B7-H4 antibodies and methods of use thereof

Abstract

The present disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to human B7-H4 (and optionally to macaque, mouse and / or rat B7-H4), as well as compositions comprising such antibodies or antigen-binding fragments thereof. In one particular aspect, the antibody specifically binding to human B7-H4, or antigen-binding fragment thereof, increases T cell proliferation, increases interferon-gamma production, and / or depletes cells expressing B7-H4 by ADCC activity. The disclosure also provides methods of treating conditions, such as cancer, by administering an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4.

Description

[0001] This application is a divisional, the application number of the parent application is 201880059953.1, the application date is August 23, 2018, and the name of the invention is “B7-H4 antibody and method of use thereof”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 550,173, filed on August 25, 2017, U.S. Provisional Application No. 62 / 579,774, filed on October 31, 2017, U.S. Provisional Application No. 62 / 607,810, filed on December 19, 2017, and U.S. Provisional Application No. 62 / 656,789, filed on April 12, 2018, each of which is incorporated herein by reference in its entirety.

[0004] References to sequence listings submitted electronically via EFS-WEB

[0005] The electronically submitted sequence listing (name: 3986.0090004_SL_ST25.txt; size: 331,354 bytes; creation date: August 3, 2018) is incorporated herein by reference in its entirety. Background of the Invention Technical Field

[0007] The present disclosure relates to antibodies that specifically bind to human B7-H4, compositions comprising such antibodies, and methods of making and using antibodies that specifically bind to B7-H4.

[0008] Description of Related Technology

[0009] B7-H4 (also known as B7x, B7-S1, and VTCN1) is an immunomodulatory molecule that shares homology with other B7 family members, including PD-L1. It is a type I transmembrane protein that contains IgV and IgC extracellular domains. Although B7-H4 expression in healthy tissues is relatively limited at the protein level, B7-H4 is expressed in several solid tumors, such as gynecological cancers such as breast cancer, ovarian cancer, and endometrial cancer. The amount of B7-H4 expressed in tumors often correlates with poor prognosis. The receptor for B7-H4 is still unknown, but it is believed to be expressed on T cells. B7-H4 is believed to directly inhibit T cell activity.

[0010] Given the expression and function of B7-H4, provided herein are antibodies that specifically bind to B7-H4 and the use of these antibodies to modulate B7-H4 activity, including in the treatment of cancer. Summary of the invention

[0011] Provided herein is an isolated antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, the isolated antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3, and a light chain variable region (VL) CDR1, CDR2, and CDR3 sequence selected from the group consisting of: SEQ ID NOs: 5-10, 15-20, 25-30, 35-40, 458-463, 45-50, 55-60, 65-70, 75-80, 85-90, 95-100, 96-110, 97-121, 98-101, 99-102, 103-104, 105-105, 106-116, 107-117, 108-118, 109-119, 110-119, 111-119, 121-122, 123-124, 125-125 NO:105-110; correspondingly, SEQ ID NO:115-120; correspondingly, SEQ ID NO:125-130; correspondingly, SEQ ID NO:135-140; correspondingly, SEQ ID NO:145-150; correspondingly, SEQ ID NO:155-160; correspondingly, SEQ ID NO:165-170; correspondingly, SEQ ID NO:175-180; correspondingly, SEQ ID NO:185-190; and correspondingly, SEQ ID NO:195-200.

[0012] In one embodiment, the antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 11, 21, 31, 41, 464, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191 or 201.

[0013] In one embodiment, the antibody or antigen-binding fragment thereof comprises a VL comprising the amino acid sequence of SEQ ID NO: 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182, 192 or 202.

[0014] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 11, 21, 31, 41, 464, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191 or 201.

[0015] Also provided herein is an isolated antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182, 192 or 202.

[0016] Also provided herein is an isolated antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, the isolated antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region comprising the following amino acid sequences, respectively: SEQ ID NOs: 11 and 12; SEQ ID NOs: 21 and 22; SEQ ID NOs: 31 and 32; SEQ ID NOs: 41 and 42; SEQ ID NOs: 464 and 42; SEQ ID NOs: 51 and 52; SEQ ID NOs: 61 and 62; SEQ ID NOs: 71 and 72; SEQ ID NOs: 81 and 82; SEQ ID NOs: 91 and 92; SEQ ID NOs: 101 and 102; SEQ ID NOs: 111 and 112; SEQ ID NOs: 121 and 122; SEQ ID NOs: 131 and 132; SEQ ID NOs: 141 and 142; SEQ ID NOs: 151 and 152; SEQ ID NOs: 161 and 162; SEQ ID NOs: 171 and 172; SEQ ID NOs: 182 and 183; SEQ ID NOs: 184 and 185; SEQ ID NOs: 186 and 187; SEQ ID NOs: 188 and 189; SEQ ID NOs: 190 and 201; SEQ ID NOs: 191 and 202; SEQ ID NOs: 192 and 203; SEQ ID NOs: 193 and 204; SEQ ID NOs: 194 and 205 NO:181 and 182; SEQ ID NO:191 and 192; or SEQ ID NO:201 and 202.

[0017] In one embodiment, the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region. In one embodiment, the heavy chain constant region is selected from human IgG 1 IgG 2 IgG 3 IgG 4 , IgA 1 and IgA 2 A group consisting of heavy chain constant regions.

[0018] In one embodiment, the antibody or antigen binding fragment further comprises a light chain constant region. In one embodiment, the light chain constant region is selected from the group consisting of human immunoglobulin IgGκ and IgGλ light chain constant regions.

[0019] In one embodiment, the antibody or antigen-binding fragment further comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is human IgG 1 A heavy chain constant region, and wherein the light chain constant region is a human IgG kappa light chain constant region.

[0020] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 13, 23, 33, 43, 469, 53, 63, 73, 83, 93, 103, 113, 123, 133, 143, 153, 163, 173, 183, 193 or 203.

[0021] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, 114, 124, 134, 144, 154, 164, 174, 184, 194 or 204.

[0022] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain comprising the following amino acid sequences, respectively: SEQ ID NOs: 13 and 14; SEQ ID NOs: 23 and 24; SEQ ID NOs: 33 and 34; SEQ ID NOs: 43 and 44; SEQ ID NOs: 469 and 44; SEQ ID NOs: 53 and 54; SEQ ID NOs: 63 and 64; SEQ ID NOs: 73 and 74; SEQ ID NOs: 83 and 84; SEQ ID NOs: 93 and 94; SEQ ID NOs: 103 and 104; SEQ ID NOs: 113 and 114; SEQ ID NOs: 123 and 124; SEQ ID NOs: 133 and 134; SEQ ID NOs: 143 and 144; SEQ ID NOs: 153 and 154; SEQ ID NOs: 163 and 164; SEQ ID NOs: 173 and 174; SEQ ID NOs: 183 and 184; SEQ ID NOs: 193 and 194; or SEQ IDNO:203 and 204.

[0023] Also provided herein is an isolated antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, wherein the antibody or antigen-binding fragment thereof comprises a VH CDR1, a VH CDR2, a VH CDR3, a VL CDR1, a VL CDR2, and a VL CDR3 of an antibody selected from the group consisting of: 15461, 20500, 20501, 20502, 20502.1, 22208, 15462, 22213, 15465, 20506, 15483, 20513, 22216, 15489, 20516, 15472, 15503, 15495, 15478, 15441, and 20496. In one embodiment, the CDRs are Kabat-defined CDRs, Chothia-defined CDRs, or AbM-defined CDRs.

[0024] Also provided herein is an isolated antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, the isolated antibody or antigen-binding fragment thereof comprising the heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 458-463, respectively. In one embodiment, (i) the antibody or antigen-binding fragment thereof comprises a variable heavy chain region comprising an amino acid sequence of SEQ ID NO: 464 and a variable light chain region comprising an amino acid sequence of SEQ ID NO: 42; or (ii) the antibody comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 469 and a light chain comprising an amino acid sequence of SEQ ID NO: 44.

[0025] Also provided herein is an isolated antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, the isolated antibody or antigen-binding fragment thereof comprising the heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 35-40, respectively. In one embodiment, (i) the antibody or antigen-binding fragment thereof comprises a variable heavy chain region comprising an amino acid sequence of SEQ ID NO: 41 and a variable light chain region comprising an amino acid sequence of SEQ ID NO: 42; or (ii) the antibody comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 43 and a light chain comprising an amino acid sequence of SEQ ID NO: 44.

[0026] Also provided herein is an isolated antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, the isolated antibody or antigen-binding fragment thereof comprising the heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 65-70, respectively. In one embodiment, (i) the antibody or antigen-binding fragment thereof comprises a variable heavy chain region comprising an amino acid sequence of SEQ ID NO: 71 and a variable light chain region comprising an amino acid sequence of SEQ ID NO: 72; or (ii) wherein the antibody comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 73 and a light chain comprising an amino acid sequence of SEQ ID NO: 74.

[0027] Also provided herein is an isolated antibody or antigen-binding fragment thereof that binds to the same epitope of human B7-H4 as that bound by the antibody or antigen-binding fragment thereof of any one of claims 1 to 20. In one embodiment, the antibody or antigen-binding fragment thereof binds to the same epitope of human B7-H4 as determined by SPR.

[0028] In one embodiment, the antibody or antigen binding fragment thereof is a human antibody or antigen binding fragment thereof. In one embodiment, the antibody or antigen binding fragment thereof is a murine, humanized or chimeric antibody or antigen binding fragment thereof.

[0029] In one embodiment, the antibody or antigen binding fragment thereof induces T cell proliferation. In one embodiment, the antibody or antigen binding fragment increases T cell proliferation by at least 21% compared to treatment with a control antibody. In one embodiment, the antibody or antigen binding fragment increases T cell proliferation by about 5% to about 35% compared to treatment with a control antibody. In one embodiment, the antibody or antigen binding fragment thereof induces CD4+T cell proliferation. In one embodiment, the antibody or antigen binding fragment thereof increases CD4+T cell proliferation by at least 9% compared to treatment with a control antibody. In one embodiment, the antibody or antigen binding fragment thereof increases CD4+T cell proliferation by about 5% to about 15% compared to treatment with a control antibody. In one embodiment, the antibody or antigen binding fragment thereof induces CD8+T cell proliferation. In one embodiment, the antibody or antigen binding fragment increases CD8+T cell proliferation by at least 11% compared to treatment with a control antibody. In one embodiment, the antibody or antigen binding fragment thereof increases CD8+T cell proliferation by about 5% to about 15% compared to treatment with a control antibody.

[0030] In one embodiment, the antibody or its antigen binding fragment induces interferon gamma (IFNγ) production. In one embodiment, the antibody or its antigen binding fragment can increase IFNγ production by at least 2 times, at least 3 times, at least 4 times and at least 5 times, at least 6 times, at least 7 times, at least 8 times, about 2 times to about 10 times, or about 3 times to about 10 times.

[0031] In one embodiment, the antibody or antigen binding fragment thereof is capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) in B7-H4 expressing cells. In one embodiment, the antibody or antigen binding fragment thereof induces specific hydrolysis of at least 20%, at least 30%, at least 40%, about 20% to about 50%, or about 30% to about 50% of cells expressing B7-H4.

[0032] In one embodiment, the antibody or antigen-binding fragment thereof inhibits tumor growth in a murine CT26 colorectal cancer model, a murine breast cancer 4T1 model, or a melanoma cell line B16-moB7-H4 / H3 model. In one embodiment, the antibody or antigen-binding fragment thereof reduces tumor growth by at least 25%, at least 30%, at least 40%, at least 45%, or at least 50% compared to treatment with a control antibody.

[0033] In one embodiment, the induction of T cell proliferation, the induction of CD4+ T cell proliferation, the induction of CD8+ T cell proliferation, the induction of IFNγ production, ADCC activity and / or the inhibition of tumor growth is dose-dependent.

[0034] In one embodiment, the antibody or antigen binding fragment thereof binds to cynomolgus monkey B7-H4. In one embodiment, the antibody or antigen binding fragment thereof binds to rat B7-H4. In one embodiment, the antibody or antigen binding fragment thereof binds to mouse B7-H4. In one embodiment, the antibody or antigen binding fragment thereof binds to human B7-H4, cynomolgus monkey B7-H4, rat B7-H4, and mouse B7-H4.

[0035] In one embodiment, the antibody or antigen-binding fragment thereof binds to the IgV domain of human B7-H4.

[0036] In one embodiment, the antibody or antigen-binding fragment thereof is afucosylated.

[0037] In one embodiment, the antibody or antigen-binding fragment thereof is a full-length antibody. In one embodiment, the antibody or antigen-binding fragment thereof is an antigen-binding fragment. In one embodiment, the antigen-binding fragment comprises Fab, Fab', F(ab') 2, single-chain Fv (scFv), disulfide-linked Fv, V-NAR domain, IgNar, intrabody, IgGΔCH2, minibody, F(ab') 3 , tetrafunctional antibody, trifunctional antibody, bifunctional antibody, single domain antibody, DVD-Ig, Fcab, mAb 2 , (scFv) 2 or scFv-Fc.

[0038] In one embodiment, the antibody or antigen-binding fragment thereof further comprises a detectable label.

[0039] Also provided herein is an isolated polynucleotide comprising a nucleic acid molecule encoding a heavy chain variable region or a heavy chain of an antibody or antigen-binding fragment thereof provided herein. In one embodiment, the nucleic acid molecule encodes a VH of SEQ ID NO: 11, 21, 31, 41, 464, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191 or 201, or a heavy chain of SEQ ID NO: 13, 23, 33, 43, 469, 53, 63, 73, 83, 93, 103, 113, 123, 133, 143, 153, 163, 173, 183, 193 or 203. In one embodiment, the nucleic acid molecule comprises a sequence of SEQ ID NO: 213, 223, 233, 243, 470, 253, 263, 273, 283, 293, 303, 313, 323, 333, 343, 353, 363, 373, 383, 393 or 403. In one embodiment, the nucleic acid molecule comprises (i) a sequence of SEQ ID NO: 213, 223, 233, 243, 470, 253, 263, 273, 283, 293, 303, 313, 323, 333, 343, 353, 363, 373, 383, 393 or 403; and (ii) a sequence of SEQ ID NO: 408.

[0040] Also provided herein is a separated polynucleotide comprising a nucleic acid molecule encoding a light chain variable region or a light chain of an antibody or antigen-binding fragment thereof provided herein. In one embodiment, the nucleic acid molecule encodes a VL of SEQ ID NO: 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182, 192 or 202, or a light chain of SEQ ID NO: 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, 114, 124, 134, 144, 154, 164, 174, 184, 194 or 204. In one embodiment, the nucleic acid molecule comprises a sequence of SEQ ID NO: 214, 224, 234, 244, 254, 264, 274, 284, 294, 304, 314, 324, 334, 344, 354, 364, 374, 384, 394 or 404. In one embodiment, the nucleic acid molecule comprises (i) a sequence of SEQ ID NO: 214, 224, 234, 244, 254, 264, 274, 284, 294, 304, 314, 324, 334, 344, 354, 364, 374, 384, 394 or 404; and (ii) a sequence of SEQ ID NO: 406.

[0041] Also provided herein is an isolated polynucleotide comprising a nucleic acid molecule encoding a heavy chain variable region or a heavy chain of an antibody or antigen-binding fragment provided herein and a light chain variable region or a light chain of the antibody or antigen-binding fragment thereof.

[0042] Also provided herein is an isolated vector comprising a polynucleotide provided herein.

[0043] A host cell is also provided herein, comprising a polynucleotide provided herein, a vector provided herein, a first vector comprising a polynucleotide provided herein (e.g., a polynucleotide comprising a variable heavy chain or a heavy chain encoding nucleic acid) and a second vector comprising a polynucleotide provided herein (e.g., a polynucleotide comprising a variable light chain or a light chain encoding nucleic acid). In one embodiment, the host cell is a cell selected from the group consisting of: Escherichia coli, Pseudomonas, Bacillus, Streptomyces, yeast, CHO, YB / 20, NSO, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, Hep G2, SP2 / 0, R1.1, BW, LM, COS1, COS 7, BSC1, BSC40, BMT10 cells, plant cells in tissue culture, insect cells and human cells. In one embodiment, the host cell is a CHO cell. In one embodiment, the host cell (eg, a mammalian host cell such as a CHO cell) lacks a functional α-1,6-fucosyltransferase gene (FUT8).

[0044] Also provided herein is a method (e.g., an in vitro method) for producing an antibody or antigen-binding fragment thereof that binds to human B7-H4, the method comprising culturing the host cell provided herein, thereby expressing the nucleic acid molecule and producing the antibody or antigen-binding fragment thereof.

[0045] Also provided herein is an isolated antibody or antigen-binding fragment thereof that specifically binds to human B7-H4 and is encoded by a polynucleotide provided herein.

[0046] Also provided herein is a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof provided herein, the polynucleotide provided herein, the vector provided herein, or the host cell provided herein; and a pharmaceutically acceptable excipient.

[0047] Also provided herein is a pharmaceutical composition comprising (i) an antibody or antigen-binding fragment provided herein and (ii) a pharmaceutically acceptable excipient, wherein at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the antibody or antigen-binding fragment thereof in the composition is afucosylated.

[0048] Also provided herein is a pharmaceutical composition comprising (i) an antibody or antigen-binding fragment provided herein and (ii) a pharmaceutically acceptable excipient, wherein at least 95% of the antibody or antigen-binding fragment thereof in the composition is afucosylated.

[0049] Also provided herein is a pharmaceutical composition comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4 and comprises heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 458-463, respectively; and (ii) a pharmaceutically acceptable excipient, wherein at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the antibody or antigen-binding fragment thereof in the composition is afucosylated. In one embodiment, (i) the antibody or antigen-binding fragment thereof comprises a variable heavy chain region comprising an amino acid sequence of SEQ ID NO: 464 and a variable light chain region comprising an amino acid sequence of SEQ ID NO: 42; or (ii) the antibody comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 469 and a light chain comprising an amino acid sequence of SEQ ID NO: 44.

[0050] Also provided herein is a pharmaceutical composition comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4 and comprises heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 458-463, respectively; and (ii) a pharmaceutically acceptable excipient, wherein at least 95% of the antibody or antigen-binding fragment thereof in the composition is afucosylated. In one embodiment, (i) the antibody or antigen-binding fragment thereof comprises a variable heavy chain region comprising an amino acid sequence of SEQ ID NO: 464 and a variable light chain region comprising an amino acid sequence of SEQ ID NO: 42; or (ii) the antibody comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 469 and a light chain comprising an amino acid sequence of SEQ ID NO: 44.

[0051] Also provided herein is a pharmaceutical composition comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4 and comprises heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 35-40, respectively; and (ii) a pharmaceutically acceptable excipient, wherein at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the antibody or antigen-binding fragment thereof in the composition is afucosylated. In one embodiment, (i) the antibody or antigen-binding fragment thereof comprises a variable heavy chain region comprising an amino acid sequence of SEQ ID NO: 41 and a variable light chain region comprising an amino acid sequence of SEQ ID NO: 42; or (ii) the antibody comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 43 and a light chain comprising an amino acid sequence of SEQ ID NO: 44.

[0052] Also provided herein is a pharmaceutical composition comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4 and comprises heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 35-40, respectively; and (ii) a pharmaceutically acceptable excipient, wherein at least 95% of the antibody or antigen-binding fragment thereof in the composition is afucosylated. In one embodiment, (i) the antibody or antigen-binding fragment thereof comprises a variable heavy chain region comprising an amino acid sequence of SEQ ID NO: 41 and a variable light chain region comprising an amino acid sequence of SEQ ID NO: 42; or (ii) the antibody comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 43 and a light chain comprising an amino acid sequence of SEQ ID NO: 44.

[0053] Also provided herein is a pharmaceutical composition comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4 and comprises heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 65-70, respectively; and (ii) a pharmaceutically acceptable excipient, wherein at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the antibody or antigen-binding fragment thereof in the composition is afucosylated. In one embodiment, (i) the antibody or antigen-binding fragment thereof comprises a variable heavy chain region comprising an amino acid sequence of SEQ ID NO: 71 and a variable light chain region comprising an amino acid sequence of SEQ ID NO: 72; or (ii) wherein the antibody comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 73 and a light chain comprising an amino acid sequence of SEQ ID NO: 74.

[0054] Also provided herein is a pharmaceutical composition comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4 and comprises heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 65-70, respectively; and (ii) a pharmaceutically acceptable excipient, wherein at least 95% of the antibody or antigen-binding fragment thereof in the composition is afucosylated. In one embodiment, (i) the antibody or antigen-binding fragment thereof comprises a variable heavy chain region comprising an amino acid sequence of SEQ ID NO: 71 and a variable light chain region comprising an amino acid sequence of SEQ ID NO: 72; or (ii) wherein the antibody comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 73 and a light chain comprising an amino acid sequence of SEQ ID NO: 74.

[0055] In one embodiment, no fucosylation is detected in the composition.

[0056] Also provided herein is a method for inducing T cell proliferation, the method comprising contacting T cells with an antibody or antigen-binding fragment thereof provided herein, a polynucleotide provided herein, a vector provided herein, a host cell provided herein, or a pharmaceutical composition provided herein. In one embodiment, T cell proliferation is reduced by at least 30%, at least 40%, at least 50%, at least 60%, at least 70% or at least 80% (e.g., compared to treatment with a control antibody).

[0057] Also provided herein is a method for inducing CD4+T cell proliferation, the method comprising contacting CD4+T cells with an antibody or antigen-binding fragment thereof provided herein, a polynucleotide provided herein, a vector provided herein, a host cell provided herein, or a pharmaceutical composition provided herein. In one embodiment, CD4+T cell proliferation is reduced by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% (e.g., compared to treatment with a control antibody).

[0058] Also provided herein is a method for inducing CD8+T cell proliferation, the method comprising contacting CD8+T cells with an antibody or antigen-binding fragment thereof provided herein, a polynucleotide provided herein, a vector provided herein, a host cell provided herein, or a pharmaceutical composition provided herein. In one embodiment, CD8+T cell proliferation is reduced by at least 30%, at least 40%, at least 50%, at least 60%, at least 70% or at least 80% (e.g., compared to treatment with a control antibody).

[0059] Also provided herein is a method for inducing interferon gamma production, the method comprising contacting a T cell with an antibody or antigen-binding fragment thereof provided herein, a polynucleotide provided herein, a vector provided herein, a host cell provided herein, or a pharmaceutical composition provided herein. In one embodiment, interferon gamma production increases by at least 30%, at least 40%, at least 50%, at least 60%, at least 70% or at least 80% (e.g., compared to treatment with a control antibody).

[0060] Also provided herein is a method for killing a cell expressing B7-H4, the method comprising contacting the cell with an antibody or antigen-binding fragment thereof provided herein, a polynucleotide provided herein, a vector provided herein, a host cell provided herein, or a pharmaceutical composition provided herein.

[0061] Also provided herein is a method for depleting cells expressing B7-H4 in a cell population, the method comprising contacting the cell population with an antibody or antigen-binding fragment thereof provided herein, a polynucleotide provided herein, a vector provided herein, a host cell provided herein, or a pharmaceutical composition provided herein.

[0062] In one embodiment, said killing or depletion occurs by ADCC.

[0063] In one embodiment, the contacting is performed in vitro. In one embodiment, the contacting is performed in vivo in a subject.

[0064] Also provided herein is a method for treating cancer in a subject, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof provided herein, a polynucleotide provided herein, a vector provided herein, a host cell provided herein, or a pharmaceutical composition provided herein. In one embodiment, the cancer is selected from the group consisting of: breast cancer, such as triple-negative breast cancer or invasive ductal carcinoma; endometrial cancer; ovarian cancer; non-small cell lung cancer; pancreatic cancer; thyroid cancer; kidney cancer; and bladder cancer. In one embodiment, the breast cancer is triple-negative breast cancer or wherein the non-small cell lung cancer is squamous cell carcinoma. In one embodiment, the non-small cell lung cancer is adenocarcinoma. In one embodiment, the cancer is selected from the group consisting of: head and neck cancer, small cell lung cancer, gastric cancer, and melanoma. In one embodiment, ovarian cancer is serous adenocarcinoma. In one embodiment, breast cancer is ductal adenocarcinoma.

[0065] In one embodiment, the cancer is a PD-1 inhibitor-inadequately responsive and / or PD-L1 inhibitor-inadequately responsive cancer. In one embodiment, the cancer expresses PD-L1 at a low level.

[0066] In one embodiment, the subject is a human.

[0067] Also provided herein is a method for detecting B7-H4 in a sample, the method comprising contacting the sample with an antibody or antigen-binding fragment thereof provided herein. In one embodiment, the sample is obtained from a cancer in a human subject.

[0068] Also provided herein is a kit comprising an antibody or antigen-binding fragment thereof provided herein, a polynucleotide provided herein, a vector provided herein, a host cell provided herein, or a pharmaceutical composition provided herein; and a) a detection reagent, b) a B7-H4 antigen, c) a notice reflecting approval for use or sale for human administration, or d) a combination thereof.

[0069] Also provided herein is an isolated antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment thereof inhibits the T cell checkpoint blocking activity of B7-H4. In one embodiment, the T cell checkpoint blocking activity is measured by an increase in IL-2 production relative to control cells.

[0070] Also provided herein is a pharmaceutical composition comprising (i) an antibody or antigen binding fragment, and (ii) a pharmaceutically acceptable excipient, wherein the antibody or antigen binding fragment inhibits the T cell checkpoint blocking activity of B7-H4. In one embodiment, the T cell checkpoint blocking activity is measured by an increase in IL-2 production relative to control cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1A The expression of B7-H4 in various tumor tissues was shown. (See Example 1.)

[0072] Figure 1B The prevalence of B7-H4 in various tumor types as determined by IHC is shown. (See Example 1.)

[0073] Figure 2A The binding of B7-H4 antibodies to HEK293T cells expressing human, cynomolgus monkey or mouse B7-H4 is shown. (See Example 6.)

[0074] Figure 2B The binding of B7-H4 antibodies to SK-BR-3 cells and to HEK293T cells expressing mouse, cynomolgus monkey or rat B7-H4 is shown. (See Example 6.)

[0075] Figure 3A B7-H4 antibody showed + T cells, CD8 + Effects on proliferation of T cells or total T cells. (See Example 7.)

[0076] Figure 3B The effect of B7-H4 antibody on interferon-γ (IFNγ) production is shown. (See Example 7.)

[0077] Figure 3C B7-H4 antibody showed + T cells, CD8 + T cell or total T cell proliferation and the effect on interferon-γ (IFNγ) production. (See Example 7.)

[0078] Figure 3D The effect of various concentrations of B7-H4 antibody on interferon-γ (IFNγ) production is shown. (See Example 7.)

[0079] Figures 4A-4D Showing the interaction between fucosylated and afucosylated antibodies and SK-BR-3 cells ( Figure 4A ) and mice expressing ( Figure 4B )、Cynomolgus macaque( Figure 4C ) or rat B7-H4 ( Figure 4D ) on HEK293T cells. (See Example 9.)

[0080] Figure 5A and 5B Binding of afucosylated and fucosylated B7-H4 antibodies (respectively) to the human Fcγ receptor IIIa (FcγRIIIa) V158 allele is shown. (See Example 10.)

[0081] Fig. 6A The T cell checkpoint blocking activity of fucosylated and afucosylated B7-H4 antibodies was shown. (See Example 11.)

[0082] Figure 6B The T cell checkpoint ligand activity of the afucosylated B7-H4 antibody as measured by IL-2 production is shown compared to cells treated with an isotype control. (See Example 11.)

[0083] Figure 7 ADCC activity of fucosylated and afucosylated B7-H4 antibodies against B7-H4 expressing cell lines is shown. (See Example 12.)

[0084] Figure 8 ADCC activity of fucosylated and afucosylated B7-H4 antibodies against cells expressing various amounts of B7-H4 is shown. (See Example 13.)

[0085] Fig. 9A The in vivo anti-tumor efficacy of B7-H4 antibody 20502 was demonstrated. (See Example 14.)

[0086] Fig. 9B The in vivo anti-tumor efficacy of B7-H4 antibody 22213 is shown. (See Example 14.)

[0087] Fig.10 The murine version of the B7-H4 antibody 20502 was shown to have in vivo anti-tumor efficacy in mice implanted with 4T1 (breast cancer) and B16 (melanoma) cells. (See Example 14.)

[0088] Fig.11 The in vivo anti-tumor efficacy of afucosylated 20502 in the MX-1 human breast cancer xenograft model is shown. Images show human B7-H4 expression in MX-1 xenograft tumors (left) and murine B7-H4 expression in 4T1 tumors (right). (See Example 14.)

[0089] Figures 12A-12C The in vivo anti-tumor efficacy of 20502-msIgG2a-F administered on the same day as anti-PD-1 antibody is shown. (See Example 15.) * indicates p<0.05; and **** indicates p<0.0001.

[0090] Fig.13 Compared with treatment with a control antibody (upper panel), treatment with afucosylated 20502 (lower panel) resulted in NK cell infiltration (left panel), T cell infiltration (middle panel), and upregulation of PD-L1 (right panel). (See Example 16.)

[0091] Fig.14A It was shown that the 20502-msIgG2a-F antibody significantly reduced tumor growth in 4T1 breast cancer cells in a dose-dependent manner. (See Example 17.)

[0092] Fig. 14B It was shown that the 20502-msIgG2a-F antibody significantly inhibited tumor growth at 30 mg / kg (p=0.0003), 20 mg / kg (p=0.0103), 10 mg / kg (p=0.0419), 3 mg / kg (p=0.0277) and 1 mg / kg (p=0.0333) as assessed by one-way ANOVA on day 30. (See Example 17.)

[0093] Fig.15A It was shown that the 20502-msIgG2a-F antibody significantly reduced the growth of B16 expressing B7-H4 / H3. (See Example 17.)

[0094] Fig. 15B It was shown that the 20502-msIgG2a-F antibody significantly inhibited tumor growth at 30 mg / kg (p=0.0085), 20 mg / kg (p=0.0041), 10 mg / kg (p=0.0017) and 3 mg / kg (p=0.0420) as assessed by one-way ANOVA on day 23. (See Example 17.) DETAILED DESCRIPTION

[0095] Antibodies (e.g., monoclonal antibodies) and antigen-binding fragments thereof that specifically bind to B7-H4 (e.g., human B7-H4) are provided herein. These B7-H4 antibodies and antigen-binding fragments thereof can, for example, cause T cell checkpoint blocking activity (e.g., measured by increased interferon gamma (IFNγ), CD4 T cell proliferation, CD8 T cell proliferation, and / or total T cell proliferation) and / or have antibody-dependent cellular cytotoxicity (ADCC activity).

[0096] Also provided herein are isolated nucleic acids (polynucleotides) encoding such antibodies and antigen-binding fragments thereof, such as complementary DNA (cDNA). Also provided are vectors (e.g., expression vectors) and cells (e.g., host cells) comprising nucleic acids (polynucleotides) encoding such antibodies and antigen-binding fragments thereof. Also provided are methods for preparing such antibodies and antigen-binding fragments thereof. In other aspects, provided herein are methods for treating certain conditions such as cancer. Also provided are related compositions (e.g., pharmaceutical compositions), kits, and detection methods.

[0097] 1.1 Terminology

[0098] As used herein, the term "B7-H4" refers to mammalian B7-H4 polypeptides, including but not limited to native B7-H4 polypeptides and isoforms of B7-H4 polypeptides. "B7-H4" encompasses full-length, unprocessed B7-H4 polypeptides as well as B7-H4 polypeptide forms produced by intracellular processing. As used herein, the term "human B7-H4" refers to a polypeptide comprising the amino acid sequence of SEQ ID NO: 1. "B7-H4 polypeptide", "B7-H4 nucleotide" or "B7-H4 nucleic acid" refers to a polynucleotide encoding B7-H4.

[0099] As used herein, the term "PD-1" refers to mammalian PD-1 polypeptides, including but not limited to native PD-1 polypeptides and isoforms of PD-1 polypeptides. PD-1 is also known as planned death protein 1 or planned cell death protein 1. "PD-1" encompasses full-length, unprocessed PD-1 polypeptides as well as PD-1 polypeptide forms obtained by intracellular processing. As used herein, the term "human PD-1" refers to a polypeptide comprising the amino acid sequence of SEQ ID NO: 439:

[0100] PGWFLDSPDR PWNPPTFSPA LLVVTEGDNA TFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPG QDCRFRVTQLPNGRDFHMSV VRARRNDSGT YLCGAISLAP KAQIKESLRAELRVTERRAEVPTAHPSPSP RPAGQFQTLV VGVVGGLLGSLVLLVWVLAV ICSRAARGTI GARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVP CVPEQTEYAT IVFPSGMGTSSPARRGSADG PRSAQPLRPEDGHCSWPL (SEQ ID NO: 439) (mature human PD-1 without signal peptide). “PD-1 polynucleotide”, “PD-1 nucleotide” or “PD-1 nucleic acid” refers to a polynucleotide encoding PD-1.

[0101] The term "antibody" means an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of the foregoing, through at least one antigen recognition site in its variable region. As used herein, the term "antibody" encompasses complete polyclonal antibodies, complete monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising antibodies, and any other modified immunoglobulin molecules, as long as these antibodies exhibit the desired biological activity. Antibodies may belong to any of the five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), which are referred to as α, δ, ε, γ, and μ, respectively, based on the properties of their heavy chain constant domains. Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional structures. Antibodies may be naked antibodies, or conjugated to other molecules, such as toxins, radioisotopes, and the like.

[0102] The term "antibody fragment" refers to a portion of a complete antibody. "Antigen binding fragment", "antigen binding domain" or "antigen binding region" refers to a portion of a complete antibody that binds to an antigen. An antigen binding fragment may contain the antigenic determining region (e.g., complementary determining region (CDR)) of a complete antibody. Examples of antigen binding fragments of antibodies include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, linear antibodies and single-chain antibodies. Antigen binding fragments of antibodies may be derived from any animal species, such as rodents (e.g., mice, rats or hamsters) and humans, or may be artificially produced.

[0103] The terms "anti-B7-H4 antibody," "B7-H4 antibody," and "antibody that binds to B7-H4" refer to an antibody that is capable of binding to B7-H4 with sufficient affinity to allow the antibody to be used as a diagnostic and / or therapeutic agent targeting B7-H4. The extent of binding of the anti-B7-H4 antibody to an unrelated, non-B7-H4 protein may be about 10% less than the binding of the antibody to B7-H4, as measured, for example, by radioimmunoassay (RIA).

[0104] The terms "anti-PD-1 antibody," "PD-1 antibody," and "antibody that binds to PD-1" refer to an antibody that is capable of binding to PD-1 with sufficient affinity to allow the antibody to be used as a diagnostic and / or therapeutic agent targeting PD-1. The extent of binding of the anti-PD-1 antibody to unrelated, non-PD-1 proteins may be about 10% less than the binding of the antibody to PD-1, as measured, for example, by radioimmunoassay (RIA).

[0105] "Monoclonal" antibody or its antigen binding fragment refers to a homogeneous group of antibodies or antigen binding fragments that participate in the highly specific recognition and binding of a single antigenic determinant or epitope. It is in contrast to polyclonal antibodies, which typically include different antibodies against different antigenic determinants. The term "monoclonal" antibody or its antigen binding fragment encompasses complete and full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single-chain (scFv) mutants, fusion proteins comprising antibody portions, and any other modified immunoglobulin molecules comprising antigen recognition sites. In addition, "monoclonal" antibody or its antigen binding fragment refers to such antibodies and antigen binding fragments thereof prepared in a variety of ways, including but not limited to hybridomas, phage selection, recombinant expression, and transgenic animals.

[0106] As used herein, the terms "variable region" or "variable domain" are used interchangeably and are commonly used in the art. A variable region typically refers to a portion of an antibody, generally a portion of a light chain or a heavy chain, typically approximately the amino terminal 110 to 120 amino acids or 110 to 125 amino acids in a mature heavy chain and about 90 to 115 amino acids in a mature light chain, the sequence of which varies significantly with the antibody and is used for the binding and specificity of a specific antibody to its specific antigen. Sequence variations are concentrated in a region called a complementary determining region (CDR), while highly conserved regions in a variable domain are called framework regions (FR). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of light and heavy chains are primarily responsible for the interaction and specificity of antibodies to antigens. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises a rodent or murine CDR and a human framework region (FR). In a specific embodiment, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable regions comprise rodent or murine CDRs and primate (eg, non-human primate) framework regions (FRs).

[0107] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody.

[0108] The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody.

[0109] The term "Kabat numbering" and similar terms are generally recognized in the art and refer to a system for numbering amino acid residues in the heavy chain and light chain variable regions of an antibody or its antigen-binding fragment. In some aspects, CDR can be determined according to the Kabat numbering system (see, for example, Kabat EA and Wu TT (1971) Ann NY Acad Sci 190: 382-391; and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, the CDR in the antibody heavy chain molecule is typically present at amino acid positions 31 to 35, and the CDR may optionally include one or two additional amino acids (referred to as 35A and 35B in the Kabat numbering scheme) (CDR1) after position 35; amino acid positions 50 to 65 (CDR2); and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, the CDRs in the antibody light chain molecule are typically present at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). In a specific embodiment, the CDRs of the antibodies described herein are determined according to the Kabat numbering scheme.

[0110] Chothia refers to the position of the structural loop (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)). When numbered using the Kabat numbering convention, the end of the Chothia CDR-H1 loop varies between H32 and H34 depending on the loop length (this is because the Kabat numbering scheme has insertions at H35A and H35B; if 35A and 35B are absent, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable region represents a compromise between the Kabat CDR and Chothia structural loops and is used by Oxford Molecular's AbM antibody modeling software.

[0111]

[0112] As used herein, the terms "constant region" or "constant domain" are used interchangeably and have their commonly used meanings in the art. The constant region is the portion of the antibody that is not directly involved in the binding of the antibody to the antigen but can exhibit various effector functions, such as interacting with Fc receptors, such as the carboxyl terminal portion of the light chain and / or heavy chain. Relative to the immunoglobulin variable domain, the constant region of the immunoglobulin molecule generally has a more conservative amino acid sequence. In certain aspects, the antibody or antigen-binding fragment comprises a constant region or portion thereof sufficient to achieve antibody-dependent cell-mediated cytotoxicity (ADCC).

[0113] As used herein, the term "heavy chain" when referring to antibodies can refer to any of the distinct types, such as α, δ, ε, γ, and μ, based on the amino acid sequence of the constant domain, thereby giving rise to the IgA, IgD, IgE, IgG, and IgM antibody classes, respectively, including the subclasses of IgG, such as IgG 1 IgG 2 IgG 3 and IgG 4 Heavy chain amino acid sequences are well known in the art. In a specific embodiment, the heavy chain is a human heavy chain.

[0114] As used herein, the term "light chain" used in reference to an antibody can refer to any distinct type, such as κ or λ, based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. In a specific embodiment, the light chain is a human light chain.

[0115] The term "chimeric" antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof whose amino acid sequence is derived from two or more species. Typically, the variable regions of the light and heavy chains correspond to the variable regions of an antibody or antigen-binding fragment thereof derived from one mammalian species (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capacity, while the constant region is homologous to the sequence in an antibody or antigen-binding fragment thereof derived from another species (usually human) to avoid causing an immune response in that species.

[0116] The term "humanized" antibody or antigen-binding fragment thereof refers to a form or antigen-binding fragment of a non-human (e.g., murine) antibody that is a specific immunoglobulin chain, a chimeric immunoglobulin, or a fragment thereof that contains minimal non-human (e.g., murine) sequences. Typically, a humanized antibody or antigen-binding fragment thereof is a human immunoglobulin in which residues from a complementary determining region (CDR) are replaced ("CDR grafted") with residues from a CDR of a non-human species (e.g., mouse, rat, rabbit, hamster) having the desired specificity, affinity, and capacity (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)). In some cases, the Fv framework region (FR) residues of a human immunoglobulin are replaced by corresponding residues in an antibody or fragment from a non-human species with desired specificity, affinity and ability. A humanized antibody or its antigen-binding fragment may be further modified by replacing additional residues in the Fv framework region and / or in the replaced non-human residues to refine and optimize the specificity, affinity and / or ability of the antibody or its antigen-binding fragment. In general, a humanized antibody or its antigen-binding fragment will comprise at least one, and typically substantially all of two or three variable domains, the domains containing all or substantially all of the CDR regions corresponding to non-human immunoglobulins, and all or substantially all of the FR regions being FR regions of human immunoglobulin common sequences. A humanized antibody or its antigen-binding fragment may also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically at least a portion of a human immunoglobulin constant region or domain. Examples of methods for producing humanized antibodies are described in U.S. Pat. No. 5,225,539; Roguska et al., Proc. Natl. Acad. Sci., USA, 91(3):969-973 (1994); and Roguska et al., Protein Eng. 9(10):895-904 (1996). In some embodiments, a "humanized antibody" is a resurfaced antibody.

[0117] The term "human" antibody or antigen-binding fragment thereof means an antibody or antigen-binding fragment thereof having an amino acid sequence derived from a human immunoglobulin locus, wherein such antibody or antigen-binding fragment is prepared using any technique known in the art. This definition of a human antibody or antigen-binding fragment thereof includes intact or full-length antibodies and fragments thereof.

[0118] An "afucosylated" antibody or antigen-binding fragment thereof or an antibody or antigen-binding fragment thereof that "lacks fucose" refers to an IgG1 or IgG3 isotype antibody or antigen-binding fragment thereof that lacks fucose in the constant region glycosylation. Glycosylation of human IgG1 or IgG3 occurs at Asn297 when the core fucosylated biantennary complex oligosaccharide glycosylation is terminated with up to 2 Gal residues. In some embodiments, the afucosylated antibody has no fucose at Asn297. Depending on the amount of terminal Gal residues, these structures are referred to as G0, G1 (a 1,6 or a 1,3) or G2 glycan residues. See, e.g., Raju, TS, BioProcess Int. 1: 44-53 (2003). CHO-type glycosylation of antibody Fc is described, e.g., in Routier, F. FL, Glycoconjugate J. 14: 201-207 (1997).

[0119] Methods for measuring fucose include any method known in the art. For the purposes of this article, fucose is detected by the method described in Example 1 of WO2015 / 017600, which is incorporated herein by reference in its entirety. In short, glycan analysis is performed by releasing glycans from antibodies (e.g., by enzymatic release), labeling glycans with anthranilic acid (2-AA), and then purifying the labeled glycans. Each glycan is separated using normal phase HPLC with fluorescence detection, and the relative amount of each glycan in the antibody is measured. Glycans can be definitively identified as lacking or including fucose by mass spectrometry. In some embodiments, fucose cannot be detected in a composition comprising a plurality of afucosylated antibodies or antigen-binding fragments thereof. In some embodiments, afucosylated antibodies or antigen-binding fragments thereof have enhanced ADCC activity, which can be measured by the analysis provided in Example 12 herein. In some embodiments, afucosylated antibodies or antigen-binding fragments thereof have enhanced affinity for FcγRIIIA. In some embodiments, the afucosylated antibody or antigen-binding fragment thereof has an enhanced affinity for FcγRIIIA (V158). In some embodiments, the afucosylated antibody or antigen-binding fragment thereof has an enhanced affinity for FcγRIIIA (F158). Affinity for FcγRIIIA or its alleles can be measured by the assay provided in Example 10 herein.

[0120] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or antigen-binding fragment thereof) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody or antigen-binding fragment thereof and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (K D Affinity can be measured and / or expressed in a variety of ways known in the art, including, but not limited to, equilibrium dissociation constant (K D ) and equilibrium association constant (K A ). D By k off / k on The quotient of K A By k on / k off Calculate the quotient of k on refers to the association rate constant of, for example, an antibody or antigen-binding fragment thereof with an antigen, and k off Refers to, for example, the dissociation of an antibody or antigen-binding fragment thereof from an antigen. on and k off It can be done by techniques known to those skilled in the art, such as or KinExA assay.

[0121] As used herein, "epitope" is a term in the art, and refers to the local region of an antigen that an antibody or its Fab can specifically bind. An epitope can be, for example, the adjacent amino acids of a polypeptide (linear or adjacent epitopes), or an epitope can be, for example, two or more non-adjacent regions (conformation, non-linear, discontinuous or non-adjacent epitopes) from one or more polypeptides. In certain embodiments, the epitope of an antibody or its Fab can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography, ELISA analysis, hydrogen / deuterium exchange in combination with mass spectrometry (e.g., liquid chromatography-electrospray mass spectrometry), array-based oligopeptide scanning analysis and / or mutagenesis localization (e.g., site-directed mutagenesis localization). For X-ray crystallography, crystallization can be achieved using any method known in the art (e.g., Giege R et al. (1994) Acta Crystallogr DBiol Crystallogr 50(Part 4):339-350; McPherson A (1990) Eur J Biochem 189:1-23; Chayen NE (1997) Structure 5:1269-1274; McPherson A (1976) J Biol Chem 251:6300-6303). Antibody / antigen-binding fragment thereof: Antigen crystals can be studied using well-known X-ray diffraction techniques and can be refined using computer software such as X-PLOR (Yale University, 1992, published by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) Vols. 114 and 115, edited by Wyckoff HW et al.; US2004 / 0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49 (Part 1): 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, edited by Carter CW; Roversi P et al. (2000) Acta Crystallogr D Biol Crystallogr 56 (Part 10): 1316-1323). Mutagenesis localization studies can be performed using any method known to those skilled in the art. For descriptions of mutagenesis techniques, including alanine scanning mutagenesis, see, for example, Champe M et al. (1995) J Biol Chem 270: 1388-1394, and Cunningham BC and Wells JA (1989) Science 244: 1081-1085.

[0122] A B7-H4 antibody that "binds to the same epitope" as a reference B7-H4 antibody refers to an antibody that binds to the same B7-H4 amino acid residue as the reference B7-H4 antibody. The ability of a B7-H4 antibody to bind to the same epitope as a reference B7-H4 antibody is determined by hydrogen / deuterium exchange analysis (see Coales et al., Rapid Commun. Mass Spectrom. 2009; 23: 639–647).

[0123] As used herein, the terms "immunospecific binding", "immunospecific recognition", "specific binding" and "specific recognition" are analogous terms in the context of antibodies or antigen-binding fragments thereof. These terms indicate that the antibody or antigen-binding fragment thereof binds to an epitope through its antigen-binding domain and that the binding requires a certain complementarity between the antigen-binding domain and the epitope. Thus, an antibody that "specifically binds" to human B7-H4 (SEQ ID NO: 1) may also bind to B7-H4 from other species (e.g., cynomolgus monkey, mouse and / or rat B7-H4) and / or B7-H4 proteins produced by other human alleles, but the antibody binds to unrelated, non-B7-H4 proteins (e.g., other B7 protein family members, such as PD-L1) to a degree that is about 10% less than its binding to B7-H4, as measured, for example, by radioimmunoassay (RIA).

[0124] In a specific embodiment, provided herein is an antibody or antigen-binding fragment thereof that binds to human, cynomolgus monkey, mouse, and rat B7-H4.

[0125] An antibody is said to "competitively inhibit" binding of a reference antibody to a given epitope if it preferentially binds to the epitope or an overlapping epitope such that it blocks binding of the reference antibody to the epitope to some extent. Competitive inhibition can be determined by any method known in the art, such as a competition ELISA assay. It is believed that an antibody can competitively inhibit binding of a reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0126] An "isolated" polypeptide, antibody, polynucleotide, vector, cell, or composition is a polypeptide, antibody, polynucleotide, vector, cell, or composition that is in a form not found in nature. An isolated polypeptide, antibody, polynucleotide, vector, cell, or composition includes a polypeptide, antibody, polynucleotide, vector, cell, or composition that has been purified to such an extent that it is no longer in the form that it is found in nature. In some embodiments, an isolated antibody, polynucleotide, vector, cell, or composition is substantially pure. As used herein, "substantially pure" means that a substance is at least 50% pure (i.e., free of contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0127] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interspersed with non-amino acids. These terms also encompass amino acid polymers modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included in the definition are, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids, etc.) and other modifications known in the art. It should be understood that since the polypeptides of the present invention are antibody-based, in certain embodiments, these polypeptides may exist in single-chain or linked chain form.

[0128] "Percent identity" refers to the degree of identity between two sequences (e.g., amino acid sequences or nucleic acid sequences). The percent identity can be determined by aligning the two sequences, introducing spaces to maximize the identity between the sequences. Alignment can be generated using programs known in the art. For purposes herein, alignment of nucleotide sequences can be performed using the blastn program with default parameters set, and alignment of amino acid sequences can be performed using the blastp program with default parameters set (see the World Wide Web of the National Center for Biotechnology Information (NCBI), ncbi.nlm.nih.gov).

[0129] As used herein, the term "host cell" can be any type of cell, such as a primary cell, a cultured cell, or a cell from a cell line. In a specific embodiment, the term "host cell" refers to a cell transfected with a nucleic acid molecule and the progeny or possible progeny of such a cell. The progeny of such a cell may be different from the parent cell transfected with the nucleic acid molecule, for example, due to mutations or environmental influences that occur in later generations or the integration of the nucleic acid molecule into the host cell genome.

[0130] The term "pharmaceutical formulation" refers to a preparation that is in a form that renders the biological activity of the active ingredient effective and does not contain other components that would be unacceptably toxic to a subject to which the formulation would be administered. The formulation may be sterile.

[0131] As used herein, the terms "administer", "administering", "administration" and similar expressions refer to methods (e.g., intravenous administration) that can be used to achieve delivery of a drug, such as an anti-B7-H4 antibody or antigen-binding fragment thereof, to a desired site of biological action. Administration techniques that can be used for the agents and methods described herein are found, for example, in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current edition, Pergamon; and Remington's, Pharmaceutical Sciences, current edition, Mack Publishing Co., Easton, Pa.

[0132] Administration "in combination with" one or more additional therapeutic agents includes simultaneous (concurrent) or consecutively in any order.

[0133] Combination therapy can provide a "synergistic effect," that is, an effect achieved when the active agents are used together that exceeds the sum of the effects resulting from the use of each active agent alone. Synergism can be achieved when: (1) the active ingredients are co-formulated and administered or delivered simultaneously in a combined unit dosage formulation; (2) the active ingredients are delivered sequentially, alternatingly, or in parallel as separate formulations; or (3) the active ingredients are administered by some other regimen. When delivered in alternation therapy, synergism can be achieved if the active agents are administered or delivered sequentially, for example by different injections with separate syringes. A "synergistic combination" produces an effect that is greater than the sum of the effects of the individual active agents in the combination.

[0134] Combination therapy may provide an "additive" effect, ie, the effect achieved when the active agents are used together is equal to the sum of the effects elicited by each active agent when used alone.

[0135] As used herein, the terms "subject" and "patient" are used interchangeably. The subject can be an animal. In some embodiments, the subject is a mammal, such as a non-human animal (e.g., a cow, pig, horse, cat, dog, rat, mouse, monkey or other primate, etc.). In some embodiments, the subject is a cynomolgus macaque. In some embodiments, the subject is a human.

[0136] The term "therapeutically effective amount" refers to an amount of a drug, such as an anti-B7-H4 antibody or antigen-binding fragment thereof, that is effective in treating a disease or condition in a subject. In the case of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells; reduce tumor size or load; inhibit (i.e., slow down to some extent and in a certain embodiment, stop) cancer cell infiltration in peripheral organs; inhibit (i.e., slow down to some extent and in a certain embodiment, stop) tumor metastasis; inhibit tumor growth to some extent; alleviate one or more cancer-related symptoms to some extent; and / or induce a favorable response, such as increasing progression-free survival (PFS), disease-free survival (DFS) or overall survival (OS), complete response (CR), partial response (PR), or in some cases stabilizing the disease (SD), alleviating progressive disease (PD), shortening the time to progression (TTP), or any combination thereof. In the case that a drug can prevent cancer cell growth and / or kill existing cancer cells, the drug can be a cytostatic and / or cytotoxic drug.

[0137] Terms such as “treating / treatment / to "treat)" or "alleviating" (alleviating / toalleviate) refers to therapeutic measures that cure, slow the progression, relieve the symptoms and / or stop the progression of the diagnosed pathological condition or disorder. Therefore, those in need of treatment include those who have been diagnosed with or suspected of having the disorder. In certain embodiments, the subject's cancer is successfully "treated" according to the methods of the present invention if the patient shows one or more of the following situations: a decrease in the number of cancer cells or the complete absence of cancer cells; a decrease in tumor size; the infiltration of cancer cells in surrounding organs, such as the spread of cancer in soft tissues and bones, is inhibited or absent; tumor metastasis is inhibited or absent; tumor growth is inhibited or absent; one or more symptoms associated with a specific cancer are alleviated; morbidity and mortality are reduced; quality of life is improved; tumorigenesis, tumorigenesis frequency or tumorigenic capacity of the tumor is reduced; the number or frequency of cancer stem cells in the tumor is reduced; tumorigenic cells differentiate into a non-tumorigenic state; progression-free survival (PFS), disease-free survival (DFS) or overall survival (OS) Increased, complete response (CR), partial response (PR), stable disease (SD), reduction in progressive disease (PD), shortened time to progression (TTP) or any combination thereof.

[0138] The terms "cancer" and "carcinoma" refer to or describe a physiological condition in which a population of cells in a mammal is characterized by unregulated cell growth. Examples of cancer include, but are not limited to, gynecological cancers (e.g., breast cancer (including triple-negative breast cancer, ductal carcinoma), ovarian cancer, and endometrial cancer), non-small cell lung cancer, pancreatic cancer, thyroid cancer, kidney cancer (e.g., renal cell carcinoma), and bladder cancer (e.g., urothelial cell carcinoma). Non-small cell non-cancers may be, for example, adenocarcinomas. Other examples of cancer include, for example, head and neck cancer, small cell lung cancer, gastric cancer, melanoma, cholangiocarcinoma, glioblastoma or glioblastoma multiforme (GBM), and merkel cell carcinoma. In one embodiment, ovarian cancer is serous adenocarcinoma. In one embodiment, breast cancer is ductal adenocarcinoma. Cancer may be a "B7-H4-expressing cancer" or a "B7-H4-expressing cancer." These terms refer to cancers that contain cells that express B7-H4. Cancer may be a primary tumor or may be an advanced or metastatic cancer.

[0139] A "refractory" cancer is one that progresses despite administration of anti-tumor treatments, such as chemotherapy, to the cancer patient.

[0140] A "recurrent" cancer is a cancer that grows back at the original site or at a distant site after responding to initial therapy.

[0141] A "relapsed" patient is one who has signs or symptoms of cancer after remission. Optionally, the patient relapses after adjuvant or neoadjuvant therapy.

[0142] "T cell checkpoint blockade activity" refers to blocking or inhibiting T cell checkpoint activity or response. T cell checkpoint blockade activity can be measured based on changes in IFNγ production in an artificial antigen presenting cell (aAPC) assay. T cell enrichment kits such as EasySep TMHuman T cell enrichment kit or similar kit) is used to enrich primary human T cells from PBMC. The enriched T cells are cultured with beads (e.g., anti-CD3 / anti-CD28 beads). After a period of time, the beads are removed magnetically, and the T cells are washed and cultured. Next, the T cells are washed and cultured with artificial antigen presenting cells (aAPC) in the presence of a dosed B7-H4 antibody. aAPC can be treated with mitomycin C, then washed thoroughly, and then added to the T cell co-culture. After co-culturing T cells, aAPC, and B7-H4 antibodies, the culture plates can be centrifuged, and the supernatant can be collected and IFNγ production can be assessed by ELISA. IFNγ production can be plotted relative to antibody concentration, and EC50 potency can be calculated using nonlinear regression curve fitting. The results can be measured with EC50+ / -STD in nM. The T cell checkpoint blocking activity of the B7-H4 antibody can be demonstrated by an increase in IFNγ production. "T cell checkpoint blocking activity" can also be measured in the analysis of cells expressing endogenous B7-H4. Primary human T cells can be enriched from HLA-A2+ donor PBMC using a T cell separation kit (e.g., human pan T cell separation kit (Human Pan T Cell Isolation Kit)). MART-1 TCR expression T cells can be produced by first activating the enriched pan T cells for 48 hours with beads (e.g., anti-CD3 / anti-CD28 Dynabead), IL-2, and IL-7. Then, activated T cells can be transduced with MART-1 TCR lentiviral particles in the presence of IL-2, IL-7, and polybrene. After transduction, MART-1 TCR+ pan T cells can be amplified for a period of time in the presence of IL-2 and IL-7. In order to produce a target cell line expressing HLA-A2, endogenous B7-H4 expression cancer cells can be transduced with HLA-A2 lentiviral particles for a period of time (e.g., 48 hours). In addition, B7-H4 can be knocked out from HLA-A2+ cell lines. Next, MART-I TCR+ all T cells can be co-cultured in the presence of various target cell lines, MART-I peptides and B7-H4 antibodies or human isotype controls at a 1:1 E: T ratio. After co-cultivation, the culture plate can be centrifuged, and the supernatant can be collected and evaluated for IL-2 production. IL-2 production can be measured by a standard immunoassay kit (such as AlphaLISA assay or similar assay).

[0143] As used in the present disclosure and claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0144] It should be understood that when various embodiments are described herein with the language "comprising", similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided. In the present invention, "comprises / comprising", "containing" and "having" and similar expressions may have their meanings in the U.S. patent law, and may mean "includes / including" and similar expressions; "consisting essentially of / consists essentially" also have their meanings in the U.S. patent law and the term is open-ended, allowing the existence of more than what is stated, as long as the basic or novel features of the stated content are not changed by the existence of more than what is stated, but does not include prior art embodiments.

[0145] Unless the context specifically states or is obvious, as used herein, the term "or" is to be understood as inclusive. The term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A," and "B." Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0146] As used herein, the terms "about" and "approximately" when used to modify a numerical value or numerical range indicate a deviation of 5% to 10% above and 5% to 10% below the value or range is within the intended meaning of the value or range.

[0147] Any composition or method provided herein can be combined with one or more of any other compositions and methods provided herein.

[0148] 1.2 Antibodies

[0149] In a specific aspect, antibodies (e.g., monoclonal antibodies, such as chimeric antibodies, humanized antibodies, or human antibodies) and antigen-binding fragments thereof that specifically bind to B7-H4 (e.g., human B7-H4) are provided herein. The amino acid sequences of human, cynomolgus monkey, murine, and rat B7-H4 are known in the art and are also provided herein, as shown in SEQ ID NOs: 1-4, respectively.

[0150] Human B7-H4:

[0151] MASLGQILFWSIISIIIILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITSKGKGNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTVVWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTESEIKRRSHLQLLNSKASLCVSSFFAISWALLPLSPYLMLK(SEQ ID NO:1)

[0152] Cynomolgus macaque B7-H4:

[0153] MASLGQILFWSIISIIFILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVIGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITSKGKGNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTVVWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTESEIKRRSHLQLLNSKASLCVSSFLAISWALLPLAPYLMLK(SEQ ID NO:2)

[0154] Murine B7-H4

[0155] MASLGQIIFWSIINIIIILAGAIALIIGFGISGKHFITVTTFTSAGNIGEDGTLSCTFEPDIKLNGIVIQWLKEGIKGLVHEFKEGKDDLSQQHEMFRGRTAVFADQVVVGNASLRLKNVQLTDAGTYTCYIRTSKGKGNANL EYKTGAFSMPEINVDYNASSESLRCEAPRWFPQPTVASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTDSEVKRRSQLQLLNSGPSPCVFSSAFVAGWALLSLSCCLMLR(SEQ ID NO:3)

[0156] Rat B7-H4

[0157] MASLGQIIFWSIINVIIILAGAIVLIIGFGISGKHFITVTTFTSAGNIGEDGTLSCTFEPDIKLNGIVIQWLKEGIKGLVHEFKEGKDDLSQQHEMFRGRTAVFADQVVVGNASLRLKNVQLTDAGTYTCYIHTSKGKGNANL EYKTGAFSMPEINVDYNASSESLRCEAPRWFPQPTVASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTDSEVKRRSQLELLNSGPSPCVSSVSAAGWALLSLSCLMLR(SEQ ID NO:4)

[0158] In certain embodiments, the antibodies or antigen binding fragments thereof described herein bind to human B7-H4. In certain embodiments, the antibodies or antigen binding fragments thereof bind to human and cynomolgus monkey B7-H4. In certain embodiments, the antibodies or antigen binding fragments thereof bind to human, murine, and rat B7-H4. In certain embodiments, the antibodies or antigen binding fragments thereof bind to human, cynomolgus monkey, murine, and rat B7-H4.

[0159] B7-H4 contains an IgC extracellular domain (amino acids 153-241 of SEQ ID NO: 1) and an IgV domain (amino acids 35-146 of SEQ ID NO: 1).

[0160] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein bind to the IgV domain of human B7-H4. Thus, provided herein are antibodies or antigen-binding fragments thereof that bind to a polypeptide consisting of amino acids 35-146 of SEQ ID NO: 1.

[0161] In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human B7-H4 and comprises six CDRs of an antibody listed in Tables 1 and 2 (i.e., three VH CDRs of the antibody listed in Table 1 and three VL CDRs of the same antibody listed in Table 2).

[0162] Table 1. VH CDR amino acid sequences 1

[0163]

[0164]

[0165] 1 The VH CDRs in Table 1 are defined according to Kabat.

[0166] Table 2. VL CDR amino acid sequences 2

[0167]

[0168]

[0169] 2 The VL CDRs in Table 2 were determined according to Kabat.

[0170] In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human B7-H4 and comprises a VH of an antibody listed in Table 3.

[0171] Table 3: Variable heavy chain (VH) amino acid sequence

[0172]

[0173]

[0174] In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human B7-H4 and comprises a VL of an antibody listed in Table 4.

[0175] Table 4: Variable light chain (VL) amino acid sequence

[0176]

[0177]

[0178] In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human B7-H4 and comprises the VH and VL of an antibody listed in Tables 3 and 4 (i.e., a VH of an antibody listed in Table 3 and a VL of the same antibody listed in Table 4).

[0179] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein bind to human B7-H4 and comprise the VH framework regions of the antibodies listed in Table 5.

[0180] Table 5. VH FR amino acid sequences 4

[0181]

[0182]

[0183]

[0184] 4 The VH framework regions described in Table 5 are determined based on the Kabat numbering system with respect to the boundaries of the CDRs. In other words, the VH CDRs are determined by Kabat and the framework regions are the amino acid residues surrounding the CDRs in the variable region in the format of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0185] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein bind to human B7-H4 and comprise the VL framework regions of the antibodies listed in Table 6.

[0186] Table 6. VL FR amino acid sequence 3

[0187]

[0188]

[0189]

[0190] 3 The VL framework regions described in Table 6 are determined based on the Kabat numbering system with respect to the boundaries of the CDRs. In other words, the VL CDRs are determined by Kabat and the framework regions are the amino acid residues surrounding the CDRs in the variable region in the form of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0191] In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human B7-H4 and comprises four VH framework regions and four VL framework regions of an antibody listed in Tables 5 and 6 (i.e., four VH framework regions of an antibody listed in Table 5 and four VL framework regions of the same antibody listed in Table 6).

[0192] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein bind to human B7-H4 and comprise the heavy chain sequence of an antibody listed in Table 7.

[0193] Table 7: Full length heavy chain amino acid sequence

[0194]

[0195]

[0196]

[0197]

[0198]

[0199] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein bind to human B7-H4 and comprise the light chain sequence of the antibodies listed in Table 8.

[0200] Table 8: Full length light chain amino acid sequence

[0201]

[0202]

[0203]

[0204] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein bind to human B7-H4 and comprise the heavy chain sequences and light chain sequences of the antibodies listed in Tables 7 and 8 (i.e., the heavy chain sequences of the antibodies listed in Table 7 and the light chain sequences of the same antibodies listed in Table 8).

[0205] In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human B7-H4, comprises six CDRs of an antibody listed in Tables 1 and 2 (i.e., three VH CDRs of an antibody listed in Table 1 and three VL CDRs of the same antibody listed in Table 2) and includes a VH comprising a sequence at least 80% identical to a VH sequence of the same antibody in Table 3 and a VL comprising a sequence at least 80% identical to a VL sequence of the same antibody in Table 4. In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human B7-H4, comprises six CDRs of an antibody listed in Tables 1 and 2 (i.e., three VH CDRs of an antibody listed in Table 1 and three VL CDRs of the same antibody listed in Table 2) and includes a VH comprising a sequence at least 85% identical to a VH sequence of the same antibody in Table 3 and a VL comprising a sequence at least 85% identical to a VL sequence of the same antibody in Table 4.

[0206] In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human B7-H4, comprises six CDRs of an antibody listed in Tables 1 and 2 (i.e., three VH CDRs of an antibody listed in Table 1 and three VL CDRs of the same antibody listed in Table 2) and includes a VH comprising a sequence at least 90% identical to a VH sequence of the same antibody in Table 3 and a VL comprising a sequence at least 90% identical to a VL sequence of the same antibody in Table 4. In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human B7-H4, comprises six CDRs of an antibody listed in Tables 1 and 2 (i.e., three VH CDRs of an antibody listed in Table 1 and three VL CDRs of the same antibody listed in Table 2) and includes a VH comprising a sequence at least 95% identical to a VH sequence of the same antibody in Table 3 and a VL comprising a sequence at least 95% identical to a VL sequence of the same antibody in Table 4.

[0207] In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human B7-H4, comprises six CDRs of an antibody listed in Tables 1 and 2 (i.e., three VH CDRs of an antibody listed in Table 1 and three VL CDRs of the same antibody listed in Table 2) and includes a VH comprising a sequence at least 96% identical to a VH sequence of the same antibody in Table 3 and a VL comprising a sequence at least 96% identical to a VL sequence of the same antibody in Table 4. In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human B7-H4, comprises six CDRs of an antibody listed in Tables 1 and 2 (i.e., three VH CDRs of an antibody listed in Table 1 and three VL CDRs of the same antibody listed in Table 2) and includes a VH comprising a sequence at least 97% identical to a VH sequence of the same antibody in Table 3 and a VL comprising a sequence at least 97% identical to a VL sequence of the same antibody in Table 4. In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human B7-H4, comprises six CDRs of an antibody listed in Tables 1 and 2 (i.e., three VH CDRs of an antibody listed in Table 1 and three VL CDRs of the same antibody listed in Table 2) and includes a VH comprising a sequence at least 98% identical to a VH sequence of the same antibody in Table 3 and a VL comprising a sequence at least 98% identical to a VL sequence of the same antibody in Table 4. In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human B7-H4, comprises six CDRs of an antibody listed in Tables 1 and 2 (i.e., three VH CDRs of an antibody listed in Table 1 and three VL CDRs of the same antibody listed in Table 2) and includes a VH comprising a sequence at least 99% identical to a VH sequence of the same antibody in Table 3 and a VL comprising a sequence at least 99% identical to a VL sequence of the same antibody in Table 4.

[0208] In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human B7-H4, comprises six CDRs of an antibody listed in Tables 1 and 2 (i.e., three VH CDRs of an antibody listed in Table 1 and three VL CDRs of the same antibody listed in Table 2), including a VH comprising a sequence at least 80% identical to a VH sequence of the same antibody in Table 3 and a VL comprising a sequence at least 80% identical to a VL sequence of the same antibody in Table 4, and binds to human, cynomolgus monkey, rat and / or mouse B7-H4. In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human B7-H4, comprises six CDRs of an antibody listed in Tables 1 and 2 (i.e., three VH CDRs of an antibody listed in Table 1 and three VLCDRs of the same antibody listed in Table 2), comprises a VH comprising a sequence at least 85% identical to a VH sequence of the same antibody in Table 3 and a VL comprising a sequence at least 85% identical to a VL sequence of the same antibody in Table 4, and binds to human, cynomolgus monkey, rat and / or mouse B7-H4.

[0209] In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human B7-H4, comprises six CDRs of an antibody listed in Tables 1 and 2 (i.e., three VH CDRs of an antibody listed in Table 1 and three VL CDRs of the same antibody listed in Table 2), including a VH comprising a sequence at least 90% identical to a VH sequence of the same antibody in Table 3 and a VL comprising a sequence at least 90% identical to a VL sequence of the same antibody in Table 4, and binds to human, cynomolgus monkey, rat and / or mouse B7-H4. In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human B7-H4, comprises six CDRs of an antibody listed in Tables 1 and 2 (i.e., three VH CDRs of an antibody listed in Table 1 and three VLCDRs of the same antibody listed in Table 2), comprises a VH comprising a sequence at least 95% identical to a VH sequence of the same antibody in Table 3 and a VL comprising a sequence at least 95% identical to a VL sequence of the same antibody in Table 4, and binds to human, cynomolgus monkey, rat and / or mouse B7-H4.

[0210] In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human B7-H4, comprises six CDRs of an antibody listed in Tables 1 and 2 (i.e., three VH CDRs of an antibody listed in Table 1 and three VL CDRs of the same antibody listed in Table 2), including a VH comprising a sequence at least 96% identical to a VH sequence of the same antibody in Table 3 and a VL comprising a sequence at least 96% identical to a VL sequence of the same antibody in Table 4, and binds to human, cynomolgus monkey, rat and / or mouse B7-H4. In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human B7-H4, comprises six CDRs of an antibody listed in Tables 1 and 2 (i.e., three VH CDRs of an antibody listed in Table 1 and three VLCDRs of the same antibody listed in Table 2), comprises a VH comprising a sequence at least 97% identical to a VH sequence of the same antibody in Table 3 and a VL comprising a sequence at least 97% identical to a VL sequence of the same antibody in Table 4, and binds to human, cynomolgus monkey, rat and / or mouse B7-H4. In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human B7-H4, comprises six CDRs of an antibody listed in Tables 1 and 2 (i.e., three VH CDRs of an antibody listed in Table 1 and three VL CDRs of the same antibody listed in Table 2), including a VH comprising a sequence at least 98% identical to a VH sequence of the same antibody in Table 3 and a VL comprising a sequence at least 98% identical to a VL sequence of the same antibody in Table 4, and binds to human, cynomolgus monkey, rat and / or mouse B7-H4. In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human B7-H4, comprises six CDRs of an antibody listed in Tables 1 and 2 (i.e., three VH CDRs of an antibody listed in Table 1 and three VL CDRs of the same antibody listed in Table 2), including a VH comprising a sequence at least 99% identical to a VH sequence of the same antibody in Table 3 and a VL comprising a sequence at least 99% identical to a VL sequence of the same antibody in Table 4, and binds to human, cynomolgus monkey, rat and / or mouse B7-H4.

[0211] In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human B7-H4, comprises six CDRs of an antibody listed in Tables 1 and 2 (i.e., three VH CDRs of an antibody listed in Table 1 and three VL CDRs of the same antibody listed in Table 2), comprises a VH comprising a sequence at least 80% identical to a VH sequence of the same antibody in Table 3 and a VL comprising a sequence at least 80% identical to a VL sequence of the same antibody in Table 4, increases T cell proliferation, increases IFNγ production and mediates ADCC activity against cells expressing B7-H4. In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human B7-H4, comprises six CDRs of an antibody listed in Tables 1 and 2 (i.e., three VH CDRs of an antibody listed in Table 1 and three VL CDRs of the same antibody listed in Table 2), comprises a VH comprising a sequence at least 85% identical to a VH sequence of the same antibody in Table 3 and a VL comprising a sequence at least 85% identical to a VL sequence of the same antibody in Table 4, increases T cell proliferation, increases IFNγ production and mediates ADCC activity against cells expressing B7-H4.

[0212] In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human B7-H4, comprises six CDRs of an antibody listed in Tables 1 and 2 (i.e., three VH CDRs of an antibody listed in Table 1 and three VL CDRs of the same antibody listed in Table 2), comprises a VH comprising a sequence at least 90% identical to a VH sequence of the same antibody in Table 3 and a VL comprising a sequence at least 90% identical to a VL sequence of the same antibody in Table 4, increases T cell proliferation, increases IFNγ production and / or mediates ADCC activity against cells expressing B7-H4. In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human B7-H4, comprises six CDRs of an antibody listed in Tables 1 and 2 (i.e., three VH CDRs of an antibody listed in Table 1 and three VL CDRs of the same antibody listed in Table 2), comprises a VH comprising a sequence at least 95% identical to a VH sequence of the same antibody in Table 3 and a VL comprising a sequence at least 95% identical to a VL sequence of the same antibody in Table 4, increases T cell proliferation, increases IFNγ production and mediates ADCC activity against cells expressing B7-H4.

[0213] In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human B7-H4, comprises six CDRs of an antibody listed in Tables 1 and 2 (i.e., three VH CDRs of an antibody listed in Table 1 and three VL CDRs of the same antibody listed in Table 2), comprises a VH comprising a sequence at least 96% identical to a VH sequence of the same antibody in Table 3 and a VL comprising a sequence at least 96% identical to a VL sequence of the same antibody in Table 4, increases T cell proliferation, increases IFNγ production and mediates ADCC activity against cells expressing B7-H4. In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human B7-H4, comprises six CDRs of an antibody listed in Tables 1 and 2 (i.e., three VH CDRs of an antibody listed in Table 1 and three VL CDRs of the same antibody listed in Table 2), comprises a VH comprising a sequence at least 97% identical to a VH sequence of the same antibody in Table 3 and a VL comprising a sequence at least 97% identical to a VL sequence of the same antibody in Table 4, increases T cell proliferation, increases IFNγ production and mediates ADCC activity against cells expressing B7-H4. In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human B7-H4, comprises six CDRs of an antibody listed in Tables 1 and 2 (i.e., three VH CDRs of an antibody listed in Table 1 and three VL CDRs of the same antibody listed in Table 2), comprises a VH comprising a sequence at least 98% identical to a VH sequence of the same antibody in Table 3 and a VL comprising a sequence at least 98% identical to a VL sequence of the same antibody in Table 4, increases T cell proliferation, increases IFNγ production and mediates ADCC activity against cells expressing B7-H4. In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human B7-H4, comprises six CDRs of an antibody listed in Tables 1 and 2 (i.e., three VH CDRs of an antibody listed in Table 1 and three VL CDRs of the same antibody listed in Table 2), comprises a VH comprising a sequence at least 99% identical to a VH sequence of the same antibody in Table 3 and a VL comprising a sequence at least 99% identical to a VL sequence of the same antibody in Table 4, increases T cell proliferation, increases IFNγ production and mediates ADCC activity against cells expressing B7-H4.

[0214] In certain aspects, the antibodies or antigen-binding fragments thereof described herein can be described by only their VL domain or only their VH domain, or only their 3 VL CDRs or only their 3 VH CDRs. See, e.g., Rader C et al. (1998) PNAS 95:8910-8915, which is incorporated herein by reference in its entirety, describing the humanization of mouse anti-αvβ3 antibodies by identifying complementary light or heavy chains from human light or heavy chain libraries, respectively, to produce humanized antibody variants with affinity as high as or higher than the affinity of the original antibody. See also Clackson T et al. (1991) Nature 352:624-628, which is incorporated herein by reference in its entirety, describing methods for generating antibodies that bind to specific antigens by using a specific VL domain (or VH domain) and screening the library for the complementary domain. This screen generated 14 new partners for specific VH domains and 13 new partners for specific VL domains that were strong binders as determined by ELISA. See also Kim SJ and Hong HJ, (2007) J Microbiol 45:572-577, which is incorporated herein by reference in its entirety, describing methods for generating antibodies that bind to a specific antigen by using a specific VH domain and screening a library (e.g., a human VL library) against a complementary VL domain; the selected VL domain can in turn be used to guide the selection of additional complementary (e.g., human) VH domains.

[0215] In certain embodiments, the CDRs of an antibody or antigen-binding fragment thereof can be identified according to the Chothia numbering scheme, which refers to the positions of immunoglobulin structural loops (see, e.g., Chothia C and Lesk AM, (1987), J Mol Biol 196:901-917; Al-Lazikani B et al. (1997) J Mol Biol 273:927-948; Chothia C et al. (1992) J Mol Biol 227:799-817; Tramontano A et al. (1990) J Mol Biol 215(1):175-82; and U.S. Pat. No. 7,709,226). Typically, when using the Kabat numbering convention, the Chothia CDR-H1 loop occurs at heavy chain amino acids 26 to 32, 33, or 34, the Chothia CDR-H2 loop occurs at heavy chain amino acids 52 to 56, and the Chothia CDR-H3 loop occurs at heavy chain amino acids 95 to 102, while the Chothia CDR-L1 loop occurs at light chain amino acids 24 to 34, the Chothia CDR-L2 loop occurs at light chain amino acids 50 to 56, and the Chothia CDR-L3 loop occurs at light chain amino acids 89 to 97. When numbered using the Kabat numbering convention, the end of the Chothia CDR-H1 loop varies between H32 and H34, depending on the loop length (this is because the Kabat numbering scheme has an insertion at H35A and H35B; if 35A and 35B are not present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34).

[0216] In certain aspects, provided herein are antibodies and antigen-binding fragments thereof that specifically bind to B7-H4 (e.g., human B7-H4) and comprise the Chothia VH and VL CDRs of the antibodies listed in Tables 3 and 4. In certain embodiments, the antibodies or antigen-binding fragments thereof that specifically bind to B7-H4 (e.g., human B7-H4) comprise one or more CDRs in which the Chothia and Kabat CDRs have the same amino acid sequence. In certain embodiments, provided herein are antibodies and antigen-binding fragments thereof that specifically bind to B7-H4 (e.g., human B7-H4) and comprise a combination of Kabat CDRs and Chothia CDRs.

[0217] In certain aspects, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to the IMGT numbering system as described in Lefranc MP, (1999) The Immunologist 7: 132-136 and Lefranc MP et al. (1999) Nucleic Acids Res 27: 209-212. According to the IMGT numbering scheme, VH-CDR1 is at positions 26 to 35, VH-CDR2 is at positions 51 to 57, VH-CDR3 is at positions 93 to 102, VL-CDR1 is at positions 27 to 32, VL-CDR2 is at positions 50 to 52, and VL-CDR3 is at positions 89 to 97. In a specific embodiment, provided herein are antibodies and antigen-binding fragments thereof that specifically bind to B7-H4 (e.g., human B7-H4) and comprise the IMGT VH and VL CDRs of the antibodies listed in Tables 3 and 4 as described in Lefranc MP (1999), supra, and Lefranc MP et al. (1999), supra.

[0218] In certain aspects, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to MacCallum RM et al., (1996) J Mol Biol 262:732-745. See also, for example, Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domains", Antibody Engineering, Kontermann and Dübel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). In a specific embodiment, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to B7-H4 (e.g., human B7-H4) and comprise the VH and VL CDRs of the antibodies listed in Tables 3 and 4 as determined by the method in MacCallum RM et al.

[0219] In certain embodiments, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to the AbM numbering scheme, which is for AbM hypervariable regions, represents a compromise between Kabat CDRs and Chothia structural loops, and is used in Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.). In a specific embodiment, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to B7-H4 (e.g., human B7-H4) and comprise the VH and VL CDRs of the antibodies listed in Tables 3 and 4 as determined by the AbM numbering scheme.

[0220] In a particular aspect, antibodies comprising heavy chains and light chains are provided herein. With respect to the heavy chain, in a particular embodiment, the heavy chain of the antibody described herein may be an α, δ, ε, γ or μ heavy chain. In another particular embodiment, the heavy chain of the antibody may comprise a human α, δ, ε, γ or μ heavy chain. In a particular embodiment, the antibody described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) comprises a heavy chain, wherein the amino acid sequence of the VH domain comprises the amino acid sequence set forth in Table 3, and wherein the constant region of the heavy chain comprises the amino acid sequence of a human γ heavy chain constant region. In a particular embodiment, the antibody described herein that specifically binds to B7-H4 (e.g., human B7-H4) comprises a heavy chain, wherein the amino acid sequence of the VH domain comprises the sequence set forth in Table 3, and wherein the constant region of the heavy chain comprises the amino acid of a human heavy chain described herein or known in the art. Non-limiting examples of human constant region sequences are described in the art, for example, see U.S. Pat. No. 5,693,780 and Kabat EA et al. (1991), supra.

[0221] With respect to the light chain, in a specific embodiment, the light chain of the antibody described herein is a kappa light chain. The constant region of a human kappa light chain may comprise the following amino acid sequence:

[0222] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 405).

[0223] The constant region of the human kappa light chain can be encoded by the following nucleotide sequence:

[0224] CGGACCGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT(SEQ ID NO:406).

[0225] In another specific embodiment, the light chain of the antibody described herein is a lambda light chain. In yet another specific embodiment, the light chain of the antibody described herein is a human kappa light chain or a human lambda light chain. In a specific embodiment, the antibody described herein that immunospecifically binds to a B7-H4 polypeptide (e.g., human B7-H4) comprises a light chain, wherein the amino acid sequence of the VL domain comprises the sequence set forth in Table 4, and wherein the constant region of the light chain comprises the amino acid sequence of a human kappa light chain constant region. In another specific embodiment, the antibody described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) comprises a light chain, wherein the amino acid sequence of the VL domain comprises the sequence set forth in Table 4, and wherein the constant region of the light chain comprises the amino acid sequence of a human lambda light chain constant region. In a specific embodiment, the antibody described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) comprises a light chain, wherein the amino acid sequence of the VL domain comprises the sequence set forth in Table 4, and wherein the constant region of the light chain comprises the amino acid sequence of a human kappa or lambda light chain constant region. Non-limiting examples of human constant region sequences are described in the art, see, eg, US Pat. No. 5,693,780 and Kabat EA et al. (1991), supra.

[0226] In a specific embodiment, an antibody described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) comprises a VH domain and a VL domain comprising any of the amino acid sequences described herein, and wherein the constant region comprises an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, or an amino acid sequence of a constant region of a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule. In another specific embodiment, an antibody described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) comprises a VH domain and a VL domain comprising any of the amino acid sequences described herein, and wherein the constant region comprises an amino acid sequence of a constant region of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class of immunoglobulin molecules (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or any subclass (e.g., IgG2a and IgG2b). In a specific embodiment, the constant regions comprise the amino acid sequence of a constant region of a human IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule, any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecules.

[0227] The constant region of a human IgG1 heavy chain may comprise the following amino acid sequence:

[0228] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:407).

[0229] Human IgG 1 The constant region of the heavy chain may be encoded by the following nucleotide sequence:

[0230] GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA(SEQ IDNO:408).

[0231] Non-limiting examples of human constant regions are described in the art, see, e.g., Kabat EA et al. (1991), supra.

[0232] In certain embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., numbered according to the Kabat numbering system (e.g., EU index in Kabat), CH2 domain (human IgG 1 residues 231-340 of human IgG) and / or CH3 domain (human IgG 1 341-447 of the antibody) and / or hinge region) to alter one or more functional properties of the antibody or antigen-binding fragment thereof, such as serum half-life, complement fixation, Fc receptor binding and / or antigen-dependent cellular cytotoxicity.

[0233] In certain embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc region (CH1 domain), such that the number of cysteine ​​residues in the hinge region is changed (e.g., increased or decreased), as described, for example, in U.S. Pat. No. 5,677,425. The number of cysteine ​​residues in the hinge region of the CH1 domain can be changed, for example, to facilitate the assembly of the light chain and the heavy chain, or to change (e.g., increase or decrease) the stability of the antibody or its antigen-binding fragment.

[0234] In some embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., CH2 domain (residues 231-340 of human IgG1) and / or CH3 domain (residues 341-447 of human IgG1) and / or hinge region) of an antibody or antigen-binding fragment thereof described herein, for example, according to the Kabat numbering system (e.g., EU index in Kabat) numbering, to increase or decrease the affinity of the antibody or antigen-binding fragment thereof for an Fc receptor on the surface of an effector cell (e.g., an activated Fc receptor). Mutations in the Fc region that decrease or increase affinity for an Fc receptor and techniques for introducing such mutations into an Fc receptor or fragment thereof are known to those of skill in the art. Examples of mutations in Fc receptors that can alter the affinity of an antibody or antigen-binding fragment thereof for an Fc receptor are described, for example, in Smith P et al. (2012) PNAS 109:6181-6186; U.S. Pat. No. 6,737,056, and International Publication Nos. WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631, which are incorporated herein by reference in their entirety.

[0235] In a specific embodiment, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into the IgG constant domain or its FcRn binding fragment (preferably Fc or hinge region-Fc domain fragment) to change (e.g., reduce or increase) the in vivo half-life of the antibody or its antigen-binding fragment. For examples of changing (e.g., reducing or increasing) the in vivo half-life of an antibody or its antigen-binding fragment, see, e.g., International Publication Nos. WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631; and U.S. Pat. Nos. 5,869,046, 6,121,022, 6,277,375, and 6,165,745. In some embodiments, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into the IgG constant domain or its FcRn binding fragment (preferably Fc or hinge region-Fc domain fragment) to reduce the in vivo half-life of the antibody or its antigen-binding fragment. In other embodiments, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into the IgG constant domain or its FcRn binding fragment (preferably Fc or hinge region-Fc domain fragment) to increase the in vivo half-life of the antibody or its antigen-binding fragment. In a specific embodiment, according to the EU index numbering in Kabat (Kabat EA et al. (1991), supra), the antibody or its antigen-binding fragment may have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231-340 of human IgG1) and / or the third constant (CH3) domain (residues 341-447 of human IgG1). In a specific embodiment, the constant region of IgG1 comprises a substitution of methionine (M) to tyrosine (Y) at position 252, a substitution of serine (S) to threonine (T) at position 254, and a substitution of threonine (T) to glutamate (E) at position 256, according to the EU index numbering in Kabat. See U.S. Pat. No. 7,658,921, which is incorporated herein by reference. Such mutant IgGs, termed "YTE mutants," have been shown to have a four-fold increase in half-life compared to the wild-type form of the same antibody (see Dall'Acqua WF et al. (2006) J Biol Chem 281: 23514-24). In certain embodiments, the antibody or antigen-binding fragment thereof comprises an IgG constant domain comprising amino acid substitutions of one, two, three or more amino acid residues at positions 251-257, 285-290, 308-314, 385-389 and 428-436 according to the EU index numbering as in Kabat.

[0236] In another embodiment, one, two or more amino acid substitutions are introduced into the Fc region of the IgG constant domain to change the effector function of the antibody or its antigen-binding fragment. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 according to the EU index numbering of Kabat can be replaced with different amino acid residues to change the affinity of the antibody or its antigen-binding fragment to the effector ligand, but maintain the antigen binding ability of the parent antibody. The effector ligand causing the relevant affinity change can be, for example, an Fc receptor or the C1 component of complement. This method is further described in detail in U.S. Patents Nos. 5,624,821 and 5,648,260. In some embodiments, the deletion or inactivation of the constant region domain (caused by point mutation or other means) can reduce the Fc receptor binding of circulating antibodies or their antigen-binding fragments, thereby enhancing tumor localization. For descriptions of mutations that delete or inactivate constant domains and thereby enhance tumor localization, see, e.g., U.S. Pat. Nos. 5,585,097 and 8,591,886. In certain embodiments, one or more amino acid substitutions may be introduced into the Fc region to remove potential glycosylation sites on the Fc region, thereby reducing Fc receptor binding (see, e.g., Shields RL et al. (2001) J Biol Chem 276: 6591-604).

[0237] In certain embodiments, one or more amino acids selected from amino acid residues 329, 331 and 322 in the constant region numbered according to the EU index of Kabat can be replaced with different amino acid residues, thereby providing the antibody or antigen-binding fragment thereof with altered C1q binding and / or reduced or abolished complement dependent cytotoxicity (CDC). This approach is further described in detail in U.S. Pat. No. 6,194,551 (Idusogie et al.). In some embodiments, one or more amino acid residues within amino acid positions 231 to 238 in the N-terminal region of the CH2 domain are altered to alter the ability of the antibody to fix complement. This approach is further described in detail in International Publication No. WO 94 / 29351. In certain embodiments, the Fc region is modified to increase the ability of the antibody or its antigen-binding fragment to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody or its antigen-binding fragment for an Fcγ receptor by one or more amino acid mutations (e.g., introduction of amino acid substitutions) at the following positions numbered according to the EU index of Kabat: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283 , 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 328, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. This method is further described in detail in International Publication No. WO 00 / 42072.

[0238] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein comprise an IgG1 constant domain having a mutation (e.g., substitution) at position 267, 328, or a combination thereof, according to the EU index numbering of Kabat. In certain embodiments, the antibodies or antigen-binding fragments thereof described herein comprise an IgG1 constant domain having a mutation (e.g., substitution) selected from the group consisting of S267E, L328F, and a combination thereof. In certain embodiments, the antibodies or antigen-binding fragments thereof described herein comprise an IgG1 constant domain having a S267E / L328F mutation (e.g., substitution). In certain embodiments, the antibodies or antigen-binding fragments thereof described herein comprising an IgG1 constant domain having a S267E / L328F mutation (e.g., substitution) have an increased binding affinity to FcγRIIA, FcγRIIB, or FcγRIIA and FcγRIIB.

[0239] It is reported that antibodies with lower fucose content have increased affinity for Fc receptors, such as FcγRIIIA. Therefore, in certain embodiments, the antibodies or antigen-binding fragments thereof described herein have lower fucose content or do not contain fucose (i.e., are "fucosylated"). These antibodies or antigen-binding fragments thereof can be produced using techniques known to those skilled in the art. For example, these antibodies or antigen-binding fragments thereof can be expressed in cells that are defective or lack fucosylation ability. In a specific example, a cell line in which two α1,6-fucosyltransferase alleles are knocked out can be used to produce antibodies or antigen-binding fragments thereof with lower fucose content. The Lonza system is an example of such a system that can be used to produce antibodies and antigen-binding fragments thereof with lower fucose content. Alternatively, antibodies or antigen-binding fragments thereof with lower fucose content or without fucose can be produced, for example, by: (i) culturing cells under conditions that prevent or reduce fucosylation; (ii) removing fucose post-translationally (e.g., using fucosidase); (iii) adding the desired carbohydrate post-translationally, for example, after recombinant expression of non-glycosylated glycoproteins; or (iv) purifying glycoproteins to select for non-fucosylated antibodies or antigen-binding fragments thereof. For methods for producing antibodies without fucose or with lower fucose content, see, for example, Longmore GD and Schachter H (1982) Carbohydr Res 100: 365-92; and Imai-Nishiya H et al., (2007) BMC Biotechnol. 7: 84. See also Example 8 herein, which describes the production of afucosylated B7-H4 antibody.

[0240] In some embodiments, the B7-H4 antibody or antigen-binding fragment thereof has enhanced in vitro ADCC activity compared to a fucosylated B7-H4 antibody or antigen-binding fragment thereof having the same amino acid sequence. In some embodiments, the specific lysis rate caused by the afucosylated B7-H4 antibody or antigen-binding fragment thereof is at least 10%, at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70% or at least 75% higher than the specific lysis rate caused by the fucosylated B7-H4 antibody.

[0241] In some embodiments, the B7-H4 antibody or antigen binding fragment thereof has enhanced affinity for FcγRIIIA compared to a fucosylated B7-H4 antibody or antigen binding fragment thereof having the same amino acid sequence. In some embodiments, the non-fucosylated B7-H4 antibody or antigen binding fragment thereof binds to FcγRIIIA with an affinity at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 7 times, at least 10 times, at least 12 times, at least 15 times, at least 17 times, or at least 20 times higher than the fucosylated B7-H4 antibody or antigen binding fragment thereof. In some embodiments, the affinity for FcγRIIIA is determined using surface plasmon resonance. In some embodiments, FcγRIIIA is selected from FcγRIIIA (V158) and FcγRIIIA (F158). In some embodiments, FcγRIIIA is FcγRIIIA (V158).

[0242] In some embodiments, the presence of fucose can be determined by a method comprising high performance liquid chromatography (HPLC), capillary electrophoresis, or MALDI-TOF mass spectrometry.

[0243] In certain embodiments, the antibody or antigen-binding fragment thereof (i) comprises the CDR sequences of 20502 (e.g., the amino acid sequences of SEQ ID NOs: 458-463), the VH and VL sequences of 20502 (the amino acid sequences of SEQ ID NOs: 464 and 42, respectively), or the heavy and light chain sequences of 20502 (the amino acid sequences of SEQ ID NOs: 469 and 44, respectively), and (ii) is afucosylated.

[0244] In certain embodiments, the composition comprises an antibody or antigen-binding fragment thereof, which (i) comprises a CDR sequence of 20502 (e.g., an amino acid sequence of SEQ ID NOs: 458-463), a VH and VL sequence of 20502 (an amino acid sequence of SEQ ID NOs: 464 and 42, respectively), or a heavy chain and light chain sequence of 20502 (an amino acid sequence of SEQ ID NOs: 469 and 44, respectively), and (ii) is afucosylated, e.g., wherein at least 95% of the antibodies in the composition are afucosylated or wherein fucosylation is not detectable in the composition.

[0245] In specific embodiments, the antibody or antigen-binding fragment thereof (i) comprises the CDR sequence of 20502.1 (e.g., the amino acid sequence of SEQ ID NOs: 35-40), the VH and VL sequences of 20502.1 (the amino acid sequences of SEQ ID NOs: 41 and 42, respectively), or the heavy chain and light chain sequences of 20502.1 (the amino acid sequences of SEQ ID NOs: 43 and 44, respectively), and (ii) is afucosylated.

[0246] In certain embodiments, the composition comprises an antibody or antigen-binding fragment thereof, which (i) comprises a CDR sequence of 20502.1 (e.g., an amino acid sequence of SEQ ID NOs: 35-40), a VH and VL sequence of 20502.1 (an amino acid sequence of SEQ ID NOs: 41 and 42, respectively), or a heavy chain and light chain sequence of 20502.1 (an amino acid sequence of SEQ ID NOs: 43 and 44, respectively), and (ii) is afucosylated, e.g., wherein at least 95% of the antibodies in the composition are afucosylated or wherein fucosylation is not detectable in the composition.

[0247] In specific embodiments, the antibody or antigen-binding fragment thereof (i) comprises the CDR sequences of 22213 (e.g., the amino acid sequences of SEQ ID NOs: 65-70), the VH and VL sequences of 22213 (the amino acid sequences of SEQ ID NOs: 71 and 72, respectively), or the heavy and light chain sequences of 22213 (the amino acid sequences of SEQ ID NOs: 73 and 74, respectively), and (ii) is afucosylated.

[0248] In certain embodiments, the composition comprises an antibody or antigen-binding fragment thereof, which (i) comprises a CDR sequence of 22213 (e.g., an amino acid sequence of SEQ ID NOs: 65-70), a VH and VL sequence of 22213 (an amino acid sequence of SEQ ID NOs: 71 and 72, respectively), or a heavy chain and light chain sequence of 22213 (an amino acid sequence of SEQ ID NOs: 73 and 74, respectively), and (ii) is afucosylated, e.g., wherein at least 95% of the antibodies in the composition are afucosylated or wherein fucosylation is not detectable in the composition.

[0249] Engineered glycoforms can be used for a variety of purposes, including but not limited to enhancing or reducing effector function. Methods for producing engineered glycoforms in the antibodies or antigen-binding fragments thereof described herein include but are not limited to, for example P et al. (1999) Nat Biotechnol 17:176-180; Davies J et al. (2001) Biotechnol Bioeng 74:288-294; Shields RL et al. (2002) J Biol Chem 277:26733-26740; Shinkawa T et al. (2003) J Biol Chem 278:3466-3473; Niwa R et al. (2004) Clin Cancer Res 1:6248-6255; Presta LG et al. (2002) Biochem Soc Trans 30:487-490; Kanda Y et al. (2007) Glycobiology 17:104-118; U.S. Pat. Nos. 6,602,684, 6,946,292 and 7,214,775; U.S. Pat. No. 4,673,674; U.S. Pat. No. 4,663,675; U.S. Pat. No. 4,663,674 Nos. 2007 / 0248600, 2007 / 0178551, 2008 / 0060092 and 2006 / 0253928; the methods disclosed in International Publication Nos. WO 00 / 61739, WO 01 / 292246, WO 02 / 311140 and WO 02 / 30954; Potillegent TM Technology (Biowa, Inc. Princeton, NJ); and Glycosylation engineering technology (Glycart biotechnology AG, Zurich, Switzerland). See also, for example, Ferrara C et al. (2006) Biotechnol Bioeng 93:851-861; International Publication Nos. WO 07 / 039818, WO 12 / 130831, WO 99 / 054342, WO 03 / 011878, and WO 04 / 065540.

[0250] In certain embodiments, any of the constant region mutations or modifications described herein can be introduced into one or both heavy chain constant regions of an antibody or antigen-binding fragment thereof described herein having two heavy chain constant regions.

[0251] In another specific embodiment, an antibody or antigen-binding fragment thereof described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) comprises a heavy chain and a light chain, wherein (i) the heavy chain comprises a VH domain comprising the VH CDR1, VH CDR2, and VH CDR3 amino acid sequences of an antibody listed in Table 1 (e.g., SEQ ID NOs: 458-460, 35-37, or 65-67); (ii) the light chain comprises a VL domain comprising the VL CDR1, VL CDR2, and VL CDR3 amino acid sequences of the same antibody listed in Table 2 (e.g., SEQ ID NOs: 461-463, 38-40, or 68-70); and (iii) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of a constant domain of a human IgG1 heavy chain; and (iv) the light chain further comprises a constant light chain domain comprising the amino acid sequence of a human kappa light chain constant domain.

[0252] In another specific embodiment, an antibody or antigen-binding fragment thereof described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) comprises a heavy chain and a light chain, wherein (i) the heavy chain comprises a VH domain comprising the amino acid sequence of an antibody listed in Table 3 (e.g., SEQ ID NO: 464, 41, or 71); (ii) the light chain comprises a VL domain comprising the amino acid sequence of the same antibody listed in Table 4 (e.g., SEQ ID NO: 42 or 73); and (iii) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of a constant domain of a human IgG1 heavy chain; (iv) the light chain further comprises a constant light chain domain comprising the amino acid sequence of a human kappa light chain constant domain.

[0253] In certain embodiments, antibodies or antigen binding fragments thereof described herein that immunospecifically bind to B7-H4 (e.g., human B7-H4) exhibit T cell checkpoint blocking activity. Exemplary methods for measuring T cell checkpoint blocking activity are provided in Examples 7 and 11 herein. In certain embodiments, antibodies or antigen binding fragments thereof described herein that immunospecifically bind to B7-H4 (e.g., human B7-H4) increase the production of interferon gamma (IFNγ) in T cells. Exemplary methods for measuring IFNγ production are provided in Examples 7 and 11 herein. In certain embodiments, antibodies or antigen binding fragments thereof described herein that immunospecifically bind to B7-H4 (e.g., human B7-H4) increase T cell proliferation. Exemplary methods for measuring T cell proliferation are provided in Example 7 herein. In certain embodiments, antibodies or antigen binding fragments thereof described herein that immunospecifically bind to B7-H4 (e.g., human B7-H4) increase CD4+T cell proliferation. Exemplary methods for measuring CD4+T cell proliferation are provided in Example 7 herein. In certain embodiments, the antibodies or antigen-binding fragments thereof described herein that immunospecifically bind to B7-H4 (eg, human B7-H4) increase CD8+ T cell proliferation. Exemplary methods for measuring CD8+ T cell proliferation are provided in Example 7 herein.

[0254] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein that immunospecifically bind to B7-H4 (e.g., human B7-H4) exhibit antibody-dependent cellular cytotoxicity (ADCC) activity. In certain embodiments, the antibodies or antigen-binding fragments thereof described herein that immunospecifically bind to B7-H4 (e.g., human B7-H4) exhibit antibody-dependent cellular cytotoxicity (ADCC) activity against a cell line (e.g., SK-BR-3 cells) that has at least 300,000 cell surface B7-H4 molecules. In certain embodiments, the antibodies or antigen-binding fragments thereof described herein that immunospecifically bind to B7-H4 (e.g., human B7-H4) exhibit antibody-dependent cellular cytotoxicity (ADCC) activity against a cell line (e.g., HCC1569 cells) that has at least 100,000 cell surface B7-H4 molecules. In certain embodiments, antibodies or antigen-binding fragments thereof described herein that immunospecifically bind to B7-H4 (e.g., human B7-H4) exhibit antibody-dependent cellular cytotoxicity (ADCC) activity against cell lines (e.g., ZR-75-1 cells) with at least 50,000 cell surface B7-H4 molecules. In certain embodiments, antibodies or antigen-binding fragments thereof described herein that immunospecifically bind to B7-H4 (e.g., human B7-H4) exhibit antibody-dependent cellular cytotoxicity (ADCC) activity against cell lines (e.g., MDA-MB-468 cells) with at least 30,000 cell surface B7-H4 molecules. In certain embodiments, antibodies or antigen-binding fragments thereof described herein that immunospecifically bind to B7-H4 (e.g., human B7-H4) exhibit antibody-dependent cellular cytotoxicity (ADCC) activity against cell lines (e.g., HCC1964 cells) with at least 15,000 cell surface B7-H4 molecules. Exemplary methods for measuring ADCC activity are provided in Example 13 herein.

[0255] In specific embodiments, the antibodies or antigen-binding fragments thereof described herein that immunospecifically bind to B7-H4 (e.g., human B7-H4) comprise framework regions (e.g., framework regions of a VH domain and / or a VL domain) that are human framework regions or derived from human framework regions. Non-limiting examples of human framework regions are described in the art, see, e.g., Kabat EA et al. (1991), supra. In certain embodiments, the antibodies or antigen-binding fragments thereof described herein comprise framework regions (e.g., framework regions of a VH domain and / or a VL domain) that are primate (e.g., non-human primate) framework regions or derived from primate (e.g., non-human primate) framework regions.

[0256] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein that specifically bind to B7-H4 (e.g., human B7-H4) comprise one, two or more VH framework regions (FRs) having an amino acid sequence described herein for the antibodies described in Table 5 above (e.g., SEQ ID NOs: 465, 466, 467 and / or 469; SEQ ID NOs: 235, 236, 237 and / or 238; or SEQ ID NOs: 265, 266, 267 and / or 268). In certain embodiments, the antibodies or antigen-binding fragments thereof described herein that specifically bind to B7-H4 (e.g., human B7-H4) comprise one, two or more VL framework regions (FRs) having an amino acid sequence described herein for the antibodies described in Table 6 above (e.g., SEQ ID NOs: 239, 240, 241 and / or 242; or SEQ ID NOs: 269, 270, 271 and / or 272). In certain embodiments, an antibody or antigen-binding fragment thereof described herein that specifically binds to B7-H4 (e.g., human B7-H4) comprises one, two, or more VH framework regions having an amino acid sequence described herein for the antibodies described in Table 5 above, and one, two, or more VL framework regions having an amino acid sequence described herein for the antibodies described in Table 6 above (e.g., (i) SEQ ID NO: 465, 466, 467, and / or 469, and SEQ ID NO: 239, 240, 241, and / or 242; (ii) SEQ ID NO: 235, 236, 237, and / or 238, and SEQ ID NO: 239, 240, 241, and / or 242; or (iii) SEQ ID NO: 265, 266, 267, and / or 268, and SEQ ID NO: 269, 270, 271, and / or 272).

[0257] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein that specifically bind to B7-H4 (e.g., human B7-H4) comprise a VH framework region (FR) having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the VH framework regions described herein above in Table 5. In certain embodiments, the antibodies or antigen-binding fragments thereof described herein that specifically bind to B7-H4 (e.g., human B7-H4) comprise a VL framework region (FR) having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the VL framework regions described herein above in Table 6. In some embodiments, an antibody or antigen-binding fragment thereof described herein that specifically binds to B7-H4 (e.g., human B7-H4) comprises a VH framework region (FR) having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the VH framework region described herein in Table 5 above, and a VL framework region (FR) having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the VL framework region described herein in Table 6 above.

[0258] In certain embodiments, an antibody or antigen-binding fragment thereof described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) comprises a VH domain having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of the VH domain of 20502 or 22213 (e.g., SEQ ID NO: 464 or 71), wherein the antibody comprises a VH CDR that is identical to the VH CDR of 20502 or 22213 (e.g., SEQ ID NO: 458-460 or 65-67).

[0259] In certain embodiments, an antibody or antigen-binding fragment thereof described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) comprises a VL domain having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of the VL domain of 20502 or 22213 (e.g., SEQ ID NO: 42 or 72), wherein the antibody comprises a VL CDR that is identical to the VL CDR of 20502 or 22213 (e.g., SEQ ID NO: 461-463 or 68-70).

[0260] In certain embodiments, an antibody or antigen-binding fragment thereof described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) comprises: (i) a VH domain having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of the VH domain of 20502 or 22213 (e.g., SEQ ID NO: 464 or 71); and (ii) a VL domain having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of the VL domain of 20502 or 22213 (e.g., SEQ ID NO: 42 or 72); wherein the antibody comprises a VH domain that is identical to the VH CDRs and VL CDRs of 20502 or 22213 (e.g., SEQ ID NO: 458-463 or SEQ ID NO: 65-70). CDR and VL CDR.

[0261] In another aspect, provided herein are antibodies or antigen-binding fragments thereof that bind to the same epitope of B7-H4 (e.g., an epitope of human B7-H4) as an antibody or antigen-binding fragment thereof described herein (e.g., 20502 or 22213).

[0262] In certain embodiments, an antibody or antigen-binding fragment thereof described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) comprises a VH domain having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of the VH domain of 20502.1 or 22213 (e.g., SEQ ID NO: 41 or 71), wherein the antibody comprises a VH CDR that is identical to the VH CDR of 20502.1 or 22213 (e.g., SEQ ID NO: 35-37 or 65-67).

[0263] In certain embodiments, an antibody or antigen-binding fragment thereof described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) comprises a VL domain having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of the VL domain of 20502.1 or 22213 (e.g., SEQ ID NO: 42 or 72), wherein the antibody comprises a VL CDR that is identical to the VL CDR of 20502.1 or 22213 (e.g., SEQ ID NO: 38-40 or 68-70).

[0264] In certain embodiments, an antibody or antigen-binding fragment thereof described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) comprises: (i) a VH domain having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of the VH domain of 20502.1 or 22213 (e.g., SEQ ID NO: 41 or 71); and (ii) a VL domain having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of the VL domain of 20502.1 or 22213 (e.g., SEQ ID NO: 42 or 72); wherein the antibody comprises a VH domain that is identical to the VH CDRs and VL CDRs of 20502.1 or 22213 (e.g., SEQ ID NO: 35-40 or SEQ ID NO: 65-70). CDR and VL CDR.

[0265] In another aspect, provided herein are antibodies or antigen-binding fragments thereof that bind to the same epitope of B7-H4 (e.g., an epitope of human B7-H4) as an antibody or antigen-binding fragment thereof described herein (e.g., 20502.1 or 22213).

[0266] A competitive binding assay can be used to determine whether two antibodies bind to overlapping epitopes. Competitive binding can be determined using an assay in which the immunoglobulin to be tested inhibits specific binding of a reference antibody to a common antigen, such as B7-H4. Various types of competitive binding assays are known, for example: direct or indirect solid phase radioimmunoassay (RIA), direct or indirect solid phase enzyme immunoassay (EIA), sandwich competition assay (see Stahli C et al. (1983) Methods Enzymol 9:242-253); direct solid phase biotin-avidin EIA (see Kirkland TN et al. (1986) J Immunol 137:3614-9); direct solid phase labeled assay, direct solid phase labeled sandwich assay (see Harlow E and Lane D, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press); direct solid phase labeled RIA using I-125 labeling (see Morel GA et al. (1988) Mol Immunol 25(1):7-15); direct solid phase biotin-avidin EIA (see Cheung RC et al., (1990) Virology 176:546-52); and directly labeled RIA (Moldenhauer G et al. (1990) Scand J Immunol 32:77-82). Typically, such assays involve the use of purified antigen (e.g., B7-H4, such as human B7-H4) bound to a solid surface or cells with either an unlabeled test immunoglobulin and a labeled reference immunoglobulin. Competitive inhibition can be measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. Typically, the test immunoglobulin is present in excess. Typically, when the competing antibody is present in excess, the competing antibody will inhibit specific binding of the reference antibody to the common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or more. Competitive binding assays can be configured in a number of different formats using labeled antigen or labeled antibodies. In one common format of this assay, the antigen is immobilized on a 96-well plate. Next, the ability of the unlabeled antibody to block the binding of the labeled antibody to the antigen is measured using a radioactive label or an enzyme label.For further details, see, e.g., Wagener C et al. (1983) J Immunol 130:2308-2315; Wagener C et al. (1984) J Immunol Methods 68:269-274; Kuroki M et al. (1990) Cancer Res 50:4872-4879; Kuroki M et al. (1992) Immunol Invest 21:523-538; Kuroki M et al. (1992) Hybridoma 11:391-407; and Antibodies: A Laboratory Manual, Harlow E and Lane D, eds., supra, pp. 386-389.

[0267] In one embodiment, the competition assay is performed using surface plasmon resonance For example, by a 'tandem approach', such as that described in Abdiche YN et al. (2009) Analytical Biochem 386: 172-180, whereby the B7-H4 antigen is immobilized on a chip surface, such as a CM5 sensor chip, and then an anti-B7-H4 antibody is flowed over the chip. To determine whether an antibody or antigen-binding fragment thereof competes with an anti-B7-H4 antibody described herein, the anti-B7-H4 antibody is first flowed over the chip surface to saturation, followed by the addition of a potential competing antibody. The binding of the competing antibody or antigen-binding fragment thereof can then be determined and quantified relative to a non-competing control.

[0268] In one embodiment, competitive inhibition of binding of another B7-H4 antibody or antigen-binding fragment thereof to B7-H4 is determined using Fortebio Octet competitive binding (e.g., as described in Example 2 below).

[0269] In another aspect, provided herein are antibodies that competitively inhibit (e.g., in a dose-dependent manner) the binding of an antibody or antigen-binding fragment thereof described herein (e.g., 20502, 20502.1 or 22213) to B7-H4 (e.g., human B7-H4) as determined using an assay known to one of skill in the art or described herein (e.g., an ELISA competitive assay or a suspension array or a surface plasmon resonance assay).

[0270] In a specific aspect, provided herein is an antibody or antigen-binding fragment thereof that competitively inhibits (e.g., in a dose-dependent manner) the binding of an antibody comprising a VH domain having the amino acid sequence set forth in SEQ ID NO:464 and a VL domain having the amino acid sequence set forth in SEQ ID NO:42 to B7-H4 (e.g., human B7-H4).

[0271] In a specific aspect, provided herein is an antibody or antigen-binding fragment thereof that competitively inhibits (e.g., in a dose-dependent manner) the binding of an antibody comprising a VH domain having the amino acid sequence set forth in SEQ ID NO:41 and a VL domain having the amino acid sequence set forth in SEQ ID NO:42 to B7-H4 (e.g., human B7-H4).

[0272] In a specific aspect, provided herein is an antibody or antigen-binding fragment thereof that competitively inhibits (e.g., in a dose-dependent manner) the binding of an antibody comprising a VH domain having the amino acid sequence set forth in SEQ ID NO:71 and a VL domain having the amino acid sequence set forth in SEQ ID NO:72 to B7-H4 (e.g., human B7-H4).

[0273] In a specific aspect, provided herein is an antibody or antigen-binding fragment thereof that competitively inhibits (e.g., in a dose-dependent manner) the binding of an antibody comprising (i) a VH domain comprising VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequence of the VH CDR listed in Table 1; and (ii) a VL domain comprising VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequence of the CDR listed in Table 2.

[0274] In certain aspects, provided herein is an antibody or antigen-binding fragment thereof that immunospecifically binds to the same B7-H4 (eg, human B7-H4) epitope as 20502, 20502.1, or 22213.

[0275] In another aspect, the antibody or antigen-binding fragment thereof immunospecifically binds to the same B7-H4 (e.g., human B7-H4) epitope as an antibody comprising (i) a VH domain comprising VH CDR1, VHCDR2, and VH CDR3 having the amino acid sequences of the CDRs listed in Table 1; and (ii) a VL domain comprising VL CDR1, VLCDR2, and VL CDR3 having the amino acid sequences of the CDRs listed in Table 2.

[0276] In a specific aspect, the antigen-binding fragments described herein that immunospecifically bind to B7-H4 (e.g., human B7-H4) are selected from the group consisting of Fab, Fab', F(ab')2, and scFv, wherein the Fab, Fab', F(ab')2, or scFv comprises a heavy chain variable region sequence and a light chain variable region sequence of an anti-B7-H4 antibody or antigen-binding fragment thereof as described herein. Fab, Fab', F(ab')2, or scFv can be produced by any technique known to those skilled in the art, including but not limited to the techniques discussed in Section 5.3 below. In certain embodiments, Fab, Fab', F(ab')2, or scFv further comprises a portion that extends the in vivo half-life of the antibody. The portion is also referred to as a "half-life extending portion". Any portion known to those skilled in the art for extending the in vivo half-life of Fab, Fab', F(ab')2, or scFv can be used. For example, the half-life extending portion can include an Fc region, a polymer, albumin, or an albumin binding protein or compound. The polymer may include natural or synthetic, optionally substituted straight or branched chain polyalkylene, polyalkenylene, polyoxyalkylene, polysaccharide, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, methoxypolyethylene glycol, lactose, amylose, dextran, glycogen or derivatives thereof. The substituent may include one or more hydroxyl groups, methyl groups or methoxy groups. In certain embodiments, Fab, Fab', F(ab')2 or scFv may be modified by adding one or more C-terminal amino acids to connect a half-life extension moiety. In certain embodiments, the half-life extension moiety is polyethylene glycol or human serum albumin. In certain embodiments, Fab, Fab', F(ab')2 or scFv is fused to the Fc region.

[0277] Anti-B7-H4 antibodies or antigen-binding fragments thereof can be fused or conjugated (e.g., covalently or non-covalently linked) to a detectable label or substance. Examples of detectable labels or substances include enzyme labels, such as glucose oxidase; radioactive isotopes, such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (121In), and technetium (99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin. These labeled antibodies or antigen-binding fragments thereof can be used to detect B7-H4 (e.g., human B7-H4) proteins. See, e.g., Section 5.5.2 below.

[0278] Antibody production

[0279] Antibodies and antigen-binding fragments thereof that immunospecifically bind to B7-H4 (e.g., human B7-H4) can be produced by any method known in the art for synthesizing antibodies and antigen-binding fragments thereof, such as by chemical synthesis or by recombinant expression techniques. Unless otherwise indicated, the methods described herein employ conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related techniques within the skill of the art. Such techniques are described, for example, in the references cited herein and fully explained in the literature. See, e.g., Sambrook J et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel FM et al. Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annual updates); Current Protocols in Immunology, John Wiley & Sons (1987 and annual updates); Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren B et al. (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

[0280] In one aspect, provided herein is a method for preparing an antibody or antigen binding fragment that immunospecifically binds to B7-H4 (e.g., human B7-H4), the method comprising culturing a cell or host cell as described herein. In one aspect, provided herein is a method for preparing an antibody or antigen binding fragment that immunospecifically binds to B7-H4 (e.g., human B7-H4), the method comprising expressing (e.g., recombinantly expressing) the antibody or antigen binding fragment using a cell or host cell as described herein (e.g., a cell or host cell comprising a polynucleotide encoding the antibody or antigen binding fragment as described herein). In a specific embodiment, the cell is an isolated cell. In a specific embodiment, an exogenous polynucleotide has been introduced into the cell. In a specific embodiment, the method further comprises the step of purifying the antibody or antigen binding fragment obtained from the cell or host cell.

[0281] Methods for producing polyclonal antibodies are known in the art (see, for example, Short Protocols in Molecular Biology, (2002) 5th edition, Ausubel FM et al., eds., John Wiley and Sons, New York, Chapter 11).

[0282] Monoclonal antibodies or their antigen-binding fragments can be prepared using a variety of techniques known in the art, including the use of hybridomas, recombinant techniques and phage display techniques, yeast-based display techniques or combinations thereof. For example, monoclonal antibodies or their antigen-binding fragments can be produced using hybridoma technology, including those known in the art and, for example, Harlow E and Lane D, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd edition, 1988); Hammerling GJ et al., Monoclonal Antibodies and T-Cell Hybridomas 563681 (Elsevier, NY, 1981), or as Kohler G and Milstein C (1975) Nature 256: Methods described in 495. Examples of yeast-based display methods that can be used to select and produce the antibodies described herein include, for example, the methods taught in WO2009 / 036379A2, WO2010 / 105256, and WO2012 / 009568, each of which is incorporated herein by reference in its entirety.

[0283] In certain embodiments, the monoclonal antibody or antigen binding fragment is an antibody or antigen binding fragment produced by a clonal cell (e.g., a hybridoma or host cell that produces a recombinant antibody or antigen binding fragment), wherein the antibody or antigen binding fragment immunospecifically binds to B7-H4 (e.g., human B7-H4) as determined, for example, by ELISA or other antigen binding assays known in the art, or in the examples provided herein. In certain embodiments, the monoclonal antibody or antigen binding fragment thereof may be a chimeric or humanized antibody or antigen binding fragment thereof. In certain embodiments, the monoclonal antibody or antigen binding fragment thereof may be a Fab fragment or a F(ab')2 fragment. The monoclonal antibodies or antigen binding fragments thereof described herein may be prepared, for example, by the hybridoma method as described in Kohler G and Milstein C (1975) Nature 256:495, or may be isolated from a phage library, for example, using techniques such as described herein. Other methods for preparing clonal cell lines and monoclonal antibodies and antigen-binding fragments thereof expressed by such cell lines are well known in the art (see, e.g., Short Protocols in Molecular Biology, (2002) 5th ed., Chapter 11, Ausubel FM et al., supra).

[0284] Antigen-binding fragments of the antibodies described herein can be produced by any technique known to those skilled in the art. For example, Fab and F(ab')2 fragments described herein can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). Fab corresponds to one of the two identical arms of a tetrameric antibody molecule and contains a complete light chain paired with a heavy chain VH and CH1 domain. F(ab')2 fragments contain two antigen-binding arms of a tetrameric antibody molecule connected by a disulfide bond in the hinge region.

[0285] In addition, the antibodies or antigen-binding fragments thereof described herein can also be produced using various phage display and / or yeast-based display methods known in the art. In phage display methods, proteins are displayed on the surface of phage particles carrying polynucleotides encoding the proteins. Specifically, DNA sequences encoding VH and VL domains are amplified from animal cDNA libraries (e.g., human or murine cDNA libraries of affected tissues). DNA encoding VH and VL domains are recombined together with scFv linkers by PCR and cloned into phagemid vectors. The vector is placed in E. coli by electroporation and E. coli is infected with helper phage. Phages used in these methods are typically filamentous phages, including fd and M13, and VH and VL domains are usually recombinantly fused with phage gene III or gene VIII. Phages expressing antibodies or antigen-binding fragments thereof that bind to specific antigens can be selected or identified using antigens, such as labeled antigens or antigens bound to or captured on solid surfaces or beads. Examples of phage display methods that can be used to prepare the antibodies or fragments described herein include those disclosed in Brinkman U et al. (1995) J Immunol Methods 182:41-50; Ames RS et al. (1995) J Immunol Methods 184:177-186; Kettleborough CA et al. (1994) Eur J Immunol 24:952-958; Persic L et al. (1997) Gene 187:9-18; Burton DR and Barbas CF (1994) Advan Immunol 57:191-280; PCT Application No. PCT / GB91 / 001134; International Publication Nos. WO 90 / 02809, WO 91 / 10737, WO 92 / 01047, WO 92 / 18619, WO 93 / 11236, WO 95 / 15982, WO 95 / 20401 and WO 97 / 13844; and U.S. Patents Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and 5,969,108.

[0286] The humanized antibody or antigen-binding fragment thereof can be selected from any immunoglobulin class, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgGl, IgG2, IgG3 and IgG4.

[0287] 1.2.1 Polynucleotides

[0288] In certain aspects, provided herein are polynucleotides comprising nucleotide sequences encoding antibodies or antigen-binding fragments thereof described herein that immunospecifically bind to B7-H4 (e.g., human B7-H4), or domains thereof (e.g., variable light chain regions and / or variable heavy chain regions); and vectors, e.g., vectors comprising these polynucleotides for recombinant expression in host cells (e.g., E. coli and mammalian cells).

[0289] In certain aspects, provided herein are polynucleotides comprising nucleotide sequences encoding antibodies or antigen-binding fragments thereof that immunospecifically bind to a B7-H4 polypeptide (e.g., human B7-H4) and comprise an amino acid sequence as described herein, as well as antibodies or antigen-binding fragments that compete with these antibodies or antigen-binding fragments for binding to a B7-H4 polypeptide (e.g., in a dose-dependent manner) or bind to the same epitope as these antibodies or antigen-binding fragments.

[0290] Also provided herein is a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising a sequence selected from the group consisting of: SEQ ID NO: 11, 12, 21, 22, 31, 32, 41, 464, 42, 51, 52, 61, 62, 71, 72, 81, 82, 91, 92, 101, 102, 111, 112, 121, 122, 131, 132, 141, 142, 151, 152, 161, 162, 171, 172, 181, 182, 191, 192, 201, and 202. In some embodiments, an antibody or antigen-binding fragment thereof comprising the polypeptide immunospecifically binds to B7-H4.

[0291] Also provided herein is a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 13, 14, 23, 24, 33, 34, 43, 469, 44, 53, 54, 63, 64, 73, 74, 83, 84, 93, 94, 103, 104, 113, 114, 123, 124, 133, 134, 143, 144, 153, 154, 163, 164, 173, 174, 183, 184, 193, 194, 203, and 204. In some embodiments, an antibody or antigen-binding fragment thereof comprising the polypeptide immunospecifically binds to B7-H4.

[0292] Also provided herein are polynucleotides comprising a nucleotide sequence encoding a variable heavy chain as shown in Table 9, e.g., an antibody or antigen-binding fragment thereof comprising the encoded variable heavy chain that binds to B7-H4.

[0293] Table 9: Polynucleotide sequences encoding variable heavy chains

[0294]

[0295]

[0296]

[0297]

[0298] Also provided herein are polynucleotides comprising a variable light chain encoding nucleotide sequence shown in Table 10, for example, an antibody or antigen-binding fragment thereof comprising the encoded variable light chain that binds to B7-H4.

[0299] Table 10: Polynucleotide sequences encoding variable light chains

[0300]

[0301]

[0302]

[0303]

[0304] Also provided herein are polynucleotides comprising the nucleotide sequence of SEQ ID NO: 213, 214, 223, 224, 233, 234, 243, 470, 244, 253, 254, 263, 264, 273, 274, 283, 284, 293, 294, 303, 304, 313, 314, 323, 324, 333, 334, 343, 344, 353, 354, 363, 364, 373, 374, 383, 384, 393, 394, 403, or 404. Also provided herein are polynucleotides comprising (i) the nucleotide sequence of SEQ ID NO: 213, 214, 223, 224, 233, 234, 243, 470, 244, 253, 254, 263, 264, 273, 274, 283, 284, 293, 294, 303, 304, 313, 314, 323, 324, 333, 334, 343, 344, 353, 354, 363, 364, 373, 374, 383, 384, 393, 394, 403 or 404; and (ii) the nucleotide sequence of SEQ ID NO: 408 or 406.

[0305] Also provided herein are polynucleotides comprising a polypeptide encoding SEQ ID NO: 11, 12, 21, 22, 31, 32, 41, 464, 42, 51, 52, 61, 62, 71, 72, 81, 82, 91, 92, 101, 102, 111, 112, 121, 122, 131, 132, 141, 142, 151, 152, 161, 162, 171, 172, 181, 182, 191, 192, 201 or 202, and respectively covalently linked to SEQ ID NO: NO:213, 214, 223, 224, 233, 234, 243, 470, 244, 253, 254, 263, 264, 273, 274, 283, 284, 293, 294, 303, 304, 313, 314, 323, 324, 333, 334, 343, 344, 353, 354, 363, 364, 373, 374, 383, 384, 393, 394, 403 or 404 has at least about 80%, 85% or 90% identical nucleotide sequences.

[0306] Also provided herein are polynucleotides comprising a polypeptide encoding SEQ ID NO: 11, 12, 21, 22, 31, 32, 41, 464, 42, 51, 52, 61, 62, 71, 72, 81, 82, 91, 92, 101, 102, 111, 112, 121, 122, 131, 132, 141, 142, 151, 152, 161, 162, 171, 172, 181, 182, 191, 192, 201 or 202, and respectively covalently linked to SEQ ID NO: NO: 213, 214, 223, 224, 233, 234, 243, 470, 244, 253, 254, 263, 264, 273, 274, 283, 284, 293, 294, 303, 304, 313, 314, 323, 324, 333, 334, 343, 344, 353, 354, 363, 364, 373, 374, 383, 384, 393, 394, 403 or 404 has at least about 95% identical nucleotide sequences.

[0307] Also provided herein are polynucleotides comprising a polypeptide encoding SEQ ID NO: 11, 12, 21, 22, 31, 32, 41, 464, 42, 51, 52, 61, 62, 71, 72, 81, 82, 91, 92, 101, 102, 111, 112, 121, 122, 131, 132, 141, 142, 151, 152, 161, 162, 171, 172, 181, 182, 191, 192, 201 or 202, and respectively covalently linked to SEQ ID NO: NO: 213, 214, 223, 224, 233, 234, 243, 470, 244, 253, 254, 263, 264, 273, 274, 283, 284, 293, 294, 303, 304, 313, 314, 323, 324, 333, 334, 343, 344, 353, 354, 363, 364, 373, 374, 383, 384, 393, 394, 403 or 404 has at least about 96% identical nucleotide sequences.

[0308] Also provided herein are polynucleotides comprising a polypeptide encoding SEQ ID NO: 11, 12, 21, 22, 31, 32, 41, 464, 42, 51, 52, 61, 62, 71, 72, 81, 82, 91, 92, 101, 102, 111, 112, 121, 122, 131, 132, 141, 142, 151, 152, 161, 162, 171, 172, 181, 182, 191, 192, 201 or 202, and respectively covalently linked to SEQ ID NO: NO: 213, 214, 223, 224, 233, 234, 243, 470, 244, 253, 254, 263, 264, 273, 274, 283, 284, 293, 294, 303, 304, 313, 314, 323, 324, 333, 334, 343, 344, 353, 354, 363, 364, 373, 374, 383, 384, 393, 394, 403 or 404 has at least about 97% identical nucleotide sequences.

[0309] Also provided herein are polynucleotides comprising a polypeptide encoding SEQ ID NO: 11, 12, 21, 22, 31, 32, 41, 464, 42, 51, 52, 61, 62, 71, 72, 81, 82, 91, 92, 101, 102, 111, 112, 121, 122, 131, 132, 141, 142, 151, 152, 161, 162, 171, 172, 181, 182, 191, 192, 201 or 202, and respectively covalently linked to SEQ ID NO: NO: 213, 214, 223, 224, 233, 234, 243, 470, 244, 253, 254, 263, 264, 273, 274, 283, 284, 293, 294, 303, 304, 313, 314, 323, 324, 333, 334, 343, 344, 353, 354, 363, 364, 373, 374, 383, 384, 393, 394, 403 or 404 has at least about 98% identical nucleotide sequences.

[0310] Also provided herein are polynucleotides comprising a polypeptide encoding SEQ ID NO: 11, 12, 21, 22, 31, 32, 41, 464, 42, 51, 52, 61, 62, 71, 72, 81, 82, 91, 92, 101, 102, 111, 112, 121, 122, 131, 132, 141, 142, 151, 152, 161, 162, 171, 172, 181, 182, 191, 192, 201 or 202, and respectively covalently linked to SEQ ID NO: NO: 213, 214, 223, 224, 233, 234, 243, 470, 244, 253, 254, 263, 264, 273, 274, 283, 284, 293, 294, 303, 304, 313, 314, 323, 324, 333, 334, 343, 344, 353, 354, 363, 364, 373, 374, 383, 384, 393, 394, 403 or 404 has at least about 99% identical nucleotide sequences.

[0311] In certain aspects, polynucleotides are provided herein, comprising nucleotide sequences encoding light or heavy chains of antibodies or antigen-binding fragments described herein. The polynucleotides may comprise nucleotide sequences encoding heavy chains containing VH FRs and CDRs (see, e.g., Tables 1 and 5) of antibodies described herein. The polynucleotides may comprise nucleotide sequences encoding light chains containing VL FRs and CDRs (see, e.g., Tables 2 and 6) of antibodies described herein.

[0312] In specific embodiments, the polynucleotides described herein encode a VH domain comprising the amino acid sequence set forth in SEQ ID NO: 464. In specific embodiments, the polynucleotides described herein encode a VL domain comprising the amino acid sequence set forth in SEQ ID NO: 42.

[0313] In specific embodiments, the polynucleotides described herein encode a VH domain comprising the amino acid sequence set forth in SEQ ID NO: 41. In specific embodiments, the polynucleotides described herein encode a VL domain comprising the amino acid sequence set forth in SEQ ID NO: 42.

[0314] In specific embodiments, the polynucleotides described herein encode a VH domain comprising the amino acid sequence set forth in SEQ ID NO: 71. In specific embodiments, the polynucleotides described herein encode a VL domain comprising the amino acid sequence set forth in SEQ ID NO: 72.

[0315] In a specific embodiment, a polynucleotide is provided herein, comprising a nucleotide sequence encoding an anti-B7-H4 antibody comprising three VH CDRs, such as VH CDR1, VH CDR2, and VH CDR3 of any antibody described herein (see, e.g., Table 1). In a specific embodiment, a polynucleotide is provided herein, comprising three VL chain CDRs, such as VL CDR1, VL CDR2, and VL CDR3 of any antibody described herein (see, e.g., Table 2). In a specific embodiment, a polynucleotide is provided herein, comprising a nucleotide sequence encoding an anti-B7-H4 antibody comprising three VH CDRs, such as VH CDR1, VH CDR2, and VH CDR3 of any antibody described herein (see, e.g., Table 1) and three VL chain CDRs, such as VL CDR1, VL CDR2, and VL CDR3 of any antibody described herein (see, e.g., Table 2).

[0316] In certain embodiments, polynucleotides are provided herein, comprising a nucleotide sequence encoding an anti-B7-H4 antibody, or antigen-binding fragment thereof, or fragment thereof, comprising a VH domain, e.g., comprising FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, comprising an amino acid sequence described herein (e.g., see Tables 1 and 5, e.g., VH CDRs and VH FRs of specific antibodies identified by name in these tables). In certain embodiments, polynucleotides are provided herein, comprising a nucleotide sequence encoding an anti-B7-H4 antibody, or antigen-binding fragment thereof, or fragment thereof, comprising a VL domain, e.g., comprising FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, comprising an amino acid sequence described herein (e.g., see Tables 2 and 6, e.g., VL CDRs and VL FRs of specific antibodies identified by name in these tables).

[0317] In certain embodiments, the polynucleotides described herein comprise a nucleotide sequence encoding a heavy chain variable region, the heavy chain variable region comprising an amino acid sequence described herein (e.g., SEQ ID NO: 464, 41, or 71), wherein an antibody comprising the heavy chain variable region immunospecifically binds to B7-H4 (e.g., human B7-H4). In a certain embodiment, the polynucleotides described herein comprise a heavy chain variable region encoding sequence provided herein (e.g., a variable region encoding portion of SEQ ID NO: 470, 243, or 273), wherein an antibody comprising the heavy chain variable region immunospecifically binds to B7-H4 (e.g., human B7-H4).

[0318] In certain embodiments, the polynucleotides described herein comprise a nucleotide sequence encoding a light chain variable region, the light chain variable region comprising an amino acid sequence described herein (e.g., SEQ ID NO: 42 or 72), wherein an antibody comprising the light chain variable region immunospecifically binds to B7-H4 (e.g., human B7-H4). In a certain embodiment, the polynucleotides described herein comprise a light chain variable region encoding sequence provided herein (e.g., a variable region encoding portion of SEQ ID NO: 244 or 274), wherein an antibody comprising the light chain variable region immunospecifically binds to B7-H4 (e.g., human B7-H4).

[0319] In a specific embodiment, the polynucleotides or polynucleotide combinations provided herein comprise a nucleotide sequence or a combination of nucleotide sequences encoding an antibody or antigen-binding fragment thereof that immunospecifically binds to B7-H4 (e.g., human B7-H4), wherein the antibody or antigen-binding fragment thereof comprises a heavy chain, wherein the heavy chain comprises a heavy chain variable domain comprising an amino acid sequence described in Table 3 (e.g., SEQ ID NO: 464, 41 or 71) and a constant region comprising the amino acid sequence of a human gamma heavy chain constant region.

[0320] In a specific embodiment, the polynucleotides or polynucleotide combinations provided herein comprise a nucleotide sequence or a combination of nucleotide sequences encoding an antibody or antigen-binding fragment thereof that immunospecifically binds to B7-H4 (e.g., human B7-H4), wherein the antibody or antigen-binding fragment thereof comprises a light chain, wherein the light chain comprises a light chain variable domain comprising an amino acid sequence described in Table 4 (e.g., SEQ ID NO: 42 or 72) and a constant region comprising the amino acid sequence of a human lambda light chain constant region.

[0321] In a specific embodiment, the polynucleotides or polynucleotide combinations provided herein comprise a nucleotide sequence or a combination of nucleotide sequences encoding an antibody or antigen-binding fragment thereof that immunospecifically binds to B7-H4 (e.g., human B7-H4), wherein the antibody or antigen-binding fragment thereof comprises (i) a heavy chain, wherein the heavy chain comprises a heavy chain variable domain comprising an amino acid sequence described in Table 3 (e.g., SEQ ID NO: 464, 41 or 71) and a constant region comprising the amino acid sequence of a human gamma heavy chain constant region; and (ii) a light chain, wherein the light chain comprises a light chain variable domain comprising an amino acid sequence described in Table 4 (e.g., SEQ ID NO: 42 or 72) and a constant region comprising the amino acid sequence of a human lambda light chain constant region.

[0322] In one embodiment, the polynucleotide combination provided herein includes a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 470 and a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 244.

[0323] In one embodiment, the polynucleotide combination provided herein includes a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 243 and a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 244.

[0324] In one embodiment, the polynucleotide combination provided herein includes a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 273 and a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 274.

[0325] In a specific embodiment, provided herein are polynucleotides comprising a nucleotide sequence encoding an anti-B7-H4 antibody or antigen-binding fragment thereof or domain thereof named herein, see, e.g., Tables 1-8.

[0326] Also provided herein are polynucleotides encoding the anti-B7-H4 antibodies or antigen-binding fragments thereof or domains thereof described herein, which are optimized, for example, by codon / RNA optimization, replacement with heterologous signal sequences, and elimination of mRNA instability elements. Methods for generating optimized nucleic acids encoding anti-B7-H4 antibodies or antigen-binding fragments thereof or domains thereof (e.g., heavy chains, light chains, VH domains, or VL domains) for recombinant expression by introducing codon changes (e.g., codon changes encoding the same amino acid due to the degeneracy of the genetic code) and / or removing inhibitory regions in mRNA can be performed by using, for example, the optimization methods described in U.S. Pat. Nos. 5,965,726, 6,174,666, 6,291,664, 6,414,132, and 6,794,498.

[0327] Polynucleotides encoding antibodies or antigen-binding fragments thereof described herein or their domains can be produced from nucleic acids from a suitable source (e.g., a hybridoma) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, genomic DNA obtained from hybridoma cells producing the antibody of interest can be used to perform PCR amplification using synthetic primers that hybridize to the 3' and 5' ends of known sequences. Such PCR amplification methods can be used to obtain nucleic acids comprising sequences encoding light chains and / or heavy chains of antibodies or their antigen-binding fragments. Such PCR amplification methods can be used to obtain nucleic acids comprising sequences encoding variable light chain regions and / or variable heavy chain regions of antibodies or their antigen-binding fragments. The amplified nucleic acids can be cloned into vectors for expression in host cells and used for further cloning, for example, to produce chimeric and humanized antibodies or their antigen-binding fragments.

[0328] The polynucleotides provided herein may be, for example, in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA, and the DNA may be double-stranded or single-stranded. If single-stranded, the DNA may be a coding strand or a non-coding (antisense) strand. In certain embodiments, the polynucleotide is a cDNA or a DNA lacking one or more endogenous introns. In certain embodiments, the polynucleotide is a non-naturally occurring polynucleotide. In certain embodiments, the polynucleotide is recombinantly produced. In certain embodiments, the polynucleotide is isolated. In certain embodiments, the polynucleotide is substantially pure. In certain embodiments, the polynucleotide is purified from natural components.

[0329] 1.2.2 Cells and vectors

[0330] In certain aspects, provided herein are vectors (e.g., expression vectors) comprising polynucleotides for recombinant expression in host cells, preferably mammalian cells, comprising nucleotide sequences encoding anti-B7-H4 antibodies and antigen-binding fragments thereof, or domains thereof. Also provided herein are cells, such as host cells, comprising such vectors for recombinant expression of anti-B7-H4 antibodies or antigen-binding fragments thereof (e.g., human or humanized antibodies or antigen-binding fragments thereof) described herein. In a specific aspect, provided herein are methods for producing antibodies or antigen-binding fragments thereof described herein, comprising expressing such antibodies or antigen-binding fragments thereof in host cells.

[0331] In certain embodiments, the recombinant expression of an antibody or antigen-binding fragment thereof or a domain thereof (e.g., a heavy chain or light chain described herein) that specifically binds to B7-H4 (e.g., human B7-H4) as described herein involves constructing an expression vector containing a polynucleotide encoding the antibody or antigen-binding fragment thereof or a domain thereof. After obtaining a polynucleotide encoding an antibody or antigen-binding fragment thereof or a domain thereof (e.g., a heavy chain or light chain variable domain) as described herein, a vector for producing the antibody or antigen-binding fragment thereof can be manufactured by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing a nucleotide sequence encoding an antibody or antigen-binding fragment thereof or a domain thereof (e.g., a light chain or heavy chain) are described herein. Expression vectors containing antibody or antigen-binding fragment thereof or a domain thereof (e.g., a light chain or heavy chain) coding sequence and appropriate transcriptional and translational control signals can be constructed using methods well known to those skilled in the art. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Replicable vectors are also provided, the value vectors comprising a nucleotide sequence of an antibody or its Fab as described herein, a heavy chain or light chain, a heavy chain or light chain variable domain, or a heavy chain or light chain CDR operably connected to a promoter. The vector may, for example, include a nucleotide sequence encoding a constant region of an antibody or its Fab (see, for example, International Publication No. WO 86 / 05807 and No. WO 89 / 01036; and U.S. Patent No. 5,122,464), and the variable domains of the antibody or its Fab may be cloned into such vectors to express a complete heavy chain, a complete light chain, or both a complete heavy chain and a light chain.

[0332] The expression vector can be transferred into cells (e.g., host cells) by conventional techniques, and the resulting cells can then be cultured by conventional techniques to produce the antibodies or antigen-binding fragments thereof described herein (e.g., antibodies or antigen-binding fragments thereof comprising the six CDRs, VH, VL, VH and VL, heavy chains, light chains, or heavy chains and light chains of 20502, 20502.1 or 22213) or domains thereof (e.g., VH, VL, VH and VL, heavy chains, or light chains of 20502, 20502.1 or 22213). Thus, provided herein are host cells containing polynucleotides encoding antibodies or antigen-binding fragments thereof described herein (e.g., antibodies or antigen-binding fragments thereof comprising six CDRs, VH, VL, VH and VL, heavy chains, light chains, or heavy and light chains of 20502, 20502.1 or 22213) or domains thereof (e.g., VH, VL, VH and VL, heavy chains, or light chains of 20502, 20502.1 or 22213), which are operably linked to promoters that allow such sequences to be expressed in host cells. In certain embodiments, for expression of double-chain antibodies or antigen-binding fragments thereof, vectors encoding heavy and light chains independently can be co-expressed in host cells to express complete immunoglobulin molecules, as described in detail below. In certain embodiments, the host cell contains a vector comprising a polynucleotide encoding the heavy and light chains of an antibody described herein (e.g., the heavy and light chains of 20502, 20502.1, or 22213) or a domain thereof (e.g., VH and VL of 20502, 20502.1, or 22213). In a specific embodiment, the host cell contains two different vectors, the first vector comprising a polynucleotide encoding the heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof described herein, and the second vector comprising a polynucleotide encoding the light chain or light chain variable region of an antibody described herein (e.g., an antibody comprising the six CDRs of 20502, 20502.1, or 22213), or a domain thereof. In other embodiments, the first host cell comprises a first vector comprising a polynucleotide encoding a heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof described herein; and the second host cell comprises a second vector comprising a polynucleotide encoding a light chain or light chain variable region of an antibody or antigen-binding fragment thereof described herein (e.g., an antibody or antigen-binding fragment thereof comprising the six CDRs of 20502, 20502.1, or 22213). In specific embodiments, the heavy chain / light chain variable region expressed by the first cell associates with the light chain / heavy chain variable region of the second cell to form an anti-B7-H4 antibody or antigen-binding fragment thereof described herein (e.g., an antibody or antigen-binding fragment thereof comprising the six CDRs of 20502, 20502.1, or 22213). In certain embodiments, a host cell population comprising the first host cell and the second host cell is provided herein.

[0333] In a specific embodiment, provided herein is a set of vectors, comprising: a first vector comprising a polynucleotide encoding a light chain / light chain variable region of an anti-B7-H4 antibody or antigen-binding fragment thereof described herein; and a second vector comprising a polynucleotide encoding a heavy chain / heavy chain variable region of an anti-B7-H4 antibody or antigen-binding fragment thereof described herein (e.g., an antibody or antigen-binding fragment thereof comprising the CDRs of 20502, 20502.1 or 22213). Alternatively, a single vector may be used that encodes and is capable of expressing both heavy and light chain polypeptides.

[0334] A variety of host-expression vector systems can be used to express the antibodies or antigen-binding fragments thereof described herein (e.g., antibodies or antigen-binding fragments thereof comprising the CDRs of 20502, 20502.1 or 22213) (see, e.g., U.S. Pat. No. 5,807,715). The host-expression system represents a vehicle with which the coding sequence of interest can be produced and subsequently purified, and also represents cells that can express the antibodies or antigen-binding fragments thereof described herein in situ when transformed or transfected with the appropriate nucleotide coding sequences. These expression systems include, but are not limited to, microorganisms such as bacteria (e.g., Escherichia coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems (e.g., green algae, such as Chlamydomonas reinhardtii) infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK) with recombinant expression constructs containing promoters derived from mammalian cell genomes (e.g., metallothionein promoters) or mammalian viruses (e.g., adenovirus late promoters; vaccinia virus 7.5K promoters). In a specific embodiment, the cells used to express the antibodies and antigen-binding fragments thereof described herein (e.g., antibodies or antigen-binding fragments thereof comprising the CDRs of 20502, 20502.1 or 22213) are CHO cells, such as those from the CHO GS System TM In a specific embodiment, the cells used to express the antibodies described herein are human cells, such as human cell lines. In a specific embodiment, the mammalian expression vector is pOptiVEC TMOr pcDNA3.3. In a specific embodiment, bacterial cells, such as Escherichia coli, or eukaryotic cells (e.g., mammalian cells) are used to express recombinant antibody molecules, especially for expressing fully recombinant antibody molecules. For example, mammalian cells such as Chinese hamster ovary (CHO) cells combined with vectors such as major immediate early gene promoter elements from human cytomegalovirus are effective antibody expression systems (Foecking MK and Hofstetter H (1986) Gene 45: 101-105; and Cockett MI et al. (1990) Biotechnology 8: 662-667). In certain embodiments, the antibody or its antigen-binding fragment described herein is produced by CHO cells or NS0 cells.

[0335] In addition, host cell lines that regulate the expression of inserted sequences or that alter and process gene products in a desired specific manner may be selected. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may contribute to protein function. To this end, eukaryotic host cells with cellular machinery for appropriate processing of primary transcripts, glycosylation, and gene product phosphorylation may be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL703O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10, and HsS78Bst cells. In certain embodiments, the anti-B7-H4 antibodies described herein (e.g., antibodies comprising the CDRs of 20502, 20502.1 or 22213, or antigen-binding fragments thereof) are produced in mammalian cells, such as CHO cells.

[0336] In certain embodiments, the anti-B7-H4 antibodies described herein (e.g., antibodies or antigen-binding fragments thereof comprising the CDRs of 20502, 20502.1 or 22213) are CHOK1 was produced in SV cells.

[0337] In some embodiments, a host cell is provided, the host cell comprising a nucleic acid encoding an anti-B7-H4 antibody or antigen-binding fragment thereof described herein, wherein the host cell lacks a functional α-1,6-fucosyltransferase gene (FUT8). In some embodiments, the host cell is a CHO cell.

[0338] Once the antibodies or antigen-binding fragments thereof described herein are produced by recombinant expression, they can be purified by any method known in the art for purifying immunoglobulin molecules, such as by chromatography (e.g., ion exchange chromatography, affinity chromatography, particularly affinity chromatography for specific antigens after protein A, and size exclusion chromatography), centrifugation, differential solubility, or by any other standard technique for purifying proteins. In addition, the antibodies or antigen-binding fragments thereof described herein can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

[0339] In specific embodiments, the antibodies or antigen-binding fragments thereof described herein are isolated or purified. Generally speaking, an isolated antibody or antigen-binding fragment thereof is substantially free of other antibodies or antigen-binding fragments thereof having antigenic specificity different from that of the isolated antibody or antigen-binding fragment thereof. For example, in a specific embodiment, the antibody or antigen-binding fragment preparations described herein are substantially free of cellular material and / or chemical precursors.

[0340] 1.3 Pharmaceutical Composition

[0341] Provided herein are compositions comprising an antibody or antigen-binding fragment thereof described herein of desired purity in a physiologically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed.

[0342] In various embodiments, the composition comprising the anti-B7-H4 antibody or antigen-binding fragment thereof is provided in the form of a formulation with a pharmaceutically acceptable carrier (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th Edition (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Edition, Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd Edition, Pharmaceutical Press (2000)).

[0343] The pharmaceutical compositions described herein can be used to block the inhibitory activity of B7-H4 against T cells and / or can be used for ADCC-dependent depletion of cells expressing B7-H4. The pharmaceutical compositions described herein can be used to treat conditions such as cancer. Examples of cancers that can be treated according to the methods described herein include, but are not limited to, breast cancer (e.g., triple-negative breast cancer, ductal carcinoma), endometrial cancer, ovarian cancer, and non-small cell lung cancer (e.g., squamous cell carcinoma), pancreatic cancer, thyroid cancer, kidney cancer (e.g., renal cell carcinoma), and bladder cancer (e.g., bladder epithelial cell carcinoma). Non-small cell non-cancer can be, for example, adenocarcinoma. Other examples of cancers that can be treated according to the methods described herein include, but are not limited to, head and neck cancer, small cell lung cancer, gastric cancer, melanoma, and bile duct epithelial carcinoma. In one embodiment, ovarian cancer is serous adenocarcinoma. In one embodiment, breast cancer is ductal adenocarcinoma.

[0344] In one embodiment, the pharmaceutical compositions described herein are used as medicaments. In one embodiment, the pharmaceutical compositions described herein are used as diagnostic agents, for example, to detect the presence of B7-H4 in a sample obtained from a patient (eg, a human patient).

[0345] Compositions intended for in vivo administration may be sterile. This is readily accomplished by filtration, for example, through sterile filtration membranes.

[0346] In some embodiments, a pharmaceutical composition is provided, wherein the pharmaceutical composition comprises an anti-B7-H4 antibody or antigen-binding fragment thereof described herein and a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition is provided, wherein the pharmaceutical composition comprises an afucosylated anti-B7-H4 antibody or antigen-binding fragment thereof described herein and a pharmaceutically acceptable carrier. In specific embodiments, the pharmaceutical composition comprises an afucosylated anti-B7-H4 antibody or antigen-binding fragment thereof, for example, wherein at least 80% of the antibodies in the composition are afucosylated. In specific embodiments, the pharmaceutical composition comprises an afucosylated anti-B7-H4 antibody or antigen-binding fragment thereof, for example, wherein at least 85% of the antibodies in the composition are afucosylated. In specific embodiments, the pharmaceutical composition comprises an afucosylated anti-B7-H4 antibody or antigen-binding fragment thereof, for example, wherein at least 90% of the antibodies in the composition are afucosylated. In specific embodiments, the pharmaceutical composition comprises an afucosylated anti-B7-H4 antibody or antigen-binding fragment thereof, for example, wherein at least 95% of the antibodies in the composition are afucosylated. In certain embodiments, the pharmaceutical composition comprises an afucosylated anti-B7-H4 antibody or antigen-binding fragment thereof, for example, wherein at least 96% of the antibodies in the composition are afucosylated. In certain embodiments, the pharmaceutical composition comprises an afucosylated anti-B7-H4 antibody or antigen-binding fragment thereof, for example, wherein at least 97% of the antibodies in the composition are afucosylated. In certain embodiments, the pharmaceutical composition comprises an afucosylated anti-B7-H4 antibody or antigen-binding fragment thereof, for example, wherein at least 98% of the antibodies in the composition are afucosylated. In certain embodiments, the pharmaceutical composition comprises an afucosylated anti-B7-H4 antibody or antigen-binding fragment thereof, for example, wherein at least 99% of the antibodies in the composition are afucosylated. In certain embodiments, the pharmaceutical composition comprises an afucosylated anti-B7-H4 antibody or antigen-binding fragment thereof, wherein no fucose is detected in the composition.

[0347] In some embodiments, the pharmaceutical composition comprises (i) an isolated antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, the antibody or antigen-binding fragment thereof comprising (a) heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3, and light chain variable region (VL) CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 458-463, respectively; (b) a variable heavy chain region comprising the amino acid sequence of SEQ ID NO: 464 and a variable light chain region comprising the amino acid sequence of SEQ ID NO: 42; or (c) a heavy chain comprising the amino acid sequence of SEQ ID NO: 469 and a light chain comprising the amino acid sequence of SEQ ID NO: 44; and (ii) a pharmaceutically acceptable excipient.

[0348] Also provided herein is a pharmaceutical composition comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4 and comprises heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3, and light chain variable region (VL) CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 458-463, respectively; and (ii) a pharmaceutically acceptable excipient, wherein at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the antibody or antigen-binding fragment thereof in the composition is afucosylated. In one embodiment, (i) the antibody or antigen-binding fragment thereof comprises a variable heavy chain region comprising an amino acid sequence of SEQ ID NO: 464 and a variable light chain region comprising an amino acid sequence of SEQ ID NO: 42; or (ii) the antibody comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 469 and a light chain comprising an amino acid sequence of SEQ ID NO: 44.

[0349] In some embodiments, the pharmaceutical composition comprises (i) an isolated antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, the antibody or antigen-binding fragment thereof comprising (a) heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3, and light chain variable region (VL) CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 35-40, respectively; (b) a variable heavy chain region comprising the amino acid sequence of SEQ ID NO: 41 and a variable light chain region comprising the amino acid sequence of SEQ ID NO: 42; or (c) a heavy chain comprising the amino acid sequence of SEQ ID NO: 43 and a light chain comprising the amino acid sequence of SEQ ID NO: 44; and (ii) a pharmaceutically acceptable excipient.

[0350] Also provided herein is a pharmaceutical composition comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4 and comprises heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3, and light chain variable region (VL) CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 35-40, respectively; and (ii) a pharmaceutically acceptable excipient, wherein at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the antibody or antigen-binding fragment thereof in the composition is afucosylated. In one embodiment, (i) the antibody or antigen-binding fragment thereof comprises a variable heavy chain region comprising an amino acid sequence of SEQ ID NO: 41 and a variable light chain region comprising an amino acid sequence of SEQ ID NO: 42; or (ii) the antibody comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 43 and a light chain comprising an amino acid sequence of SEQ ID NO: 44.

[0351] In some embodiments, the pharmaceutical composition comprises (i) an isolated antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, the antibody or antigen-binding fragment thereof comprising (a) heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3, and light chain variable region (VL) CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 65-70, respectively, (b) a variable heavy chain region comprising the amino acid sequence of SEQ ID NO: 71 and a variable light chain region comprising the amino acid sequence of SEQ ID NO: 72, or (c) a heavy chain comprising the amino acid sequence of SEQ ID NO: 73 and a light chain comprising the amino acid sequence of SEQ ID NO: 74; and (ii) a pharmaceutically acceptable excipient.

[0352] Also provided herein is a pharmaceutical composition comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4 and comprises heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3, and light chain variable region (VL) CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 65-70, respectively; and (ii) a pharmaceutically acceptable excipient, wherein at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the antibody or antigen-binding fragment thereof in the composition is afucosylated. In one embodiment, (i) the antibody or antigen-binding fragment thereof comprises a variable heavy chain region comprising an amino acid sequence of SEQ ID NO: 71 and a variable light chain region comprising an amino acid sequence of SEQ ID NO: 72; or (ii) the antibody comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 73 and a light chain comprising an amino acid sequence of SEQ ID NO: 74.

[0353] 1.4 Purpose and Method

[0354] 1.4.1 Therapeutic uses and methods

[0355] In one aspect, provided herein are methods for modulating one or more immune functions in a subject, the methods comprising administering to a subject in need thereof an anti-B7-H4 antibody or antigen-binding fragment thereof described herein, or a pharmaceutical composition thereof as described above and herein.

[0356] In another embodiment, an anti-B7-H4 antibody or its antigen binding fragment, or a pharmaceutical composition is administered to a patient (e.g., a human patient) to increase the proliferation of T cells, CD4+T cells, or CD8+T cells in the patient. In another embodiment, an anti-B7-H4 antibody or its antigen binding fragment, or a pharmaceutical composition is administered to a patient (e.g., a human patient) to increase the production of interferon gamma (IFNγ) in the patient. In another embodiment, an anti-B7-H4 antibody or its antigen binding fragment, or a pharmaceutical composition is administered to a patient (e.g., a human patient) to block the inhibitory activity of B7-H4 against T cells in the patient. In another embodiment, an anti-B7-H4 antibody or its antigen binding fragment, or a pharmaceutical composition is administered to a patient (e.g., a human patient) to deplete cancer cells expressing B7-H4 in the patient. In another embodiment, an anti-B7-H4 antibody or its antigen binding fragment, or a pharmaceutical composition is administered to achieve two or more of the above effects.

[0357] In one embodiment, provided herein is a method for treating cancer, such as a cancer expressing B7-H4. The method for treating cancer may include administering an anti-B7-H4 antibody or antigen-binding fragment thereof provided herein, or a pharmaceutical composition comprising an anti-B7-H4 antibody or antigen-binding fragment thereof provided herein to a patient in need thereof (e.g., a human patient).

[0358] In certain embodiments, provided herein are methods for treating cancer, the cancer being selected from the group consisting of: breast cancer (e.g., triple negative breast cancer, ductal carcinoma), endometrial cancer, ovarian cancer, non-small cell lung cancer (e.g., squamous cell carcinoma), pancreatic cancer, thyroid cancer, kidney cancer (e.g., renal cell carcinoma), and bladder cancer (e.g., bladder epithelial carcinoma). In certain embodiments, provided herein are methods for treating non-small cell lung cancer, the non-small cell lung cancer being adenocarcinoma. In certain embodiments, provided herein are methods for treating cancer, the cancer being selected from the group consisting of: head and neck cancer, small cell lung cancer, gastric cancer, melanoma, and bile duct epithelial carcinoma. In one embodiment, ovarian cancer is serous adenocarcinoma. In one embodiment, breast cancer is ductal adenocarcinoma. In some embodiments, the method comprises administering to a patient in need thereof (e.g., a human patient) an anti-B7-H4 antibody or antigen-binding fragment thereof provided herein, or a pharmaceutical composition comprising an anti-B7-H4 antibody or antigen-binding fragment thereof provided herein. In some embodiments, the cancer is a cancer expressing B7-H4.

[0359] In a certain embodiment, a method for treating a PD-1 / PD-L1 inhibitor-inadequately responsive cancer is provided herein. A PD-1 / PD-L1 inhibitor-inadequately responsive cancer may have previously responded to a PD-1 / PD-L1 inhibitor, but may become less responsive to a PD-1 / PD-L1 inhibitor, or the cancer may have never responded to a PD-1 / PD-L1 inhibitor. Inadequate response to a PD-1 / PD-L1 inhibitor means that all aspects of a cancer that is expected to improve after a standard dose of a PD-1 / PD-L1 inhibitor are not improved, and / or improvement occurs only when a dose exceeding the standard dose is administered. In some embodiments, a subject with a PD-1 / PD-L1 inhibitor-inadequately responsive cancer has experienced, or is experiencing, an insufficient response to a PD-1 / PD-L1 inhibitor after receiving a standard dose for at least two weeks, at least three weeks, at least four weeks, at least six weeks, or at least twelve weeks. A "standard" dose is determined by a medical professional and may depend on the subject's age, health history, severity of the disease, frequency of administration, etc. In some embodiments, the subject with a PD-1 / PD-L1 inhibitor-inadequately responsive cancer has experienced, or is experiencing, an inadequate response to an anti-PD-1 antibody and / or an anti-PD-L1 antibody. In some embodiments, the subject with a PD-1 / PD-L1 inhibitor-inadequately responsive cancer has experienced, or is experiencing, an inadequate response to AMP-224. In some embodiments, the subject with a PD-1 / PD-L1 inhibitor-inadequately responsive cancer has experienced, or is experiencing, an inadequate response to a PD-1 / PD-L1 inhibitor selected from nivolumab, pembrolizumab, and atezolizumab.

[0360] In a certain embodiment, a method for treating a cancer that expresses a lower amount of PD-L1 is provided herein. In some embodiments, a cancer that expresses a "lower amount of PD-L1" or a cancer that "expresses PD-L1 at a low level" indicates that the content of PD-L1 is lower than the expression of a cancer suitable for treatment with a PD-1 or PD-L1 antagonist, wherein the patient for treatment is selected based on the PD-L1 expression. In some embodiments, a "lower amount of PD-L1" is a tumor in which less than 1% of cells have membrane staining. In some embodiments, a "lower amount" of PD-L1 is less than 1% of tumor cells, for example, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or 0% of cells are stained. In some embodiments, the PD-L1 expression can be measured by chromogenic IHC or immunofluorescence IHC (Aqua score). In certain embodiments, 5% or less (including tumor and / or immune cells) of PD-L1 staining can indicate that the sample expresses a "lower amount of PD-L1". In certain embodiments, PD-L1 staining of 10% or less (including tumor and / or immune cells) can indicate that the sample expresses "lower amounts of PD-L1." Unless otherwise indicated herein, a 5% threshold is used herein (i.e., 5% or less indicates "lower amounts of PD-L1").

[0361] In another embodiment, an anti-B7-H4 antibody or antigen binding fragment thereof, or a pharmaceutical composition is administered to a patient (e.g., a human patient) diagnosed with cancer to increase the proliferation of T cells, CD4+T cells, or CD8+T cells in the patient. In another embodiment, an anti-B7-H4 antibody or antigen binding fragment thereof, or a pharmaceutical composition is administered to a patient (e.g., a human patient) diagnosed with cancer to increase the production of interferon gamma (IFNγ) in the patient. In another embodiment, an anti-B7-H4 antibody or antigen binding fragment thereof, or a pharmaceutical composition is administered to a patient (e.g., a human patient) diagnosed with cancer to block the inhibitory activity of B7-H4 against T cells in the patient. In another embodiment, an anti-B7-H4 antibody or antigen binding fragment thereof, or a pharmaceutical composition is administered to a patient (e.g., a human patient) diagnosed with cancer to deplete cancer cells expressing B7-H4 in the patient.

[0362] In other embodiments, an anti-B7-H4 antibody or antigen-binding fragment thereof, or a pharmaceutical composition, is administered to a patient as provided above in combination with an additional therapeutic agent, such as a chemotherapeutic agent or an immunomodulatory agent, such as a T cell checkpoint inhibitor.

[0363] In an exemplary embodiment, the additional therapeutic agent is a PD-1 antagonist, such as an antagonist PD-1 antibody. Suitable PD-1 antibodies include, for example, OPDIVO (nivolumab), KEYTRUDA (pembrolizumab) or MEDI-0680 (AMP-514; WO2012 / 145493). Suitable PD-1 antibodies also include, for example, camrelizumab (SHR-1210), tislelizumab (BGB-A317) or spartalizumab (NPVPDR001, NVS240118, PDR001). The additional therapeutic agent may also include pidilizumab (CT-011). A recombinant protein composed of the extracellular domain of PD-L2 (B7-DC) fused to the Fc portion of IgG1, called AMP-224, can also be used to antagonize the PD-1 receptor.

[0364] In another exemplary embodiment, the additional therapeutic agent is a PD-L1 antagonist, such as an antagonist PD-L1 antibody. Suitable PD-L1 antibodies include, for example, TECENTRIQ (atezolizumab), durvalumab (MEDI4736), BMS-936559 (WO2007 / 005874), MSB0010718C (WO2013 / 79174), or rHigM12B7.

[0365] In yet another exemplary embodiment, the additional therapeutic agent is a GITR agonist, such as an agonistic GITR antibody. Suitable GITR antibodies include, for example, TRX-518 (WO06 / 105021, WO09 / 009116), MK-4166 (WO11 / 028683), or GITR antibodies disclosed in WO2015 / 031667. In another embodiment, the additional therapeutic agent is a GITR antibody disclosed in WO2017 / 015623. In a specific embodiment, the GITR antibody is selected from: a) an antibody comprising a GITR binding domain (GITR-BD) comprising a CDR1 comprising the sequence of SEQ ID NO: 411, a CDR2 comprising the sequence of SEQ ID NO: 412, and a CDR3 comprising the sequence of SEQ ID NO: 413; b) an antibody comprising a GITR-BD comprising the sequence of SEQ ID NO: 414; c) a tetravalent molecule comprising two copies of a polypeptide having the structure (GITR-BD)-Linker-(GITR-BD)-Linker-Hinge-Fc, wherein (i) the GITR-BD comprises a CDR1 comprising the sequence of SEQ ID NO: 411, a CDR2 comprising the sequence of SEQ ID NO: 412, and a CDR3 comprising the sequence of SEQ ID NO: 413; NO:413; (ii) the linker is a polypeptide; (iii) the hinge is a polypeptide derived from an immunoglobulin hinge region; and (iv) the Fc is an immunoglobulin Fc polypeptide; d) a tetravalent molecule comprising two copies of a polypeptide having the structure (GITR-BD)-linker-(GITR-BD)-linker-hinge-Fc, wherein (i) GITR-BD comprises an amino acid sequence comprising SEQ ID NO:414; (ii) the linker is a polypeptide; (iii) the hinge is a polypeptide derived from an immunoglobulin hinge region; and (iv) the Fc is an immunoglobulin Fc polypeptide; and e) a tetravalent molecule comprising two copies of a polypeptide comprising a sequence of SEQ ID NO:415. In any of the above embodiments of the tetravalent molecule, the hinge region may comprise the sequence of SEQ ID NO:416, 417 or 418. In any of the above embodiments of the tetravalent molecule, the linker may comprise an amino acid sequence selected from GG, GGG, and SEQ ID NOs:419-425. In certain embodiments, the hinge comprises SEQ ID NO: 417 and the linker comprises any one of SEQ ID NOs: 419-423.

[0366] In yet another exemplary embodiment, the additional therapeutic agent is a CD80 extracellular domain (CD80 ECD), such as SEQ ID NO: 426; or a CD80 ECD fusion molecule comprising a CD80 ECD and a fusion partner as disclosed in WO2017 / 079117. In certain embodiments, the CD80 ECD fusion molecule is a CD80 ECD-Fc fusion protein. In a specific embodiment, the CD80 ECD-Fc fusion protein comprises the sequence of SEQ ID NO: 427 or 428.

[0367] In yet another exemplary embodiment, the additional therapeutic agent is an anti-CSF1R antibody disclosed in US8,182,813, US8,206,715, US8,263,079, US8,513,199, or US9,221,910. In a specific embodiment, the anti-CSF1R antibody is selected from: a) an antibody comprising a heavy chain comprising the sequence of SEQ ID NO: 429 and a light chain comprising the sequence of SEQ ID NO: 430; b) an antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain (HC) complementarity determining region 1 (CDR1) comprising the sequence of SEQ ID NO: 431, a HC CDR2 comprising the sequence of SEQ ID NO: 432, and a HC CDR3 comprising the sequence of SEQ ID NO: 433, and the light chain comprises a light chain (LC) CDR1 comprising the sequence of SEQ ID NO: 434, a LC CDR2 comprising the sequence of SEQ ID NO: 435, and a LC CDR3 comprising the sequence of SEQ ID NO: 436; and c) an antibody comprising a heavy chain comprising the sequence of SEQ ID NO: 437 and a light chain comprising the sequence of SEQ ID NO: 438.

[0368] Typically, the patient is a human, but non-human mammals, including transgenic mammals, can also be treated.

[0369] In some embodiments, the present invention relates to an antibody or its antigen binding fragment or pharmaceutical composition provided herein for use as a medicament. In some aspects, the present invention relates to an antibody or its antigen binding fragment or pharmaceutical composition provided herein for use in a method for treating cancer. In some aspects, the present invention relates to an antibody or its antigen binding fragment or pharmaceutical composition provided herein for use in a method for treating a subject's cancer, the method comprising administering to the subject an effective amount of an antibody or its antigen binding fragment or pharmaceutical composition provided herein.

[0370] 1.4.1.1 Route of administration and dosage

[0371] The antibodies or antigen-binding fragments thereof or compositions described herein can be delivered to a subject by a variety of routes, such as parenteral, subcutaneous, intravenous, intradermal, transdermal, intranasal, intratumoral, and administration to tumor-draining lymph nodes. In one embodiment, the antibody or antigen-binding fragment thereof or compositions are administered by intravenous route.

[0372] The amount of the antibody or antigen-binding fragment thereof or composition effective to treat the condition will depend on the nature of the disease. The precise dose employed in the composition will also depend on the route of administration and the severity of the disease.

[0373] 1.4.2 Detection and diagnostic purposes

[0374] Anti-B7-H4 antibodies or antigen-binding fragments thereof described herein (see Section 5.2) can be used to analyze the B7-H4 protein content in biological samples using classical methods known to those skilled in the art, including immunoassays, such as enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, or Western blotting. Suitable antibody analytical labels are known in the art and include enzyme labels, such as glucose oxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (121In), and technetium (99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine; and biotin. Such labels can be used to label the antibodies or antigen-binding fragments thereof described herein. Alternatively, another antibody or antigen-binding fragment thereof that recognizes the anti-B7-H4 antibodies or antigen-binding fragments thereof described herein can be labeled and used in combination with the anti-B7-H4 antibodies or antigen-binding fragments thereof to detect B7-H4 protein content.

[0375] Analysis of the amount of B7-H4 protein expression is expected to include qualitatively or quantitatively measuring or estimating the amount of B7-H4 protein in a first biological sample, directly (e.g., by determining or estimating the absolute protein level) or relatively (e.g., by comparing to the amount of a disease-associated protein in a second biological sample). The amount of B7-H4 polypeptide expression in a first biological sample can be measured or estimated and compared to a standard B7-H4 protein level, which is obtained from a second biological sample obtained from an individual who does not have the disease or is determined by averaging the levels of a population of individuals who do not have the disease. As is known in the art, once the "standard" B7-H4 polypeptide level is known, that level can be used repeatedly as a comparison standard.

[0376] As used herein, the term "biological sample" refers to any biological sample obtained from a subject, cell line, tissue or other cell source that may express B7-H4. Methods for obtaining tissue biopsies and body fluids from animals (e.g., humans) are well known in the art. Biological samples include peripheral mononuclear blood cells. The biological sample can also be a blood sample, in which circulating tumor cells (or "CTCs") may express B7-H4 and be detected.

[0377] The anti-B7-H4 antibodies described herein can be used for predictive, diagnostic, monitoring and screening applications, including standard in vitro and in vivo applications well known to those skilled in the art and based on the present invention. Predictive, diagnostic, monitoring and screening assays and kits for in vitro assessment and evaluation of immune system status and / or immune response can be used for prediction, diagnosis and monitoring to evaluate samples including patients known to have or suspected of having immune system dysfunction or cancer. Such predictive and diagnostic monitoring and evaluation have been used in practice using antibodies against the HER2 protein in breast cancer (HercepTest™, Dako), where the assays have also been used to evaluate the use of In vivo applications include targeted cell therapy as well as immune system modulation and radiological imaging of immune responses.

[0378] The anti-B7-H4 antibodies and antigen-binding fragments thereof described herein may carry a detectable or functional label. When a fluorescent label is used, currently available microscopy and fluorescence activated cell sorter analysis (FACS) or a combination of the two methods and procedures known in the art can be used to identify and quantify specific binding members. The anti-B7-H4 antibodies and antigen-binding fragments thereof described herein may carry a fluorescent label. Exemplary fluorescent labels include, for example, reactive and conjugated probes such as aminocoumarins, fluorescein and Texas Red, Alexa Fluor dyes, Cy dyes, and DyLight dyes. The anti-B7-H4 antibodies may carry a radioactive label, such as the isotopes 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 67Cu, 90Y, 99Tc, 111In, 117Lu, 121I, 124I, 125I, 131I, 198Au, 211At, 213Bi, 225Ac, and 186Re. When a radioactive label is used, specific binding of the anti-B7-H4 antibody or antigen-binding fragment to B7-H4 (e.g., human B7-H4) can be identified and quantified using currently available counting procedures known in the art. In instances where the label is an enzyme, detection can be achieved by currently used colorimetric, spectrophotometric, fluorospectrophotometric, amperometric, or gas quantitative analysis techniques known in the art. This can be achieved by contacting a sample or control sample with an anti-B7-H4 antibody or antigen-binding fragment thereof under conditions that allow for the formation of a complex between the antibody or antigen-binding fragment thereof and B7-H4. Any complexes formed between the antibody or antigen-binding fragment thereof and B7-H4 in the sample and control are detected and compared. Insofar as the antibodies or antigen-binding fragments described herein specifically bind to B7-H4, the antibodies or antigen-binding fragments thereof can be used to specifically detect expression of B7-H4 on the surface of cells. B7-H4 can also be purified by immunoaffinity purification using the antibodies or antigen-binding fragments thereof described herein.

[0379] Also included herein is an assay system that can be prepared in the form of a test kit for quantitative analysis of, for example, the extent of the presence of B7-H4. The system or test kit can include a labeled component, such as a labeled antibody or antigen-binding fragment, and one or more additional immunochemical reagents. For more details on the kit, see, for example, Section 5.6 below.

[0380] In some aspects, provided herein are methods for in vitro detection of B7-H4 in a sample, the method comprising contacting the sample with an antibody or an antigen binding fragment thereof. In some aspects, provided herein are uses of antibodies or antigen binding fragments thereof provided herein for in vitro detection of B7-H4 in a sample. In one aspect, provided herein are antibodies or antigen binding fragments thereof or pharmaceutical compositions provided herein for detecting B7-H4 in a subject or a sample obtained from a subject. In one aspect, provided herein are antibodies or antigen binding fragments thereof or pharmaceutical compositions provided herein for use as diagnostic agents. In a preferred embodiment, the antibody comprises a detectable label. In a preferred embodiment, the B7-H4 is human B7-H4. In a preferred embodiment, the subject is human.

[0381] 1.5 Kit

[0382] Provided herein is a kit comprising one or more antibodies or Fabs thereof as described herein or their conjugates. In a specific embodiment, a pharmaceutical package or kit is provided herein, comprising one or more containers, which are filled with one or more components of a pharmaceutical composition as described herein, such as one or more antibodies or Fabs thereof provided herein. Optionally associated with such containers may be instructions for the manufacture, use or sale of prescribed drugs or biological products in a governmental agency prescribed form, which instructions reflect the approval of the agency to manufacture, use or sell for human administration.

[0383] Also provided herein are kits that can be used in diagnostic methods. In one embodiment, the kit comprises an antibody or antigen-binding fragment thereof described herein in one or more containers, preferably a purified antibody or antigen-binding fragment thereof. In a specific embodiment, the kit described herein contains a substantially isolated B7-H4 antigen (e.g., human B7-H4), which can be used as a control. In another specific embodiment, the kit described herein further comprises a control antibody or antigen-binding fragment thereof that does not react with the B7-H4 antigen. In another specific embodiment, the kit described herein contains one or more components for detecting the binding of an antibody or antigen-binding fragment thereof to a B7-H4 antigen (e.g., the antibody or antigen-binding fragment thereof can be conjugated to a detectable matrix, such as a fluorescent compound, an enzyme substrate, a radioactive compound, or a luminescent compound, or a second antibody that recognizes a first antibody or antigen-binding fragment thereof can be conjugated to a detectable matrix). In a specific embodiment, the kit provided herein can include a recombinantly produced or chemically synthesized B7-H4 antigen. The B7-H4 antigen provided in the kit can also be attached to a solid support. In a more specific embodiment, the detection member of the above-mentioned kit includes a solid support to which the B7-H4 antigen is attached. Such a kit may also include an anti-human antibody or antigen-binding fragment thereof, or an anti-mouse / rat antibody or antigen-binding fragment thereof to which a reporter label is not attached. In this embodiment, the binding of the antibody or antigen-binding fragment thereof to the B7-H4 antigen can be detected by the binding of the reporter-labeled antibody or antigen-binding fragment thereof.

[0384] The following examples are provided for purposes of illustration and not limitation.

[0385] Examples

[0386] The examples in this section (ie, Section 6) are provided for illustrative purposes and not for limiting purposes.

[0387] Example 1: Assessment of the prevalence of B7-H4 expression in multiple indications

[0388] The presence of B7-H4 on archival samples, mixtures of whole sections, and tumor microarrays was detected using the B7-H4 mouse monoclonal antibody A57.1 (ATCC catalog number PTA-5180). Samples were treated with primary antibodies and detected using a polymer detection system attached to DAB (Ventana Medical Systems).

[0389] B7-H4 was readily detected in the cell membrane and cytosol of tumor tissues collected from multiple cancer patients, including invasive ductal carcinoma, triple-negative breast cancer, ovarian cancer, non-small cell lung cancer, and endometrial cancer. (Figure 1) In addition, the frequency of expression was also high in the indications listed in Table 11.

[0390] Table 11: Detection of B7-H4 in tumors

[0391]

[0392] B7-H4 is expressed in other cancers, such as kidney cancer (e.g., renal cell carcinoma), bladder cancer (e.g., urothelial cell carcinoma), pancreatic cancer, and thyroid cancer. See, e.g., Zhu, J. et al., Asian Pacific J. Cancer Prev. 14:3011-3015 (2011); Krambeck A et al., PNAS 103:10391-10396 (2006); Fan, M. et al., Int. J. Clin. Exp. Pathol. 7:6768-6775 (2014); Xu, H. et al., Oncology Letters 11:1841-1846 (2016); and Liu, W. et al., Oncology Letters 8:2527-2534 (2014).

[0393] In subsequent experiments, the prevalence of B7-H4 in various tumor types was assessed by immunohistochemistry (IHC). Figure 1B The following tumor types and disease stages were tested: endometrial cancer, breast invasive ductal carcinoma (IDC), triple negative breast cancer (TN), triple negative breast cancer (advanced), ovarian cancer, bladder cancer (early), non-small cell lung cancer (squamous cell (sq), early), non-small cell lung cancer (sq, advanced), head and neck cancer, bladder cancer (advanced), small cell lung cancer, cholangiocarcinoma, non-small cell lung cancer (adenocarcinoma (ad)), thyroid cancer, pancreatic cancer, gastric cancer, melanoma, glioblastoma or glioblastoma multiforme (GBM), and Merkel cell carcinoma. The expression of B7-H4 surface protein on tumor cells was evaluated for tumor samples obtained from various tumor types and disease stages (early (I / II), advanced (III / IV)) where available. The IHC intensity of antibody A57.1 was scored according to a 0 to 3 classification, and a single IHC score was assigned to the sample by observing the highest intensity in a minimum of 10% of all tumor cells in each whole section. A total of 50 to 100 samples were evaluated for each tumor type.

[0394] Example 2: Generation of novel antibodies against human B7-H4

[0395] B7-H4-specific antibodies were isolated from full-length human IgG1 natural antibody libraries using an in vitro yeast display system. These libraries were designed to mimic immune systems; these immune systems do not contain predefined heavy and light chain pairs. Multiple rounds of positive and negative selection strategies were performed on the libraries using B7-H4 protein to enrich for IgGs that cross-reacted with human, cynomolgus macaque, and murine B7-H4 targets. After four rounds of selection, the resulting IgGs were sequenced and unique antibodies were generated and evaluated for expected and monomeric binding affinities to the recombinant B7-H4 extracellular domain, for epitope grouping, and for target-specific cellular binding.

[0396] A more detailed description of the selection, affinity maturation and analytical methods used to generate and characterize B7-H4 antibodies is discussed below.

[0397] Materials and methods

[0398] Antigens were biotinylated using the EZ-Link Sulfo-NHS-biotinylation kit from Pierce. Goat F(ab')2 anti-human κ-FITC (LC-FITC), ExtrAvidin-PE (EA-PE), and Streptavidin-AF633 (SA-633) were obtained from Southern Biotech, Sigma, and Molecular Probes, respectively. Streptavidin microbeads and MACS LC separation columns were purchased from Miltenyi Biotec. Goat anti-human IgG-PE (Human-PE) was obtained from SouthernBiotech.

[0399] Natural Discovery

[0400] As previously described (see Y. Xu et al., Addressing polyspecificity of antibodies selected from an in vitro yeast presentation system: a FACS-based, high-throughput selection and analytical tool. PEDS 26.10, 663-70 (2013); WO2009036379; WO2010105256; and WO2012009568), about 10 9Eight natural human synthetic yeast libraries with high diversity were propagated. For the first two rounds of selection, magnetic bead sorting was performed using the Miltenyi MACS system as described previously (see Siegel et al., High efficiency recovery and epitope-specific sorting of an scFv yeast display library." J Immunol Methods 286 (1-2), 141-153 (2004)). Briefly, yeast cells (approximately 10 10 Cells / library) were incubated with 10 ml of 10 nM biotinylated Fc fusion antigen in wash buffer (phosphate buffered saline (PBS) / 0.1% bovine serum albumin (BSA)) for 30 minutes. After washing once with 40 ml of ice-cold wash buffer, the cell pellet was resuspended in 20 mL of wash buffer, and streptavidin microbeads (500 μl) were added to the yeast and incubated at 4°C for 15 minutes. Next, the yeast was aggregated, resuspended in 20 mL of wash buffer, and loaded onto a Miltenyi LS column. After all 20 mL were loaded, the column was washed 3 times with 3 ml of wash buffer. Next, the column was removed from the magnetic field, and the yeast was eluted with 5 mL of growth medium and then grown overnight. Subsequent rounds of selection were performed using flow cytometry. Approximately 2×10 7Yeast were pelleted, washed three times with wash buffer, and incubated at 30° C. with decreasing concentrations of biotinylated Fc fusion antigen (10 to 1 nM) under equilibrium conditions, with 10 nM biotinylated Fc fusion antigens of different species (to obtain species cross-reactivity), or with poly-specificity depletion reagent (PSR) (to remove non-specific antibodies from selection). For PSR depletion, the library was incubated with a 1:10 dilution of biotinylated PSR reagent as previously described (see Y. Xu et al., addressing polyspecificity of antibodies selected from an in vitro yeast presentation system: a FACS-based, high-throughput selection and analytical tool. PEDS 26.10, 663-70 (2013)). Next, the yeast was washed twice with wash buffer and stained with LC-FITC (1:100 dilution) and SA-633 (1:500 dilution) or EAPE (1:50 dilution) secondary reagent for 15 minutes at 4°C. After washing twice with wash buffer, the cell mass was resuspended in 0.3 mL wash buffer and transferred to a filter-capped sorting tube. Sorting was performed using a FACS ARIA sorter (BD Biosciences), and the sorting gate was determined to select antibodies with desired characteristics. The selection cycle was repeated until a population with all desired characteristics was obtained. After the last round of sorting, yeast was spread and individual colonies were selected for characterization.

[0401] Antibody Optimization

[0402] As described below, optimization of antibodies was performed by light chain batch shuffling followed by introducing diversity into the heavy and light chain variable regions. A combination of some of these methods was used for each antibody.

[0403] Light chain batch shuffling: The heavy chain plasmid from the natural selection output was extracted from yeast by smash and grab, amplified in E. coli, purified from E. coli, and transformed into 5×10 6 The selection was performed using one round of MACS and four rounds of FACS under the same conditions as for natural discovery.

[0404] CDRH1 and CDRH2 selection: CDRH3 recombinants from single antibodies at a diversity of 1 × 10 8The selections were performed using one round of MACS and four rounds of FACS in a pre-prepared library with CDRH1 and CDRH2 variants as described in Natural Discovery. For each round of FACS, the library was checked for PSR binding, species cross-reactivity, and affinity pressure, and sorting was performed to obtain a population with the desired characteristics. For these selections, affinity pressure was applied by using decreasing concentrations of biotinylated HIS-B7-H4 antigen (100 to 1 nM) at 30°C under equilibrium conditions.

[0405] VH Mut Selection: The heavy chain variable region (VH) was mutagenized by error-prone PCR. Next, the library was generated by transforming the mutagenized VH and heavy chain expression vectors into yeast that already contained the parental light chain plasmid. Two rounds of selection were performed in a similar manner to the previous cycle using FACS sorting. For each round of FACS, the library was checked for PSR binding and affinity pressure, and sorting was performed to obtain a population with the desired characteristics. The affinity pressures for these selections were performed as described above in the CDRH1 and CDRH2 selections.

[0406] CDRL1 and CDRL2 selection: CDRL3 of a single antibody was recombined to a diversity of approximately 5 × 10 5 Three rounds of selection were performed in a similar manner to the previous cycles using pre-prepared libraries of CDRL1 and CDRL2 variants and sorted using FACS. For each round of FACS, the library was checked for PSR binding and affinity pressure, and sorting was performed to obtain a population with the desired characteristics. The affinity pressure for these selections was performed as described above in the CDRH1 and CDRH2 selections.

[0407] Antibody production and purification

[0408] Yeast clones were grown to saturation and then induced at 30°C for 48 hours under shaking. After induction, yeast cells were agglomerated and supernatants were collected for purification. IgG was purified using a protein A column and eluted with acetic acid (pH 2.0). Fab fragments were produced by papain digestion and purified by KappaSelect (GE Healthcare LifeSciences).

[0409] ForteBio K D Measurement

[0410] ForteBio affinity measurements were performed on Octet RED384 as described previously (see Estep et al., High throughput solution-based measurement of antibody-antigen affinity and epitopebinning. Mabs 5(2), 270-278 (2013)). Briefly, ForteBio affinity measurements were performed by loading IgG online onto the AHQ sensor. The sensor was equilibrated offline for 30 minutes in the assay buffer, followed by online monitoring for 60 seconds to establish a baseline. The sensor loaded with IgG was exposed to 100 nM antigen for 3 minutes, then transferred to the assay buffer and kept for 3 minutes to measure the dissociation rate. For monovalent affinity assessment, Fab was used instead of IgG. For this assessment, non-biotinylated Fc fusion antigen was loaded online onto the AHQ sensor. The sensor was equilibrated offline for 30 minutes in the assay buffer, followed by online monitoring for 60 seconds to establish a baseline. Antigen loaded sensors were exposed to 100 nM Fab for 3 min and then transferred to assay buffer for 3 min to measure the off-rate. All kinetics were analyzed using a 1:1 binding model.

[0411] ForteBio Epitope Grouping / Ligand Blocking

[0412] Epitope grouping / ligand blocking was performed using a standard sandwich cross-blocking assay. A control anti-target IgG was loaded onto the AHQ sensor and unoccupied Fc binding sites on the sensor were blocked with an irrelevant human IgG1 antibody. Next, the sensor was sequentially exposed to 100 nM target antigen and another anti-target antibody or ligand. After antigen association, additional binding of the other antibody or ligand indicated an unoccupied epitope (non-competitor), while no binding indicated epitope blocking (competitor or ligand blocking).

[0413] Four non-competing antibodies ("Grouping Antibodies" 1, 2, 3, and 4) were used to identify four different portions of the B7-H4 extracellular domain by SPR. All antibodies generated during this experiment were then evaluated for competing with the four grouping antibodies for binding to the B7-H4 extracellular domain. If a B7-H4 antibody competes with one of the four grouping antibodies (e.g., Grouping Antibody 1), then the B7-H4 antibody is determined to be in that group (e.g., Group 1). If a B7-H4 antibody competes with two of the four grouping antibodies (e.g., Grouping Antibodies 2 and 3), then the B7-H4 antibody is determined to be in both groups (e.g., Group 2 / 3).

[0414] IgG antibodies belonged to at least four binding groups, with >90% of antibodies binding to human / cynomolgus monkey or mouse B7-H4 with affinities ranging between >0.1 nm and <100 nM. Of the recombinant protein binders, about 75% also bound to B7-H4 on cells.

[0415] Cell binding assay

[0416] 100,000 cells overexpressing antigen were washed with wash buffer and incubated with 100 μl of 100 nM IgG for 5 minutes at room temperature. Then, cells were washed twice with wash buffer and incubated with 100 μl of 1:100 anti-human IgG PE on ice for 15 minutes. Then, cells were washed twice with wash buffer and analyzed on a FACS Canto II analyzer (BD Biosciences).

[0417] PSR binding assay

[0418] PSR analysis was performed as previously described (see Xu Y et al. (2013) Addressing polyspecificity of antibodies selected from an in vitro yeast presentation system: AFACS-based, high-throughput selection and analytical tool. Protein Eng Des Sel 26(10): 663–670). Briefly, soluble membrane proteins were prepared from CHO cells. The enriched membrane fraction was biotinylated using NHS-LC Biotin (Pierce, ThermoFisher). This polyspecific reagent was incubated with IgG displaying yeast and subsequently washed. Next, a secondary labeling mixture (Extravidin-R-PE, anti-human LC-FITC, and propidium iodide) was added to the mixture. Samples were analyzed on a FACSCanto II analyzer (BD Biosciences) using an HTS sample injector. Flow cytometry data were analyzed for mean fluorescence intensity (MFI) in the R-PE channel to assess nonspecific binding. MFI values ​​were normalized within 0 to 1 based on three reference antibodies exhibiting low, medium and high PSR MFI values.

[0419] Dynamic scanning fluorimetry

[0420] 10 μL of 20x Sypro Orange was added to 20 μL of 0.2-1 mg / mL mAb or Fab solution. The sample plate temperature was ramped from 40°C to 95°C in 0.5°C increments using an RT-PCR instrument (BioRad CFX96 RT PCR), with 2 minutes of equilibrium at each temperature. Tm was extracted using the first negative derivative of the raw data.

[0421] AC-SINS

[0422] AC-SINS was performed as previously described (see Liu Y et al. (2014) High-throughput screening for developability during early-stage antibody discovery using self-interaction nanoparticle spectroscopy. MAbs 6(2): 483–492). Briefly, gold nanoparticles (Ted Pella Inc.) were coated with 80% capture anti-human goat IgG Fc (Jackson ImmunoResearch) and 20% polyclonal goat nonspecific antibody (Jackson ImmunoResearch). The antibody of interest was then incubated with the particles for 2 hours, and the wavelength shift was measured using a Molecular Devices SpectraMax M2 with SoftMax Pro6 software. Self-interaction clones showed a wavelength shift farther from the PBS sample.

[0423] Example 3: Structural Characterization of Human Monoclonal Antibodies Against B7-H4

[0424] The B7-H4 antibody is a human IgG1 / κ isotype. The variable heavy chain (VH) region contains alleles from the germline genes VH1, VH3 and VH4, and the variable light chain (VL) region contains alleles from the germline genes containing Vk1 and Vk3. Some antibody subgroups have high consistency in the corresponding CDR regions of VH and VL, and the consistency can be in the range of 72-100%. Antibodies with the same germline alleles have high consistency in their VH and VL framework (FR) regions, which is in the range of 88-100%. The sequence of the B7-H4 antibody is provided in Tables 1-10.

[0425] Example 4: Characterization of Monoclonal Antibody Binding to B7-H4 Protein

[0426] The binding affinity of the anti-B7-H4 antibodies to the B7-H4 extracellular domain (ECD) was determined by biolayer interferometry (BLI). The ForteBio affinity analysis is described in more detail in Example 2 above. Briefly, recombinant human B7-H4-huIgG1 (SEQ ID NO: 409) protein was immobilized on a protein A tip, followed by the addition of isotype control hIgG1 to saturate any remaining binding sites on the capture tip. Next, the binding of the anti-B7-H4 antibodies was evaluated. In addition, the binding of the antibodies to cynomolgus monkey B7-H4 (SEQ ID NO: 2) and mouse B7-H4 (SEQ ID NO: 3) was also evaluated using the same protocol. The results are summarized in Table 12.

[0427] B7-H4-huIgG1:

[0428] MASLGQILFWSIISIIIILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTD AGTYKCYIITSKGKGNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTVVWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTESEIKRR SHLQLLNSKASGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:409)

[0429] Table 12: Binding of B7-H4 Antibodies to the Extracellular Domain as Determined by Biofilm Interferometry

[0430]

[0431]

[0432] In addition, the binding affinity of the selected anti-B7-H4 antibodies to the human B7-H4 N-terminal domain (B7-H4 IgV-huIgG1; SEQ ID NO: 410) was determined by surface plasmon resonance (SPR). Briefly, anti-human Fab antibodies were immobilized on a carboxyl-derivatized SPR chip surface, and anti-B7-H4 antibodies were captured on the resulting surface at 5 μg / ml for 30 seconds. Then, various concentrations (0nM, 3.7nM, 11.1nM, 33.3nM, 100nM, and 300nM) of B7-H4 IgV-huIgG1 were flowed over the surface and allowed to bind to the anti-B7-H4 antibodies during association, followed by washing with buffer during dissociation. The data were fitted using a 1:1 binding model, and the results are summarized in Table 13.

[0433] B7-H4 IgV-huIgG1:

[0434] MASLGQILFWSIISIIIILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITSKGKGNANLEYKTGAFSGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:410)

[0435] Table 13: Binding of B7-H4 Antibodies to the N-terminal Domain as Determined by Surface Plasmon Resonance

[0436]

[0437] Thus, multiple analyses confirmed that the antibody binds to B7-H4.

[0438] Example 5: Epitope Mapping

[0439] Anti-B7-H4 antibody binding groups were determined by biolayer interferometry (BLI). Epitope mapping analysis has been described in more detail in Example 2 above. In brief, a primary anti-B7-H4 antibody was captured on a Protein A tip, followed by the addition of a control huIgG1 antibody to saturate additional binding sites on the tip. Next, the sensor tip was exposed to antigen (B7-H4-huIgG1) (SEQ ID NO: 409), followed by the addition of a secondary anti-B7-H4 antibody, which was then evaluated for binding. The epitope group was determined by comparison to binding following the use of a non-B7-H4 antibody as the primary antibody. The results are summarized in Table 12.

[0440] Example 6: Characterization of Monoclonal Antibody Binding to B7-H4 Expressed on the Cell Surface

[0441] HEK293T cell lines were transfected to express full-length human, cynomolgus monkey, mouse, or rat B7-H4. In addition, SK-BR-3 breast cancer cells expressing endogenous human B7-H4 were used to assess the ability of the antibodies to bind B7-H4 expressed on the cell surface. 5 The parental HEK293T cell line or the transfected HEK293T cell line was incubated with 100 nM B7-H4 antibody. After incubation, the cells were pelleted, washed, and incubated with the secondary labeled antibody, and the samples were collected on a flow cytometer. The fold binding (FOP) relative to the parental cell line was calculated as follows: MFI transfected HKE293T cells / MFI parental HEK293T cells. FOP values ​​greater than 10 were considered to show specific binding.

[0442] To evaluate the cell binding potency of B7-H4 antibody, 1×10 5 SK-BR-3 cells or 293 cells transfected to express cynomolgus monkey, mouse or rat B7-H4 were incubated with titrated doses of B7-H4 antibodies. After incubation, cells were pelleted, washed, and incubated with secondary labeled antibodies, and samples were collected on a flow cytometer. Data were analyzed using FlowJo software and MFI was plotted against antibody concentration. EC50 cell binding potency was calculated using nonlinear regression curve fitting (GraphPad Prism).

[0443] All B7-H4 antibodies demonstrated binding to HEK293T cells expressing human B7-H4, cynomolgus monkey B7-H4, or mouse B7-H4. The antibodies also demonstrated potent dose-dependent binding to SK-BR-3 cells endogenously expressing human B7-H4 on the cell surface or HEK293T cells transfected to express cynomolgus monkey, mouse, or rat B7-H4 on the cell surface. These data demonstrate that most B7-H4 antibodies fully cross-react with cynomolgus monkey, mouse, and rat B7-H4 ( Figure 2A and 2B , Table 14).

[0444] Table 14: Binding of B7-H4 Antibodies to Cell Surface B7-H4

[0445]

[0446] Example 7: Characterization of monoclonal antibody T cell checkpoint blockade activity

[0447] To characterize the T cell checkpoint blockade activity of the B7-H4 antibody, EasySep TM Primary human T cells were enriched from peripheral blood mononuclear cells (PBMC) using the Human T Cell Enrichment Kit according to the manufacturer's instructions. The enriched T cells were cultured at 2 × 10 5 1 × 10 cells / mL were incubated with anti-CD3 / anti-CD28 Dynabeads at a ratio of one bead per cell. After six days, the beads were removed magnetically, and the T cells were washed and incubated at 37°C for 1 × 10 6 Four days later, T cells were washed and incubated at 37°C at 1×10 6 cells / mL and 2×10 6 Artificial antigen presenting cells (aAPC) were cultured together with 10 μg / mL of antibody or titrated doses of antibody. aAPC is a HEK293T cell line transfected to co-express the scFv form of anti-human CD3 clone OKT3 and full-length human B7-H4 on the cell surface. aAPC was treated with mitomycin C for one hour at 37°C and then washed thoroughly before being added to T cell co-cultures. 72 hours after co-culturing T cells, aAPC and B7-H4 antibodies, the culture plates were centrifuged, the supernatant was collected and IFNγ production was evaluated by ELISA, and the cells were collected and stained to evaluate proliferation by FACS (specifically, the cells in each well were cultured with antibodies against CD4 and CD8). Then, the cells were washed, fixed, permeabilized, and cultured with Edu-Click reagent according to the manufacturer's instructions. The cells were washed and the number of proliferating CD4+T cells, CD8+T cells or total T cells was measured using flow cytometry. Data were analyzed using FlowJo software. For samples treated with only a single concentration of antibody, data were calculated and reported as fold increase relative to control-huIgG treated samples. For samples treated with dose titrated antibodies, IFNγ production was plotted versus antibody concentration and EC50 potency was calculated using nonlinear regression curve fitting (GraphPad Prism).

[0448] As measured by IFNγ production and / or CD4 + T cells, CD8 + All Group 2 / 3 and Group 3 antibodies reproducibly elicited T cell checkpoint blockade activity as measured by increased proliferation of either WT T cells or total T cells. Figure 3A Antibody 15441 was shown to increase T cell proliferation by at least 3% and CD4+ and CD8+ T cell proliferation by at least 2%. Figure 3A It was also shown that antibody 15461 increased T cell proliferation by at least 21%, CD4+ T cell proliferation by at least 9%, and CD8+ T cell proliferation by at least 11%. Figure 3B-3D Other dose-dependent T cell checkpoint blockade activities were demonstrated.

[0449] Table 15: B7-H4 Antibody-T Cell Checkpoint Blockade Activity

[0450]

[0451] Table 16: Binding of B7-H4 Antibodies to Cell Surface B7-H4

[0452]

[0453]

[0454] Example 8: Generation of afucosylated and fucosylated monoclonal antibodies

[0455] Compared to fully fucosylated antibodies, antibodies containing Fc regions with lower fucose content in the glycan moiety can exhibit higher ADCC activity (Niwa R et al., Clinical Cancer Research 11(6):2327-36 (2005)). B7-H4 antibodies were produced in CHO-x cells that produce normally fucosylated antibodies (Yamane-Ohnuki N et al., Biotechnology and Bioengineering 87(5):614-22 (2004)) and in a CHO cell line engineered to produce afucosylated antibody (CHO-y cells) (supra).

[0456] Example 9: Characterization of binding of afucosylated and fucosylated monoclonal antibodies

[0457] Fucosylated and afucosylated anti-B7-H4 antibodies were characterized by SPR following the protocol described in Example 4. These antibodies showed similar binding to human B7-H4 protein and, therefore, glycosylation had no effect on binding (Table 17).

[0458] Table 17: Binding of afucosylated and fucosylated antibodies

[0459]

[0460]

[0461] Fucosylated and afucosylated anti-B7-H4 antibodies were also characterized by flow cytometry. In these experiments, HEK293T cell lines were transfected to express full-length human, cynomolgus monkey, mouse, or rat B7-H4. In addition, the SK-BR-3 breast cancer cell line expressing endogenous human B7-H4 was used to assess the cell binding potency of B7-H4 antibodies. 1×10 5 SK-BR-3 cells or 293 cells transfected to express full-length human, cynomolgus monkey, mouse or rat B7-H4 were incubated with titrated doses of B7-H4 antibodies. After incubation, cells were pelleted, washed, incubated with secondary labeled antibodies, and run on a flow cytometer. Data were analyzed using FlowJo software. MFI was plotted against antibody concentration and EC50 cell binding potency was calculated using nonlinear regression curve fitting (GraphPad Prism).

[0462] The B7-H4 antibodies showed potent dose-dependent binding to SK-BR-3 cells that endogenously express B7-H4 on the cell surface or to 293 cell lines transfected to express cynomolgus monkey, mouse, or rat B7-H4 on the cell surface. Whether the antibody was fucosylated or afucosylated did not affect binding potency and species cross-reactivity ( Figures 4A-4D and Table 17).

[0463] Example 10: Binding of afucosylated and fucosylated monoclonal antibodies to the human Fcγ receptor IIIa (FcγRIIIa) V158 allele

[0464] Antibody 20502 was generated in fucosylated (Ab-F) and afucosylated (Ab-A) forms and the binding affinity of the Fc region to FcgRIIIa (V158) was tested by surface plasmon resonance (SPR). Briefly, protein A was covalently attached to a dextran chip using an amine coupling kit with 100 mM sodium borate buffer pH 8.0 containing 100 mM ethylenediamine as a blocking reagent. Ab-A or Ab-F were captured on separate flow cells at 2 densities, and a protein A-derivatized flow cell was used as a reference control. FcγRIIIA (V158) was diluted in HBS-P + running buffer and injected in duplicate at 6 concentrations (0 nM, 1.37 nM, 12.3 nM, 37 nM, 111 nM, 333 nM, and 1000 nM). The association constant, dissociation constant and affinity for Ab-A binding were calculated using the Biacore T200 evaluation software 1:1 binding model. The affinity constants for Ab-A and Ab-F binding were determined using the Biacore T200 evaluation software steady-state affinity model. The affinity of the afucosylated B7-H4 antibody (Ab-A) for Fcγ receptor IIIA (V158) was 140-fold higher than that of the same antibody with fucosylated Fc (Ab-F) ( Figure 5A and 5B , Table 18).

[0465] Table 18: Fcγ receptor IIIa (FcγRIIIa) V158 allele binding

[0466] ka(1 / Ms) kd(1 / s) <![CDATA[K D (nM)]]> Ab-A 6.46E+05 9.54E-10 15 Ab-F N / A N / A 210

[0467] Example 11: T cell checkpoint blocking activity of afucosylated and fucosylated monoclonal antibodies

[0468] Using EasySep TM Primary human T cells were enriched from PBMC using the Human T Cell Enrichment Kit according to the manufacturer's instructions. The enriched T cells were cultured at 2 × 10 5 1 × 10 cells / mL were incubated with anti-CD3 / anti-CD28 Dynabeads at a ratio of one bead per cell. After six days, the beads were removed magnetically, and the T cells were washed and cultured at 37°C at 1 × 10 6 Four days later, T cells were washed and incubated at 37°C at 1×10 6 cells / mL and 2×10 6Artificial antigen presenting cells (aAPC) at a concentration of 10 cells / mL were incubated together in the presence of titrated doses of B7-H4 antibodies. aAPCs were treated with mitomycin C for one hour at 37°C, followed by extensive washing and then added to T cell co-cultures. 72 hours after co-culturing T cells, aAPCs and B7-H4 antibodies, the culture plates were centrifuged and the supernatants were collected and IFNγ production was assessed by ELISA. IFNγ production was plotted against antibody concentration and EC50 potency was calculated using nonlinear regression curve fitting (GraphPad Prism).

[0469] The B7-H4 antibody exhibited potent T cell checkpoint blockade activity as measured by increased IFNγ production. In addition, there was no significant difference in potency between the afucosylated and fucosylated antibodies ( Fig. 6A and Table 19).

[0470] Table 19: T cell checkpoint blockade efficacy

[0471]

[0472]

[0473] Next, primary human T cells were enriched from HLA-A2+ donor PBMCs using the Human Pan T Cell Isolation Kit following the manufacturer's instructions. 7 5×10 cells were activated with anti-CD3 / anti-CD28 Dynabeads, 100 ng / mL IL-2 and 15 ng / mL IL-7. 6 T cells expressing MART-I TCR were generated by 48 hours of enriched whole T cells. Then, 5×10 cells were transduced with MART-I TCR lentiviral particles in the presence of 200 ng / mL IL-2, 30 ng / mL IL-7, and 10 μg / mL polybrene. 6Activated T cells. 24 hours after transduction, in the presence of 33.3ng / mL IL-2 and 5ng / mL IL-7, MART-I TCR+ full T cells were expanded for 15 days. In order to produce a target cell line expressing HLA-A2, HLA-A2 lentiviral particles were used to transduce human breast cancer cell lines MDA-MB-468 and SK-BR-3 expressing endogenous B7-H4 for 48 hours. In addition, B7-H4 was knocked out from the HLA-A2+SK-BR-3 cell line. MART-I TCR+ full T cells were co-cultured in the presence of various target cells at a 1:1E:T ratio, 500μg / mL MART-I peptides and 67nM B7-H4 antibody 20502 (no fucosylation) or human isotype controls. 24 hours after co-cultivation, the culture dish was centrifuged and the supernatant was collected, and IL-2 production was assessed by AlphaLisa according to the manufacturer's instructions.

[0474] like Figure 6B As shown in , when endogenously expressed at physiological amounts, B7-H4 exhibits T cell checkpoint ligand activity in this assay as IL-2 production is reduced in wells containing SK-BR-3B7-H4+ cells relative to SK-BR-3KO cells. In addition, the B7-H4 antibody also exhibits T cell checkpoint blocking activity ( Figure 6B ). Thus, this data confirms the T cell checkpoint blocking activity of B7-H4 antibody 20502 (afucosylated) and shows that B7-H4 antibody 20502 provides T cell checkpoint blocking activity in cells that endogenously express B7-H4.

[0475] Example 12: ADCC activity of afucosylated and fucosylated monoclonal antibodies

[0476] The ADCC activity of B7-H4 antibodies against target cell lines expressing B7-H4 was evaluated. Specifically, 1×10 6Primary human PBMC cells at 10 cells / mL were used for cytokine activation. The next day, cells were washed and incubated with SK-BR-3 target cells labeled with Calcein-AM at an effector cell: target ratio of 40:1. Target cell lysis was quantified using a fluorimeter 4 hours after incubation. Triton / X-treated samples were used as maximum lysis control samples, while samples treated with medium alone were used as background lysis control samples. The percentage (%) of specific lysis rate was calculated as follows: [1-((sample-medium control) / (maximum lysis-medium control))]×100. The percentage (%) of specific lysis rate was plotted against antibody concentration, and the EC50 potency was calculated using nonlinear regression curve fitting (GraphPad Prism).

[0477] The B7-H4 antibody exhibited potent dose-dependent ADCC activity against the breast cell line SK-BR-3 expressing endogenous B7-H4. In addition, the afucosylated antibody exhibited significantly more potent ADCC activity compared to the fucosylated antibody ( Figure 7 and Table 20).

[0478] Table 20: ADCC activity

[0479]

[0480] Example 13: Correlation between ADCC activity and receptor density

[0481] B7-H4 density on the surface of SK-BR-3, HCC1569, ZR-75-1, MDA-MB-48, and HCC1964 cells was quantified by FACS according to the manufacturer's instructions. Specifically, 1×10 5 The cells were incubated with 15 μg / mL B7-H4 antibody for 25 minutes. TM Simply Cellular (QSC) microspheres (pre-coated with increasing concentrations of anti-mouse IgG capture antibody) were incubated with 15 μg / mL B7-H4 antibody on ice for 25 minutes. After incubation, cells and QSC microspheres were pelleted and washed, and samples were acquired on a flow cytometer. Data were analyzed using FlowJo software. The mean fluorescence intensity (MFI) was calculated and entered A regression is automatically calculated that relates the fluorescence channel value of each bead to its pre-assigned antibody binding capacity (ABC) value. The ABC value is also assigned after the MFI value of the labeled cells is also added to the template.

[0482] The ADCC activity of B7-H4 antibodies against target cell lines expressing B7-H4 with different B7-H4 cell surface density values ​​was evaluated. Specifically, 1×10 4 SK-BR-3, HCC1569, ZR-75-1, MDA-MB-468, or HCC1964 target cells were co-incubated with titrated doses of B7-H4 antibody. After 25 minutes, a single-use vial containing Jurkat-huCD16 reporter cells from Promega was thawed and 7.5 × 10 4 Cells were added to the target cell / B7-H4 antibody mixture and incubated at 37°C. After 24 hours, samples were allowed to reach room temperature (RT) and incubated with Bio-Glo buffer. Substrate and luminescence were quantified on an EnVision multi-label reader. Data were plotted as luminescence versus antibody concentration and EC50 potency was calculated using nonlinear regression curve fitting (GraphPad Prism).

[0483] B7-H4 antibody ADCC activity depends on B7-H4 cell surface density: when the number of cell surface molecules decreases, the amount of maximal ADCC activity also decreases. In addition, afucosylated antibodies exhibit improved ADCC activity compared to fucosylated antibodies, especially against target cells with low B7-H4 cell surface density ( Figure 8 ).

[0484] Example 14: In vivo anti-tumor efficacy

[0485] Seven-week-old female BALB / c mice were purchased from Charles River Laboratories (Hollister, CA) and acclimated for three weeks before the start of the study. The murine colorectal cancer cell line CT26 was engineered to express a chimeric protein consisting of the murine B7-H4 extracellular domain and the murine B7H3 transmembrane domain. These tumor cells were inoculated at 1.0 × 10 per mouse. 6 Cells / 200 μl were implanted subcutaneously into the right flank of mice. Prior to inoculation, cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 2 mM L-glutamine for no more than three passages. Cells were incubated at 37°C with 5% CO 2 After reaching 80-85% confluence, cells were harvested and plated at 5 × 10 6 Cells / mL were resuspended in a 1:1 mixture of serum-free RPMI 1640 and Matrigel.

[0486] After cell implantation, mice were monitored for tumor growth twice a week. For tumor measurement, the length and width of each tumor were measured using a caliper, and the volume was calculated according to the following formula: tumor volume (mm3) = (width (mm) × length (mm2)) / 2. On the day of treatment start, all tumors were measured, tumors that did not meet the requirements were excluded, and mice were randomly divided into treatment groups. For anti-B7-H4 treatment, the antibodies used included 20502 ( Fig. 9A ) and 22213( Fig. 9B As a control, polyclonal human IgG (Bio X Cell, BE0092) or mouse IgG2a (Bio X Cell, BE0085) was administered to mice. Antibodies were administered four times twice a week via intravenous (iv) injection starting on day 4 or 5 after inoculation.

[0487] Tumors were measured at least twice a week until the tumor volume exceeded 10% of the animal's body weight, or reached approximately 2000 mm3. Changes in tumor size were shown by plotting individual tumors against the day the animal was inoculated with CT26 cells. Unpaired, two-tailed t-test analysis was used to calculate the P value for the tumor volume calculated on each day of the study. When administered at doses between 10-20 mg / kg, treatment with 20502 or 22213 significantly slowed tumor growth compared to human IgG controls (p<0.05) ( Fig. 9A and 9B ).

[0488] Similar experiments were performed using mice implanted with 4T1 cells (murine breast cancer cell line) or B16 (murine melanoma cell line). These mice were treated with 20 mg / kg of a mouse surrogate of 20502 called 20502-msIgG2a-F, which contains a fusion of the 20502 variable region with fucosylated mouse IgG2a; or with a murine IgG control antibody. In both the 4T1 breast cancer and B16 melanoma models, 20502-msIgG2a-F treatment significantly slowed tumor growth compared to the murine IgG control ( Fig.10 ).

[0489] In addition, additional experiments were performed using afucosylated 20502 in the MX-1 human breast cancer xenograft model. Female NSG (NOD-scid, IL2Rγnull) mice were purchased from Jackson Laboratory (Bar Harbor, ME) and acclimated for one week prior to the start of the study. It has been previously shown that the human breast cancer cell line MX-1 endogenously expresses the cell surface B7H4 protein. 5×10 6 These tumor cells were cultured in a 1:1 mixture of serum-free RPMI 1640 and Matrigel at a rate of 1.0 × 10 cells / mL per mouse. 6cells / 100 μl were implanted subcutaneously into the right flank of mice.

[0490] After cell implantation, mice were monitored for tumor growth twice a week. For tumor measurement, the length and width of each tumor were measured using a caliper, and the volume was calculated according to the following formula: Tumor volume (mm3) = (width (mm) × length (mm)2) / 2. On day 7 after inoculation, all tumors were measured, tumors that did not meet the requirements were excluded, and mice were randomly divided into treatment groups. All animals were administered a DNA construct to induce constitutive expression of human IL-15 in the circulation for subsequent studies. On day 9, half of the mice received an intravenous (iv) injection of 20×10 6 Human peripheral blood mononuclear cells (PBMCs) were obtained from StemCell Technologies (Tukwila, WA). Mice were administered afucosylated 20502 (20 mg / kg) or saline as a negative control starting on day 11. The treatments were administered four times twice a week by intravenous (iv) injection.

[0491] Tumor measurements continued at least twice weekly until tumor volume exceeded 10% of the animal's body weight or until the animal demonstrated a 15% or greater percentage loss of initial body weight. On day 28, P values ​​were calculated using one-way ANOVA to compare the mean tumor volume of all treatment groups. Fig.11 As shown in , when administered to mice pre-injected with human PBMCs, afucosylated 20502 treatment significantly slowed tumor growth compared to saline controls (p<0.05).

[0492] Example 15: In vitro anti-tumor efficacy in combination with anti-PD-1 antibodies

[0493] method

[0494] Eight-week-old female BALB / c mice were purchased from Charles River Laboratories (Hollister, CA) and acclimatized for two weeks before the start of the study. The murine breast cancer cell line 4T1 was engineered to express a chimeric protein containing the extracellular domain of murine B7-H4 and the transmembrane domain of murine B7H3. Tumor cells were plated at 0.5 × 10 per mouse. 5 Cells / 50 μl were implanted in situ into the mammary fat pad of mice. Prior to inoculation, cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) for no more than three generations. At 37°C, cells were grown in a humidified atmosphere containing 5% CO2. After reaching 80-85% confluence, cells were collected and resuspended in serum-free RPMI 1640 and implanted into the mammary fat pad via the flank of each mouse.

[0495] After cell implantation, mice were monitored for tumor growth twice a week. The length and width of each tumor were measured using a caliper, and the volume was calculated according to the following formula: Tumor volume (mm3) = (width (mm) × length (mm)2) / 2. On the day of treatment start, all tumors were measured, tumors that did not meet the requirements were excluded, and mice were randomly divided into treatment groups. For anti-B7-H4 treatment, a mouse substitute for 20502, called 20502-msIgG2a-F, was used, which contained a fusion of the 20502 variable region and fucosylated mouse IgG2a. As a control, msIgG2a (anti-HEL) was administered to mice. Starting from the 11th day after inoculation, 20502-msIgG2a-F or msIgG2a was administered four times by intravenous (iv) injection twice a week. Starting from the 11th day after inoculation, anti-PD-1 (modified RMP1-14 (Bio X Cell) containing an Fc-silencing msIgG2a domain) was administered three times twice a week by intraperitoneal (ip) injection. Continue to measure tumors at least twice a week until tumor volume exceeds 10% of the animal's body weight or reaches approximately 2000 mm. 3 .

[0496] result

[0497] The changes in tumor size, mean tumor volume, and survival percentage are shown in Fig. 12A , 12B and 12C. Treatment with 20502-msIgG2a-F or anti-PD-1 significantly slowed tumor growth compared to msIgG2a control (p<0.05). Compared to any monotherapy, the co-administration of 20502-msIgG2a-F and anti-PD-1 significantly enhanced tumor growth inhibition (p<0.05). In addition, the combination therapy caused complete tumor regression in 5 of the 12 mice. Using one-way analysis of variance, the P value of the tumor volume calculated on each day of the study and the multiple comparisons between the groups were calculated.

[0498] Example 16: Anti-B7-H4 Antibodies Increase NK Cell and T Cell Infiltration and Upregulate PD-L1 Eight-week-old female BALB / c mice were purchased from Charles River Laboratories (Hollister, CA) and 4T1+moB7-H4 / H3 cells were inoculated at 0.5×10 5 Cells / 50 μl were orthotopically implanted into the mammary fat pad of mice. On the day of treatment initiation, all tumors were measured, tumors that did not meet the requirements were excluded, and mice were randomly divided into treatment groups. Mice were administered 20 mg / kg huIgG1 (anti-HEL) or afucosylated 20502 twice (days 11 and 14 after inoculation) by intravenous (iv) injection.

[0499] Twenty-four hours after the second dose, mice were euthanized and perfused with phosphate-buffered saline (PBS). Tumors were then extracted, fixed in 10% formalin for five hours, rinsed in PBS, and placed in 30% sucrose solution for at least 24 hours. OCT Compound and cut into 20 μm sections in a -15°C chamber (cryostat). Tissue-loaded slides were rinsed in PBS containing 0.3% Triton, blocked with 5% normal goat serum, and stained overnight with primary antibodies (anti-NKp46, anti-CD3, or anti-PD-L1; 1:500). Two consecutive sections from each tumor were stained.

[0500] After overnight incubation, sections were rinsed in 0.3% Triton, then incubated with secondary antibody (1:400) for three hours in a dark humid chamber and rinsed. Tissues were then fixed in 1% paraformaldehyde and rinsed in PBS. Seal the slides with DAPI and use Cytoseal TM Seal and allow to dry in a dark chamber. Images were acquired manually using a fluorescence microscope and camera. Fig.13 As shown in Figure 2, afucosylated 20502 treatment increased natural killer (NK) cells (as measured by anti-NKp46 antibody) and CD3+ T cells at the tumor margin, with a modest increase in CD3+ T cells in the tumor core. Increased PD-L1 expression was also observed.

[0501] Example 17: Anti-B7-H4 Antibodies Significantly Reduce Tumor Growth in Vivo in a Dose-Dependent Manner in 4T1- and B16-moB7-H4 / H3 Models

[0502] Female BALB / c or C57Bl / 6 mice, six to eight weeks old, were purchased from Charles River Laboratories (Hollister, CA) and acclimated for up to three weeks prior to the start of the study. Murine breast cancer cell line 4T1 and melanoma cell line B16-F10 were engineered to express a chimeric protein consisting of the extracellular domain of murine B7-H4 and the transmembrane domain of murine B7-H3. For 4T1+moB7-H4 / H3, these tumor cells were grown at 0.05 × 10 per mouse. 6 Cells / 50 μl were implanted orthotopically into the mammary fat pad, or for B16+moB7-H4 / H3, 0.5×10 cells / 50 μl were implanted per mouse. 6Cells / 100 μl were implanted subcutaneously into the right flank of mice. Prior to inoculation, cells were cultured in RPMI-1640 or DMEM medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 2 mM L-glutamine for no more than three passages. Cells were maintained at 37°C in a 5% CO 2 Grows in a humid atmosphere.

[0503] After cell implantation, mice were monitored for tumor growth twice a week. For tumor measurement, the length and width of each tumor were measured using a caliper and the volume was calculated according to the following formula: Tumor volume (mm3) = (width (mm) × length (mm)2) / 2. On the day of treatment start, all tumors were measured, tumors that did not meet the requirements were excluded, and mice were randomly divided into treatment groups. Starting from day 11 (4T1+moB7-H4 / H3) or day 6 (B16+moB7-H4 / H3) after inoculation, mice were administered 20502-msIgG2a-F antibody (anti-B7-H4, msIgG2a, fucosylated (also known as "cmFPA150-F") or mouse IgG2a control antibody four times by intravenous (iv) injection twice a week, except for the 20 mg / kg group, which was continuously administered twice a week until the end of the study.

[0504] Tumor measurements continued at least twice weekly until tumor volume exceeded 10% of the animal's body weight, or reached approximately 2000 mm3. Changes in tumor size were shown by plotting mean tumor volume versus the day the animal was inoculated. Fig.14A and 14B ) and at 3 mg / kg or higher in the B16+moB7-H4 / H3 model ( Fig.15A and 15B ), 20502-msIgG2a-F treatment significantly slowed tumor growth compared to mouse IgG2a control (p<0.05). One-way ANOVA was used to compare all treatment groups with msIgG2a and calculate statistical significance.

[0505] Fig.14A and 14BIt was shown that the 20502-msIgG2a-F antibody significantly slowed the growth of 4T1 expressing B7-H4 / H3. BALB / c mice were orthotopically inoculated with 4T1 breast cancer cells engineered to express a fusion of murine B7-H4 ECD and B7-H3 TM. Starting from day 11 after inoculation, the mice were administered 20502-msIgG2a-F antibody (anti-B7-H4, msIgG2a, fucosylated) twice a week. As assessed by one-way ANOVA on day 30, the 20502-msIgG2a-F antibody showed a dose-dependent slowing of tumor growth at 30 mg / kg (p=0.0003), 20 mg / kg (p=0.0103), 10 mg / kg (p=0.0419), 3 mg / kg (p=0.0277) and 1 mg / kg (p=0.0333), as well as significant tumor growth inhibition.

[0506] Fig.15A and 15B It was shown that the 20502-msIgG2a-F antibody significantly slowed the growth of B16 expressing B7-H4 / H3. C57Bl / 6 mice were subcutaneously inoculated with B16-F10 melanoma cells engineered to express a fusion of murine B7-H4 ECD and B7-H3 TM. Starting on day 6 after inoculation, the mice were administered 20502-msIgG2a-F antibody (anti-B7-H4, msIgG2a, fucosylated) twice a week. As assessed by one-way ANOVA on day 23, the 20502-msIgG2a-F antibody showed a dose-dependent slowing of tumor growth at 30 mg / kg (p=0.0085), 20 mg / kg (p=0.0041), 10 mg / kg (p=0.0017) and 3 mg / kg (p=0.0420). Fig.15A ), and significant tumor growth inhibition ( Fig. 15B ).

[0507] Thus, in both different cancer models, the 20502-msIgG2a-F antibody significantly reduced tumor size in a dose-dependent manner. This data in breast cancer and melanoma cell lines indicates that the 20502-msIgG2a-F antibody can be used to treat cancer patients.

[0508] Sequence Listing

[0509] GITR antibody sequences

[0510]

[0511] CD80 sequence

[0512]

[0513]

[0514] CSFR1 sequence

[0515]

[0516]

[0517] PD-1 (Nivolumab) antibody sequence

[0518]

[0519]

[0520] ***

[0521] The scope of the present invention is not limited by the specific embodiments described herein. In fact, various modifications of the present invention other than those described will be apparent to those skilled in the art from the foregoing description. These modifications are intended to be within the scope of the appended claims.

[0522] All references (e.g., publications or patents or patent applications) cited herein are incorporated by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0523] Other embodiments are within the scope of the following claims.

[0524] The present invention also includes the following exemplary embodiments:

[0525] 1. An isolated antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, the isolated antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3, and a light chain variable region (VL) CDR1, CDR2, and CDR3 sequence selected from the group consisting of:

[0526] (a) correspondingly, SEQ ID NO:458-463;

[0527] (b) correspondingly, SEQ ID NO: 5-10;

[0528] (c) correspondingly, SEQ ID NO: 15-20;

[0529] (d) correspondingly, SEQ ID NO: 25-30;

[0530] (e) correspondingly, SEQ ID NO:35-40;

[0531] (f) correspondingly, SEQ ID NO:45-50;

[0532] (g) correspondingly, SEQ ID NO:55-60;

[0533] (h) correspondingly, SEQ ID NO: 65-70;

[0534] (i) correspondingly, SEQ ID NO:75-80;

[0535] (j) correspondingly, SEQ ID NO:85-90;

[0536] (k) correspondingly, SEQ ID NO:95-100;

[0537] (l) correspondingly, SEQ ID NO: 105-110;

[0538] (m) correspondingly, SEQ ID NO: 115-120;

[0539] (n) correspondingly, SEQ ID NO: 125-130;

[0540] (o) correspondingly, SEQ ID NO: 135-140;

[0541] (p) correspondingly, SEQ ID NO: 145-150;

[0542] (q) correspondingly, SEQ ID NO: 155-160;

[0543] (r) correspondingly, SEQ ID NO: 165-170;

[0544] (s) correspondingly, SEQ ID NO: 175-180;

[0545] (t) correspondingly, SEQ ID NO: 185-190; and

[0546] (u) correspondingly, SEQ ID NO:195-200.

[0547] 2. The antibody or antigen-binding fragment thereof according to embodiment 1, wherein the antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 11, 21, 31, 41, 464, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191 or 201.

[0548] 3. The antibody or antigen-binding fragment thereof of embodiment 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises a VL comprising the amino acid sequence of SEQ ID NO: 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182, 192 or 202.

[0549] 4. An isolated antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 11, 21, 31, 41, 464, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191 or 201.

[0550] 5. An isolated antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182, 192 or 202.

[0551] 6. An isolated antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, the isolated antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region comprising the following amino acid sequence:

[0552] (a) SEQ ID NOs: 464 and 42, respectively;

[0553] (b) correspondingly, SEQ ID NOs: 11 and 12;

[0554] (c) correspondingly, SEQ ID NOs: 21 and 22;

[0555] (d) correspondingly, SEQ ID NOs: 31 and 32;

[0556] (e) correspondingly, SEQ ID NOs: 41 and 42;

[0557] (f) correspondingly, SEQ ID NOs: 51 and 52;

[0558] (g) correspondingly, SEQ ID NOs: 61 and 62;

[0559] (h) correspondingly, SEQ ID NOs: 71 and 72;

[0560] (i) correspondingly, SEQ ID NOs: 81 and 82;

[0561] (j) correspondingly, SEQ ID NOs: 91 and 92;

[0562] (k) correspondingly, SEQ ID NOs: 101 and 102;

[0563] (l) correspondingly, SEQ ID NO: 111 and 112;

[0564] (m) correspondingly, SEQ ID NOs: 121 and 122;

[0565] (n) correspondingly, SEQ ID NOs: 131 and 132;

[0566] (o) correspondingly, SEQ ID NOs: 141 and 142;

[0567] (p) SEQ ID NOs: 151 and 152, respectively;

[0568] (q) SEQ ID NOs: 161 and 162, respectively;

[0569] (r) correspondingly, SEQ ID NOs: 171 and 172;

[0570] (s) SEQ ID NOs: 181 and 182, respectively;

[0571] (t) SEQ ID NOs: 191 and 192, respectively; or

[0572] (u) correspondingly, SEQ ID NO: 201 and 202.

[0573] 7. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 6, wherein the antibody or antigen-binding fragment further comprises a heavy chain constant region.

[0574] 8. The antibody or antigen-binding fragment thereof according to embodiment 7, wherein the heavy chain constant region is selected from the group consisting of human immunoglobulin IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2 heavy chain constant regions.

[0575] 9. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 8, wherein the antibody or antigen-binding fragment further comprises a light chain constant region.

[0576] 10. The antibody or antigen-binding fragment thereof according to embodiment 9, wherein the light chain constant region is selected from the group consisting of human immunoglobulin IgGκ and IgGλ light chain constant regions.

[0577] 11. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 6, wherein the antibody or antigen-binding fragment further comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is a human IgG1 heavy chain constant region, and wherein the light chain constant region is a human IgGκ light chain constant region.

[0578] 12. The antibody or antigen-binding fragment thereof of any one of embodiments 1 to 6, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 13, 23, 33, 43, 469, 53, 63, 73, 83, 93, 103, 113, 123, 133, 143, 153, 163, 173, 183, 193 or 203.

[0579] 13. The antibody or antigen-binding fragment thereof of any one of embodiments 1 to 6 or 12, wherein the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, 114, 124, 134, 144, 154, 164, 174, 184, 194 or 204.

[0580] 14. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 6, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain comprising the following amino acid sequence:

[0581] (a) SEQ ID NOs: 469 and 44, respectively;

[0582] (b) correspondingly, SEQ ID NOs: 13 and 14;

[0583] (c) correspondingly, SEQ ID NOs: 23 and 24;

[0584] (d) correspondingly, SEQ ID NOs: 33 and 34;

[0585] (e) correspondingly, SEQ ID NOs: 43 and 44;

[0586] (f) SEQ ID NOs: 53 and 54, respectively;

[0587] (g) SEQ ID NOs: 63 and 64, respectively;

[0588] (h) SEQ ID NOs: 73 and 74, respectively;

[0589] (i) correspondingly, SEQ ID NOs: 83 and 84;

[0590] (j) correspondingly, SEQ ID NOs: 93 and 94;

[0591] (k) correspondingly, SEQ ID NOs: 103 and 104;

[0592] (l) correspondingly, SEQ ID NOs: 113 and 114;

[0593] (m) correspondingly, SEQ ID NOs: 123 and 124;

[0594] (n) correspondingly, SEQ ID NOs: 133 and 134;

[0595] (o) correspondingly, SEQ ID NOs: 143 and 144;

[0596] (p) SEQ ID NOs: 153 and 154, respectively;

[0597] (q) SEQ ID NOs: 163 and 164, respectively;

[0598] (r) correspondingly, SEQ ID NOs: 173 and 174;

[0599] (s) SEQ ID NOs: 183 and 184, respectively;

[0600] (t) SEQ ID NOs: 193 and 194, respectively; or

[0601] (u) correspondingly, SEQ ID NOs: 203 and 204.

[0602] 15. An isolated antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, wherein the antibody or antigen-binding fragment thereof comprises VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2 and VL CDR3 of an antibody selected from the group consisting of: 15461, 20500, 20501, 20502, 20502.1, 22208, 15462, 22213, 15465, 20506, 15483, 20513, 22216, 15489, 20516, 15472, 15503, 15495, 15478, 15441 and 20496.

[0603] 16. The antibody or antigen-binding fragment thereof according to embodiment 15, wherein the CDR is a Kabat-defined CDR, a Chothia-defined CDR, or an AbM-defined CDR.

[0604] 17. An isolated antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, wherein the antibody or antigen-binding fragment thereof comprises VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2 and VL CDR3 according to the Chothia definition of 20502.1 or the AbM definition.

[0605] 18. An isolated antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, the isolated antibody or antigen-binding fragment thereof comprising the heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 458-463, respectively.

[0606] 19. An antibody or antigen-binding fragment thereof according to embodiment 18, wherein (i) the antibody or antigen-binding fragment thereof comprises a variable heavy chain region comprising the amino acid sequence of SEQ ID NO: 464 and a variable light chain region comprising the amino acid sequence of SEQ ID NO: 42; or (ii) the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 469 and a light chain comprising the amino acid sequence of SEQ ID NO: 44.

[0607] 20. An isolated antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, the isolated antibody or antigen-binding fragment thereof comprising the heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 35-40, respectively.

[0608] 21. An antibody or antigen-binding fragment thereof according to embodiment 20, wherein (i) the antibody or antigen-binding fragment thereof comprises a variable heavy chain region comprising the amino acid sequence of SEQ ID NO:41 and a variable light chain region comprising the amino acid sequence of SEQ ID NO:42; or (ii) the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:43 and a light chain comprising the amino acid sequence of SEQ ID NO:44.

[0609] 22. An isolated antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, the isolated antibody or antigen-binding fragment thereof comprising the heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 65-70, respectively.

[0610] 23. An antibody or antigen-binding fragment thereof according to embodiment 22, wherein (i) the antibody or antigen-binding fragment thereof comprises a variable heavy chain region comprising the amino acid sequence of SEQ ID NO:71 and a variable light chain region comprising the amino acid sequence of SEQ ID NO:72; or (ii) wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:73 and a light chain comprising the amino acid sequence of SEQ ID NO:74.

[0611] 24. An isolated antibody or antigen-binding fragment thereof that binds to the same epitope of human B7-H4 as the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 23.

[0612] 25. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 24, wherein the antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof.

[0613] 26. The antibody or antigen-binding fragment thereof according to any one of embodiments 1, 15 to 20, 22 or 24, wherein the antibody or antigen-binding fragment thereof is a murine, humanized or chimeric antibody or antigen-binding fragment thereof.

[0614] 27. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 26, wherein the antibody or antigen-binding fragment thereof induces T cell proliferation.

[0615] 28. The antibody or antigen-binding fragment thereof according to embodiment 27, wherein the antibody or antigen-binding fragment increases T cell proliferation by at least 21% compared to treatment with a control antibody.

[0616] 29. The antibody or antigen-binding fragment thereof according to embodiment 27, wherein the antibody or antigen-binding fragment increases T cell proliferation by about 5% to about 35% compared to treatment with a control antibody.

[0617] 30. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 29, wherein the antibody or antigen-binding fragment thereof induces CD4+ T cell proliferation.

[0618] 31. The antibody or antigen-binding fragment thereof according to embodiment 30, wherein the antibody or antigen-binding fragment thereof increases CD4+ T cell proliferation by at least 9% compared to treatment with a control antibody.

[0619] 32. The antibody or antigen-binding fragment thereof according to embodiment 30, wherein the antibody or antigen-binding fragment thereof increases CD4+ T cell proliferation by about 5% to about 15% compared to treatment with a control antibody.

[0620] 33. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 32, wherein the antibody or antigen-binding fragment thereof induces CD8+ T cell proliferation.

[0621] 34. The antibody or antigen-binding fragment thereof according to embodiment 33, wherein the antibody or antigen-binding fragment thereof increases CD8+ T cell proliferation by at least 11% compared to treatment with a control antibody.

[0622] 35. The antibody or antigen-binding fragment thereof according to embodiment 34, wherein the antibody or antigen-binding fragment thereof increases CD8+ T cell proliferation by about 5% to about 15% compared to treatment with a control antibody.

[0623] 36. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 35, wherein the antibody or antigen-binding fragment thereof induces interferon-γ (IFNγ) production.

[0624] 37. An antibody or antigen-binding fragment thereof according to embodiment 36, wherein the antibody or antigen-binding fragment thereof is capable of increasing IFNγ production by at least 2 times, at least 3 times, at least 4 times and at least 5 times, at least 6 times, at least 7 times, at least 8 times, about 2 times to about 10 times, or about 3 times to about 10 times.

[0625] 38. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 37, wherein the antibody or antigen-binding fragment thereof is capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) in cells expressing B7-H4.

[0626] 39. The antibody or antigen-binding fragment thereof of embodiment 38, wherein the antibody or antigen-binding fragment thereof induces at least 20%, at least 30%, at least 40%, about 20% to about 50%, or about 30% to about 50% specific lysis of cells expressing B7-H4.

[0627] 40. The antibody or antigen-binding fragment thereof of any one of embodiments 1 to 39, wherein the antibody or antigen-binding fragment thereof inhibits tumor growth in a murine CT26 colorectal cancer model, a murine breast cancer 4T1 model, or a melanoma cell line B16-moB7-H4 / H3 model.

[0628] 41. The antibody or antigen-binding fragment thereof of embodiment 40, wherein the antibody or antigen-binding fragment thereof reduces tumor growth by at least 25%, at least 30%, at least 40%, at least 45% or at least 50% compared to treatment with a control antibody.

[0629] 42. An antibody or antigen-binding fragment thereof according to any one of embodiments 27 to 41, wherein the induction of T cell proliferation, the induction of CD4+T cell proliferation, the induction of CD8+T cell proliferation, the induction of IFNγ production, the ADCC activity and / or the inhibition of tumor growth are dose-dependent.

[0630] 43. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 42, wherein the antibody or antigen-binding fragment thereof binds to cynomolgus monkey B7-H4.

[0631] 44. The antibody or antigen-binding fragment thereof of any one of embodiments 1 to 43, wherein the antibody or antigen-binding fragment thereof binds to rat B7-H4.

[0632] 45. The antibody or antigen-binding fragment thereof of any one of embodiments 1 to 44, wherein the antibody or antigen-binding fragment thereof binds to mouse B7-H4.

[0633] 46. ​​The antibody or antigen-binding fragment thereof of any one of embodiments 1 to 45, wherein the antibody or antigen-binding fragment thereof binds to the IgV domain of human B7-H4.

[0634] 47. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 46, wherein the antibody or antigen-binding fragment thereof is afucosylated.

[0635] 48. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 47, which is a full-length antibody.

[0636] 49. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 47, which is an antigen-binding fragment.

[0637] 50. An antigen binding fragment according to embodiment 49, wherein the antigen binding fragment comprises Fab, Fab', F(ab')2, single-chain Fv (scFv), disulfide-linked Fv, V-NAR domain, IgNar, intrabody, IgGΔCH2, minibody, F(ab')3, tetrafunctional antibody, trifunctional antibody, bifunctional antibody, single domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2 or scFv-Fc.

[0638] 51. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 50, further comprising a detectable label.

[0639] 52. An isolated polynucleotide comprising a nucleic acid molecule encoding the heavy chain variable region or the heavy chain of the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 51.

[0640] 53. The isolated polynucleotide of embodiment 52, wherein the nucleic acid molecule encodes the VH of SEQ ID NO: 11, 21, 31, 41, 464, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, or 201, or the heavy chain of SEQ ID NO: 13, 23, 33, 43, 469, 53, 63, 73, 83, 93, 103, 113, 123, 133, 143, 153, 163, 173, 183, 193, or 203.

[0641] 54. The isolated polynucleotide of embodiment 52, wherein the nucleic acid molecule comprises the sequence of SEQ ID NO: 213, 223, 233, 243, 470, 253, 263, 273, 283, 293, 303, 313, 323, 333, 343, 353, 363, 373, 383, 393 or 403.

[0642] 55. An isolated polynucleotide according to embodiment 52, wherein the nucleic acid molecule comprises (i) a sequence of SEQ ID NO: 213, 223, 233, 243, 470, 253, 263, 273, 283, 293, 303, 313, 323, 333, 343, 353, 363, 373, 383, 393 or 403; and (ii) a sequence of SEQ ID NO: 408.

[0643] 56. An isolated polynucleotide comprising a nucleic acid molecule encoding the light chain variable region or the light chain of the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 51.

[0644] 57. The isolated polynucleotide of embodiment 56, wherein the nucleic acid molecule encodes the VL of SEQ ID NO: 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182, 192, or 202, or the light chain of SEQ ID NO: 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, 114, 124, 134, 144, 154, 164, 174, 184, 194, or 204.

[0645] 58. An isolated polynucleotide according to embodiment 56, wherein the nucleic acid molecule comprises the sequence of SEQ ID NO:214, 224, 234, 244, 254, 264, 274, 284, 294, 304, 314, 324, 334, 344, 354, 364, 374, 384, 394 or 404.

[0646] 59. An isolated polynucleotide according to embodiment 56, wherein the nucleic acid molecule comprises (i) a sequence of SEQ ID NO: 214, 224, 234, 244, 254, 264, 274, 284, 294, 304, 314, 324, 334, 344, 354, 364, 374, 384, 394 or 404; and (ii) a sequence of SEQ ID NO: 406.

[0647] 60. An isolated polynucleotide comprising a nucleic acid molecule encoding a heavy chain variable region or a heavy chain of an antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 51 and a light chain variable region or a light chain of an antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 51.

[0648] 61. An isolated vector comprising the polynucleotide according to any one of embodiments 52 to 60.

[0649] 62. A host cell comprising a polynucleotide according to any one of embodiments 52 to 60, a vector according to embodiment 61, or a first vector comprising a polynucleotide according to any one of embodiments 52 to 55 and a second vector comprising a polynucleotide according to any one of embodiments 56 to 59.

[0650] 63. According to the host cell described in embodiment 62, the host cell is selected from the group consisting of: Escherichia coli, Pseudomonas, Bacillus, Streptococcus, yeast, CHO, YB / 20, NSO, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, Hep G2, SP2 / 0, R1.1, BW, LM, COS1, COS 7, BSC1, BSC40, BMT10 cells, plant cells in tissue culture, insect cells and human cells.

[0651] 64. The host cell of embodiment 62 or 63, wherein the host cell lacks a functional α-1,6-fucosyltransferase gene (FUT8).

[0652] 65. A method of producing an antibody or antigen-binding fragment thereof that binds to human B7-H4, the method comprising culturing the host cell of any one of embodiments 62 to 64, thereby expressing nucleic acid and producing the antibody or antigen-binding fragment thereof.

[0653] 66. An isolated antibody or antigen-binding fragment thereof that specifically binds to human B7-H4 and is encoded by the polynucleotide of any one of embodiments 52 to 60.

[0654] 67. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 51 or 66, and a pharmaceutically acceptable excipient.

[0655] 68. A pharmaceutical composition comprising (i) an antibody or antigen-binding fragment according to any one of embodiments 1 to 51 or 66, and (ii) a pharmaceutically acceptable excipient, wherein at least 95% of the antibody or antigen-binding fragment thereof in the composition is afucosylated.

[0656] 69. A pharmaceutical composition comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4 and comprises the heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 458-460, respectively; and (ii) a pharmaceutically acceptable excipient, wherein at least 95% of the antibody or antigen-binding fragment thereof in the composition is afucosylated.

[0657] 70. A pharmaceutical composition according to embodiment 69, wherein (i) the antibody or antigen-binding fragment thereof comprises a variable heavy chain region comprising the amino acid sequence of SEQ ID NO: 464 and a variable light chain region comprising the amino acid sequence of SEQ ID NO: 42; or (ii) the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 469 and a light chain comprising the amino acid sequence of SEQ ID NO: 44.

[0658] 71. A pharmaceutical composition comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4 and comprises the heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2 and CDR3 sequences of SEQ ID NO:35-40, respectively; and (ii) a pharmaceutically acceptable excipient, wherein at least 95% of the antibody or antigen-binding fragment thereof in the composition is afucosylated.

[0659] 72. A pharmaceutical composition according to embodiment 71, wherein (i) the antibody or antigen-binding fragment thereof comprises a variable heavy chain region comprising the amino acid sequence of SEQ ID NO:41 and a variable light chain region comprising the amino acid sequence of SEQ ID NO:42; or (ii) the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:43 and a light chain comprising the amino acid sequence of SEQ ID NO:44.

[0660] 73. A pharmaceutical composition comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4 and comprises the heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 65-70, respectively; and (ii) a pharmaceutically acceptable excipient, wherein at least 95% of the antibody or antigen-binding fragment thereof in the composition is afucosylated.

[0661] 74. A pharmaceutical composition according to embodiment 73, wherein (i) the antibody or antigen-binding fragment thereof comprises a variable heavy chain region comprising the amino acid sequence of SEQ ID NO:71 and a variable light chain region comprising the amino acid sequence of SEQ ID NO:72; or (ii) wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:73 and a light chain comprising the amino acid sequence of SEQ ID NO:74.

[0662] 75. The pharmaceutical composition of any one of embodiments 67 to 74, wherein fucosylation is not detectable in the composition.

[0663] 76. The pharmaceutical composition of any one of Embodiments 67 to 75, further comprising an anti-PD-1 antibody or an antigen-binding fragment thereof.

[0664] 77. The pharmaceutical composition of embodiment 76, wherein the anti-PD-1 antibody or antigen-binding fragment thereof is nivolumab or pembrolizumab.

[0665] 78. The pharmaceutical composition according to embodiment 76, wherein the anti-PD-1 antibody or antigen-binding fragment thereof is AMP-514, carrelizumab, tislelizumab and sparlivizumab.

[0666] 79. The pharmaceutical composition of any one of Embodiments 67 to 75, further comprising an anti-PD-L1 antibody or an antigen-binding fragment thereof.

[0667] 80. A method for inducing T cell proliferation, the method comprising contacting the T cells with the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 51 or 66, or the pharmaceutical composition according to any one of embodiments 67 to 79.

[0668] 81. The method of embodiment 80, wherein T cell proliferation is reduced by at least 30%, at least 40%, at least 50%, at least 60%, at least 70% or at least 80%.

[0669] 82. A method for inducing CD4+ T cell proliferation, the method comprising contacting CD4+ T cells with the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 51 or 66, or the pharmaceutical composition according to any one of embodiments 67 to 79.

[0670] 83. The method of embodiment 82, wherein CD4+ T cell proliferation is reduced by at least 30%, at least 40%, at least 50%, at least 60%, at least 70% or at least 80%.

[0671] 84. A method for inducing CD8+ T cell proliferation, the method comprising contacting CD8+ T cells with the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 51 or 66, or the pharmaceutical composition according to any one of embodiments 67 to 79.

[0672] 85. The method of embodiment 84, wherein CD8+ T cell proliferation is reduced by at least 30%, at least 40%, at least 50%, at least 60%, at least 70% or at least 80%.

[0673] 86. A method for inducing interferon gamma production, the method comprising contacting T cells with the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 51 or 66, or the pharmaceutical composition according to any one of embodiments 67 to 79.

[0674] 87. The method of embodiment 86, wherein interferon gamma production is increased by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80%.

[0675] 88. A method for killing a cell expressing B7-H4, the method comprising contacting the cell with the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 51 or 66, or the pharmaceutical composition according to any one of embodiments 67 to 79.

[0676] 89. A method for depleting cells expressing B7-H4 in a cell population, the method comprising contacting the cell population with an antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 51 or 66, or a pharmaceutical composition according to any one of embodiments 67 to 79.

[0677] 90. The method of embodiment 88 or 89, wherein said killing or said depletion occurs by ADCC.

[0678] 91. According to any one of embodiments 80 to 90, the method further comprises contacting the T cell, the CD4+T cell, the CD8+T cell, the cell or the cell population with an anti-PD-1 antibody or an antigen-binding fragment thereof.

[0679] 92. The method of embodiment 91, wherein the contacting with the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 51 or 66 or the pharmaceutical composition according to any one of embodiments 67 to 78 and the contacting with the anti-PD-1 antibody or antigen-binding fragment thereof occur simultaneously.

[0680] 93. The method of embodiment 91, wherein the contacting with the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 51 or 66 or the pharmaceutical composition according to any one of embodiments 67 to 78 and the contacting with the anti-PD-1 antibody or antigen-binding fragment thereof occur sequentially.

[0681] 94. The method of any one of embodiments 80 to 90, further comprising contacting the T cell, the CD4+T cell, the CD8+T cell, the cell or the cell population with an anti-PD-L1 antibody or an antigen-binding fragment thereof.

[0682] 95. The method of embodiment 94, wherein said contacting with the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 51 or 66 or the pharmaceutical composition according to any one of embodiments 67 to 75 and said contacting with the anti-PD-L1 antibody or antigen-binding fragment thereof occur simultaneously.

[0683] 96. The method of embodiment 94, wherein the contacting with the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 51 or 66 or the pharmaceutical composition according to any one of embodiments 67 to 75 and the contacting with the anti-PD-L1 antibody or antigen-binding fragment thereof occur sequentially.

[0684] 97. The method of any one of embodiments 80 to 96, wherein the contacting is performed in vitro.

[0685] 98. The method of any one of embodiments 80 to 96, wherein the contacting is performed in a subject.

[0686] 99. A method of treating a B7-H4-expressing cancer in a subject, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 51 or 66, or the pharmaceutical composition according to any one of Embodiments 67 to 79.

[0687] 100. The method of embodiment 99, wherein the cancer is selected from the group consisting of breast cancer, ductal carcinoma, endometrial cancer, ovarian cancer, non-small cell lung cancer, pancreatic cancer, thyroid cancer, kidney cancer, and bladder cancer.

[0688] 101. The method of embodiment 100, wherein the breast cancer is triple-negative breast cancer or wherein the non-small cell lung cancer is squamous cell carcinoma.

[0689] 102. The method of embodiment 100, wherein the non-small cell lung cancer is adenocarcinoma.

[0690] 103. The method of embodiment 99, wherein the cancer is selected from the group consisting of head and neck cancer, small cell lung cancer, gastric cancer, and melanoma.

[0691] 104. The method of embodiment 100, wherein the cancer is ovarian cancer and the ovarian cancer is serous adenocarcinoma.

[0692] 105. The method of embodiment 101, wherein the cancer is breast cancer and the breast cancer is ductal adenocarcinoma.

[0693] 106. The method of any one of embodiments 99 to 105, wherein the cancer is a PD-1 inhibitor-inadequately responsive cancer.

[0694] 107. The method of any one of embodiments 99 to 106, wherein the cancer is a PD-L1 inhibitor-inadequately responsive cancer.

[0695] 108. The method of any one of embodiments 99 to 107, wherein the cancer expresses a lower amount of PD-L1.

[0696] 109. The method of any one of embodiments 99 to 108, wherein the subject is human.

[0697] 110. The method of any one of embodiments 99 to 109, further comprising administering an anti-PD-1 antibody or an antigen-binding fragment thereof to the subject.

[0698] 111. The method according to embodiment 110, wherein administration of the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 51 or 66 or the pharmaceutical composition according to any one of embodiments 67 to 78 occurs simultaneously with administration of the anti-PD-1 antibody or antigen-binding fragment thereof.

[0699] 112. The method according to embodiment 110, wherein administration of the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 51 or 66 or the pharmaceutical composition according to any one of embodiments 67 to 78 occurs sequentially with administration of the anti-PD-1 antibody or antigen-binding fragment thereof.

[0700] 113. The method of embodiment 112, wherein administration of the anti-PD-1 antibody or antigen-binding fragment thereof occurs after administration of the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 51 or 66 or the pharmaceutical composition according to any one of embodiments 67 to 78.

[0701] 114. The method of any one of embodiments 91 to 93, 97, 98, and 110 to 113, wherein the anti-PD-1 antibody or antigen-binding fragment thereof is nivolumab or pembrolizumab.

[0702] 115. The method of any one of embodiments 91 to 93, 97, 98, and 110 to 113, wherein the anti-PD-1 antibody or antigen-binding fragment thereof is AMP-514, camrelizumab, tislelizumab, or sparlivizumab.

[0703] 116. A method for detecting B7-H4 in a sample, the method comprising contacting the sample with the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 51 or 66.

[0704] 117. A method according to embodiment 116, wherein the sample is obtained from a cancer in a human subject.

[0705] 118. A kit comprising the antibody or antigen-binding fragment thereof of any one of embodiments 1 to...

Claims

1. An isolated antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, the isolated antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3, and a light chain variable region (VL) CDR1, CDR2, and CDR3 sequence selected from the group consisting of: (a) correspondingly, SEQ ID NO:458-463; (b) correspondingly, SEQ ID NO: 5-10; (c) correspondingly, SEQ ID NO: 15-20; (d) correspondingly, SEQ ID NO: 25-30; (e) correspondingly, SEQ ID NO:35-40; (f) correspondingly, SEQ ID NO:45-50; (g) correspondingly, SEQ ID NO:55-60; (h) correspondingly, SEQ ID NO: 65-70; (i) correspondingly, SEQ ID NO:75-80; (j) correspondingly, SEQ ID NO:85-90; (k) correspondingly, SEQ ID NO:95-100; (l) correspondingly, SEQ ID NO: 105-110; (m) correspondingly, SEQ ID NO: 115-120; (n) correspondingly, SEQ ID NO: 125-130; (o) correspondingly, SEQ ID NO: 135-140; (p) correspondingly, SEQ ID NO: 145-150; (q) correspondingly, SEQ ID NO: 155-160; (r) correspondingly, SEQ ID NO: 165-170; (s) correspondingly, SEQ ID NO: 175-180; (t) correspondingly, SEQ ID NO: 185-190; and (u) correspondingly, SEQ ID NO:195-200.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 11, 21, 31, 41, 464, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191 or 201.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises a VL comprising the amino acid sequence of SEQ ID NO: 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182, 192 or 202.

4. An isolated antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 11, 21, 31, 41, 464, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191 or 201.

5. An isolated antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182, 192 or 202.

6. An isolated antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, the isolated antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region comprising the following amino acid sequence: (a) SEQ ID NOs: 464 and 42, respectively; (b) correspondingly, SEQ ID NOs: 11 and 12; (c) correspondingly, SEQ ID NOs: 21 and 22; (d) correspondingly, SEQ ID NOs: 31 and 32; (e) correspondingly, SEQ ID NOs: 41 and 42; (f) correspondingly, SEQ ID NOs: 51 and 52; (g) correspondingly, SEQ ID NOs: 61 and 62; (h) correspondingly, SEQ ID NOs: 71 and 72; (i) correspondingly, SEQ ID NOs: 81 and 82; (j) correspondingly, SEQ ID NOs: 91 and 92; (k) correspondingly, SEQ ID NOs: 101 and 102; (l) correspondingly, SEQ ID NO: 111 and 112; (m) correspondingly, SEQ ID NOs: 121 and 122; (n) correspondingly, SEQ ID NOs: 131 and 132; (o) correspondingly, SEQ ID NOs: 141 and 142; (p) SEQ ID NOs: 151 and 152, respectively; (q) SEQ ID NOs: 161 and 162, respectively; (r) correspondingly, SEQ ID NOs: 171 and 172; (s) SEQ ID NOs: 181 and 182, respectively; (t) SEQ ID NOs: 191 and 192, respectively; or (u) correspondingly, SEQ ID NO: 201 and 202.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antibody or antigen-binding fragment further comprises a heavy chain constant region.

8. The antibody or antigen-binding fragment thereof according to claim 7, wherein the heavy chain constant region is selected from the group consisting of human immunoglobulin IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2 heavy chain constant regions.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the antibody or antigen-binding fragment further comprises a light chain constant region.

10. The antibody or antigen-binding fragment thereof according to claim 9, wherein the light chain constant region is selected from the group consisting of human immunoglobulin IgGκ and IgGλ light chain constant regions.

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