acid residue that is not one encoded by the genetic code, (ii) the mature polypeptide is fused with another compound such as polyethylene glycol, or (iii) additional amino acids are fused to the protein, such as a leader or secretory sequence or a sequence for purification of the protein.

[0060] Typical modifications include, but are not limited to, acetylation, acylation, ADP- ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

[0061] Modifications are made anywhere in antigen-binding proteins described herein, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini. Certain common peptide modifications that are useful for modification of antigen-binding proteins include glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, blockage of the amino or carboxyl group in a polypeptide, or both, by a covalent modification, and ADP-ribosylation.

Polynucleotides Encoding Antigen Binding Proteins

[0062] Also provided, in some embodiments, are polynucleotide molecules encoding an antigen binding protein described herein. In some embodiments, the polynucleotide molecules are provided as a DNA construct. In other embodiments, the polynucleotide molecules are provided as a messenger RNA transcript. [0063] The polynucleotide molecules are constructed by known methods such as by combining the genes encoding the three binding domains either separated by peptide linkers or, in other embodiments, directly linked by a peptide bond, into a single genetic construct operably linked to a suitable promoter, and optionally a suitable transcription terminator, and expressing it in bacteria or other appropriate expression system such as, for example CHO cells. In the embodiments where the target binding domain is a small molecule, the polynucleotides contain genes encoding the domains that bind to an innate immune cell and the HSA. In the

embodiments where the half-life extension domain is a small molecule, the polynucleotides contain genes encoding the domains that bind to the innate immune cell and the target antigen. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. The promoter is selected such that it drives the expression of the polynucleotide in the respective host cell.

[0064] In some embodiments, the polynucleotide is inserted into a vector, preferably an expression vector, which represents a further embodiment. This recombinant vector can be constructed according to known methods. Vectors of particular interest include plasmids, phagemids, phage derivatives, virii (e.g., retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, lentiviruses, and the like), and cosmids.

[0065] A variety of expression vector/host systems may be utilized to contain and express the polynucleotide encoding the polypeptide of the described antigen-binding protein. Examples of expression vectors for expression in E.coli are pSKK (Le Gall et al., J Immunol Methods. (2004) 285(1): 111-27) or pcDNA5 (Invitrogen) for expression in mammalian cells.

[0066] Thus, the antigen-binding proteins as described herein, in some embodiments, are produced by introducing a vector encoding the protein as described above into a host cell and culturing said host cell under conditions whereby the protein domains are expressed, may be isolated and, optionally, further purified.

Pharmaceutical Compositions

[0067] Also provided, in some embodiments, are pharmaceutical compositions comprising an antigen binding protein described herein, a vector comprising the polynucleotide encoding the polypeptide of the antigen-binding proteins or a host cell transformed by this vector and at least one pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the ingredients and that is not toxic to the patient to whom it is

administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Such carriers can be formulated by conventional methods and can be administered to the subject at a suitable dose. Preferably, the compositions are sterile. These compositions may also contain adjuvants such as preservative, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents.

[0068] In some embodiments of the pharmaceutical compositions, the antigen binding protein described herein is encapsulated in nanoparticles. In some embodiments, the nanoparticles are fullerenes, liquid crystals, liposome, quantum dots, superparamagnetic nanoparticles, dendrimers, or nanorods. In other embodiments of the pharmaceutical compositions, the antigen binding protein is attached to liposomes. In some instances, the antigen binding protein are conjugated to the surface of liposomes. In some instances, the antigen binding protein are encapsulated within the shell of a liposome. In some instances, the liposome is a cationic liposome.

[0069] The antigen-binding proteins described herein are contemplated for use as a medicament. Administration is effected by different ways, e.g. by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. In some embodiments, the route of administration depends on the kind of therapy and the kind of compound contained in the pharmaceutical composition. The dosage regimen will be determined by the attending physician and other clinical factors. Dosages for any one patient depends on many factors, including the patient^'s size, body surface area, age, sex, the particular compound to be administered, time and route of administration, the kind of therapy, general health and other drugs being administered concurrently. An "effective dose" refers to amounts of the active ingredient that are sufficient to affect the course and the severity of the disease, leading to the reduction or remission of such pathology and may be determined using known methods.

Methods of treatment

[0070] Also provided herein, in some embodiments, are methods and uses for stimulating the immune system of an individual in need thereof comprising administration of an antigen binding protein described herein. In some instances, the administration of an antigen binding protein described herein induces and/or sustains cytotoxicity towards a cell expressing a target antigen where the cell expressing the target antigen is in a microenvironment with increased levels of protease activity. In some instances, the cell expressing a target antigen is a cancer or tumor cell, a virally infected cell, a bacterially infected cell, an autoreactive T or B cell, damaged red blood cells, arterial plaques, or fibrotic tissue. In some instances, the blood of the subject is rich in proteases. [0071] Also provided herein are methods and uses for a treatment of a disease, disorder or condition associated with a target antigen comprising administering to an individual in need thereof an antigen binding protein described herein. Diseases, disorders or conditions associated with a target antigen include, but are not limited to, viral infection, bacterial infection, autoimmune disease, transplant rejection, atherosclerosis, or fibrosis. In other embodiments, the disease, disorder or condition associated with a target antigen is a proliferative disease, a tumorous disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, a graft-versus-host disease or a host-versus-graft disease. In one embodiment, the disease, disorder or condition associated with a target antigen is cancer. In one instance, the cancer is a hematological cancer. In another instance, the cancer is a solid tumor cancer.

[0072] As used herein, in some embodiments, "treatment" or "treating" or "treated" refers to therapeutic treatment wherein the object is to slow (lessen) an undesired physiological condition, disorder or disease, or to obtain beneficial or desired clinical results. For the purposes described herein, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of the condition, disorder or disease; stabilization (i.e., not worsening) of the state of the condition, disorder or disease; delay in onset or slowing of the progression of the condition, disorder or disease; amelioration of the condition, disorder or disease state; and remission (whether partial or total), whether detectable or undetectable, or enhancement or improvement of the condition, disorder or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment. In other embodiments, "treatment" or "treating" or "treated" refers to prophylactic measures, wherein the object is to delay onset of or reduce severity of an undesired physiological condition, disorder or disease, such as, for example is a person who is predisposed to a disease (e.g., an individual who carries a genetic marker for a disease such as breast cancer).

[0073] In some embodiments of the methods described herein, the antigen-binding proteins are administered in combination with an agent for treatment of the particular disease, disorder or condition. Agents include but are not limited to, therapies involving antibodies, small molecules (e.g., chemotherapeutics), hormones (steroidal, peptide, and the like), radiotherapies (γ-rays, X- rays, and/or the directed delivery of radioisotopes, microwaves, UV radiation and the like), gene therapies (e.g., antisense, retroviral therapy and the like) and other immunotherapies. In some embodiments, the antigen-binding proteins are administered in combination with anti -diarrheal agents, anti-emetic agents, analgesics, opioids and/or non-steroidal anti-inflammatory agents. In some embodiments, the antigen-binding proteins are administered before, during, or after surgery.

Certain Definitions

[0074] As used herein, "elimination half-time" is used in its ordinary sense, as is described in Goodman and Gillman's The Pharmaceutical Basis of Therapeutics 21-25 (Alfred Goodman Gilman, Louis S. Goodman, and Alfred Gilman, eds., 6th ed. 1980). Briefly, the term is meant to encompass a quantitative measure of the time course of drug elimination. The elimination of most drugs is exponential (i.e., follows first-order kinetics), since drug concentrations usually do not approach those required for saturation of the elimination process. The rate of an exponential process may be expressed by its rate constant, k, which expresses the fractional change per unit of time, or by its half-time, ti_/2 the time required for 50% completion of the process. The units of these two constants are time^-1 and time, respectively. A first-order rate constant and the half- time of the reaction are simply related (kxti_/2=0.693) and may be interchanged accordingly. Since first-order elimination kinetics dictates that a constant fraction of drug is lost per unit time, a plot of the log of drug concentration versus time is linear at all times following the initial distribution phase (i.e. after drug absorption and distribution are complete). The half-time for drug elimination can be accurately determined from such a graph.

EXAMPLES

Example 1 : Construction of an Exemplary Antigen Binding Protein to CD20

Generation of a scFv CD 16 binding domain

[0075] The human CD16 canonical sequence is Uniprot Accession No. P08637. Antibodies against CD 16 are generated via known technologies such as affinity maturation. Where murine anti-CD 16 antibodies are used as a starting material, humanization of murine anti-CD 16 antibodies is desired for the clinical setting, where the mouse-specific residues may induce a human-anti -mouse antigen (HAMA) response in subjects who receive treatment of an antigen binding protein described herein. Humanization is accomplished by grafting CDR regions from murine anti-CD 16 antibody onto appropriate human germline acceptor frameworks, optionally including other modifications to CDR and/or framework regions. As provided herein, antibody and antibody fragment residue numbering follows Kabat (Kabat E. A. et al, 1991; Chothia et al, 1987).

[0076] Human or humanized anti-CD 16 antibodies are therefore used to generate scFv sequences for CD 16 binding domains of an antigen-binding protein. DNA sequences coding for human or humanized VL and VH domains are obtained, and the codons for the constructs are, optionally, optimized for expression in cells from Homo sapiens. A protease cleavage site is included between the VH and VL domains. The order in which the VL and VH domains appear in the scFv is varied (i.e., VL-VH, or VH-VL orientation), and three copies of the "G4S" or "G₄S" subunit (G₄S)₃ connect the variable domains to create the scFv domain. Anti-CD 16 scFv plasmid constructs can have optional Flag, His or other affinity tags, and are electroporated into HEK293 or other suitable human or mammalian cell lines and purified. Validation assays include binding analysis by FACS, kinetic analysis using Proteon, and staining of CD16- expressing cells.

Generation of a scFv CD 20 binding domain

[0077] CD20 is one of the cell surface proteins present on B-lymphocytes. CD20 antigen is found in normal and malignant pre-B and mature B lymphocytes, including those in over 90% of B-cell non-Hodgkin's lymphomas (NHL). The antigen is absent in hematopoietic stem cells, activated B lymphocytes (plasma cells) and normal tissue. As such, several antibodies mostly of murine origin have been described: 1F5, 2B8/C2B8, 2H7, and 1H4.

[0078] A scFv binding domain to CD20 is generated similarly to the above method for generation of a scFv binding domain to CD 16.

Cloning of DNA expression constructs encoding the antigen-binding protein

[0079] The anti-CD 16 scFv with protease cleavage site domains are used to construct an antigen binding protein in combination with an anti-CD20 scFv domain and a half-life extension domain (e.g., a HSA binding peptide or VH domain), with the domains organized as shown Figure 1. For expression of an antigen binding protein in CHO cells, coding sequences of all protein domains are cloned into a mammalian expression vector system. In brief, gene sequences encoding the CD 16 binding domain, half-life extension domain, and CD20 binding domain along with peptide linkers LI and L2 are separately synthesized and subcloned. The resulting constructs are then ligated together in the order of 'CD20 binding domain - LI - VH CD 16 binding domain - L2 - protease cleavage domain - L3 - VL CD 16 binding domain - L4 - anti- CD20 scFv - L5 - half-life extension domain to yield a final construct. All expression constructs are designed to contain coding sequences for an N-terminal signal peptide and a C- terminal hexahistidine (6xHis)-tag to facilitate protein secretion and purification, respectively. Expression of antigen-binding proteins in stably transfected CHO cells

[0080] A CHO cell expression system (Flp-In®, Life Technologies), a derivative of CHO-K1 Chinese Hamster ovary cells (ATCC, CCL-61) (Kao and Puck, Proc. Natl. Acad Sci USA 1968;60(4): 1275-81), is used. Adherent cells are subcultured according to standard cell culture protocols provided by Life Technologies. [0081] For adaption to growth in suspension, cells are detached from tissue culture flasks and placed in serum-free medium. Suspension-adapted cells are cryopreserved in medium with 10% DMSO.

[0082] Recombinant CHO cell lines stably expressing secreted antigen-binding proteins are generated by transfection of suspension-adapted cells. During selection with the antibiotic Hygromycin B viable cell densities are measured twice a week, and cells are centrifuged and resuspended in fresh selection medium at a maximal density of 0. lxlO⁶ viable cells/mL. Cell pools stably expressing antigen-binding proteins are recovered after 2-3 weeks of selection at which point cells are transferred to standard culture medium in shake flasks. Expression of recombinant secreted proteins is confirmed by performing protein gel electrophoresis or flow cytometry. Stable cell pools are cryopreserved in DMSO containing medium.

[0083] Antigen-binding proteins are produced in 10-day fed-batch cultures of stably transfected CHO cell lines by secretion into the cell culture supernatant. Cell culture supernatants are harvested after 10 days at culture viabilities of typically >75%. Samples are collected from the production cultures every other day and cell density and viability are assessed. On day of harvest, cell culture supernatants are cleared by centrifugation and vacuum filtration before further use.

[0084] Protein expression titers and product integrity in cell culture supernatants are analyzed by SDS-PAGE.

Purification of antigen-binding proteins

[0085] Antigen-binding proteins are purified from CHO cell culture supernatants in a two-step procedure. The constructs are subjected to affinity chromatography in a first step followed by preparative size exclusion chromatography (SEC) on Superdex 200 in a second step. Samples are buffer-exchanged and concentrated by ultrafiltration to a typical concentration of >1 mg/mL. Purity and homogeneity (typically >90%) of final samples are assessed by SDS PAGE under reducing and non-reducing conditions, followed by immunoblotting using an anti-HSA or anti idiotype antibody as well as by analytical SEC, respectively. Purified proteins are stored at aliquots at -80°C until use.

Example 2: Determination of antigen affinity by flow cytometry

[0086] The antigen-binding proteins of Example 1 are tested for their binding affinities to human CD16⁺ and CD20⁺ cells and cynomolgus CD16⁺ and CD20⁺ cells.

[0087] CD16⁺ and CD20⁺ cells are incubated with 100 μΙ_, of serial dilutions of the antigen- binding proteins of Example 1 and at least one protease. After washing three times with FACS buffer the cells are incubated with 0.1 mL of 10 μg/mL mouse monoclonal anti -idiotype antibody in the same buffer for 45 min on ice. After a second washing cycle, the cells are incubated with 0.1 mL of 15 μ§/ιηΙ. FITC-conjugated goat anti-mouse IgG antibodies under the same conditions as before. As a control, cells are incubated with the anti-His IgG followed by the FITC-conjugated goat anti-mouse IgG antibodies without the antigen-binding proteins. The cells were then washed again and resuspended in 0.2 mL of F ACS buffer containing 2 μg/mL propidium iodide (PI) in order to exclude dead cells. The fluorescence of lxlO⁴ living cells is measured using a Beckman-Coulter FC500 MPL flow cytometer using the MXP software (Beckman-Coulter, Krefeld, Germany) or a Millipore Guava EasyCyte flow cytometer using the Incyte software (Merck Millipore, Schwalbach, Germany). Mean fluorescence intensities of the cell samples are calculated using CXP software (Beckman-Coulter, Krefeld, Germany) or Incyte software (Merck Millipore, Schwalbach, Germany). After subtracting the fluorescence intensity values of the cells stained with the secondary and tertiary reagents alone the values are them used for calculation of the K_D values with the equation for one-site binding (hyperbola) of the GraphPad Prism (version 6.00 for Windows, GraphPad Software, La Jolla California USA).

[0088] CD 16 binding affinity and crossreactivity are evaluated in titration and flow cytometric experiments on CD16⁺ Jurkat cells and the cynomolgus CD16⁺ HSC-F cell line (JCRB, cat.: JCRBl 164). CD20 binding and crossreactivity are assessed on the human CD20⁺ tumor cell lines. The K_D ratio of crossreactivity is calculated using the K_D values determined on the CHO cell lines expressing either recombinant human or recombinant cynomolgus antigens.

Example 3 : Cytotoxicity Assay

[0089] The antigen binding protein of Example 1 is evaluated in vitro on its mediation of T cell dependent cytotoxicity to CD20⁺ target cells.

[0090] Fluorescence labeled CD20⁺ REC-1 cells (a Mantle cell lymphoma cell line, ATCC CRL-3004) are incubated with isolated PBMC of random donors or CB15 T-cells (standardized T-cell line) as effector cells in the presence of the antigen binding protein of Example 1 and at least one protease. After incubation for 4 h at 37°C in a humidified incubator, the release of the fluorescent dye from the target cells into the supernatant is determined in a spectrofluorimeter. Target cells incubated without the antigen binding protein of Example land target cells totally lysed by the addition of saponin at the end of the incubation serve as negative and positive controls, respectively.

[0091] Based on the measured remaining living target cells, the percentage of specific cell lysis is calculated according to the following formula: [1 -(number of living targetS_(Sampie₎/number of living targetS(_Spontaneous))] x 100%. Sigmoidal dose response curves and EC₅₀ values are calculated by non-linear regression/4-parameter logistic fit using the GraphPad Software. The lysis values obtained for a given antibody concentration are used to calculate sigmoidal dose- response curves by 4 parameter logistic fit analysis using the Prism software. Example 3 : Pharmacokinetics of Antigen-binding Proteins

[0092] The antigen binding protein of Example 1 is evaluated for half-time elimination in animal studies.

[0093] The antigen binding protein is administered to cynomolgus monkeys as a 0.5 mg/kg bolus injection into the saphenous vein. Another cynomolgus monkey group receives a comparable protein in size with binding domains to CD 16 and CD20, but lacking a half-life extension domain. A third and fourth group receive a protein with CD 16 and half-life extension domains and a protein with CD20 and half-life extension domains respectively, and both comparable in size to the antigen-binding protein. Each test group consists of 5 monkeys.

Serum samples are taken at indicated time points, serially diluted, and the concentration of the proteins is determined using a binding ELISA to CD 16 and/or CD20.

[0094] Pharmacokinetic analysis is performed using the test article plasma concentrations.

Group mean plasma data for each test article conforms to a multi-exponential profile when plotted against the time post-dosing. The data are fit by a standard two-compartment model with bolus input and first-order rate constants for distribution and elimination phases. The general equation for the best fit of the data for i.v. administration is: c(t)=Ae^~at+Be^~pt, where c(t) is the plasma concentration at time t, A and B are intercepts on the Y-axis, and a and β are the apparent first-order rate constants for the distribution and elimination phases, respectively. The a-phase is the initial phase of the clearance and reflects distribution of the protein into all extracellular fluid of the animal, whereas the second or β-phase portion of the decay curve represents true plasma clearance. Methods for fitting such equations are well known in the art. For example, A=D/V(a-k21)/(a-p), B=D/V(p-k21)/(a-p), and a and β (for α>β) are roots of the quadratic equation: r²+(kl2+k21+kl0)r+k21kl0=0 using estimated parameters of V=volume of distribution, kl0=elimination rate, kl2=transfer rate from compartment 1 to compartment 2 and k21=transfer rate from compartment 2 to compartment 1, and D=the administered dose.

[0095] Data analysis: Graphs of concentration versus time profiles are made using

KaleidaGraph (KaleidaGraph™ V. 3.09 Copyright 1986-1997. Synergy Software. Reading, Pa.). Values reported as less than reportable (LTR) are not included in the PK analysis and are not represented graphically. Pharmacokinetic parameters are determined by compartmental analysis using WinNonlin software (WinNonlin® Professional V. 3.1 WinNonlin™ Copyright 1998- 1999. Pharsight Corporation. Mountain View, Calif). Pharmacokinetic parameters are computed as described in Ritschel W A and Kearns G L, 1999, EST: Handbook Of Basic Pharmacokinetics Including Clinical Applications, 5th edition, American Pharmaceutical Assoc., Washington, D C. [0096] It is expected that the antigen binding protein of Example 1 has improved pharmacokinetic parameters such as an increase in elimination half-time as compared to proteins lacking a half-life extension domain.

Example 4: Xenograft Tumor Model

[0097] The antigen binding protein of Example 1 is evaluated in a xenograft model.

[0098] Female immune-deficient NOD/scid mice are sub-lethally irradiated (2 Gy) and subcutaneously inoculated with 4xl0⁶ Ramos RA1 cells into the right dorsal flank. When tumors reach 100 to 200 mm³, animals are allocated into 3 treatment groups. Groups 2 and 3 (8 animals each) are intraperitoneally injected with 1.5xl0⁷ activated human T-cells. Three days later, animals from Group 3 are subsequently treated with a total of 9 intravenous doses of 50 μg antigen binding protein of Example 1 (qdx9d). Groups 1 and 2 are only treated with vehicle. Body weight and tumor volume are determined for 30 days.

[0099] It is expected that animals treated with the antigen binding protein of Example 1 have a statistically significant delay in tumor growth in comparison to the respective vehicle-treated control group.

Example 5: Proof-of-Concept Clinical Trial Protocol for Administration of the Antigen Binding Protein of Example 1 to B-cell Lymphoma Patients

[00100] This is a Phase VII clinical trial for studying the antigen binding protein of

Example 1 as a treatment for B-cell Lymphoma.

[00101] Study Outcomes:

[00102] Primary : Maximum tolerated dose of antigen binding protein of Example 1

[00103] Secondary : To determine whether in vitro response of antigen binding protein of Example 1 is associated with clinical response

[00104] Phase I

[00105] The maximum tolerated dose (MTD) will be determined in the phase I section of the trial.

1.1 The maximum tolerated dose (MTD) will be determined in the phase I section of the trial.

1.2 Patients who fulfill eligibility criteria will be entered into the trial to antigen binding protein of Example 1.

1.3 The goal is to identify the highest dose of antigen binding protein of Example 1 that can be administered safely without severe or unmanageable side effects in participants. The dose given will depend on the number of participants who have been enrolled in the study prior and how well the dose was tolerated. Not all participants will receive the same dose. [00106] Phase II

2.1 A subsequent phase II section will be treated at the MTD with a goal of determining if therapy with therapy of antigen binding protein of Example 1 results in at least a 20% response rate.

Primary Outcome for the Phase II— To determine if therapy of antigen binding protein of Example 1 results in at least 20% of patients achieving a clinical response (blast response, minor response, partial response, or complete response)

[00107] Eligibility:

Histologically confirmed newly diagnosed aggressive B-cell lymphoma according to the current World Health Organisation Classification, from 2001 to 2007

Any stage of disease.

Treatment with R-CHOP or R-CHOP like regimens (+/- transplant).

Age > 18 years

Karnofsky performance status > 50% or ECOG performance status 0-2

Life expectancy > 6 weeks

[00108] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. An antigen-binding protein, comprising a single polypeptide chain comprising two or more inactive innate immune cell binding domains, two or more target antigen binding domains, one or more half-life extension domains, and one or more protease cleavage domains;

wherein upon protease cleavage of the protease cleavage domain, the innate immune cell binding domain becomes active and binds to an innate immune cell.

2. The antigen-binding protein of claim 1, wherein upon binding of the target antigens by the target antigen binding domains, the innate immune cell binding domain becomes active and binds to an innate immune cell.

3. The antigen-binding protein of claim 1 or claim 2, wherein the innate immune cell binding domains bind to a cell surface antigen restricted to a specific innate immune cell lineage and activates an innate immune cell selected from dendritic cells, plasmacytoid dendritic cells, natural killer cells, natural killer T cells, monocytes, neutrophils, basophils, and eosinophils, wherein the innate immune cell is not a T cell.

4. The antigen-binding protein of claim 3, wherein the cell surface antigen is selected from CDlc, CD83, CD141, CD209, MHC II, CD123, CD303, CD304, CD16, CD56, CDld, CD 160, PLZF, KG2d, CD94- KG2A/C/E , CD 14, CD 16, CD64, CD 15 CD 16, 2D7 antigen, CD123, CD203c, FcsRIa, CDl lb, CD193, EMR1, and Siglec-8.

5. The antigen-binding protein of any one of claims 1 to 4, wherein the target antigen binding domains bind to the same target antigen.

6. The antigen-binding protein of any one of claims 1 to 4, wherein the target antigen binding domains bind to different target antigens.

7. The antigen-binding protein of any one of claims 1 to 6, wherein the protease cleavage domain is cleaved by at least one of a serine protease, a cysteine protease, an aspartate protease, a threonine protease, a glutamic acid protease, a metalloproteinase, a gelatinase, and a asparagine peptide lyase.

8. The antigen-binding protein of any one of claims 1 to 7, wherein the protease cleavage domain is cleaved by at least one of a Cathepsin B, a Cathepsin C, a Cathepsin D, a Cathepsin E, a Cathepsin K, a Cathepsin L, a kallikrein, a hKl, a hK10, a hK15, a plasmin, a collagenase, a Type IV collagenase, a stromelysin, a Factor Xa, a chymotrypsin-like protease, a trypsin-like protease, a elastase-like protease, a subtili sin-like protease, an actinidain, a bromelain, a calpain, a caspase, a caspase-3, a Mirl-CP, a papain, a HIV-1 protease, a HSV protease, a CMV protease, a chymosin, a renin, a pepsin, a matriptase, a legumain, a plasmepsin, a nepenthesin, a metalloexopeptidase, a metalloendopeptidase, a matrix metalloprotease (MMP), a MMPl, a MMP2, a MMP3, a MMP8, a MMP9, a MMP10, a MMPl 1, a MMPl 2, a MMP13, a MMP14, an ADAM10, an ADAM12, an urokinase plasminogen activator (uPA), an

enterokinase, a prostate-specific antigen (PSA, hK3), an interleukin-ΐβ converting enzyme, a thrombin, a FAP (FAP-a), a dipeptidyl peptidase, and a dipeptidyl peptidase IV (DPPIV/CD26).

9. The antigen-binding protein of any one of claims 1 to 8, wherein the protease cleavage domain is cleaved at the site of a tumor.

10. The antigen-binding protein of claim 9, wherein the protease is expressed by a cell in a microenvironment of the tumor.

11. The antigen binding protein of any one of claims 1 to 10, wherein the protease cleavage domain is cleaved in the blood of a subject.

12. The antigen binding protein of any one of claims 1 to 11, wherein the protein further comprises two or more protease cleavage domains.

13. The antigen-binding protein of any one of claims 1 to 12, wherein one or more innate immune cell binding domains comprise a polypeptide derived from a single-chain variable fragment (scFv) specific to a cell surface marker on an innate immune cell.

14. The antigen-binding protein of any one of claims 1 to 13, wherein one or more innate immune cell binding domains are specific for a dendritic cell..

15. The antigen-binding protein of any one of claims 1 to 13, wherein one or more innate immune cell binding domains are specific for a plasmacytoid dendritic cell.

16. The antigen-binding protein of any one of claims 1 to 13, wherein one or more innate immune cell binding domains are specific for a natural killer cell.

17. The antigen binding protein of any one of claims 1 to 13, wherein one or more innate immune cell binding domains are specific for a natural killer T cell.

18. The antigen binding protein of any one of claims 1 to 13, wherein one or more innate immune cell binding domains are specific for a monocyte.

19. The antigen binding protein of any one of claims 1 to 13, wherein one or more innate immune cell binding domains are specific for a neutrophil.

20. The antigen binding protein of any one of claims 1 to 13, wherein one or more innate immune cell binding domains are specific for a basophil.

21. The antigen binding protein of any one of claims 1 to 13, wherein one or more innate immune cell binding domains are specific for an eosinophil.

22. The antigen-binding protein of any one of claims 1 to 21, wherein one or more innate immune cell binding domains comprise complementary determining regions (CDRs) selected from the group consisting of Lorvotuzumab, 3C12C, CSL362, 3G8, rMil2, E4, NNC141-0100.

23. The antigen-binding protein of any one of claims 1 to 22, wherein one or more innate immune cell binding domains are humanized.

24. The antigen-binding protein of any one of claims 1 to 23, wherein one or more activated innate immune cell binding domains have a K_D binding 1000 nM or less to innate immune cells.

25. The antigen-binding protein of any one of claims 1 to 23, wherein one or more activated innate immune cell binding domains have a K_D binding 100 nM or less to innate immune cell innate immune cells.

26. The antigen-binding protein of any one of claims 1 to 23, wherein one or more activated innate immune cell binding domains have a K_D binding 10 nM or less to innate immune cells.

27. The antigen-binding protein of any one of claims 1 to 26, wherein one or more innate immune cell binding domains have crossreactivity with cynomolgus innate immune cells.

28. The antigen-binding protein of any one of claims 1 to 27, wherein one or more innate immune cell binding domains comprise an amino acid sequence provided herein.

29. The antigen-binding protein of any one of claims 1 to 28, wherein one or more half- life extension domains comprise a binding domain to human serum albumin.

30. The antigen-binding protein of any one of claims 1 to 29, wherein one or more half- life extension domains comprise a scFv, a variable heavy domain (VH), a variable light domain (VL), a nanobody, a peptide, a ligand, or a small molecule.

31. The antigen-binding protein of any one of claims 1 to 30, wherein one or more half- life extension domains comprise a scFv.

32. The antigen-binding protein of any one of claims 1 to 30, wherein one or more half- life extension domains comprise a VH domain.

33. The antigen-binding protein of any one of claims 1 to 30, wherein one or more half- life extension domains comprise a VL domain.

34. The antigen-binding protein of any one of claims 1 to 30, wherein one or more half- life extension domains comprise a nanobody.

35. The antigen-binding protein of any one of claims 1 to 30, wherein one or more half- life extension domains comprise a peptide.

36. The antigen-binding protein of any one of claims 1 to 30, wherein one or more half- life extension domains comprise a ligand.

37. The antigen-binding protein of any one of claims 1 to 30, wherein one or more half- life extension domains comprise a small molecule.

38. The antigen-binding protein of any one of claims 1 to 30, wherein one or more half- life extension domains comprise an F_c domain.

39. The antigen-binding protein of any one of claims 1 to 38, wherein at least one half- life extension domain is at the N-terminus of the protein prior to protease cleavage.

40. The antigen-binding protein of any one of claims 1 to 39, wherein at least one half- life extension domain is at the C-terminus of the protein prior to protease cleavage.

41. The antigen-binding protein of any one of claims 1 to 38, wherein at least one half- life extension domain is not at the C-terminus or the N-terminus of the protein prior to protease cleavage.

42. The antigen-binding protein of any one of claims 1 to 41, wherein the protease cleavage domain is in the half-life extension domain or the innate immune cell binding domain.

43. The antigen-binding protein of any one of claims 1 to 41, wherein the protease cleavage domain is not in the half-life extension domain or the innate immune cell binding domain.

44. The antigen-binding protein of any one of claims 1 to 43, wherein the target antigen binding domains independently comprise a scFv, a VH domain, a VL domain, a non-Ig domain, or a ligand that specifically binds to the target antigen.

45. The antigen-binding protein of any one of claims 1 to 44, wherein at least one target antigen binding domains specifically bind to a cell surface molecule.

46. The antigen-binding protein of any one of claims 1 to 45, wherein at least one target antigen binding domains specifically bind to a tumor antigen.

47. The antigen-binding protein of any one of claims 1 to 46, wherein the target antigen binding domains specifically and independently bind to an antigen selected from at least one of EpCAM, EGFR, HER-2, HER-3, cMet, CEA, and FoIR.

48. The antigen-binding protein of any one of claims 1 to 47, wherein the target antigen binding domains comprise a scFv, a variable heavy domain (VH), a variable light domain (VL), or a nanobody.

49. The antigen-binding protein of any one of claims 1 to 48, wherein the target antigen binding domains specifically and independently bind to two different antigens, wherein at least one of the antigens is selected from one of EpCAM, EGFR, HER-2, HER-3, cMet, CEA, and FoIR.

50. The antigen binding protein of any one of claims 1 to 49, wherein the protein prior to cleavage of the protease cleavage domain is less than about 100 kDa.

51. The antigen-binding protein of any one of claims 1 to 50, wherein the protein after cleavage of the protease cleavage domain is about 25 to about 75 kDa.

52. The antigen-binding protein of any one of claims 1 to 51, wherein the protein prior to protease cleavage has a size that is above the renal threshold for first-pass clearance.

53. The antigen-binding protein of any one of claims 1 to 52, wherein the protein prior to protease cleavage has an elimination half-time of at least about 50 hours.

54. The antigen-binding protein of any one of claims 1 to 53, wherein the protein prior to protease cleavage has an elimination half-time of at least about 100 hours.

55. The antigen-binding protein of any one of claims 1 to 54, wherein the protein has increased tissue penetration or tissue distribution as compared to an IgG to the same target antigen.

56. The antigen-binding protein of any one of claims 1 to 55, wherein the protein has improved pharmacokinetics as compared to an IgG to the same target antigen.

57. The antigen-binding protein of any one of claims 1 to 56, wherein the protein has reduced or eliminated target mediated drug disposition through innate immune cell binding as compared to an IgG to an innate immune cell.

58. The antigen-binding protein of any one of claims 1 to 57, wherein the protein has a shallower alpha phase and higher exposure in the beta phase as compared to an IgG to the same target antigen.

59. The antigen-binding protein of any one of claims 1 to 58, wherein the protein has a larger therapeutic window with smaller peak/trough differences in exposure as compared to an IgG to the same target antigen.

60. A polynucleotide encoding an antigen-binding protein according to any one of claims

1 to 59.

61. A vector comprising the polynucleotide of claim 60.

62. A host cell transformed with the vector according to claim 61.

63. A pharmaceutical composition comprising (i) the antigen-binding protein according to any one of claims 1 to 59, the polynucleotide according to claim 60, the vector according to claim 61 or the host cell according to claim 62 and (ii) a pharmaceutically acceptable carrier.

64. A process for the production of an antigen-binding protein of claim 1, said process comprising culturing a host transformed or transfected with a vector comprising a nucleic acid sequence encoding an antigen-binding protein of claim 1 under conditions allowing the expression of the protein and recovering and purifying the produced protein from the culture.

65. A method for the treatment or amelioration of a proliferative disease, a tumorous disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, a graft-versus-host disease or a host-versus-graft disease comprising the administration of an antigen-binding protein of claim 1 to a subject in need of such a treatment or amelioration.

66. The method according to claim 65, wherein the subject is a human.

67. The method according to claim 65, wherein the method further comprises administration of an agent in combination with the antigen-binding protein of claim 1.

68. An antigen-binding protein, wherein the protein comprises a single polypeptide chain comprising a protease cleavage domain (P) separating the chain into a first and second region; wherein the first region comprises an anti- innate immune cell V_H binding domain (IV_H) and a target antigen binding domain (Ti) and the second region comprises an anti- innate immune cell V_L binding domain (IV_L) and a target antigen binding domain (T₂); wherein the protein optionally comprises a half-life extension domain (H) in the first region, second region, or both regions, and

wherein upon activation by protease cleavage of P, the first and second regions associate to form a complete anti- innate immune cell V_L/V_H binding domain that binds innate immune cells.

69. The antigen-binding protein of claim 68, wherein upon and binding the target antigen by Ti and T₂, the first and second regions associate to form a complete anti- innate immune cell V_L/V_H binding domain that binds innate immune cells.

70. An antigen-binding protein, wherein the protein comprises a single polypeptide chain comprising a protease cleavage domain (P) separating the chain into a first and second region; wherein the first region comprises an anti- innate immune cell V_L binding domain (IV_L) and a target antigen binding domain (Ti) and the second region comprises an anti- innate immune cell V_H binding domain (IV_H) and a target antigen binding domain (T₂); wherein the protein optionally comprises a half-life extension domain (H) in the first region, second region, or both regions, and

wherein upon activation by protease cleavage of P and binding of the target antigen by Ti and T₂, the first and second regions associate to form a complete anti- innate immune cell V_L/V_H binding domain that binds innate immune cells.

71. An antigen-binding protein, wherein the protein comprises a single polypeptide chain comprising a first and second region;

wherein the first region comprises an anti- innate immune cell V_H binding domain (IV_H), an inactive anti- innate immune cell V_L binding domain (IV_L,) which associates with IV_H and a target antigen binding domain (Ti);

wherein the second region comprises a an anti- innate immune cell V_L binding domain (IV_L); an inactive anti- innate immune cell V_H binding domain (IV_H,) which associates with IV_H and a target antigen binding domain (T₂); wherein the protein optionally comprises a half-life extension domain (H) in the first region, second region, or both regions, and

wherein IV_L, and IV_H, each comprise at least one protease cleavage domains; and

wherein upon activation by protease cleavage the protease cleavage domains and binding the target antigen by Ti and T₂, the first and second regions associate to form a complete anti- innate immune cell VL/VH binding domain that binds innate immune cells.

72. An antigen-binding protein, wherein the protein comprises a single polypeptide chain comprising a first and second region;

wherein the first region comprises an anti- innate immune cell VL binding domain (IVL), an inactive anti- innate immune cell VH binding domain (IVH_;) which associates with IVL and a target antigen binding domain (Ti);

wherein the second region comprises a an anti- innate immune cell V_H binding domain (IVH); an inactive anti- innate immune cell VL binding domain (IVL_;) which associates with IVL and a target antigen binding domain (T₂); wherein the protein optionally comprises a half-life extension domain (H) in the first region, second region, or both regions, and

wherein TV_Ll and IVH; each comprise at least one protease cleavage domains; and wherein upon activation by protease cleavage the protease cleavage domains and binding the target antigen by Ti and T₂, the first and second regions associate to form a complete anti- innate immune cell VL/V_h binding domain that binds innate immune cells.