B7-H4 antibody and method of use thereof

Abstract

To provide a B7-H4 antibody and a method for using it. [Solution] This disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to human B7-H4 (and optionally to cynomolgus monkey, mouse, and / or rat B7-H4), as well as compositions comprising such antibodies or antigen-binding fragments. In certain embodiments, antibodies or antigen-binding fragments that specifically bind to human B7-H4 increase T cell proliferation, increase interferon-γ production, and / or deplete B7-H4 expressing cells via ADCC activity. This disclosure also provides a method for treating disorders such as cancer by administering antibodies or antigen-binding fragments that specifically bind to human B7-H4.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits of U.S. Provisional Application No. 62 / 550,173 filed on 25 August 2017, U.S. Provisional Application No. 62 / 579,774 filed on 31 October 2017, U.S. Provisional Application No. 62 / 607,810 filed on 19 December 2017, and U.S. Provisional Application No. 62 / 656,789 filed on 12 April 2018, which are incorporated herein by reference in their entirety.

[0002] Reference to sequence listings submitted electronically via EFS-WEB The contents of the electronically submitted sequence listing (filename: 3986.0090004_SL_ST25.txt; size: 331,354 bytes; creation date: August 3, 2018) are incorporated herein by reference in their entirety. [Background technology]

[0003] Technical field This disclosure relates to antibodies that specifically bind to human B7-H4 compositions containing such antibodies, and to methods for producing and using antibodies that specifically bind to B7-H4.

[0004] Explanation of related technologies 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. This molecule is a type I transmembrane protein containing both IgV and IgC external domains. While B7-H4 expression at protein levels is relatively limited in healthy tissues, it is expressed in several solid tumors, such as gynecological cancers including breast, ovarian, and endometrial cancers. B7-H4 expression in tumors tends to correlate with poor prognosis. The receptor for B7-H4 is unknown, but it is thought to be expressed on T cells. B7-H4 is thought to directly inhibit T cell activity.

[0005] Taking into account the expression and function of B7-H4, this specification provides antibodies that specifically bind to B7-H4, and the use of these antibodies to modulate B7-H4 activity, including, for example, in the treatment of cancer. [Overview of the project] [Means for solving the problem]

[0006] In this specification, the following are selected from the group including: 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; 105-110; 115-120; 125-130; 135-140; 145-150; 155-160; 165-170; 175-180; 185-190; and 195-200, respectively: Heavy Chain Variable Region (VH) Complementarity Determination Region (CDR) 1, VH CDR 2, VH The present invention provides an isolated antibody or its antigen-binding fragment that specifically binds to human B7-H4, having CDR3 and light chain variable regions (VL) CDR1, CDR2, and CDR3 sequences.

[0007] In one embodiment, the antibody or its antigen-binding fragment contains a VH having the amino acid sequence of SEQ ID NOs: 11, 21, 31, 41, 464, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, or 201.

[0008] In one embodiment, the antibody or its antigen-binding fragment includes a VL having the amino acid sequence of SEQ ID NOs: 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182, 192, or 202.

[0009] In one embodiment, the antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region having the amino acid sequence of SEQ ID NOs: 11, 21, 31, 41, 464, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, or 201.

[0010] This specification also provides isolated antibodies or antigen-binding fragments thereof that specifically bind to human B7-H4, the antibodies comprising a heavy chain variable region and a light chain variable region, the light chain variable region having the amino acid sequence of SEQ ID NOs: 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182, 192, or 202.

[0011] The Specified Specification also provides isolated antibodies or antigen-binding fragments thereof that specifically bind to human B7-H4, comprising heavy chain variable regions and light chain variable regions having the amino acid sequences of: 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: 181 and 182; SEQ ID NOs: 191 and 192; or SEQ ID NOs: 201 and 202, respectively.

[0012] In one embodiment, the antibody or its antigen-binding fragment further comprises a heavy chain constant region. In one embodiment, 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.

[0013] 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.

[0014] In one embodiment, the antibody or antigen-binding fragment further comprises a heavy chain constant region and a light chain constant region, the heavy chain constant region is a human IgG1 heavy chain constant region, and the light chain constant region is a human IgGκ light chain constant region.

[0015] In one embodiment, the antibody or its antigen-binding fragment comprises a heavy chain having 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.

[0016] In one embodiment, the antibody or its antigen-binding fragment comprises a light chain having 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.

[0017] In one embodiment, the antibody or its antigen-binding fragment is: SEQ ID NO: 13 and 14 respectively; SEQ ID NO: 23 and 24 respectively; SEQ ID NO: 33 and 34 respectively; SEQ ID NO: 43 and 44 respectively; SEQ ID NO: 469 and 44 respectively; SEQ ID NO: 53 and 54 respectively; SEQ ID NO: 6 and 64 respectively; SEQ ID NO: 73 and 74 respectively; SEQ ID NO: 83 and 84 respectively; SEQ ID NO: 93 and 94 respectively; SEQ ID NO: 103 and 104 respectively; SEQ ID NO: 113 and 114 respectively; SEQ ID NO: 123 and 124 respectively; SEQ ID NO: 133 and 134 respectively; SEQ ID NO: 143 and 144 respectively; SEQ ID NO: 153 and 154 respectively; SEQ ID NO: 163 and 164 respectively; SEQ ID NO: 173 and 174 respectively; SEQ ID NO: 183 and 184 respectively; SEQ ID NO: 193 and 194 respectively; or SEQ ID NO: 203 and 204 respectively, and comprises heavy and light chains having the amino acid sequences.

[0018] This specification also provides isolated antibodies or antigen-binding fragments thereof that specifically bind to human B7-H4, and these antibodies or antigen-binding fragments include VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of antibodies 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, CDR is a Kabat-defined CDR, a Chothia-defined CDR, or an AbM-defined CDR.

[0019] Furthermore, this specification provides isolated antibodies or antigen-binding fragments thereof that specifically bind to human B7-H4, each having 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, respectively, as of SEQ ID NOs.458 to 463. In one embodiment, (i) the antibody or antigen-binding fragment comprises a variable heavy chain region having the amino acid sequence of SEQ ID NO.464 and a variable light chain region having the amino acid sequence of SEQ ID NO.42, or (ii) the antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO.469 and a light chain having the amino acid sequence of SEQ ID NO.44.

[0020] This specification also provides isolated antibodies or antigen-binding fragments thereof that specifically bind to human B7-H4, each having 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, respectively, as of SEQ ID NOs.35 to 40. In one embodiment, (i) the antibody or antigen-binding fragment comprises a variable heavy chain region having the amino acid sequence of SEQ ID NOs.41 and a variable light chain region having the amino acid sequence of SEQ ID NOs.42, or (ii) the antibody comprises a heavy chain having the amino acid sequence of SEQ ID NOs.43 and a light chain having the amino acid sequence of SEQ ID NOs.44.

[0021] This specification also provides isolated antibodies or antigen-binding fragments thereof that specifically bind to human B7-H4, each having 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, respectively, as of SEQ ID NOs.65 to 70.In one embodiment, (i) the antibody or antigen-binding fragment comprises a variable heavy chain region having the amino acid sequence of SEQ ID NOs.71 and a variable light chain region having the amino acid sequence of SEQ ID NOs.72, or (ii) the antibody comprises a heavy chain having the amino acid sequence of SEQ ID NOs.73 and a light chain having the amino acid sequence of SEQ ID NOs.74.

[0022] This specification also provides an isolated antibody or antigen-binding fragment thereof that binds to the same human B7-H4 epitope as the antibody or antigen-binding fragment described in any one of claims 1 to 20. In one embodiment, the antibody or antigen-binding fragment binds to the same human B7-H4 epitope determined by SPR.

[0023] In one embodiment, the antibody or its antigen-binding fragment is a human antibody or its antigen-binding fragment. In another embodiment, the antibody or its antigen-binding fragment is a mouse, humanized, or chimeric antibody or its antigen-binding fragment.

[0024] In one embodiment, the antibody or its antigen-binding fragment 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 approximately 5% to approximately 35% compared to treatment with a control antibody. In one embodiment, the antibody or its antigen-binding fragment induces CD4+ T cell proliferation. In one embodiment, the antibody or its antigen-binding fragment increases CD4+ T cell proliferation by at least 9% compared to treatment with a control antibody. In one embodiment, the antibody or its antigen-binding fragment increases CD4+ T cell proliferation by approximately 5% to approximately 15% compared to treatment with a control antibody. In one embodiment, the antibody or its antigen-binding fragment induces CD8+ T cell proliferation. In one embodiment, the antibody or its 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 its antigen-binding fragment increases CD8+ T cell proliferation by approximately 5% to approximately 15% compared to treatment with a control antibody.

[0025] 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 twofold, at least threefold, at least fourfold, and at least fivefold, at least sixfold, at least sevenfold, at least eightfold, about twofold to about tenfold, or about threefold to about tenfold.

[0026] In one embodiment, an antibody or its antigen-binding fragment can induce antibody-dependent cell-mediated cytotoxicity (ADCC) in B7-H4 expressing cells. In one embodiment, the antibody or its antigen-binding fragment induces specific lysis in at least 20%, at least 30%, at least 40%, about 20% to about 50%, or about 30% to about 50% of B7-H4 expressing cells.

[0027] In one embodiment, the antibody or its antigen-binding fragment inhibits tumor growth in a mouse CT26 colorectal cancer model, a mouse mammary cancer 4T1 model, or a melanoma cell line B16-mouse B7-H4 / H3 model. In one embodiment, the antibody or its antigen-binding fragment 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.

[0028] In one embodiment, the induction of T cell proliferation, CD4+ T cell proliferation, CD8+ T cell proliferation, induction of IFNγ production, ADCC activity, and / or inhibition of tumor growth are dose-dependent.

[0029] In one embodiment, the antibody or its antigen-binding fragment binds to cynomolgus monkey B7-H4. In another embodiment, the antibody or its antigen-binding fragment binds to rat B7-H4. In another embodiment, the antibody or its antigen-binding fragment binds to mouse B7-H4. In yet another embodiment, the antibody or its antigen-binding fragment binds to human B7-H4, cynomolgus monkey B7-H4, rat B7-H4, and mouse B7-H4.

[0030] In one embodiment, the antibody or its antigen-binding fragment binds to the IgV domain of human B7-H4.

[0031] In one embodiment, the antibody or its antigen-binding fragment is afucosylated.

[0032] In one embodiment, the antibody or its antigen-binding fragment is a full-length antibody. In one embodiment, the antibody or its antigen-binding fragment is an antigen-binding fragment. In one embodiment, the antigen-binding fragment contains Fab, Fab', F(ab')2, single-chain Fv(scFv), disulfide-bonded Fv, V-NAR domain, IgNar, intracellular antibody, IgGΔCH2, minibody, F(ab')3, tetrabody, triabody, diabody, single-domain antibody, DVD-Ig, Fcab, mAb 2 This includes (scFv)2 or scFv-Fc.

[0033] In one embodiment, the antibody or its antigen-binding fragment further has a detectable label.

[0034] This specification also provides isolated polynucleotides comprising a nucleic acid molecule encoding a heavy chain variable region or heavy chain of an antibody or antigen-binding fragment provided herein. In one embodiment, the nucleic acid molecule encodes the VH of SEQ ID NOs: 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 NOs: 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 has the sequence of SEQ ID NOs: 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 has (i) the sequence of SEQ ID NOs: 213, 223, 233, 243, 470, 253, 263, 273, 283, 293, 303, 313, 323, 333, 343, 353, 363, 373, 383, 393, or 403, and (ii) the sequence of SEQ ID NO: 408.

[0035] This specification also provides isolated polynucleotides comprising a light chain variable region or a nucleic acid molecule encoding a light chain of an antibody or antigen-binding fragment provided herein. In one embodiment, the nucleic acid molecule encodes the VL of SEQ ID NOs: 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 NOs: 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 has the sequence of SEQ ID NOs: 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 has (i) the sequence of SEQ ID NOs: 214, 224, 234, 244, 254, 264, 274, 284, 294, 304, 314, 324, 334, 344, 354, 364, 374, 384, 394, or 404, and (ii) the sequence of SEQ ID NO: 406.

[0036] This specification also provides isolated polynucleotides comprising nucleic acid molecules encoding the heavy chain variable region or heavy chain of an antibody or antigen-binding fragment provided herein, and the light chain variable region or light chain of an antibody or antigen-binding fragment.

[0037] This specification also provides isolated vectors containing polynucleotides provided herein.

[0038] This specification also provides a host cell comprising a polynucleotide provided herein, a vector provided herein, a first vector having one polynucleotide provided herein (e.g., a polynucleotide containing a variable heavy chain or a nucleic acid encoding a heavy chain), and a second vector having another polynucleotide provided herein (e.g., a polynucleotide containing a variable light chain or a nucleic acid encoding a light chain). In one embodiment, the host cell is a cell selected from the group consisting of E. coli, Pseudomonas, Bacillus, Streptomyces, yeast, CHO, YB / 20, NS0, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, HepG2, SP2 / 0, R1.1, BW, LM, COS1, COS7, BSC1, BSC40, BMT10 cells, plant cells, insect cells, and human cells in tissue culture. In one embodiment, the host cell is a CHO cell. In one embodiment, the host cell (e.g., a mammalian host cell such as a CHO cell) lacks the functional α-1,6-fucosyltransferase gene (FUT8).

[0039] This specification also provides a method (e.g., an in vitro method) for producing an antibody or antigen-binding fragment that binds to human B7-H4, comprising culturing a host cell provided herein to express a nucleic acid molecule and thereby produce an antibody or an antigen-binding fragment thereof.

[0040] This specification also provides isolated antibodies or antigen-binding fragments thereof that specifically bind to human B7-H4 and are encoded by the polynucleotides provided herein.

[0041] This specification also provides pharmaceutical compositions comprising antibodies or antigen-binding fragments thereof, polynucleotides thereof, vectors thereof, or host cells thereof; and pharmaceutically acceptable excipients.

[0042] This specification also provides pharmaceutical compositions 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 in the composition is afcosylated.

[0043] This specification also provides 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 in the composition is afcosylated.

[0044] This specification also refers to (i) heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, and VH CDR2, respectively, which specifically bind to human B7-H4 and correspond to sequence numbers 458-463. The present invention provides a pharmaceutical composition comprising (ii) an antibody or antigen-binding fragment having CDR3 and light chain variable regions (VL) CDR1, CDR2, and CDR3 sequences, 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 in the composition is afcosylated. In one embodiment, (i) the antibody or antigen-binding fragment comprises a variable heavy chain region having the amino acid sequence of SEQ ID NO: 464 and a variable light chain region having the amino acid sequence of SEQ ID NO: 42, or (ii) the antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 469 and a light chain having the amino acid sequence of SEQ ID NO: 44.

[0045] This specification also refers to (i) heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, and VH CDR2, respectively, which specifically bind to human B7-H4 and correspond to sequence numbers 458-463. The present invention provides a pharmaceutical composition comprising (ii) an antibody or its antigen-binding fragment having CDR3 and light chain variable regions (VL) CDR1, CDR2, and CDR3 sequences, and (ii) a pharmaceutically acceptable excipient, wherein at least 95% of the antibody or its antigen-binding fragment in the composition is afcosylated. In one embodiment, (i) the antibody or its antigen-binding fragment comprises a variable heavy chain region having the amino acid sequence of SEQ ID NO: 464 and a variable light chain region having the amino acid sequence of SEQ ID NO: 42, or (ii) the antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 469 and a light chain having the amino acid sequence of SEQ ID NO: 44.

[0046] This specification also provides a pharmaceutical composition comprising (i) an antibody or antigen-binding fragment having 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 to 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 in the composition is afucosylated. In one embodiment, (i) the antibody or antigen-binding fragment comprises a variable heavy chain region having the amino acid sequence of SEQ ID NO: 41 and a variable light chain region having the amino acid sequence of SEQ ID NO: 42, or (ii) the antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 43 and a light chain having the amino acid sequence of SEQ ID NO: 44.

[0047] This specification also provides pharmaceutical compositions comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4 and has 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, respectively, of SEQ ID NOs. 35 to 40, and (ii) a pharmaceutically acceptable excipient, wherein at least 95% of the antibody or antigen-binding fragment in the composition is afcosylated. In one embodiment, (i) the antibody or antigen-binding fragment comprises a variable heavy chain region having the amino acid sequence of SEQ ID NO: 41 and a variable light chain region having the amino acid sequence of SEQ ID NO: 42, or (ii) the antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 43 and a light chain having the amino acid sequence of SEQ ID NO: 44.

[0048] This specification also provides pharmaceutical compositions comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4 and has 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, respectively, of SEQ ID NOs. 65 to 70, 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 in the composition is afucosylated. In one embodiment, (i) the antibody or antigen-binding fragment comprises a variable heavy chain region having the amino acid sequence of SEQ ID NO: 71 and a variable light chain region having the amino acid sequence of SEQ ID NO: 72, or (ii) the antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 73 and a light chain having the amino acid sequence of SEQ ID NO: 74.

[0049] This specification also provides pharmaceutical compositions comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4 and has 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, respectively, of SEQ ID NOs. 65 to 70, and (ii) a pharmaceutically acceptable excipient, wherein at least 95% of the antibody or antigen-binding fragment in the composition is afcosylated. In one embodiment, (i) the antibody or antigen-binding fragment comprises a variable heavy chain region having the amino acid sequence of SEQ ID NO: 71 and a variable light chain region having the amino acid sequence of SEQ ID NO: 72, or (ii) the antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 73 and a light chain having the amino acid sequence of SEQ ID NO: 74.

[0050] In one embodiment, fucosylation is undetectable in the composition.

[0051] This specification also provides a method for inducing T cell proliferation, 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).

[0052] This specification also provides a method for inducing CD4+ T cell proliferation, 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).

[0053] This specification also provides a method for inducing CD8+ T cell proliferation, 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).

[0054] This specification also provides a method for inducing interferon-gamma production, 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, 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% (e.g., compared to treatment with a control antibody).

[0055] This specification also provides a method for killing B7-H4 expressing cells, comprising contacting B7-H4 expressing 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.

[0056] This specification also provides a method for depleting B7-H4 expressing cells from a cell population, 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.

[0057] In one embodiment, killing or depletion occurs via ADCC.

[0058] In one embodiment, contact is in vitro. In another embodiment, contact is inside the subject.

[0059] This specification also provides a method for treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody or its antigen-binding fragment 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 trinegative 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 trinegative breast cancer, or 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, the ovarian cancer is serous adenocarcinoma. In one embodiment, the breast cancer is ductal adenocarcinoma.

[0060] In one embodiment, the cancer is an inadequate responder to PD-1 inhibitors and / or PD-L1 inhibitors. In one embodiment, the cancer expresses low levels of PD-L1.

[0061] In one embodiment, the subject is a human.

[0062] This specification also provides a method for detecting B7-H4 in a sample, comprising contacting the sample with an antibody or an antigen-binding fragment thereof provided herein. In one embodiment, the sample is obtained from a cancer in a human subject.

[0063] This specification also provides antibodies or antigen-binding fragments thereof, polynucleotides provided herein, vectors provided herein, host cells provided herein, or pharmaceutical compositions provided herein, and kits comprising a) a detection reagent, b) a B7-H4 antigen, c) a notice reflecting approval for use or sale of human administration, or d) a combination thereof.

[0064] This specification also provides isolated antibodies or antigen-binding fragments, which inhibit the T-cell checkpoint blocking activity of B7-H4. In one embodiment, T-cell checkpoint blocking activity is measured by an increase in IL-2 production compared to control cells.

[0065] This specification also provides pharmaceutical compositions 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 compared to a control cell. [Brief explanation of the drawing]

[0066] [Figure 1A] It exhibits B7-H4 expression in various tumor tissues. (See Example 1). [Figure 1B] This shows the distribution rate of B7-H4 in various tumor types as determined by IHC (see Example 1). [Figure 2A] This demonstrates the binding of B7-H4 antibodies to human, cynomolgus monkey, or mouse B7-H4 expressing HEK293T cells. (See Example 6). [Figure 2B] This demonstrates the binding of B7-H4 antibodies to SK-BR-3 cells and HEK293T cells expressing mouse, cynomolgus monkey, or rat B7-H4. (See Example 6). [Figure 3A] The effect of the B7-H4 antibody on CD4+, CD8+, or total T cell proliferation is demonstrated. (See Example 7). [Figure 3B]This demonstrates the effect of the B7-H4 antibody on interferon-gamma (IFNγ) production. (See Example 7). [Figure 3C] The effect of the B7-H4 antibody on CD4+, CD8+, or total T cell proliferation and interferon-γ (IFNγ) production is demonstrated. (See Example 7). [Figure 3D] The effects of various concentrations of B7-H4 antibody on interferon-gamma (IFNγ) production are demonstrated. (See Example 7). [Figure 4] (Figure 4A) Shows the binding of fucosylation and afucosylation antibodies to B7-H4 on SK-BR-3 cells. (See Example 9). (Figure 4B) Shows the binding of fucosylation and afucosylation antibodies to HEK293T cells expressing mouse B7-H4. (See Example 9). (Figure 4C) Shows the binding of fucosylation and afucosylation antibodies to HEK293T cells expressing cynomolgus monkey B7-H4. (See Example 9). (Figure 4D) Shows the binding of fucosylation and afucosylation antibodies to HEK293T cells expressing rat B7-H4. (See Example 9). [Figure 5] (Figure 5A) Shows the binding of afucosylated B7-H4 antibody to the human Fcγ receptor IIIa (FcγRIIIa)V158 allele. (See Example 10). (Figure 5B) Shows the binding of fucosylated B7-H4 antibody to the human Fcγ receptor IIIa (FcγRIIIa)V158 allele. (See Example 10). [Figure 6A] The fucosylated and afucosylated B7-H4 antibodies exhibit T cell checkpoint blocking activity. (See Example 11). [Figure 6B] The T cell checkpoint ligand activity of the afucosylated B7-H4 antibody compared to isotype control-treated cells is demonstrated by measuring IL-2 production. (See Example 11). [Figure 7] The ADCC activity of fucosylated and afucosylated B7-H4 antibodies against B7-H4 expressing cell lines is demonstrated. (See Example 12). [Figure 8] The ADCC activity of fucosylated and afucosylated B7-H4 antibodies against cells with various B7-H4 expression levels is demonstrated (see Example 13). [Figure 9A] The in vivo antitumor efficacy of the B7-H4 antibody 20502 is demonstrated (see Example 14). [Figure 9B] The in vivo antitumor efficacy of B7-H4 antibody 22213 is demonstrated (see Example 14). [Figure 10] This demonstrates the in vivo antitumor efficacy of the mouse version of the B7-H4 antibody 20502 in 4T1 (breast cancer) and B16 (melanoma) cell-transplanted mice. (See Example 14). [Figure 11] This demonstrates the in vivo antitumor efficacy of afucosylated 20502 in an MX-1 human breast cancer xenograft model. The image shows human B7-H4 expression in MX-1 xenograft tumors (left) and mouse B7-H4 expression in 4T1 tumors (right). (See Example 14). [Figure 12] (Figure 12A) Shows the in vivo antitumor efficacy of 20502-mouse IgG2a-F administered on the same day as the anti-PD-1 antibody. (See Example 15). * indicates p<0.05; **** indicates p<0.0001. (Figure 12B) Shows the in vivo antitumor efficacy of 20502-mouse IgG2a-F administered on the same day as the anti-PD-1 antibody. (See Example 15). * indicates p<0.05; **** indicates p<0.0001. (Figure 12C) Shows the in vivo antitumor efficacy of 20502-mouse IgG2a-F administered on the same day as the anti-PD-1 antibody. (See Example 15). * indicates p<0.05; **** indicates p<0.0001. [Figure 13] Treatment with afucosylated 20502 (bottom panel) results in NK cell infiltration (left panel), T cell infiltration (center panel), and PD-L1 upregulation (right panel) compared to treatment with a control antibody (top panel). (See Example 16). [Figure 14](Figure 14A) The 20502-mouse IgG2a-F antibody significantly reduces tumor growth of 4T1 breast cancer cells in a dose-dependent manner. (See Example 17). (Figure 14B) One-way ANOVA at day 30 shows that the 20502-mouse IgG2a-F antibody significantly inhibits tumor growth at doses of 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). (See Example 17). [Figure 15] (Figure 15A) The 20502-mouse IgG2a-F antibody significantly reduces the proliferation of B16 expressing B7-H4 / H3. (See Example 17). (Figure 15B) One-way ANOVA at day 23 shows that the 20502-mouse IgG2a-F antibody significantly inhibits tumor growth at doses of 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) (See Example 17). [Modes for carrying out the invention]

[0067] This specification provides antibodies (e.g., monoclonal antibodies) that specifically bind to B7-H4 (e.g., human B7-H4) and their antigen-binding fragments. Anti-B7-H4 antibodies and their antigen-binding fragments may, for example, induce T cell checkpoint blocking activity (e.g., by measurement in increased interferon-γ (IFNγ), CD4 T cell proliferation, CD8 T cell proliferation, and / or total T cell proliferation) and / or antibody-dependent cell-mediated cytotoxicity (ADCC activity).

[0068] We also provide isolated nucleic acids (polynucleotides), such as complementary DNA (cDNA) encoding such antibodies and their antigen-binding fragments. Furthermore, we provide vectors (e.g., expression vectors) and cells (e.g., host cells) containing such antibodies and nucleic acids (polynucleotides) encoding such antibodies and their antigen-binding fragments. We also provide methods for producing such antibodies and their antigen-binding fragments. In other embodiments, we provide methods for treating specific pathological conditions, such as cancer. We also provide related compositions (e.g., pharmaceutical compositions), kits, and detection methods.

[0069] 1.1 Terminology As used herein, the term "B7-H4" refers to mammalian B7-H4 polypeptides, including but not limited to the naturally occurring B7-H4 polypeptide and its isoforms. "B7-H4" encompasses the full-length, unprocessed B7-H4 polypeptide and the forms of B7-H4 polypeptides resulting from intracellular processing. As used herein, the term "human B7-H4" refers to the polypeptide having the amino acid sequence of SEQ ID NO: 1. "B7-H4 polynucleotide," "B7-H4 nucleotide," or "B7-H4 nucleic acid" refers to the polynucleotide encoding B7-H4.

[0070] As used herein, the term "PD-1" refers to mammalian PD-1 polypeptides, including but not limited to natural PD-1 polypeptides and isoforms of PD-1 polypeptides. PD-1 is also known as programmed death protein 1 or programmed cell death protein 1. "PD-1" encompasses the full-length, unprocessed PD-1 polypeptide and the PD-1 polypeptide forms resulting from intracellular processing. As used herein, the term "human PD-1" refers to the polypeptide having the amino acid sequence of SEQ ID NO: 439: PGWFLDSPDR PWNPPTFSPA LLVVTEGDNA TFTCSFSNTS ESFVLNWYRM SPSNQTDKLA AFPEDRSQPG QDCRFRVTQL PNGRDFHMSV VRARRNDSGT YLCGAISLAP KAQIKESLRA ELRVTERRAE VPTAHPSPSP RPAGQFQTLV VGVVGGLLGS LVLLVWVLAV ICSRAARGTI GARRTGQPLK EDPSAVPVFS VDYGELDFQW REKTPEPPVP CVPEQTEYAT IVFPSGMGTS SPARRGSADG PRSAQPLRPE DGHCSWPL (Sequence ID 439) (Mature human PD-1 without a signal sequence). "PD-1 polynucleotide," "PD-1 nucleotide," or "PD-1 nucleic acid" refers to the polynucleotide that codes for PD-1.

[0071] 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 thereof, via at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term “antibody” includes intact polyclonal antibodies, intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, antibody-containing fusion proteins, and any other modified immunoglobulin molecules, insofar as the antibody exhibits the desired biological activity. Antibodies may be one of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of their heavy chain constant domains, which are called α, δ, ε, γ, and μ, respectively. Different classes of immunoglobulins have different well-known subunit structures and three-dimensional arrangements. Antibodies can be kept as they are, or they can be bound to other molecules such as toxins or radioisotopes.

[0072] The term "antibody fragment" refers to a portion of an intact antibody. "Antigen-binding fragment," "antigen-binding domain," or "antigen-binding region" refers to the portion of an intact antibody that binds to an antigen. Antigen-binding fragments can contain the antigen-determining region (e.g., the complementarity-determining region (CDR)) of an intact antibody. Examples of antibody antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, and single-chain antibodies. Antibody antigen-binding fragments can be derived from animal species such as rodents (mice, rats, hamsters, etc.) or humans, or they can be artificially produced.

[0073] The terms "anti-B7-H4 antibody," "B7-H4 antibody," and "antibody that binds to B7-H4" refer to antibodies that can bind to B7-H4 with sufficient affinity and are therefore useful as diagnostic and / or therapeutic agents targeting B7-H4. The degree of binding of anti-B7-H4 antibodies to unrelated non-B7-H4 proteins may be less than approximately 10% of the binding of antibodies to B7-H4, as measured, for example, by radioimmunoassay (RIA).

[0074] The terms "anti-PD-1 antibody," "PD-1 antibody," and "antibody that binds to PD-1" refer to antibodies that can bind to PD-1 with sufficient affinity and are therefore useful as diagnostic and / or therapeutic agents targeting PD-1. The degree of binding of anti-PD-1 antibodies to unrelated non-PD-1 proteins may be less than approximately 10% of the binding of antibodies to PD-1, as measured, for example, by radioimmunoassay (RIA).

[0075] A “monoclonal” antibody or its antigen-binding fragment refers to a homogeneous population of antibodies or antigen-binding fragments involved in the highly specific recognition and binding of a single antigenic determinant or epitope. This is generally in contrast to polyclonal antibodies, which contain different antibodies against different antigenic determinants. The term “monoclonal” antibody or its antigen-binding fragment includes both intact and complete monoclonal antibodies, as well as antibody fragments (Fab, Fab', F(ab')2, Fv, etc.), single-chain (scFv) variants, fusion proteins containing antibody portions, and other modified immunoglobulin molecules containing antigen recognition sites. Furthermore, “monoclonal” antibody or its antigen-binding fragment refers to such antibodies and their antigen-binding fragments produced by many methods, including but not limited to hybridomas, phage selection, recombinant expression, and transgenic animals.

[0076] As used herein, the terms “variable region” or “variable domain” are used interchangeably and are common in the art. The variable region typically refers to a portion of an antibody, generally a portion of the light or heavy chain, usually the amino-terminus of the mature heavy chain (approximately 110-120 or 110-125 amino acids) and the mature light chain (approximately 90-115 amino acids), which differ significantly in sequence between antibodies and are used for the binding and specificity of a particular antibody to a particular antigen. Sequence variability is concentrated in a region called the complementarity-determining region (CDR), while more highly conserved regions within the variable domain are called the framework region (FR). While we do not wish to dwell on specific mechanisms or theories, it is believed that the CDRs of the light and heavy chains are primarily responsible for antibody-antigen interaction and specificity. In certain embodiments, the variable region is the human variable region. In certain embodiments, the variable region includes rodent or mouse CDRs and human framework regions (FRs). In certain embodiments, the variable region is the primate (e.g., non-human primate) variable region. In certain embodiments, the variable region includes a rodent or mouse CDR and a primate (e.g., non-human primate) framework region (FR).

[0077] The terms "VL" and "VL domain" are used interchangeably and refer to the variable region of the antibody light chain.

[0078] The terms "VH" and "VH domain" are used interchangeably and refer to the variable region of the antibody's heavy chain.

[0079] The term "Kabat numbering" and similar terms are recognized in the art and refer to a numbering system for amino acid residues within the heavy and light chain variable regions of an antibody or its antigen-binding fragment. In certain embodiments, the CDR can be determined according to the Kabat numbering system (e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment). (See *of Health and Human Services*, NIH Publication No. 91-3242). Using the Kabat numbering system, CDRs within an antibody heavy chain molecule are typically located at amino acid positions 31–35 (possibly including one or two additional amino acids following position 35 (referred to as 35A and 35B in the Kabat numbering scheme)) (CDR1), 50–65 (CDR2), and 95–102 (CDR3). Using the Kabat numbering system, CDRs within an antibody light chain molecule are typically located at amino acid positions 24–34 (CDR1), 50–56 (CDR2), and 89–97 (CDR3). In certain embodiments, the CDRs of the antibodies described herein are determined according to the Kabat numbering scheme.

[0080] Instead, Chothia refers to the location of the structural loop (Chothia and Lesk, J.Mol.Biol.196:901-917(1987)). The ends of the Chothia CDR-H1 loop, numbered using Kabat numbering rules, vary considerably from H32 to H34 depending on the loop length (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B is 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). The AbM hypervariable region represents a compromise between the Kabat CDR and the Chothia structural loop and is used in Oxford Molecular's AbM antibody modeling software. [Table A]

[0081] As used herein, the terms “constant region” or “constant domain” are used interchangeably and are well known in the art. The constant region is an antibody moiety, e.g., the carboxyl-terminal portion of the light and / or heavy chain, which does not directly participate in the binding of the antibody to the antigen but can exhibit various effector functions, e.g., interaction with Fc receptors. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence than the immunoglobulin variable domain. In certain embodiments, an antibody or antigen-binding fragment contains a constant region or portion sufficient for antibody-dependent cell-mediated cytotoxicity (ADCC).

[0082] As used herein, the term “heavy chain” in relation to antibodies may refer to alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ) that, based on the amino acid sequence of the constant domain, produce antibodies of any distinct type, e.g., IgA, IgD, IgE, IgG, and IgM classes (including subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4). Heavy chain amino acid sequences are well known in the art. In certain embodiments, the heavy chain is a human heavy chain.

[0083] As used herein, the term “light chain,” when used in reference to antibodies, may refer to any distinct type, such as kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. In certain embodiments, the light chain is a human light chain.

[0084] The term "chimeric" refers to an antibody or antigen-binding fragment whose amino acid sequence originates from two or more species. Typically, the variable regions of both the light and heavy chains correspond to the variable regions of the antibody or antigen-binding fragment derived from one mammalian species (e.g., mouse, rat, rabbit) with the desired specificity, affinity, and ability, while the constant region is homologous to the sequence of the antibody or antigen-binding fragment derived from another species (usually human), thus avoiding the induction of an immune response in that species.

[0085] The term "humanized" antibody or its antigen-binding fragment refers to a form of non-human (e.g., mouse) antibody or antigen-binding fragment that is a specific immunoglobulin chain, chimeric immunoglobulin, or fragment thereof containing minimal non-human (e.g., mouse) sequences. Typically, a humanized antibody or its antigen-binding fragment is a human immunoglobulin with desirable specificity, affinity, and capability, in which residues from the complementarity-determining region (CDR) have been replaced with residues from the CDR of a non-human species (e.g., mouse, rat, rabbit, hamster) ("CDR graft") (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 human immunoglobulin are replaced with corresponding residues of an antibody or fragment derived from a non-human species having the desired specificity, affinity, and capability. The humanized antibody or its antigen-binding fragment can be further modified by substituting additional residues within the Fv framework region and / or within the substituted non-human residues to refine and optimize the specificity, affinity, and / or function of the antibody or its antigen-binding fragment. Generally, a humanized antibody or its antigen-binding fragment contains substantially at least one, usually two or three, variable domains, including all or substantially all CDR regions corresponding to the non-human immunoglobulin, while all or substantially all FR regions are the FR regions of the human immunoglobulin consensus sequence. The humanized antibody or its antigen-binding fragment can also contain at least a portion of the constant region or domain (Fc) of an immunoglobulin, usually human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Patent 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, the “humanized antibody” is a resurfaced antibody.

[0086] The term “human” antibody or its antigen-binding fragment means an antibody or its antigen-binding fragment having an amino acid sequence derived from a human immunoglobulin locus, and such antibody or antigen-binding fragment is prepared using any technique known in the art. This definition of human antibody or its antigen-binding fragment includes intact or full-length antibodies and their fragments.

[0087] An "afcosylated" antibody or its antigen-binding fragment, or a "fucose-deficient" antibody or its antigen-binding fragment, refers to an IgG1 or IgG3 isotype antibody or its antigen-binding fragment that lacks fucose in its constant-region glycosylation. Glycosylation of human IgG1 or IgG3 occurs as a core-fucosylated branched complex oligosaccharide glycosylation terminated at Asn297 by up to two Gal residues. In some embodiments, aafcosylated antibodies lack fucose at Asn297. These structures are referred to as G0, G1 (1,6 or 1,3), or G2 glycan residues, depending on the amount of terminal Gal residues. See, for example, Raju, TS, BioProcess Int. 1:44-53 (2003). CHO-type glycosylation of antibody Fc is described, for example, in Routier, F.FL, Glycoconjugate J. 14:201-207 (1997).

[0088] Methods for measuring fucose include any method known in the art. For the purposes of this specification, fucose is detected by the method described in Example 1 of WO2015 / 017600, which is incorporated herein by reference in its entirety. Briefly, glycan analysis is performed by releasing glycans from an antibody (e.g., by enzymatic release), labeling the glycans with anthranilic acid (2-AA), and purifying the labeled glycans. The glycans are separated using normal-phase HPLC with fluorescence detection, and the relative amount of each glycan in the antibody is measured. Mass spectrometry may clearly identify whether the glycans lack or contain fucose. In some embodiments, fucose is undetectable in compositions comprising multiple afucosylated antibodies or their antigen-binding fragments. In some embodiments, the afucosylated antibodies or their antigen-binding fragments have enhanced ADCC activity, which can be measured by the assay provided in Example 12 herein. In some embodiments, the afucosylated antibodies or their antigen-binding fragments have improved affinity for FcγRIIIA. In some embodiments, the afucosylated antibody or its antigen-binding fragment exhibits enhanced affinity for FcγRIIIA(V158). The affinity for FcγRIIIA or its allele may be measured by the assay provided in Example 10 of this specification.

[0089] "Binding affinity" generally refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or its antigen-binding fragment) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antibody or its antigen-binding fragment and an antigen). The affinity of molecule X for its partner Y is usually expressed by the dissociation constant (K). D It can be expressed as (K). The affinity is the equilibrium dissociation constant (K D ), and equilibrium association constant (K AIt can be measured and / or expressed in many ways known in the art, including but not limited to these. K D is k off / k on calculated from the quotient of, while K A is k on / k off calculated from the quotient of. k on refers to, for example, the association rate constant of an antibody or an antigen-binding fragment thereof to an antigen, and k off refers to, for example, the dissociation of an antibody or an antigen-binding fragment thereof from an antigen. k on and k off can be determined by techniques known to those skilled in the art, such as BIAcore® or KinExA.

[0090] As used herein, "epitope" is a term in the art and refers to the localized region of an antigen to which an antibody or an antigen-binding fragment thereof can specifically bind. An epitope can be, for example, a continuous amino acid of a polypeptide (linear or contiguous epitope), or an epitope can be, for example, a combination of two or more non-adjacent regions of a polypeptide (conformation, non-linear, discontinuous, or non-contiguous epitope). In certain embodiments, the epitope to which an antibody or an antigen-binding fragment thereof binds can be determined, for example, by NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange combined with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scan assays, and / or mutation mapping (e.g., site-specific mutation mapping). In the case of X-ray crystallography, crystallization may be achieved using any of the methods known in the art (e.g., Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 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). Antibodies / their antigen-binding fragments: Antigen crystals can be tested using well-known X-ray diffraction techniques and refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff HW et al.,; US2004 / 0014194), and BUSTER (see Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1):37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, ed Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10):1316-1323). Mutagenesis mapping tests can be achieved using any method known to those skilled in the art. For a description of mutagenesis techniques, including alanine scanning mutagenesis, see, for example, Champe M et al., (1995) J Biol Chem 270:1388-1394 and Cunningham BC & Wells JA (1989) Science 244:1081-1085.

[0091] 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 B7-4 antibody to bind to the same epitope as a reference B7-4 antibody is measured by a hydrogen / deuterium exchange assay (see Coales et al. Rapid Commun. Mass Spectrom. 2009;23:639-647).

[0092] As used herein, the terms “immunely binding,” “immunely recognizing,” “specifically binding,” and “specifically recognizing” are synonymous terms in the context of antibodies or their antigen-binding fragments. These terms indicate that an antibody or its antigen-binding fragment binds to an epitope via its antigen-binding domain, and that the binding involves some degree of complementarity between the antigen-binding domain and the epitope. Therefore, an antibody that “specifically binds” to human B7-H4 (SEQ ID NO: 1) may also bind to B7-H4 proteins produced from other species (e.g., cynomolgus monkey, mouse, and / or rat B7-H4) and / or other human alleles, but the degree of binding to unrelated non-B7-H4 proteins (e.g., other B7 protein family members such as PD-L1) is less than approximately 10% of the binding of the antibody to B7-H4 as measured, for example, by radioimmunoassay (RIA).

[0093] In certain embodiments, this specification provides antibodies or antigen-binding fragments that bind to human, cynomolgus monkey, mouse, and rat B7-H4.

[0094] An antibody is said to "competitively inhibit" the binding of a reference antibody to a given epitope if it preferentially binds to that epitope or a duplicate epitope to such an extent that it blocks, to some degree, the binding of the reference antibody to that epitope. Competitive inhibition may be measured by any method known in the art, such as a competitive ELISA assay. An antibody may be said to competitively inhibit the binding of the reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0095] "Isolated" polypeptides, antibodies, polynucleotides, vectors, cells, or compositions are polypeptides, antibodies, polynucleotides, vectors, cells, or compositions in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells, or compositions include those that have been purified to such an extent that they are no longer found in nature. In some embodiments, the isolated antibodies, polynucleotides, vectors, cells, or compositions are substantially pure. As used herein, "substantially pure" means 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.

[0096] The terms “polypeptide,” “peptide,” and “protein” are used herein to mean the same thing and refer to polymers of amino acids of any length. Polymers can be linear or branched, may contain modified amino acids, and may contain non-amino acids. The term also includes amino acid polymers modified naturally or by intervention; for example, by other operations or modifications such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or binding with labeling components. Furthermore, the definition also includes polypeptides containing, for example, one or more analogues of amino acids (including, for example, non-natural amino acids), and other modifications known in the art. Since the polypeptides of the present invention are antibody-based, it is understood that in certain embodiments, polypeptides may arise as single-chain or linked chains.

[0097] "Identity ratio" refers to the degree of identity between two sequences (e.g., amino acid sequences or nucleic acid sequences). The identity ratio is determined by aligning the two sequences and introducing gaps to maximize the identity between them. Alignments can be generated using programs known in the art. For the purposes of this specification, nucleotide sequence alignment can be performed using the blastn program configured with default parameters, and amino acid sequence alignment can be performed using the blastp program configured with default parameters (see National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov).

[0098] As used herein, the term “host cell” can refer to any type of cell, e.g., primary cells, cultured cells, or any cell derived from a cell line. In certain embodiments, the term “host cell” refers to cells into which nucleic acid molecules have been transfected and their offspring or potential offspring. Such offspring may not be identical to the parent cells into which the nucleic acid molecules were transfected, for example, due to mutations that may occur in subsequent generations, environmental influences, or the integration of the nucleic acid molecules into the host cell genome.

[0099] The term "pharmaceutical preparation" refers to a preparation that is in a form that enables the biological activity of the active ingredient and does not contain additional ingredients that would be unacceptably toxic to the subject to whom the preparation will be administered. The preparation may be sterile.

[0100] As used herein, the terms “administer,” “give delivery,” and “dosage” refer to methods that may be used to enable the delivery of a drug, such as an anti-B7-H4 antibody or its antigen-binding fragment, to a desired site of action (e.g., intravenous administration). Dosage techniques that may be used in conjunction with the agents and methods described herein can be 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.

[0101] "Combined" administration with one or more additional therapeutic agents includes simultaneous (concurrent) administration or sequential administration in any order.

[0102] Combination therapy can provide a “synergistic effect,” that is, the effect of using active agents together is greater than the sum of the effects of using the active agents separately. Synergistic effects are obtained when active agents are: (1) co-formulated in combination as unit-dose formulations and administered or delivered simultaneously; (2) as separate formulations, sequentially, alternately, or in parallel; or (3) delivered by several other regimens. In the case of alternating therapy, for example, by administering or delivering the active agents sequentially by different injections in separate syringes, synergistic effects can be achieved. A “synergistic combination” produces an effect greater than the sum of the individual effects of the combined active agents.

[0103] Combination therapy can provide an "additive" effect, meaning that the effect of using active agents together is equal to the sum of the effects resulting from using the active agents separately.

[0104] As used herein, the terms “subject” and “patient” are used interchangeably. The subject may 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). In some embodiments, the subject is a cynomolgus macaque. In some embodiments, the subject is a human.

[0105] The term “therapeutic dose” refers to a certain amount of a drug, e.g., an anti-B7-H4 antibody or its antigen-binding fragment, that is effective in treating a disease or disorder in a subject. In the case of cancer, a therapeutic dose of a drug may reduce the number of cancer cells; reduce the size or burden of the tumor; inhibit (i.e., delay to some extent, and in certain embodiments, halt) the invasion of cancer cells into peripheral organs; inhibit (i.e., delay to some extent, and in certain embodiments, halt) tumor metastasis; suppress tumor growth to some extent; alleviate to some extent one or more symptoms associated with cancer; and / or result in a favorable response such as an increase in progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS), a complete response (CR), a partial response (PR), or in some cases, a reduction in stable disease (SD), progressive disease (PD), a reduction in tumor arrest time (TTP), or any combination thereof. A drug may be cell proliferation inhibitory and / or cytotoxic insofar as it can prevent and / or kill the growth of existing cancer cells.

[0106] The terms “to treat,” “to cure,” “to alleviate,” and “to reduce” refer to therapeutic means that result in the cure, slowing, reduction of symptoms, and / or cessation of progression of a diagnosed pathological condition or disorder. Therefore, people who need treatment include those who have already been diagnosed with or are suspected of having a disorder. In certain embodiments, a patient is "treated" for cancer according to the method of the present invention if: a patient exhibits: a reduction or complete disappearance of the number of cancer cells; a reduction in tumor size; inhibition or cessation of cancer cell invasion into peripheral organs, including, for example, the spread of cancer to soft tissues and bones; inhibition or cessation of tumor metastasis; inhibition or cessation of tumor growth; relief of one or more symptoms associated with a particular cancer; a reduction in morbidity and mortality; an improvement in quality of life; a decrease in the tumor's tumorigenicity, tumorigenicity frequency, or tumorigenic capacity; a decrease in the number or frequency of cancer stem cells within the tumor; differentiation of tumorigenic cells into a non-tumoric state; an increase in progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS); a complete response (CR), a partial response (PR), stable disease (SD), a decrease in progressive disease (PD), a decrease in tumor arrest time (TTP), or any combination thereof.

[0107] The terms “cancer” and “of cancer” refer to or represent a physiological condition in mammals characterized by uncontrolled cell proliferation in a population of cells. Examples of cancer include, but are not limited to, gynecological cancers (e.g., breast cancer (including tertiary-negative breast cancer and ductal carcinoma), ovarian cancer, and endometrial cancer), non-small cell lung cancer, pancreatic cancer, thyroid cancer, renal cancer (e.g., renal cell carcinoma), and bladder cancer (e.g., urothelial carcinoma). Non-small cell lung cancer may be, for example, adenocarcinoma. Further 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 carcinoma. Cancer may be “B7-H4 expressing cancer” or “B7-H4 expressing cancer.” Such terms refer to cancer containing cells that express B7-H4. The cancer may be a primary tumor, an advanced cancer, or a metastatic cancer.

[0108] "Refractory" cancer is cancer that progresses even when cancer patients are given antitumor treatments such as chemotherapy.

[0109] "Recurrent" cancer is cancer that has grown again, either at the original site or a distant site, after responding to initial treatment.

[0110] A "relapsed" patient is one who has signs or symptoms of cancer after achieving remission. In some cases, patients experience a relapse after adjuvant therapy or neoadjuvant therapy.

[0111] "T cell checkpoint blocking activity" refers to the blocking or inhibition of T cell checkpoint activity or response. T cell checkpoint blocking activity can be measured by an artificial antigen-presenting cell (aAPC) assay based on changes in IFNγ production. Primary human T cells can be enriched from PBMCs using a T cell enrichment kit (EasySep® Human T Cell Enrichment Kit or a similar kit). The enriched T cells are incubated in the presence of beads (e.g., anti-CD3 / anti-CD28 beads). After a certain period, the beads are magnetically removed, the T cells are washed, and incubated again. The T cells are then washed and incubated with artificial antigen-presenting cells (aAPCs) in the presence of dose-escalating B7-H4 antibody. The aAPCs can be treated with mitomycin C and then thoroughly washed before being added to the T cell co-culture. After co-culture of T cells, aAPCs, and B7-H4 antibody, the plate can be centrifuged, the supernatant collected, and IFNγ production can be evaluated by ELISA. IFNγ production can be plotted against antibody concentration, and EC50 efficacy can be calculated using a nonlinear regression curve approximation method. The results can be measured as EC50 ± standard deviation in nM. T cell checkpoint blocking activity by B7-H4 antibody can be indicated by an increase in IFNγ production. "T cell checkpoint blocking activity" can also be measured by assays using cells that endogenously express B7-H4. Primary human T cells can be enriched from HLA-A2+ donor PBMCs using a T cell isolation kit (e.g., Human Pan T Cell Isolation Kit). MART-I TCR-expressing T cells can be generated by first activating the enriched pan T cells with beads (e.g., anti-CD3 / anti-CD28 Dynabeads), IL-2, and IL-7 for 48 hours. Subsequently, MART-I TCR lentiviral particles can be transduced into the activated T cells in the presence of IL-2, IL-7, and polybrenes. After transduction, MART-I TCR+ pan-T cells can be proliferated for a certain period in the presence of IL-2 and IL-7.To generate target cell lines expressing HLA-A2, cancer cell lines expressing endogenous B7-H4 can be transduced with HLA-A2 lentiviral particles for a specified period (e.g., 48 hours). Furthermore, B7-H4 can be knocked out from HLA-A2+ cell lines. Subsequently, MART-I TCR+ pan-T cells can be co-cultured in the presence of various target cell lines with a 1:1 E:T ratio, MART-I peptide, and B7-H4 antibody or human isotype control. After co-incubation, plates can be centrifuged and the supernatant collected to evaluate IL-2 production. IL-2 production can be measured using a standard immunoassay kit (such as the AlphaLISA assay or a similar assay).

[0112] As used in this disclosure and claims, the singular forms "a," "an," and "the" include the plural form unless the context explicitly indicates otherwise.

[0113] Whenever an embodiment is described in this specification using the word “comprises,” other similar embodiments described using the terms “consist of” and / or “essentially from” are also provided. In this disclosure, “comprises,” “comprising,” “containing,” and “having,” etc., may have the meanings attributed to them in U.S. patent law and may mean “includes,” “including,” etc.; “consisting essentially of” or “consists essentially” may similarly have the meanings set forth in U.S. patent law, and the term is non-restrictive and may allow for more than described, provided that the existence of more does not alter the fundamental or novel features of what is described, except for embodiments of the prior art.

[0114] Where used herein, the term “or” is understood to be inclusive unless specifically stated or evident from the context. Where used herein in phrases such as “A and / or B,” the term “and / or” includes both “A and B,” “A or B,” and “A” and “B.” Similarly, where used in phrases such as “A, B, and / or C,” the term “and / or” includes 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).

[0115] As used herein, the terms “about” and “approximately” mean, within the intended meaning of the value or range described, that when used to modify a number or range of numbers, there remains a deviation of 5% to 10% above and 5% to 10% below the value or range.

[0116] Any composition or method provided herein may be combined with one or more other compositions and methods provided herein.

[0117] 1.2 Antibodies In certain embodiments, this specification provides antibodies (e.g., monoclonal antibodies such as chimeric, humanized, or human antibodies) that specifically bind to B7-H4 (e.g., human B7-H4) and antigen-binding fragments thereof. The amino acid sequences of human, cynomolgus monkey, mouse, and rat B7-H4 are known in the art and are provided herein as represented by SEQ ID NOs: 1 to 4, respectively. Human B7-H4: MASLGQILFWSIISIIIILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITSKGKGNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTVVWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTESEIKRRSHLQLLNSKASLCVSSFFAISWALLPLSPYLMLK (SEQ ID NO: 1) Cynomolgus monkey B7 - H4: MASLGQILFWSIISIIFILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVIGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITSKGKGNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTVVWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTESEIKRRSHLQLLNSKASLCVSSFLAISWALLPLAPYLMLK (SEQ ID NO: 2) Mouse B7 - H4: MASLGQIIFWSIINIIIILAGAIALIIGFGISGKHFITVTTFTSAGNIGEDGTLSCTFEPDIKLNGIVIQWLKEGIKGLVHEFKEGKDDLSQQHEMFRGRTAVFADQVVVGNASLRLKNVQLTDAGTYTCYIRTSKGKGNANLEYKTGAFSMPEINVDYNASSESLRCEAPRWFPQPTVAWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTDSEVKRRSQLQLLNSGPSPCVFSSAFVAGWALLSLSCCLMLR (SEQ ID NO: 3) Rat B7 - H4: MASLGQIIFWSIINVIIILAGAIVLIIGFGISGKHFITVTTFTSAGNIGEDGTLSCTFEPDIKLNGIVIQWLKEGIKGLVHEFKEGKDDLSQQHEMFRGRTAVFADQVVVGNASLRLKNVQLTDAGTYTCYIHTSKGKGNANLEYKTGAFSMPEINVDYNASSESLRCEAPRWFPQPTVAWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTDSEVKRRSQLELLNSGPSPCVSSVSAAGWALLSLSCCLMLR (Sequence ID 4)

[0118] In certain embodiments, the antibody or its antigen-binding fragment described herein binds to human B7-H4. In certain embodiments, the antibody or its antigen-binding fragment binds to human and cynomolgus monkey B7-H4. In certain embodiments, the antibody or its antigen-binding fragment binds to human, mouse, and rat B7-H4. In certain embodiments, the antibody or its antigen-binding fragment binds to human, cynomolgus monkey, mouse, and rat B7-H4.

[0119] B7-H4 contains an IgC external domain (amino acids 153-241 of SEQ ID NO: 1) and an IgV domain (amino acids 35-146 of SEQ ID NO: 1).

[0120] In certain embodiments, the antibody or its antigen-binding fragment described herein binds to the IgV domain of human B7-H4. Accordingly, this specification provides an antibody and its antigen-binding fragment that bind to a polypeptide consisting of amino acids 35-146 of SEQ ID NO: 1.

[0121] In certain embodiments, the antibodies or antigen-binding fragments described herein are conjugated to human B7-H4 and comprise six CDRs of the antibodies listed in Tables 1 and 2 (i.e., three VH CDRs of the antibodies listed in Table 1 and three VL CDRs of the same antibodies listed in Table 2). [Table 1-1] [Table 1-2] [Table 2-1] [Table 2-2]

[0122] 2 The VL CDR in Table 2 is determined according to Kabat.

[0123] In certain embodiments, the antibody or its antigen-binding fragment described herein is bound to human B7-H4 and contains the VH of the antibody described in Table 3. [Table 3-1] [Table 3-2] [Table 3-3]

[0124] In certain embodiments, the antibody or its antigen-binding fragment described herein is bound to human B7-H4 and comprises the VL of the antibody described in Table 4. [Table 4-1] [Table 4-2]

[0125] In certain embodiments, the antibodies or antigen-binding fragments described herein are bound to human B7-H4 and include the VH and VL of the antibodies described in Tables 3 and 4 (i.e., the VH of the antibody described in Table 3 and the VL of the same antibody described in Table 4).

[0126] In certain embodiments, the antibody or antigen-binding fragment described herein is bound to human B7-H4 and includes the VH framework region of the antibody described in Table 5. [Table 5-1] [Table 5-2] [Table 5-3]

[0127] In certain embodiments, the antibody or antigen-binding fragment described herein is bound to human B7-H4 and includes the VL framework region of the antibody described in Table 6. [Table 6-1] [Table 6-2] [Table 6-3]

[0128] In certain embodiments, the antibodies or antigen-binding fragments described herein are bound to human B7-H4 and include four VH framework regions and four VL framework regions of the antibodies described in Tables 5 and 6 (i.e., four VH framework regions of the antibodies described in Table 5 and four VL framework regions of the same antibodies described in Table 6).

[0129] In certain embodiments, the antibody or antigen-binding fragment described herein is bound to human B7-H4 and comprises the heavy chain sequence of the antibody described in Table 7. [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5]

[0130] In certain embodiments, the antibody or antigen-binding fragment described herein is bound to human B7-H4 and includes the light chain sequence of the antibody described in Table 8. [Table 8-1] [Table 8-2] [Table 8-3]

[0131] In certain embodiments, the antibody or antigen-binding fragment described herein is bound to human B7-H4 and comprises the heavy chain and light chain sequences of the antibody described in Tables 7 and 8 (i.e., the heavy chain sequence of the antibody described in Table 7 and the light chain sequence of the same antibody described in Table 8).

[0132] In certain embodiments, the antibody or antigen-binding fragment described herein is bound to human B7-H4 and comprises six CDRs of the antibody described in Tables 1 and 2 (i.e., three VH CDRs of the antibody described in Table 1 and three VL CDRs of the same antibody described in Table 2), and also comprises a VH having at least 80% identical sequence to the VH sequence of the same antibody in Table 3, and a VL having at least 80% identical sequence to the VL sequence of the same antibody in Table 4.

[0133] In certain embodiments, the antibody or antigen-binding fragment described herein is conjugated to human B7-H4 and comprises six CDRs of the antibody described in Tables 1 and 2 (i.e., three VH CDRs of the antibody described in Table 1 and three VL CDRs of the same antibody described in Table 2), and also comprises a VH having a sequence at least 90% identical to the VH sequence of the same antibody in Table 3, and a VL having a sequence at least 90% identical to the VL sequence of the same antibody in Table 4. In certain embodiments, the antibody or antigen-binding fragment described herein is conjugated to human B7-H4 and comprises six CDRs of the antibody described in Tables 1 and 2 (i.e., three VH CDRs of the antibody described in Table 1 and three VL CDRs of the same antibody described in Table 2), and also comprises a VH having a sequence at least 95% identical to the VH sequence of the same antibody in Table 3, and a VL having a sequence at least 95% identical to the VL sequence of the same antibody in Table 4.

[0134] In certain embodiments, the antibody or antigen-binding fragment described herein is conjugated to human B7-H4 and comprises six CDRs of the antibody described in Tables 1 and 2 (i.e., three VH CDRs of the antibody described in Table 1 and three VL CDRs of the same antibody described in Table 2), and also comprises a VH having a sequence at least 96% identical to the VH sequence of the same antibody in Table 3, and a VL having a sequence at least 96% identical to the VL sequence of the same antibody in Table 4. In certain embodiments, the antibody or antigen-binding fragment described herein is conjugated to human B7-H4 and comprises six CDRs of the antibody described in Tables 1 and 2 (i.e., three VH CDRs of the antibody described in Table 1 and three VL CDRs of the same antibody described in Table 2), and also comprises a VH having a sequence at least 97% identical to the VH sequence of the same antibody in Table 3, and a VL having a sequence at least 97% identical to the VL sequence of the same antibody in Table 4. In certain embodiments, the antibody or antigen-binding fragment described herein is conjugated to human B7-H4 and comprises six CDRs of the antibody described in Tables 1 and 2 (i.e., three VH CDRs of the antibody described in Table 1 and three VL CDRs of the same antibody described in Table 2), and also comprises a VH having a sequence at least 98% identical to the VH sequence of the same antibody in Table 3, and a VL having a sequence at least 98% identical to the VL sequence of the same antibody in Table 4. In certain embodiments, the antibody or antigen-binding fragment described herein is conjugated to human B7-H4 and comprises six CDRs of the antibody described in Tables 1 and 2 (i.e., three VH CDRs of the antibody described in Table 1 and three VL CDRs of the same antibody described in Table 2), and also comprises a VH having a sequence at least 99% identical to the VH sequence of the same antibody in Table 3, and a VL having a sequence at least 99% identical to the VL sequence of the same antibody in Table 4.

[0135] In certain embodiments, the antibodies or antigen-binding fragments described herein bind to human B7-H4 and comprise six CDRs of the antibodies listed in Tables 1 and 2 (i.e., three VH CDRs of the antibodies listed in Table 1 and three VL CDRs of the same antibodies listed in Table 2), and also comprise a VH having at least 80% identical sequence to the VH sequence of the same antibody in Table 3, and a VL having at least 80% identical sequence to the VL sequence of the same antibody in Table 4, and bind to human, cynomolgus monkey, rat, and / or mouse B7-H4. In certain embodiments, the antibodies or antigen-binding fragments described herein bind to human B7-H4 and comprise six CDRs of the antibodies listed in Tables 1 and 2 (i.e., three VH CDRs of the antibodies listed in Table 1 and three VL CDRs of the same antibodies listed in Table 2), and also comprise a VH having at least 85% identical sequence to the VH sequence of the same antibody in Table 3, and a VL having at least 85% identical sequence to the VL sequence of the same antibody in Table 4, and bind to human, cynomolgus monkey, rat, and / or mouse B7-H4.

[0136] In certain embodiments, the antibodies or antigen-binding fragments described herein bind to human B7-H4 and comprise six CDRs of the antibodies listed in Tables 1 and 2 (i.e., three VH CDRs of the antibodies listed in Table 1 and three VL CDRs of the same antibodies listed in Table 2), and also comprise a VH having a sequence at least 90% identical to the VH sequence of the same antibody in Table 3, and a VL having a sequence at least 90% identical to the VL sequence of the same antibody in Table 4, and bind to human, cynomolgus monkey, rat, and / or mouse B7-H4. In certain embodiments, the antibodies or antigen-binding fragments described herein bind to human B7-H4 and comprise six CDRs of the antibodies listed in Tables 1 and 2 (i.e., three VH CDRs of the antibodies listed in Table 1 and three VL CDRs of the same antibodies listed in Table 2), and also comprise a VH having a sequence at least 95% identical to the VH sequence of the same antibody in Table 3, and a VL having a sequence at least 95% identical to the VL sequence of the same antibody in Table 4, and bind to human, cynomolgus monkey, rat, and / or mouse B7-H4.

[0137] In certain embodiments, the antibodies or antigen-binding fragments described herein bind to human B7-H4 and comprise six CDRs of the antibodies listed in Tables 1 and 2 (i.e., three VH CDRs of the antibodies listed in Table 1 and three VL CDRs of the same antibodies listed in Table 2), and also comprise a VH having a sequence at least 96% identical to the VH sequence of the same antibody in Table 3, and a VL having a sequence at least 96% identical to the VL sequence of the same antibody in Table 4, and bind to human, cynomolgus monkey, rat, and / or mouse B7-H4. In certain embodiments, the antibodies or antigen-binding fragments described herein bind to human B7-H4 and comprise six CDRs of the antibodies listed in Tables 1 and 2 (i.e., three VH CDRs of the antibodies listed in Table 1 and three VL CDRs of the same antibodies listed in Table 2), and also comprise a VH having a sequence at least 97% identical to the VH sequence of the same antibody in Table 3, and a VL having a sequence at least 97% identical to the VL sequence of the same antibody in Table 4, and bind to human, cynomolgus monkey, rat, and / or mouse B7-H4. In certain embodiments, the antibodies or antigen-binding fragments described herein bind to human B7-H4 and comprise six CDRs of the antibodies listed in Tables 1 and 2 (i.e., three VH CDRs of the antibodies listed in Table 1 and three VL CDRs of the same antibodies listed in Table 2), and also comprise a VH having a sequence at least 98% identical to the VH sequence of the same antibody in Table 3, and a VL having a sequence at least 98% identical to the VL sequence of the same antibody in Table 4, and bind to human, cynomolgus monkey, rat, and / or mouse B7-H4. In certain embodiments, the antibodies or antigen-binding fragments described herein bind to human B7-H4 and comprise six CDRs of the antibodies listed in Tables 1 and 2 (i.e., three VH CDRs of the antibodies listed in Table 1 and three VL CDRs of the same antibodies listed in Table 2), and also comprise a VH having a sequence at least 99% identical to the VH sequence of the same antibody in Table 3, and a VL having a sequence at least 99% identical to the VL sequence of the same antibody in Table 4, and bind to human, cynomolgus monkey, rat, and / or mouse B7-H4.

[0138] In certain embodiments, the antibodies or antigen-binding fragments described herein bind to human B7-H4 and comprise six CDRs of the antibodies listed in Tables 1 and 2 (i.e., three VH CDRs of the antibodies listed in Table 1 and three VL CDRs of the same antibodies listed in Table 2), and also comprise a VH having at least 80% identical sequence to the VH sequence of the same antibody in Table 3, and a VL having at least 80% identical sequence to the VL sequence of the same antibody in Table 4, thereby increasing T cell proliferation, increasing IFNγ production, and mediating ADCC activity against B7-H4 expressing cells. In certain embodiments, the antibodies or antigen-binding fragments described herein bind to human B7-H4 and comprise six CDRs of the antibodies listed in Tables 1 and 2 (i.e., three VH CDRs of the antibodies listed in Table 1 and three VL CDRs of the same antibodies listed in Table 2), and also comprise a VH having at least 85% identical sequence to the VH sequence of the same antibody in Table 3, and a VL having at least 85% identical sequence to the VL sequence of the same antibody in Table 4, thereby increasing T cell proliferation, increasing IFNγ production, and mediating ADCC activity against B7-H4 expressing cells.

[0139] In certain embodiments, the antibodies or antigen-binding fragments described herein bind to human B7-H4 and comprise six CDRs of the antibodies listed in Tables 1 and 2 (i.e., three VH CDRs of the antibodies listed in Table 1 and three VL CDRs of the same antibodies listed in Table 2), and also comprise a VH having at least 90% identical sequence to the VH sequence of the same antibody in Table 3, and a VL having at least 90% identical sequence to the VL sequence of the same antibody in Table 4, thereby increasing T cell proliferation, increasing IFNγ production, and mediating ADCC activity against B7-H4 expressing cells. In certain embodiments, the antibodies or antigen-binding fragments described herein bind to human B7-H4 and comprise six CDRs of the antibodies listed in Tables 1 and 2 (i.e., three VH CDRs of the antibodies listed in Table 1 and three VL CDRs of the same antibodies listed in Table 2), and also comprise a VH having at least 95% identical sequence to the VH sequence of the same antibody in Table 3, and a VL having at least 95% identical sequence to the VL sequence of the same antibody in Table 4, thereby increasing T cell proliferation, increasing IFNγ production, and mediating ADCC activity against B7-H4 expressing cells.

[0140] In certain embodiments, the antibodies or antigen-binding fragments described herein bind to human B7-H4 and comprise six CDRs of the antibodies listed in Tables 1 and 2 (i.e., three VH CDRs of the antibodies listed in Table 1 and three VL CDRs of the same antibodies listed in Table 2), and also comprise a VH having at least 96% identical sequence to the VH sequence of the same antibody in Table 3, and a VL having at least 96% identical sequence to the VL sequence of the same antibody in Table 4, thereby increasing T cell proliferation, increasing IFNγ production, and mediating ADCC activity against B7-H4 expressing cells. In certain embodiments, the antibodies or antigen-binding fragments described herein bind to human B7-H4 and comprise six CDRs of the antibodies listed in Tables 1 and 2 (i.e., three VH CDRs of the antibodies listed in Table 1 and three VL CDRs of the same antibodies listed in Table 2), and also comprise a VH having at least 97% identical sequence to the VH sequence of the same antibody in Table 3, and a VL having at least 97% identical sequence to the VL sequence of the same antibody in Table 4, thereby increasing T cell proliferation, increasing IFNγ production, and mediating ADCC activity against B7-H4 expressing cells. In certain embodiments, the antibodies or antigen-binding fragments described herein bind to human B7-H4 and comprise six CDRs of the antibodies listed in Tables 1 and 2 (i.e., three VH CDRs of the antibodies listed in Table 1 and three VL CDRs of the same antibodies listed in Table 2), and also comprise a VH having at least 98% identical sequence to the VH sequence of the same antibody in Table 3, and a VL having at least 98% identical sequence to the VL sequence of the same antibody in Table 4, thereby increasing T cell proliferation, increasing IFNγ production, and mediating ADCC activity against B7-H4 expressing cells. In certain embodiments, the antibodies or antigen-binding fragments described herein bind to human B7-H4 and comprise six CDRs of the antibodies listed in Tables 1 and 2 (i.e., three VH CDRs of the antibodies listed in Table 1 and three VL CDRs of the same antibodies listed in Table 2), and also comprise a VH having a sequence at least 99% identical to the VH sequence of the same antibody in Table 3, and a VL having a sequence at least 99% identical to the VL sequence of the same antibody in Table 4, thereby increasing T cell proliferation, increasing IFNγ production, and mediating ADCC activity against B7-H4 expressing cells.

[0141] In certain embodiments, the antibodies or antigen-binding fragments described herein may be described by their VL domain alone, their VH domain alone, their three VL CDRs alone, or their three VH CDRs alone. For example, see Rader C et al., (1998) PNAS 95:8910-8915, incorporated herein by reference in its entirety, which describes the humanization of a mouse anti-αvβ3 antibody by identifying complementary light chains or heavy chains obtained from human light chain or heavy chain libraries, respectively, and resulting in a humanized antibody variant having affinity equal to or greater than that of the original antibody. See also Clackson T et al., (1991) Nature 352:624-628, incorporated herein by reference in its entirety, which describes a method for producing antibodies that bind to a specific antigen using a specific VL domain (or VH domain) and screening a library against complementary variable domains. This screening generated 14 novel partners for specific VH domains and 13 novel partners for specific VL domains, which were robust binders according to ELISA measurements. See also Kim SJ & Hong HJ, (2007) J Microbiol 45:572-577, which is incorporated herein by reference in its entirety and describes a method for producing antibodies that bind to specific antigens using specific VH domains and screening a library (e.g., a human VL library) against complementary VL domains; the selected VL domains can then be used to guide the selection of additional complementary (e.g., human) VH domains.

[0142] In certain embodiments, the CDR of an antibody or its antigen-binding fragment can be determined according to the Chothia numbering scheme, which points to the position of the immunoglobulin structural loop (e.g., Chothia C & 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 (See et al., (1990) J Mol Biol 215(1):175-82; and U.S. Patent No. 7,709,226). Typically, when using Kabat numbering rules, the Chothia CDR-H1 loop is located at heavy chain amino acids 26-32, 33, or 34; the Chothia CDR-H2 loop is located at heavy chain amino acids 52-56; the Chothia CDR-H3 loop is located at heavy chain amino acids 95-102; the Chothia CDR-L1 loop is located at light chain amino acids 24-34; the Chothia CDR-L2 loop is located at light chain amino acids 50-56; and the Chothia CDR-L3 loop is located at light chain amino acids 89-97. The end of a Chothia CDR-H1 loop, numbered using the Kabat numbering rules, will vary between H32 and H34 depending on the loop length (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B exists, the loop ends at 32; if only 35A exists, the loop ends at 33; and if both 35A and 35B exist, the loop ends at 34).

[0143] In certain embodiments, this specification provides antibodies and antigen-binding fragments thereof that specifically bind to B7-H4 (e.g., human B7-H4) and include Chothia VH and VL CDRs of the antibodies listed in Tables 3 and 4. In certain embodiments, the antibody or antigen-binding fragment that specifically binds to B7-H4 (e.g., human B7-H4) includes one or more CDRs, in which case Chothia and Kabat CDRs have the same amino acid sequence. In certain embodiments, this specification provides antibodies and antigen-binding fragments thereof that specifically bind to B7-H4 (e.g., human B7-H4) and include a combination of Kabat CDR and Chothia CDR.

[0144] In certain embodiments, the CDRs of an antibody or its antigen-binding fragment can be determined according to the IMGT numbering system 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 located at positions 26-35, VH-CDR2 at positions 51-57, VH-CDR3 at positions 93-102, VL-CDR1 at positions 27-32, VL-CDR2 at positions 50-52, and VL-CDR3 at positions 89-97. In certain embodiments, as described above, for example, in Lefranc MP (1999) and Lefranc MP et al., (1999), the Specified herein provides antibodies and their antigen-binding fragments that specifically bind to B7-H4 (e.g., human B7-H4) and include IMGT VH and VL CDR of the antibodies listed in Tables 3 and 4.

[0145] In certain embodiments, the CDR of the antibody or its antigen-binding fragment is MacCallum RM This can be determined according to et al., (1996) J Mol Biol 262:732-745. See also, for example, Martin A. “Protein Sequence and Structure Analysis of Antibody Variable Domains,” in Antibody Engineering, Kontermann and Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). In certain embodiments, this specification provides antibodies or antigen-binding fragments thereof that specifically bind to B7-H4 (e.g., human B7-H4) and include the VH and VL CDRs of the antibodies listed in Tables 3 and 4 as determined by the method of MacCallum RM et al.

[0146] In certain embodiments, the CDR of an antibody or its antigen-binding fragment can be determined according to an AbM numbering scheme, which refers to an AbM hypervariable region representing a compromise between the Kabat CDR and the Chothia structural loop, used in Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.). In certain embodiments, this specification provides an antibody or its antigen-binding fragment that specifically binds to B7-H4 (e.g., human B7-H4) and includes the VH and VL CDRs of the antibodies listed in Tables 3 and 4, determined by the AbM numbering scheme.

[0147] In certain embodiments, antibodies comprising heavy and light chains are provided herein. With respect to the heavy chain, in certain embodiments, the heavy chain of the antibody described herein may be an alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In another particular embodiment, the heavy chain of the antibody described herein may comprise a human alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In certain embodiments, the antibody described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) comprises a heavy chain, in which case the amino acid sequence of the VH domain has the amino acid sequence shown in Table 3, and the constant region of the heavy chain has the amino acid sequence of the human gamma (γ) heavy chain constant region. In certain embodiments, the antibodies described herein that specifically bind to B7-H4 (e.g., human B7-H4) include a heavy chain, in which case the amino acid sequence of the VH domain has the sequence shown in Table 3, and the constant region of the heavy chain has the amino acids of a human heavy chain described herein or known in the art. Non-limiting examples of human constant region sequences have been described in the art; see, for example, U.S. Patent No. 5,693,780 and Kabat EA et al., (1991) above.

[0148] With respect to the light chain, in certain embodiments, the light chain of the antibody described herein is a κ light chain. The constant region of the human κ light chain may have the following amino acid sequence:

[0149] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 405).

[0150] The constant region of the human κ light chain can be encoded by the following nucleotide sequence:

[0151] CGGACCGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGT GTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO: 406)

[0152] In another specific embodiment, the light chain of the antibody described herein is a λ light chain. In yet another specific embodiment, the light chain of the antibody described herein is a human κ light chain or a human λ 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, in which case the amino acid sequence of the VL domain has the sequence shown in Table 4, and the constant region of the light chain has the amino acid sequence of the human κ 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, in which case the amino acid sequence of the VL domain has the sequence shown in Table 4, and the constant region of the light chain has the amino acid sequence of the human λ 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, in which case the amino acid sequence of the VL domain has the sequence shown in Table 4, and the constant region of the light chain has the amino acid sequence of the human κ or λ light chain constant region. Non-limiting examples of human constant region sequences have been described in the Art; see, for example, U.S. Patent No. 5,693,780 and Kabat EA et al., (1991) above.

[0153] In certain embodiments, the antibody described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) comprises a VH domain and a VL domain having any amino acid sequence described herein, in which case the constant region has the amino acid sequence of the constant region of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, or a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule. In another particular embodiment, the antibody described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) comprises a VH domain and a VL domain having any amino acid sequence described herein, in which case the constant region has the amino acids of the constant region of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class of an immunoglobulin molecule (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b). In certain embodiments, the constant region has amino acids in the constant region of a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class of immunoglobulin molecule (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b).

[0154] The constant region of human IgG monohelic acid may have the following amino acid sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 407).

[0155] The constant region of human IgG monochain can be encoded by the following nucleotide sequence: GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGGCCCCTGACCAGCGGCTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCT GCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTG GAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCAGTACCGGGTGGTCAGCTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCATCGGAAAACCATCTCCAAAGCCAAAGCCAGTCAGCCCTGACCCTGCCTGGCTGGTCCAAAGGCCTTCTCTCTCTCCACAGCAAAGACTACAAGACCACGCCTCCCGTGACTCCGACGGCTCCTTCTCTCTCTCTCCACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCGGGAACGTCTTCTCATGCTCCCGTGATGCATGAGGCTCTTGCACAACCACTACACGCGAAGAGCCTCTCCCTGTCTCCCGGGAAAAA (SEQ ID NO: 408).

[0156] Non-restrictive examples of the human steady-state region have been described in this art; see, for example, Kabat EA et al., (1991) mentioned above.

[0157] In certain embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of the antibody or antigen-binding fragment described herein (e.g., numbered by the Kabat numbering system (e.g., Kabat's EU index), the CH2 domain (residues 231-340 of human IgG1) and / or the CH3 domain (residues 341-447 of human IgG1) and / or the hinge region) to alter one or more functional properties of the antibody or antigen-binding fragment, such as serum half-life, complement binding, Fc receptor binding, and / or antigen-dependent cytotoxicity.

[0158] In certain embodiments, for example, as described in U.S. Patent No. 5,677,425, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc region (CH1 domain) to alter the number of cysteine ​​residues in the hinge region (e.g., increase or decrease). The number of cysteine ​​residues in the hinge region of the CH1 domain may be altered, for example, to facilitate the assembly of the light and heavy chains or to alter the stability of the antibody or its antigen-binding fragment (e.g., increase or decrease).

[0159] In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of the antibody or antigen-binding fragment described herein (e.g., numbered by the Kabat numbering system (e.g., Kabat's EU index), the CH2 domain (residues 231-340 of human IgG1) and / or the CH3 domain (residues 341-447 of human IgG1) and / or the hinge region) to increase or decrease the affinity of the antibody or antigen-binding fragment to an Fc receptor (e.g., activated Fc receptor) on the surface of effector cells. Mutations within the Fc region that decrease or increase affinity to an Fc receptor, and techniques for introducing such mutations into an Fc receptor or fragment are known to those skilled in the art. Examples of mutations in the Fc receptor that can alter the affinity of antibodies or antigen-binding fragments to the Fc receptor are described, for example, in Smith P et al., (2012) PNAS 109:6181-6186, U.S. Patent No. 6,737,056, and International Publications WO02 / 060919; WO98 / 23289; and WO97 / 34631, which are incorporated herein by reference.

[0160] In certain 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 an Fc or hinged Fc domain fragment) to alter (e.g., decrease or increase) the half-life of the antibody or its antigen-binding fragment in vivo. For examples of mutations that alter (e.g., decrease or increase) the half-life of the antibody or its antigen-binding fragment in vivo, see, for example, International Publications WO02 / 060919; WO98 / 23289; and WO97 / 34631, and U.S. Patents 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 the Fc or hinge-Fc domain fragment) to decrease the half-life of the antibody or its antigen-binding fragment in vivo. 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 the Fc or hinge-Fc domain fragment) to increase the half-life of the antibody or its antigen-binding fragment in vivo. In certain embodiments, 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), numbered according to the Kabat EU index (Kabat EA et al., (1991) above). In certain embodiments, the constant region of IgG1 has, in Kabat EU index numbering, a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256. See U.S. Patent No. 7,658,921, incorporated herein by reference.This type of mutant IgG, called the "YTE variant," has been shown to exhibit a four-fold half-life compared to the wild-type version of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281:23514-24). In certain embodiments, the antibody or its antigen-binding fragment contains an IgG constant domain with one, two, three or more amino acid substitutions at amino acid residues at positions 251–257, 285–290, 308–314, 385–389 and 428–436, numbered according to the Kabat EU index.

[0161] In further embodiments, one, two, or more amino acid substitutions are introduced into the IgG constant domain Fc region to alter the effector function(s) 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, numbered by the Kabat EU index, can be replaced with different amino acid residues so that the antibody or its antigen-binding fragment alters its affinity for an effector ligand while retaining the antigen-binding ability of the parent antibody. The effector ligand that alters affinity may be, for example, the Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patents 5,624,821 and 5,648,260. In some embodiments, deletion or inactivation (by point mutation or other means) of the constant region domain reduces the Fc receptor binding of the circulating antibody or its antigen-binding fragment, thereby increasing tumor localization. For a description of mutations that delete or inactivate the constant domain, thereby increasing tumor localization, see, for example, U.S. Patents 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 and reduce Fc receptor binding (see, for example, Shields RL et al., (2001) J Biol Chem 276:6591-604).

[0162] In certain embodiments, one or more amino acids selected from amino acid residues 329, 331, and 322 of the constant region of the antibody or its antigen-binding fragment can be replaced with different amino acid residues, numbered according to the Kabat EU index, to alter C1q binding and / or reduce or eliminate complement-dependent cytotoxicity (CDC). This approach is described in more detail in U.S. Patent No. 6,194,551 (Idusogie et al). In some embodiments, one or more amino acid residues at positions 231-238 of the N-terminal region of the CH2 domain are altered, thereby changing the antibody's ability to bind complement. This approach is described in International Publication No. WO94 / 29351. In certain embodiments, the Fc region is modified by mutating one or more amino acids (e.g., introducing amino acid substitutions) at the following positions to enhance the ability of the antibody or its antigen-binding fragment to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and / or to increase the affinity of the antibody or its antigen-binding fragment to the Fcγ receptor: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 27, according to Kabat's EU index numbering. 0, 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 approach is further described in International Publication No. WO00 / 42072.

[0163] In certain embodiments, the antibody or antigen-binding fragment described herein includes a constant domain of IgG1 having a mutation (e.g., substitution) at position 267, 328, or a combination thereof, according to Kabat's EU index numbering. In certain embodiments, the antibody or antigen-binding fragment described herein includes a constant domain of IgG1 having a mutation (e.g., substitution) selected from the group consisting of S267E, L328F, and combinations thereof. In certain embodiments, the antibody or antigen-binding fragment described herein includes a constant domain of IgG1 having the S267E / L328F mutation (e.g., substitution). In certain embodiments, the antibody or antigen-binding fragment described herein, including a constant domain of IgG1 having the S267E / L328F mutation (e.g., substitution), has high binding affinity to FcγRIIA, FcγRIIB, or FcγRIIA and FcγRIIB.

[0164] Antibodies with reduced fucose content have been reported to have increased affinity for Fc receptors, such as FcγRIIIA. Therefore, in certain embodiments, the antibodies or antigen-binding fragments described herein have low fucose content or are fucose-deficient (i.e., "afcosylated"). Such antibodies or antigen-binding fragments can be produced using techniques known to those skilled in the art. For example, they may be expressed in cells with impaired or deficient fucosylation ability. In certain examples, antibodies or antigen-binding fragments with reduced fucose content can be produced using cell lines in which both alleles of α1,6-fucosyltransferase are knocked out. The Potelligent® system (Lonza) is an example of a system that can be used to produce antibodies and antigen-binding fragments with reduced fucose content. Alternatively, antibodies or antigen-binding fragments with reduced or no fucose content can be produced, for example, by: (i) cell culture under conditions that prevent or reduce fucosylation; (ii) post-translational removal of fucose (e.g., by a fucosidase enzyme); (iii) post-translational addition of a desired carbohydrate after recombinant expression of a non-glycosylated glycoprotein (e.g.); or (iv) purification of glycoproteins to select an unfucosylated antibody or antigen-binding fragment. For methods of producing antibodies with no or reduced fucose content, see, for example, Longmore GD & Schachter H (1982) Carbohydr Res 100:365-92 and Imai-Nishiya H et al., (2007) BMC Biotechnol. 7:84. See also Example 8 of this specification, which describes the production of an afucosylated B7-H4 antibody.

[0165] In some embodiments, the B7-H4 antibody or its antigen-binding fragment exhibits higher in vitro ADCC activity compared to the fucosylated B7-H4 antibody or its antigen-binding fragment having the same amino acid sequence. In some embodiments, the fucosylated B7-H4 antibody or its antigen-binding fragment induces specific lysis at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 65, at least 70, or at least 75 percent points higher than specific lysis by the fucosylated B7-H4 antibody.

[0166] In some embodiments, the B7-H4 antibody or its antigen-binding fragment has a higher affinity for FcγRIIIA than the fucosylated B7-H4 antibody or its antigen-binding fragment having the same amino acid sequence. In some embodiments, the fucosylated B7-H4 antibody or its antigen-binding fragment binds to FcγRIIIA with 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 affinity than the fucosylated B7-H4 antibody or its antigen-binding fragment. 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).

[0167] In some embodiments, the presence of fucose can be determined by methods including high-performance liquid chromatography (HPLC), capillary electrophoresis, or MALDI-TOF mass spectrometry.

[0168] In certain embodiments, the antibody or its antigen-binding fragment has (i) a CDR sequence of 20502 (e.g., the amino acid sequences of SEQ ID NOs. 458-463), VH and VL sequences of 20502 (the amino acid sequences of SEQ ID NOs. 464 and 42, respectively), or heavy and light chain sequences of 20502 (the amino acid sequences of SEQ ID NOs. 469 and 44, respectively), and (ii) is afucosylated.

[0169] In certain embodiments, the composition comprises (i) a CDR sequence of 20502 (e.g., the amino acid sequences of SEQ ID NOs. 458-463), VH and VL sequences of 20502 (e.g., the amino acid sequences of SEQ ID NOs. 464 and 42, respectively), or heavy and light chain sequences of 20502 (e.g., the amino acid sequences of SEQ ID NOs. 469 and 44, respectively), and (ii) an afucosylated antibody or an antigen-binding fragment thereof, in which case, for example, at least 95% of the antibody in the composition is afucosylated, or fucosylation is undetectable in the composition.

[0170] In certain embodiments, the antibody or its antigen-binding fragment (i) has a CDR sequence of 20502.1 (e.g., the amino acid sequences of SEQ ID NOs. 35-40), VH and VL sequences of 20502.1 (the amino acid sequences of SEQ ID NOs. 41 and 42, respectively), or heavy and light chain sequences of 20502.1 (the amino acid sequences of SEQ ID NOs. 43 and 44, respectively), and (ii) is afucosylated.

[0171] In certain embodiments, the composition comprises (i) an antibody or antigen-binding fragment having (i) a CDR sequence of 20502.1 (e.g., the amino acid sequences of SEQ ID NOs. 35-40), VH and VL sequences of 20502.1 (e.g., the amino acid sequences of SEQ ID NOs. 41 and 42, respectively), or heavy and light chain sequences of 20502.1 (e.g., the amino acid sequences of SEQ ID NOs. 43 and 44, respectively), and (ii) an afucosylated antibody or antigen-binding fragment thereof, in which case, for example, at least 95% of the antibody in the composition is afucosylated, or fucosylation is undetectable in the composition.

[0172] In certain embodiments, the antibody or its antigen-binding fragment has (i) a 22213 CDR sequence (e.g., the amino acid sequences of SEQ ID NOs. 65-70), a 22213 VH and VL sequences (the amino acid sequences of SEQ ID NOs. 71 and 72, respectively), or a 22213 heavy chain and light chain sequence (the amino acid sequences of SEQ ID NOs. 73 and 74, respectively), and (ii) is afucosylated.

[0173] In certain embodiments, the composition comprises (i) an antibody or antigen-binding fragment having (i) a CDR sequence of 22213 (e.g., the amino acid sequences of SEQ ID NOs. 65-70), VH and VL sequences of 22213 (e.g., the amino acid sequences of SEQ ID NOs. 71 and 72, respectively), or heavy and light chain sequences of 22213 (e.g., the amino acid sequences of SEQ ID NOs. 73 and 74, respectively), and (ii) an afucosylated antibody or antigen-binding fragment thereof, in which case, for example, at least 95% of the antibody in the composition is afucosylated, or fucosylation is undetectable in the composition.

[0174] Modified glycoforms may be useful for a variety of purposes, including but not limited to enhancing or reducing effector function. Methods for generating modified glycoforms in antibodies or their antigen-binding fragments described herein include, for example, Umana 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. Patent Nos. 6,602,684, 6,946,292, and 7,214,775; U.S. Patent Publication Nos. US2007 / 0248600, 2007 / 0178551, 2008 / 0060092, and 2006 / 0253928; International Publication Nos. WO00 / 61739, WO01 / 292246, WO02 / 311140, and WO02 / 30954; Potillegent™ Technology (Biowa, Inc.) Examples include, but are not limited to, the methods disclosed in Princeton, NJ, and GlycoMAb® Glycosylation Engineering Technology (Glycart biotechnology AG, Zurich, Switzerland). See also, for example, Ferrara C et al., (2006) Biotechnol Bioeng 93:851-861; International Publications WO07 / 039818; WO12 / 130831; WO99 / 054342; WO03 / 011878; and WO04 / 065540.

[0175] 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 the antibody or antigen-binding fragment described herein, which has two heavy chain constant regions.

[0176] In another specific embodiment, the antibody or antigen-binding fragment described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) comprises a heavy chain and a light chain, in which (i) the heavy chain comprises a VH domain having the VH CDR1, VL CDR2, and VL CDR3 amino acid sequences of the 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 having the VL CDR1, VH CDR2, and VH CDR3 amino acid sequences of the same antibody listed in Table 2 (e.g., SEQ ID NOs. 461-463, 38-40, or 68-70); (iii) the heavy chain further comprises a constant heavy chain domain having the amino acid sequence of the constant domain of the human IgG1 heavy chain; and the light chain further comprises a constant light chain domain having the amino acid sequence of the constant domain of the human κ light chain.

[0177] In another specific embodiment, the antibody or antigen-binding fragment described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) comprises a heavy chain and a light chain, in which (i) the heavy chain comprises a VH domain having the amino acid sequence of the antibody listed in Table 3 (e.g., SEQ ID NO: 464, 41, or 71); (ii) the light chain comprises a VL domain having the amino acid sequence of the same antibody listed in Table 4 (e.g., SEQ ID NO: 42 or 73); (iii) the heavy chain further comprises a constant heavy chain domain having the amino acid sequence of the constant domain of the human IgG1 heavy chain; and the light chain further comprises a constant light chain domain having the amino acid sequence of the constant domain of the human κ light chain.

[0178] In certain embodiments, the antibody or antigen-binding fragment described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) exhibits T-cell checkpoint blocking activity. Exemplary methods for measuring T-cell checkpoint blocking activity are shown in Examples 7 and 11 of this specification. In certain embodiments, the antibody or antigen-binding fragment described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) increases interferon-gamma (IFNγ) production in T cells. Exemplary methods for measuring IFNγ production are shown in Examples 7 and 11 of this specification. In certain embodiments, the antibody or antigen-binding fragment described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) increases T-cell proliferation. Exemplary methods for measuring T-cell proliferation are shown in Example 7 of this specification. In certain embodiments, the antibody or antigen-binding fragment described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) increases CD4+ T-cell proliferation. Exemplary methods for measuring CD4+ T-cell proliferation are shown in Example 7 of this specification. In certain embodiments, antibodies or antigen-binding fragments described herein that immunospecifically bind to B7-H4 (e.g., human B7-H4) increase CD8+ T cells. An exemplary method for measuring CD8+ T cell proliferation is shown in Example 7 herein.

[0179] In certain embodiments, the antibody or antigen-binding fragment described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) exhibits antibody-dependent cell-mediated cytotoxicity (ADCC) activity. In certain embodiments, the antibody or antigen-binding fragment described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) exhibits antibody-dependent cell-mediated cytotoxicity (ADCC) activity against cell lines having at least 300,000 cell surface B7-H4 molecules (e.g., SK-BR-3 cells). In certain embodiments, the antibody or antigen-binding fragment described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) exhibits antibody-dependent cell-mediated cytotoxicity (ADCC) activity against cell lines having at least 100,000 cell surface B7-H4 molecules (e.g., HCC1569 cells). In certain embodiments, the antibody or antigen-binding fragment described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) exhibits antibody-dependent cell-mediated cytotoxicity (ADCC) activity against cell lines having at least 50,000 cell surface B7-H4 molecules (e.g., ZR-75-1 cells). In certain embodiments, the antibody or antigen-binding fragment described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) exhibits antibody-dependent cell-mediated cytotoxicity (ADCC) activity against cell lines having at least 30,000 cell surface B7-H4 molecules (e.g., MDA-MB-468 cells). In certain embodiments, the antibody or antigen-binding fragment described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) exhibits antibody-dependent cell-mediated cytotoxicity (ADCC) activity against cell lines having at least 15,000 cell surface B7-H4 molecules (e.g., HCC1964 cells). An exemplary method for measuring ADCC activity is shown in Example 13 herein.

[0180] In certain embodiments, the antibody or antigen-binding fragment described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) is a human framework region or comprises a framework region derived from a human framework region (e.g., a framework region of the VH domain and / or VL domain). Non-limiting examples of human framework regions are described in the Art, for example, in Kabat EA et al. See al., (1991). In certain embodiments, the antibody or antigen-binding fragment described herein is a primate (e.g., non-human primate) framework region or comprises a framework region derived from a primate (e.g., non-human primate) framework region (e.g., a framework region of the VH domain and / or VL domain).

[0181] In certain embodiments, the antibody or antigen-binding fragment described herein that specifically binds to B7-H4 (e.g., human B7-H4) includes one, two, or more VH framework regions (FRs) having the amino acid sequences described herein (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) for the antibodies listed in Table 5 above. In some embodiments, the antibody or antigen-binding fragment described herein that specifically binds to B7-H4 (e.g., human B7-H4) includes one, two, or more VL framework regions (FRs) having the amino acid sequences described herein (e.g., SEQ ID NOs. 239, 240, 241, and / or 242; or SEQ ID NOs. 269, 270, 271, and / or 272) for the antibodies listed in Table 6 above. In certain embodiments, the antibody or antigen-binding fragment described herein that specifically binds to B7-H4 (e.g., human B7-H4) comprises one, two or more VH framework regions having the amino acid sequences described herein for the antibodies listed in Table 5 above, and also comprises one, two or more VL framework regions having the amino acid sequences described herein for the same antibodies listed in Table 6 above (e.g., (i) SEQ ID NOs: 465, 466, 467, and / or 469, and SEQ ID NOs: 239, 240, 241, and / or 242; (ii) SEQ ID NOs: 235, 236, 237, and / or 238, and SEQ ID NOs: 239, 240, 241, and / or 242, or (iii) SEQ ID NOs: 265, 266, 267, and / or 268, and SEQ ID NOs: 269, 270, 271, and / or 272).

[0182] In certain embodiments, the antibody or antigen-binding fragment described herein that specifically binds to B7-H4 (e.g., human B7-H4) includes 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 with respect to the VH framework region described in Table 5 above. In certain embodiments, the antibody or antigen-binding fragment described herein that specifically binds to B7-H4 (e.g., human B7-H4) includes 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 with respect to the VL framework region described in Table 6 above. In some embodiments, the antibody or antigen-binding fragment 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 with respect to the VH framework region described 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 with respect to the VL framework region described in Table 6 above,

[0183] In certain embodiments, the antibody or antigen-binding fragment 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 with respect to the amino acid sequence of the VH domain of 20502 or 22213 (e.g., SEQ ID NOs. 464 or 71), in which case the antibody comprises a VH CDR identical to that of the VH CDR of 20502 or 22213 (e.g., SEQ ID NOs. 458-460 or 65-67).

[0184] In certain embodiments, the antibody or antigen-binding fragment 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 with respect to the amino acid sequence of the VL domain of 20502 or 22213 (e.g., SEQ ID NOs. 42 or 72), in which case the antibody or antigen-binding fragment comprises a VL CDR identical to the VL CDR of 20502 or 22213 (e.g., SEQ ID NOs. 461-463 or 68-70).

[0185] In certain embodiments, the antibody or antigen-binding fragment 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 with respect 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 with respect to the amino acid sequence of the VL domain of 20502 or 22213 (e.g., SEQ ID NO: 42 or 72), in which case the antibody comprises the same VH CDR and VL CDR as the VH CDR and VL CDR of 20502 or 22213 (e.g., SEQ ID NOs: 458-463 or SEQ ID NOs: 65-70). Includes CD-R.

[0186] In another embodiment, the Specified herein provides an antibody or antigen-binding fragment that binds to the same B7-H4 epitope (e.g., human B7-H4 epitope) as the antibody or antigen-binding fragment described herein (e.g., 20502 or 22213).

[0187] In certain embodiments, the antibody or antigen-binding fragment 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 with respect to the amino acid sequence of the VH domain of 20502.1 or 22213 (e.g., SEQ ID NOs. 41 or 71), in which case the antibody comprises a VH CDR identical to that of the VH CDR of 20502.1 or 22213 (e.g., SEQ ID NOs. 35-37 or 65-67).

[0188] In certain embodiments, the antibody or antigen-binding fragment 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 with respect to the amino acid sequence of the VL domain of 20502.1 or 22213 (e.g., SEQ ID NOs. 42 or 72), in which case the antibody or antigen-binding fragment comprises a VL CDR identical to the VL CDR of 20502.1 or 22213 (e.g., SEQ ID NOs. 38-40 or 68-70).

[0189] In certain embodiments, the antibody or antigen-binding fragment 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 with 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 with the amino acid sequence of the VL domain of 20502.1 or 22213 (e.g., SEQ ID NO: 42 or 72), in which case the antibody is identical to the VH CDR and VL CDR of 20502.1 or 22213 (e.g., SEQ ID NO: 35-40 or SEQ ID NO: 65-70). Includes CDR and VL CDR.

[0190] In another embodiment, the Specified herein provides an antibody or antigen-binding fragment that binds to the same B7-H4 epitope (e.g., human B7-H4 epitope) as the antibody or antigen-binding fragment described herein (e.g., 20502.1 or 22213).

[0191] Competitive binding assays can be used to determine whether two antibodies bind to a duplicate epitope. Competitive binding can be measured in assays in which the immunoglobulin under test inhibits the specific binding of a reference antibody to a common antigen such as B7-H4. Many types of competitive binding assays are known, for example: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competitive assays (see Stahli C et al., (1983) Methods Enzymol 9:242-253); solid-phase direct biotin-avidin EIA (see Kirkland TN et al., (1986) J Immunol 137:3614-9); solid-phase direct labeling assays, solid-phase direct labeling sandwich assays (see Harlow E & Lane These include D, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct-labeled RIA using I-125 labeling (Morel GA et al., (1988) Mol Immunol 25(1):7-15); solid-phase direct biotin-avidin EIA (Cheung RC et al., (1990) Virology 176:546-52); and direct-labeled RIA (Moldenhauer G et al., (1990) Scand J Immunol 32:77-82). Typically, such assays involve the use of purified antigens (e.g., B7-H4 such as human B7-H4) bound to a solid surface or cell holding either the unlabeled test immunoglobulin or the labeled reference immunoglobulin. Competitive inhibition can be measured by determining the amount of label bound to the solid surface or cell in the presence of the test immunoglobulin. Typically, the test immunoglobulin is present in excess. Typically, when competing antibodies are present in excess, the specific binding of the reference antibody to the common antigen is inhibited by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or more. Competitive binding assays can be constructed in numerous different forms using either labeled antigens or labeled antibodies. In a common version of this assay, the antigen is immobilized in a 96-well plate. The ability of an unlabeled antibody to block the binding of the labeled antibody to the antigen is then measured, using radiolabeling or enzymatic labeling. For further details, see, for example, 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,Ed Harlow E & Lane D editors See supra, pp.386-389.

[0192] In one embodiment, a competitive assay is performed using surface plasmon resonance (BIAcore®) by a “tandem approach,” such as described by Abdiche YN et al., (2009) Analytical Biochem 386:172-180, thereby immobilizing the B7-H4 antigen on the surface of a chip, such as a CM5 sensor chip, and then flowing an anti-B7-H4 antibody onto the chip. To determine whether the antibody or its antigen-binding fragment competes with the anti-B7-H4 antibody described herein, the anti-B7-H4 antibody is first flowed onto the chip surface to achieve saturation, and then the potentially competing antibody is added. The binding of the competing antibody or its antigen-binding fragment can then be measured and quantified in comparison to a non-competing control.

[0193] In one embodiment, Fortebio Octet competitive binding (for example, as described in Example 2 below) is used to determine that a B7-H4 antibody or its antigen-binding fragment competitively inhibits the binding of another B7-H4 antibody or antigen-binding fragment to B7-H4.

[0194] In another embodiment, this specification provides antibodies that competitively inhibit (e.g., dose-dependently) the binding of the antibodies or their antigen-binding fragments (e.g., 20502, 20502.1, or 22213) described herein to B7-H4 (e.g., human B7-H4) by measurement using assays known to those skilled in the art or described herein (e.g., competitive ELISA assay, or suspension array or surface plasmon resonance assay).

[0195] In certain embodiments, the Specified provides an antibody or antigen-binding fragment that competitively inhibits (e.g., dose-dependently) the binding of an antibody comprising a VH domain having the amino acid sequence shown in SEQ ID NO: 464 and a VL domain having the amino acid sequence shown in SEQ ID NO: 42 to B7-H4 (e.g., human B7-H4).

[0196] In certain embodiments, the Specified provides an antibody or antigen-binding fragment that competitively inhibits (e.g., dose-dependently) the binding of an antibody comprising a VH domain having the amino acid sequence shown in SEQ ID NO: 41 and a VL domain having the amino acid sequence shown in SEQ ID NO: 42 to B7-H4 (e.g., human B7-H4).

[0197] In certain embodiments, the Specified provides an antibody or antigen-binding fragment that competitively inhibits (e.g., dose-dependently) the binding of an antibody comprising a VH domain having the amino acid sequence shown in SEQ ID NO: 71 and a VL domain having the amino acid sequence shown in SEQ ID NO: 72 to B7-H4 (e.g., human B7-H4).

[0198] In certain embodiments, the Specified provides an antibody or antigen-binding fragment that competitively inhibits binding to B7-H4 (e.g., human B7-H4) (e.g., in a dose-dependent manner) using an antibody comprising (i) a VH domain having the amino acid sequence of the VH CDR described in Table 1, and (ii) a VL domain having the amino acid sequence of the CDR described in Table 2, and VL domain having VL CDR1, VL CDR2, and VL CDR3.

[0199] In certain embodiments, the Specified herein provides antibodies or antigen-binding fragments that immunospecifically bind to the same B7-H4 (e.g., human B7-H4) epitope as described in 20502, 20502.1, or 22213.

[0200] In another specific embodiment, the antibody or antigen-binding fragment described herein immunospecifically binds to the same B7-H4 (e.g., human B7-H4) epitope as an antibody epitope comprising (i) a VH domain including VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of the CDRs listed in Table 1, and (ii) a VL domain including VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of the CDRs listed in Table 2.

[0201] In certain embodiments, the antigen-binding fragment described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) is selected from the group consisting of Fab, Fab', F(ab')2, and scFv, in which case Fab, Fab', F(ab')2, or scFv has a heavy chain variable region sequence and a light chain variable region sequence of an anti-B7-H4 antibody or its antigen-binding fragment, as described herein. Fab, Fab', F(ab')2, or scFv can be generated 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 includes a portion that extends the half-life of the antibody in vivo. This portion is also called the “half-life extension portion”. Any portion known to those skilled in the art can be used to extend the half-life of Fab, Fab', F(ab')2, or scFv in vivo. For example, the half-life extension portion may include an Fc region, a polymer, albumin, or an albumin-binding protein or albumin-binding compound. The polymer may include natural or synthetic linear or branched polyalkylenes, polyalkenes, polyoxyalkylenes, polysaccharides, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, methoxypolyethylene glycol, lactose, amylose, dextran, glycogen, or derivatives thereof, as optionally substituted. Substituents may include one or more hydroxy, methyl, or methoxy groups. In certain embodiments, Fab, Fab', F(ab')2, or scFv may be modified by the addition of one or more C-terminal amino acids to attach the half-life extension portion. In certain embodiments, the half-life extension portion is polyethylene glycol or human serum albumin. In certain embodiments, Fab, Fab', F(ab')2, or scFv is fused to the Fc region.

[0202] Anti-B7-H4 antibodies or their antigen-binding fragments can be fused to or conjugated (e.g., covalently or noncovalently linked) with detectable labels or substances. 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); luminescence labels such as luminol; and fluorescent labels such as fluorescein and rhodamine, as well as biotin. B7-H4 (e.g., human B7-H4) proteins can be detected using such labeled antibodies or their antigen-binding fragments. See, for example, section 5.5.2 below.

[0203] Antibody production

[0204] Antibodies and antigen-binding fragments that immunospecifically bind to B7-H4 (e.g., human B7-H4) can be produced by any method known in the art for the synthesis of antibodies and antigen-binding fragments, either by chemical synthesis or recombinant expression techniques. Unless otherwise specified, the methods described herein utilize prior art in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields in the art. These techniques are described and fully explained in the references cited herein, for example, 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 Report);Current Protocols in Immunology,John Wiley & Sons(1987 and Annual Report)Gait(ed.)(1984)Oligonucleotide Synthesis:A Practical Approach,IRL See 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.

[0205] In certain embodiments, this specification provides a method for producing an antibody or antigen-binding fragment that immunospecifically binds to B7-H4 (e.g., human B7-H4), comprising culturing cells or host cells described herein. In certain embodiments, this specification provides a method for producing an antibody or antigen-binding fragment that immunospecifically binds to B7-H4 (e.g., human B7-H4), comprising expressing (e.g., recombinantly expressing) the antibody or antigen-binding fragment using cells or host cells described herein (e.g., cells or host cells containing a polynucleotide encoding the antibody or antigen-binding fragment described herein). In certain embodiments, the cells are isolated cells. In certain embodiments, an exogenous polynucleotide is introduced into the cells. In certain embodiments, this method further includes a step of purifying the antibody or antigen-binding fragment obtained from the cells or host cells.

[0206] Methods for producing polyclonal antibodies are known in the art (e.g., Short See Chapter 11 of Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., eds., John Wiley and Sons, New York.

[0207] Monoclonal antibodies or their antigen-binding fragments can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombination, and phage display techniques, yeast-based presentation techniques, or combinations thereof. For example, monoclonal antibodies or their antigen-binding fragments are known in the art, for example, Harlow E & Lane D, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling GJ et al., in: Monoclonal Antibodies and T-Cell Hybridomas These antibodies can be produced using hybridoma techniques, including the method shown in 563 681 (Elsevier, NY, 1981) or the method described in Kohler G & Milstein C (1975) Nature 256:495. Examples of yeast-based presentation methods that can be used to select and generate the antibodies described herein include, for example, the methods disclosed in WO2009 / 036379A2; WO2010 / 105256; and WO2012 / 009568, each of which is incorporated herein by reference in its entirety.

[0208] 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 producing a recombinant antibody or antigen-binding fragment), in which case the antibody or antigen-binding fragment immunospecifically binds to B7-H4 (e.g., human B7-H4) as measured, for example, by ELISA or other antigen-binding assays known in the art or provided herein. In certain embodiments, the monoclonal antibody or its antigen-binding fragment may be a chimeric or humanized antibody or its antigen-binding fragment. In certain embodiments, the monoclonal antibody or its antigen-binding fragment may be a Fab fragment or an F(ab')2 fragment. The monoclonal antibodies or their antigen-binding fragments described herein are, for example, Kohler G & Milstein C (1975) Nature They can be prepared by the hybridoma method described in 256:495, or isolated from a phage library using, for example, the techniques described herein. Other methods for preparing clonal cell lines and the monoclonal antibodies and their antigen-binding fragments expressed therein are well known in the art (see, for example, Chapter 11 of Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al.).

[0209] The antigen-binding fragments of the antibodies described herein can be produced by any technique known to those skilled in the art. For example, the Fab and F(ab')2 fragments described herein can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (which produces the Fab fragment) or pepsin (which produces the F(ab')2 fragment). The Fab fragment corresponds to one of the two identical arms of the tetrameric antibody molecule and includes a complete light chain paired with the VH and CH1 domains of the heavy chain. The F(ab')2 fragment includes the two antigen-binding arms of the tetrameric antibody molecule linked by a disulfide bond within a hinge region.

[0210] Furthermore, the antibodies or antigen-binding fragments described herein can also be generated using various phage display and / or yeast-based presentation methods known in the art. In phage display methods, proteins are displayed on the surface of phage particles possessing the encoding polynucleotide sequence. In particular, the DNA sequences encoding the VH and VL domains are amplified from an animal cDNA library (e.g., a human or mouse cDNA library of diseased tissue). The DNA encoding the VH and VL domains is recombined with an scFv linker by PCR and cloned into a phagemide vector. The vector is electroporated into E. coli to infect E. coli with helper phages. The phages used in these methods are typically filamentous phages such as fd and M13, and are usually fused by recombination with either phage gene III or gene VIII to the VH and VL domains. Phages expressing antibodies or antigen-binding fragments that bind to a specific antigen can be selected or identified by antigen, for example, using labeled antigens or antigens bound to or captured on a solid surface or beads.Examples of phage display methods that can be used to produce the antibodies or fragments described herein include: 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 & Barbas CF (1994) Advan Immunol 57:191-280; PCT application PCT / GB91 / 001134; International publications WO90 / 02809, WO91 / 10737, WO92 / 01047, WO92 / 18619, WO93 / 11236, WO95 / 15982, WO95 / 20401, and WO97 / 13844; and U.S. Patent Nos. 5,698,426, 5,2 Examples include the methods disclosed in Nos. 23,409, 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.

[0211] The humanized antibody or its antigen-binding fragment can be selected from any class of immunoglobulin, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4.

[0212] 1.2.1 Polynucleotides In certain embodiments, the Specified Public Service provides polynucleotides comprising nucleotide sequences encoding the antibodies or antigen-binding fragments thereof or their domains (e.g., variable light chain regions and / or variable heavy chain regions) described herein, and B7-H4 (e.g., human B7-H4) antigens that bind immunospecifically, and vectors, such as vectors comprising such polynucleotides for recombinant expression in host cells (e.g., Escherichia coli and mammalian cells).

[0213] In certain embodiments, the Specified provides a polynucleotide having a nucleotide sequence encoding an antibody or an antigen-binding fragment, which immunospecifically binds to a B7-H4 polypeptide (e.g., human B7-H4) and has the amino acid sequence described herein, as well as an antibody or antigen-binding fragment that competes with such an antibody or antigen-binding fragment for binding to a B7-H4 polypeptide (e.g., in a dose-dependent manner), or that binds to the same epitope as such an antibody or antigen-binding fragment.

[0214] This specification also provides polynucleotides having nucleotide sequences encoding a polypeptide having sequences selected from the group consisting of SEQ ID NOs: 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 containing the polypeptide is immunospecifically bound to B7-H4.

[0215] The present specification also provides a polynucleotide having a nucleotide sequence encoding a polypeptide having a sequence selected from the group consisting of SEQ ID NOs: 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 an antigen-binding fragment thereof containing the polypeptide binds immunospecifically to B7-H4.

[0216] The present specification also provides a polynucleotide having a nucleotide sequence encoding a variable heavy chain shown in Table 9, wherein, for example, an antibody or an antigen-binding fragment thereof containing the encoded variable heavy chain binds to B7-H4.

Table 9-1

Table 9-2

Table 9-3

Table 9-4

[0217] The present specification also provides a polynucleotide having a nucleotide sequence encoding a variable light chain shown in Table 10, wherein, for example, an antibody or an antigen-binding fragment thereof containing the encoded variable light chain binds to B7-H4.

Table 10-1

Table 10-2

Table 10-3

Table 10-4

[0218] This specification also provides polynucleotides having the nucleotide sequences of Sequence IDs 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. This specification also provides (i) nucleotide sequences of SEQ ID NOs: 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) polynucleotides having the nucleotide sequence of SEQ ID NOs: 408 or 406.

[0219] This specification also codes for sequence numbers 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 also sequence numbers 213, 214, 223, 224, 233, 234, The present invention provides polynucleotides having nucleotide sequences that are at least approximately 80%, 85%, or 90% identical to 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.

[0220] This specification also codes for sequence numbers 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 also sequence numbers 213, 214, 223, 224, 23 The present invention provides polynucleotides having a nucleotide sequence that is at least approximately 95% identical to 3, 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.

[0221] This specification also codes for sequence numbers 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 also sequence numbers 213, 214, 223, 224, 23 The present invention provides polynucleotides having a nucleotide sequence that is at least approximately 96% identical to 3, 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.

[0222] This specification also codes for sequence numbers 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 also sequence numbers 213, 214, 223, 224, 23 The present invention provides polynucleotides having a nucleotide sequence that is at least approximately 97% identical to 3, 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.

[0223] This specification also codes for sequence numbers 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 also sequence numbers 213, 214, 223, 224, 23 The present invention provides polynucleotides having a nucleotide sequence that is at least approximately 98% identical to 3, 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.

[0224] This specification also codes for sequence numbers 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 also sequence numbers 213, 214, 223, 224, 23 The present invention provides polynucleotides having nucleotide sequences that are at least approximately 99% identical to 3, 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.

[0225] In certain embodiments, this specification provides polynucleotides having nucleotide sequences encoding the light chain or heavy chain of an antibody or antigen-binding fragment described herein. The polynucleotide may have a nucleotide sequence encoding the heavy chain, including the VH FR and CDR of an antibody described herein (see, for example, Tables 1 and 5). The polynucleotide may have a nucleotide sequence encoding the light chain, including the VL FR and CDR of an antibody described herein (see, for example, Tables 2 and 6).

[0226] In certain embodiments, the polynucleotide described herein encodes a VH domain having the amino acid sequence described in SEQ ID NO: 464. In certain embodiments, the polynucleotide described herein encodes a VL domain having the amino acid sequence described in SEQ ID NO: 42.

[0227] In certain embodiments, the polynucleotide described herein encodes a VH domain having the amino acid sequence described in SEQ ID NO: 41. In certain embodiments, the polynucleotide described herein encodes a VL domain having the amino acid sequence described in SEQ ID NO: 42.

[0228] In certain embodiments, the polynucleotide described herein encodes a VH domain having the amino acid sequence described in SEQ ID NO: 71. In certain embodiments, the polynucleotide described herein encodes a VL domain having the amino acid sequence described in SEQ ID NO: 72.

[0229] In certain embodiments, this specification provides a polynucleotide having a nucleotide sequence encoding an anti-B7-H4 antibody comprising three VH chain CDRs, for example, VH CDR1, VH CDR2, and VH VL CDR3 of any of the antibodies described herein (see, for example, Table 1). In certain embodiments, this specification provides a polynucleotide comprising three VL chain CDRs, for example, VL CDR1, VL CDR2, and VL CDR3 of any one of the antibodies described herein (see, for example, Table 2). In certain embodiments, this specification provides a polynucleotide having a nucleotide sequence encoding an anti-B7-H4 antibody comprising three VH chain CDRs, for example, VH CDR1, VH CDR2, and VH CDR3 of any one of the antibodies described herein (see, for example, Table 1), and three VL chain CDRs, for example, VL CDR1, VL CDR2, and VL CDR3 of any one of the antibodies described herein (see, for example, Table 2).

[0230] In certain embodiments, this specification provides a polynucleotide having a nucleotide sequence encoding an anti-B7-H4 antibody or its antigen-binding fragment, or a fragment thereof comprising a VH domain including FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 having, for example, the amino acid sequence described herein (see, for example, Tables 1 and 5, e.g., VH CDR and VH FR of a particular antibody identified by the name in the table). In certain embodiments, this specification provides a polynucleotide having a nucleotide sequence encoding an anti-B7-H4 antibody or its antigen-binding fragment, or a fragment thereof comprising a VL domain including FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 having, for example, the amino acid sequence described herein (see, for example, Tables 2 and 6, e.g., VL CDR and VL FR of a particular antibody identified by the name in the table).

[0231] In certain embodiments, the polynucleotide described herein has a nucleotide sequence encoding a heavy chain variable region having the amino acid sequence described herein (e.g., SEQ ID NOs. 464, 41, or 71), in which case the antibody containing the heavy chain variable region binds immunospecifically to B7-H4 (e.g., human B7-H4). In certain embodiments, the polynucleotide described herein has a sequence encoding a heavy chain variable region provided herein (e.g., the portion encoding the variable region of SEQ ID NOs. 470, 243, or 273), and the antibody containing the heavy chain variable region binds immunospecifically to B7-H4 (e.g., human B7-H4).

[0232] In certain embodiments, the polynucleotides described herein have a nucleotide sequence (e.g., SEQ ID NO: 42 or 72) encoding a light chain variable region having the amino acid sequence described herein, and in that case, an antibody comprising the light chain variable region immunospecifically binds to B7-H4 (e.g., human B7-H4). In certain embodiments, the polynucleotides described herein have a sequence encoding the light chain variable region provided herein (e.g., the portion encoding the variable region of SEQ ID NO: 244 or 274), and an antibody comprising the heavy chain variable region immunospecifically binds to B7-H4 (e.g., human B7-H4).

[0233] In certain embodiments, the polynucleotides or combinations of polynucleotides provided herein have a nucleotide sequence or combination of nucleotide sequences encoding an antibody or an antigen-binding fragment thereof that immunospecifically binds to B7-H4 (e.g., human B7-H4), and in that case, the antibody or antigen-binding fragment thereof comprises a heavy chain, and the heavy chain comprises a heavy chain variable domain having the amino acid sequence shown in Table 3 (e.g., SEQ ID NO: 464, 41, or 71) and a constant region having the amino acid sequence of the human gamma (γ) heavy chain constant region.

[0234] In certain embodiments, the polynucleotides or combinations of polynucleotides provided herein have a nucleotide sequence or combination of nucleotide sequences encoding an antibody or an antigen-binding fragment thereof that immunospecifically binds to B7-H4 (e.g., human B7-H4), and in that case, the antibody or antigen-binding fragment thereof comprises a light chain, and the light chain comprises a light chain variable domain having the amino acid sequence shown in Table 4 (e.g., SEQ ID NO: 42 or 72) and a constant region having the amino acid sequence of the human lambda (λ) light chain constant region.

[0235] In certain embodiments, the polynucleotides or combinations of polynucleotides provided herein have a nucleotide sequence or combination of nucleotide sequences encoding an antibody or antigen-binding fragment thereof that immunospecifically binds to B7-H4 (e.g., human B7-H4), in which case the antibody or antigen-binding fragment comprises (i) a heavy chain, the heavy chain comprising a heavy chain variable domain having the amino acid sequence shown in Table 3 (e.g., SEQ ID NO: 464, 41, or 71) and a constant region having the amino acid sequence of the human gamma (γ) heavy chain constant region, and (ii) a light chain, the light chain comprising a light chain variable domain having the amino acid sequence shown in Table 4 (e.g., SEQ ID NO: 42 or 72) and a constant region having the amino acid sequence of the human λ light chain constant region.

[0236] In one embodiment, the polynucleotide combination provided herein includes a polynucleotide having the nucleotide sequence of SEQ ID NO: 470 and a polynucleotide having the nucleotide sequence of SEQ ID NO: 244.

[0237] In one embodiment, the polynucleotide combination provided herein includes a polynucleotide having the nucleotide sequence of SEQ ID NO: 243 and a polynucleotide having the nucleotide sequence of SEQ ID NO: 244.

[0238] In one embodiment, the polynucleotide combination provided herein includes a polynucleotide having the nucleotide sequence of SEQ ID NO: 273 and a polynucleotide having the nucleotide sequence of SEQ ID NO: 274.

[0239] In certain embodiments, this specification provides polynucleotides having nucleotide sequences encoding an anti-B7-H4 antibody or its antigen-binding fragment or its domain, as specified herein; see, for example, Tables 1-8.

[0240] This specification also provides polynucleotides encoding anti-B7-H4 antibodies or their antigen-binding fragments or domains, which are optimized, for example, by codon / RNA optimization, substitution with heterologous signal sequences, and removal of mRNA-destabilizing elements. Thus, methods for generating nucleic acids encoding anti-B7-H4 antibodies or their antigen-binding fragments or domains (e.g., heavy chain, light chain, VH domain, or VL domain) optimized for recombinant expression by introducing codon changes (e.g., codon changes encoding the same amino acid due to degeneracy of the gene code), and / or methods for removing inhibitory regions of mRNA, can be carried out by adapting the optimization methods described in, for example, U.S. Patents 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498.

[0241] The polynucleotides encoding antibodies or their antigen-binding fragments or domains described herein can be generated from nucleic acids derived from a suitable source (e.g., hybridomas) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of known sequences can be performed using genomic DNA obtained from hybridoma cells producing the antibody of interest. Using such PCR amplification methods, nucleic acids having sequences encoding the light and / or heavy chains of the antibody or its antigen-binding fragment can be obtained. Using such PCR amplification methods, nucleic acids having sequences encoding variable light and / or variable heavy chain regions of the antibody or its antigen-binding fragment can be obtained. The amplified nucleic acids can be cloned into vectors for expression in host cells and further cloning to generate, for example, chimeric and humanized antibodies or their antigen-binding fragments.

[0242] The polynucleotides provided herein may be, for example, in the form of RNA or DNA. DNA includes cDNA, genomic DNA, and synthetic DNA, and DNA may be double-stranded or single-stranded. If single-stranded, DNA may be a coding strand or a non-coding (antisense) strand. In certain embodiments, the polynucleotide is cDNA or DNA lacking one or more endogenous introns. In certain embodiments, the polynucleotide is a non-natural 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 a natural component.

[0243] 1.2.2 Cells and Vectors In certain embodiments, this specification provides a vector (e.g., an expression vector) comprising an anti-B7-H4 antibody and a polynucleotide having a nucleotide sequence encoding the antigen-binding fragment or its domain for recombinant expression in host cells, preferably mammalian cells. This specification also provides cells, e.g., host cells, comprising such a vector for recombinant expression of the anti-B7-H4 antibody or its antigen-binding fragment (e.g., a human or humanized antibody or its antigen-binding fragment) described herein. In certain embodiments, this specification provides a method for producing the antibody or its antigen-binding fragment described herein, comprising expressing such an antibody or its antigen-binding fragment in host cells.

[0244] In certain embodiments, recombinant expression of the antibody or its antigen-binding fragment or domain (e.g., the heavy or light chain described herein) that specifically binds to B7-H4 (e.g., human B7-H4) involves constructing an expression vector containing a polynucleotide encoding the antibody or its antigen-binding fragment or domain. After obtaining the polynucleotide encoding the antibody or its antigen-binding fragment or domain (e.g., heavy or light chain variable domain) described herein, a vector for producing the antibody or its antigen-binding fragment can be produced by recombinant DNA technology using techniques well known in the art. Therefore, a method for preparing a protein by expressing a polynucleotide containing an antibody or its antigen-binding fragment or domain (e.g., light or heavy chain) encoding a nucleotide sequence is described herein. Using methods well known to those skilled in the art, an expression vector containing the sequence encoding the antibody or its antigen-binding fragment or domain (e.g., light or heavy chain) and appropriate transcription and translational regulatory signals can be constructed. These methods include, for example, in vitro recombinant DNA technology, synthesis technology, and in vitro recombinant DNA technology. This includes vivo genetic recombination. The Specified Specified Specified Reproducible Vectors also provide a nucleotide sequence encoding an antibody or its antigen-binding fragment, a heavy chain or light chain, a heavy chain or light chain variable domain, or a heavy chain or light chain CDR operably linked to a promoter. Such vectors may, for example, have a nucleotide sequence encoding a constant region of the antibody or its antigen-binding fragment (see, for example, International Publications WO86 / 05807 and WO89 / 01036; and U.S. Patent No. 5,122,464), and the variable domain of the antibody or its antigen-binding fragment can be cloned into such vectors for the expression of the entire heavy chain, the entire light chain, or both the entire heavy chain and the entire light chain.

[0245] By conventional technology, an expression vector can be transferred into cells (e.g., host cells), and then the resulting cells can be cultured by conventional technology to produce the antibodies or antigen-binding fragments thereof described herein (e.g., antibodies or antigen-binding fragments thereof containing six CDRs of 20502, 20502.1, or 22213, VH, VL, VH and VL, heavy chain, light chain, or heavy chain and light chain) or their domains (e.g., VH, VL, VH and VL, heavy chain or light chain of 20502, 20502.1, or 22213). Accordingly, the Specified provides host cells comprising a polynucleotide encoding an antibody or its antigen-binding fragment (e.g., an antibody or its antigen-binding fragment comprising six CDRs, VH, VL, VH and VL, heavy chain, light chain, or heavy chain and light chain of 20502, 20502.1, or 22213) or a domain (e.g., VH, VL, VH and VL, heavy chain, light chain, or heavy chain or light chain of 20502, 20502.1, or 22213) operably linked to a promoter for the expression of such sequences. In certain embodiments, for the expression of a double-chain antibody or its antigen-binding fragment, as detailed below, a vector encoding both the heavy chain and light chain separately can be co-expressed in the host cell for the expression of the entire immunoglobulin. In certain embodiments, the host cell has a vector comprising a polynucleotide encoding both the heavy and light chains of the antibody described herein (e.g., the heavy and light chains of 20502, 20502.1, or 22213) or their domains (e.g., the VH and VL of 20502, 20502.1, or 22213). In certain embodiments, the host cell has two different vectors, namely a first vector comprising a polynucleotide encoding the heavy chain or heavy chain variable region of the antibody described herein or its antigen-binding fragment, and a second vector comprising a polynucleotide encoding the light chain or light chain variable region of the antibody described herein or its antigen-binding fragment (e.g., the antibody comprising six CDRs of 20502, 20502.1, or 22213) or its domains.In other embodiments, a first host cell comprises a first vector comprising a polynucleotide encoding the heavy chain or heavy chain variable region of the antibody or antigen-binding fragment described herein, and a second host cell comprises a second vector comprising a polynucleotide encoding the light chain or light chain variable region of the antibody or antigen-binding fragment described herein (e.g., an antibody or antigen-binding fragment comprising six CDRs of 20502, 20502.1, or 22213). In certain embodiments, the heavy chain / heavy chain variable region expressed by the first cell is associated with the light chain / light chain variable region of the second cell to form the anti-B7-H4 antibody or antigen-binding fragment described herein (e.g., an antibody or antigen-binding fragment comprising six CDRs of 20502, 20502.1, or 22213). In certain embodiments, a population of host cells comprising such a first host cell and such a second host cell is provided herein.

[0246] In certain embodiments, this specification provides a vector population comprising a first vector containing a polynucleotide encoding the light chain / light chain variable region of the anti-B7-H4 antibody or its antigen-binding fragment as described herein, and a second vector containing a polynucleotide encoding the heavy chain / heavy chain variable region of the anti-B7-H4 antibody or its antigen-binding fragment as described herein (e.g., an antibody or its antigen-binding fragment containing the CDR of 20502, 20502.1, or 22213). Alternatively, a single vector capable of encoding and expressing both heavy chain and light chain polypeptides can be used.

[0247] Various host expression vector systems can be used to express the antibodies and their antigen-binding fragments described herein (e.g., antibodies or their antigen-binding fragments containing CDRs 20502, 20502.1, or 22213) (see, for example, U.S. Patent No. 5,807,715). Such host expression systems represent vehicles that can produce and subsequently purify the desired coding sequence, but also represent cells that, upon transformation or translocation with a suitable nucleotide coding sequence, can express the antibodies or their antigen-binding fragments described herein in situ. These include bacteria (e.g., E. coli and B. subtilis) transformed with expression vectors containing recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA with antibody coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell lines infected with recombinant virus expression vectors containing antibody coding sequences (e.g., baculovirus); and plant cell lines (e.g., Chlamydomonas) infected with recombinant virus expression vectors (cauliflower mosaic virus CaMV; tobacco mosaic virus TMV) or transformed with recombinant plasmid expression vectors containing antibody coding sequences (e.g., Ti plasmid). This includes, but is not limited to, microorganisms such as green algae (e.g., reinhardtii); or mammalian cell lines having recombinant expression constructs containing mammalian cell genome-derived promoters (e.g., metallothionein promoter) or mammalian virus-derived promoters (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter) (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20 and BMT10 cells).In certain embodiments, the cells for expressing the antibodies and their antigen-binding fragments described herein (e.g., antibodies or their antigen-binding fragments containing CDRs of 20502, 20502.1, or 22213) are CHO cells, e.g., CHO cells derived from CHO GS System® (Lonza). In certain embodiments, the cells for expressing the antibodies described herein are human cells, e.g., human cell lines. In certain embodiments, the mammalian expression vector is pOptiVEC® or pcDNA3.3. In certain embodiments, bacterial cells such as Escherichia coli, or eukaryotic cells (e.g., mammalian cells) are used for the expression of recombinant antibody molecules, particularly for the expression of the entire recombinant antibody molecule. Mammalian cells such as Chinese hamster ovary (CHO) cells combined with a vector, e.g., a major mid-to-early gene promoter element derived from human cytomegalovirus, are an effective antibody expression system (Foecking MK & Hofstetter H (1986) Gene 45:101-105; and Cockett MI et al.). al., (1990) Biotechnology 8:662-667). In certain embodiments, the antibodies or antigen-binding fragments described herein are produced by CHO cells or NS0 cells.

[0248] Furthermore, host cell lines can be selected that regulate the expression of the inserted sequence or modify and process the gene product in a desired manner. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may contribute to protein function. For this purpose, eukaryotic host cells can be used that possess cellular mechanisms for appropriate processing, glycosylation, and phosphorylation of the primary transcript of the gene product. Examples of such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK293, NIH3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (mouse myeloma cell line that does not endogenously produce immunoglobulin chains), CRL7O3O, 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 antibody described herein (e.g., an antibody or its antigen-binding fragment containing the CDR of 20502, 20502.1, or 22213) is produced in mammalian cells such as CHO cells.

[0249] In certain embodiments, the anti-B7-H4 antibody described herein (e.g., an antibody or its antigen-binding fragment containing CDR 20502, 20502.1, or 22213) is produced in Potelligent® CHOK1SV cells.

[0250] In some embodiments, host cells are provided that contain nucleic acids encoding the B7-H4 antibody or its antigen-binding fragment as described herein, in which case the host cells lack the functional α-1,6-fucosyltransferase gene (FUT8). In some embodiments, the host cells are CHO cells.

[0251] After the antibodies or antigen-binding fragments described herein are produced by recombinant expression, they can be purified by any method known in the art for the purification of immunoglobulin molecules, such as chromatography (e.g., ion exchange, affinity, particularly affinity for a specific antigen after protein A, and sizing column chromatography), centrifugation, solubility testing, or other standard methods for protein purification. Furthermore, the antibodies or antigen-binding fragments described herein can be fused to heterologous polypeptide sequences described herein or known in the art to facilitate purification.

[0252] In certain embodiments, the antibody or its antigen-binding fragment described herein is isolated or purified. Generally, the isolated antibody or its antigen-binding fragment is substantially free of other antibodies or its antigen-binding fragment having different antigen specificity than the isolated antibody or its antigen-binding fragment. For example, in certain embodiments, the preparation of the antibody or its antigen-binding fragment described herein is substantially free of cellular material and / or chemical precursors.

[0253] 1.3 Pharmaceutical Compositions This specification provides compositions comprising an antibody or antigen-binding fragment described herein having a desired purity in a physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1990), Mack Publishing Co., Easton, PA). The acceptable carrier, excipient, or stabilizer is harmless to the recipient at the dose and concentration used.

[0254] In various embodiments, compositions comprising an anti-B7-H4 antibody or its antigen-binding fragment are provided in formulations comprising a pharmaceutically acceptable carrier (see, for example, Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed., Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd ed., Pharmaceutical Press (2000)).

[0255] The pharmaceutical compositions described herein may be useful for blocking the inhibitory activity of B7-H4 against T cells and / or for ADCC-dependent depletion of B7-H4 expressing cells. The pharmaceutical compositions described herein may be useful for treating conditions such as cancer. Examples of cancers that can be treated by the methods described herein include, but are not limited to, breast cancer (e.g., trinegative 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., urothelial carcinoma). Non-small cell lung cancer may be, for example, adenocarcinoma. Further examples of cancers that can be treated by the methods described herein include, but are not limited to, head and neck cancer, small cell lung cancer, gastric cancer, melanoma, and cholangiocarcinoma. In one embodiment, ovarian cancer is serous adenocarcinoma. In one embodiment, breast cancer is ductal carcinoma.

[0256] In one embodiment, the pharmaceutical composition described herein is a composition for use as a pharmaceutical agent. In one embodiment, the pharmaceutical composition described herein is a composition for use as a diagnostic agent for detecting the presence of B7-H4 in a sample obtained from a patient (e.g., a human patient).

[0257] Compositions used for in vivo administration can be sterile. This can be easily achieved, for example, by filtering through a sterile filtration membrane.

[0258] In some embodiments, a pharmaceutical composition is provided, in which case the pharmaceutical composition comprises an anti-B7-H4 antibody or its antigen-binding fragment as described herein and a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition is provided, in which case the pharmaceutical composition comprises an afucosylated anti-B7-H4 antibody or its antigen-binding fragment as described herein and a pharmaceutically acceptable carrier. In certain embodiments, such a pharmaceutical composition comprises, for example, an afucosylated anti-B7-H4 antibody or antigen-binding fragment in which at least 80% of the antibody in the composition is afucosylated. In certain embodiments, such a pharmaceutical composition comprises, for example, an afucosylated anti-B7-H4 antibody or antigen-binding fragment in which at least 85% of the antibody in the composition is afucosylated. In certain embodiments, such a pharmaceutical composition comprises, for example, an afucosylated anti-B7-H4 antibody or antigen-binding fragment in which at least 90% of the antibody in the composition is afucosylated. In certain embodiments, such a pharmaceutical composition includes, for example, an afucosylated anti-B7-H4 antibody or antigen-binding fragment in which at least 95% of the antibody in the composition is afucosylated. In certain embodiments, such a pharmaceutical composition includes, for example, an afucosylated anti-B7-H4 antibody or antigen-binding fragment in which at least 96% of the antibody in the composition is afucosylated. In certain embodiments, such a pharmaceutical composition includes, for example, an afucosylated anti-B7-H4 antibody or antigen-binding fragment in which at least 97% of the antibody in the composition is afucosylated. In certain embodiments, such a pharmaceutical composition includes, for example, an afucosylated anti-B7-H4 antibody or antigen-binding fragment in which at least 98% of the antibody in the composition is afucosylated. In certain embodiments, such a pharmaceutical composition includes, for example, an afucosylated anti-B7-H4 antibody or antigen-binding fragment in which at least 99% of the antibody in the composition is afucosylated. In certain embodiments, such a pharmaceutical composition comprises an afucosylated anti-B7-H4 antibody or antigen-binding fragment in which fucose cannot be detected in the composition.

[0259] In some embodiments, the pharmaceutical composition comprises (i) (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 to 463, respectively; (b) a variable heavy chain region having the amino acid sequence of SEQ ID NO. 464 and a variable light chain region having the amino acid sequence of SEQ ID NO. 42; or (c) a heavy chain having the amino acid sequence of SEQ ID NO. 469 and a light chain having the amino acid sequence of SEQ ID NO. 44; and (ii) a pharmaceutically acceptable excipient, comprising an isolated antibody or antigen-binding fragment that specifically binds to human B7-H4.

[0260] This specification also refers to (i) heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, and VH CDR2, respectively, which specifically bind to human B7-H4 and correspond to sequence numbers 458-463. The present invention provides a pharmaceutical composition comprising (ii) an antibody or its antigen-binding fragment having CDR3 and light chain variable regions (VL) CDR1, CDR2, and CDR3 sequences, 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 its antigen-binding fragment in the composition is afcosylated. In one embodiment, (i) the antibody or its antigen-binding fragment comprises a variable heavy chain region having the amino acid sequence of SEQ ID NO: 464 and a variable light chain region having the amino acid sequence of SEQ ID NO: 42, or (ii) the antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 469 and a light chain having the amino acid sequence of SEQ ID NO: 44.

[0261] In some embodiments, the pharmaceutical composition comprises (i) (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 to 40, respectively; (b) a variable heavy chain region having the amino acid sequence of SEQ ID NO. 41 and a variable light chain region having the amino acid sequence of SEQ ID NO. 42; or (c) a heavy chain having the amino acid sequence of SEQ ID NO. 43 and a light chain having the amino acid sequence of SEQ ID NO. 44; and (ii) a pharmaceutically acceptable excipient, comprising an isolated antibody or antigen-binding fragment thereof that specifically binds to human B7-H4.

[0262] This specification also provides pharmaceutical compositions comprising (i) an antibody or antigen-binding fragment 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, respectively, of SEQ ID NOs. 35 to 40, 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 in the composition is afucosylated. In one embodiment, (i) the antibody or antigen-binding fragment comprises a variable heavy chain region having the amino acid sequence of SEQ ID NO: 41 and a variable light chain region having the amino acid sequence of SEQ ID NO: 42, or (ii) the antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 43 and a light chain having the amino acid sequence of SEQ ID NO: 44.

[0263] In some embodiments, the pharmaceutical composition comprises (i) (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 to 70, respectively; (b) a variable heavy chain region having the amino acid sequence of SEQ ID NO. 71 and a variable light chain region having the amino acid sequence of SEQ ID NO. 72, or (c) a heavy chain having the amino acid sequence of SEQ ID NO. 73 and a light chain having the amino acid sequence of SEQ ID NO. 74; and (ii) a pharmaceutically acceptable excipient, comprising an isolated antibody or antigen-binding fragment that specifically binds to human B7-H4.

[0264] This specification also provides pharmaceutical compositions comprising (i) an antibody or antigen-binding fragment 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, respectively, as of SEQ ID NOs. 65 to 70, 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 in the composition is afucosylated. In one embodiment, (i) the antibody or antigen-binding fragment comprises a variable heavy chain region having the amino acid sequence of SEQ ID NO: 71 and a variable light chain region having the amino acid sequence of SEQ ID NO: 72, or (ii) the antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 73 and a light chain having the amino acid sequence of SEQ ID NO: 74.

[0265] 1.4 Use and Method 1.4.1 Therapeutic Use and Methods In one embodiment, this specification describes a method for modulating one or more immune functions of a subject, comprising administering an anti-B7-H4 antibody or its antigen-binding fragment, or a pharmaceutical composition thereof, to a subject requiring such antibody, as described above and in this specification.

[0266] 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 the patient's T cells, CD4+ T cells, or CD8+ T cells. 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 patient's interferon-gamma (IFNγ) production. 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 the patient's T cells. 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 the patient's cancer cells that express B7-H4. 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.

[0267] In certain embodiments, this specification provides a method for treating cancer, for example, cancer expressing B7-H4. The method for treating cancer may include administering an anti-B7-H4 antibody or its antigen-binding fragment, or a pharmaceutical composition containing an anti-B7-H4 antibody or its antigen-binding fragment, to a patient in need (e.g., a human patient).

[0268] In certain embodiments, methods for treating cancer selected from the group consisting of breast cancer (e.g., trine-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., urothelial carcinoma) are provided. In certain embodiments, methods for treating non-small cell lung cancer that is adenocarcinoma are provided. In certain embodiments, methods for treating cancer selected from the group consisting of head and neck cancer, small cell lung cancer, gastric cancer, melanoma, and cholangiocarcinoma are provided. In one embodiment, ovarian cancer is serous adenocarcinoma. In one embodiment, breast cancer is ductal carcinoma. In some embodiments, such methods include administering an anti-B7-H4 antibody or its antigen-binding fragment, or a pharmaceutical composition comprising an anti-B7-H4 antibody or its antigen-binding fragment, to a patient in need (e.g., a human patient). In some embodiments, the cancer is a cancer that expresses B7-H4.

[0269] In certain embodiments, this specification provides a method for treating cancers that are inadequately responsive to PD-1 / PD-L1 inhibitors. Cancers that are inadequately responsive to PD-1 / PD-L1 inhibitors may have previously responded to PD-1 / PD-L1 inhibitors but have become less responsive to them, or the cancer may have never responded to PD-1 / PD-L1 inhibitors at all. Inadequately responsive to PD-1 / PD-L1 inhibitors means that the aspects of the cancer that are expected to improve after administration of a standard dose of a PD-1 / PD-L1 inhibitor do not improve, and / or improvement occurs only with administration exceeding the standard dose. In some embodiments, subjects with cancers that are inadequately responsive to PD-1 / PD-L1 inhibitors have experienced, or are experiencing, an inadequate response to PD-1 / PD-L1 inhibitors for at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 6 weeks, or at least 12 weeks after administration of at least the standard dose. The “standard” dose is determined by a healthcare professional and may depend on the subject’s age, weight, medical history, disease severity, and frequency of administration. In some embodiments, subjects with cancer that is inadequately responsive to PD-1 / PD-L1 inhibitors have experienced or are experiencing an inadequate response to anti-PD-1 antibodies and / or anti-PD-L1 antibodies. In some embodiments, subjects with cancer that is inadequately responsive to PD-1 / PD-L1 inhibitors have experienced or are experiencing an inadequate response to AMP-224. In some embodiments, subjects with cancer that is inadequately responsive to PD-1 / PD-L1 inhibitors have experienced or are experiencing an inadequate response to PD-1 / PD-L1 inhibitors selected from nivolumab, pembrolizumab, and atezolizumab.

[0270] In certain embodiments, this specification provides methods for treating cancers expressing low levels of PD-L1. In some embodiments, "low-level PD-L1" cancer or "low-level PD-L1" cancer means that the level of PD-L1 is below the expression level of cancers indicated for treatment with PD-1 or PD-L1 antagonists, in which case patients are selected for treatment based on their PD-L1 expression level. In some embodiments, "low-level PD-L1" means that less than 1% of the cells in the tumor are stained at the membrane. In some embodiments, "low level" for PD-L1 means less than 1% staining, e.g., 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0% of tumor cells are stained. In some embodiments, the PD-L1 expression level can be measured by chromogenic IHC or immunofluorescence IHC (Aqua scoring). In certain embodiments, PD-L1 staining of 5% or less (including tumor and / or immune cells) may indicate that a sample expresses "low levels of PD-L1". In certain embodiments, PD-L1 staining of 10% or less (including tumor and / or immune cells) may indicate that a sample expresses "low levels of PD-L1". Unless otherwise specified herein, a threshold of 5% is used (i.e., 5% or less indicates "low levels of PD-L1").

[0271] In another embodiment, an anti-B7-H4 antibody or its antigen-binding fragment, or a pharmaceutical composition, is administered to a patient diagnosed with cancer (e.g., a human patient) to increase the proliferation of the patient's T cells, CD4+ T cells, or CD8+ T cells. In yet another embodiment, an anti-B7-H4 antibody or its antigen-binding fragment, or a pharmaceutical composition, is administered to a patient diagnosed with cancer (e.g., a human patient) to increase the patient's interferon-gamma (IFN-gamma) production. In yet another embodiment, an anti-B7-H4 antibody or its antigen-binding fragment, or a pharmaceutical composition, is administered to a patient diagnosed with cancer (e.g., a human patient) to block the inhibitory activity of B7-H4 against the patient's T cells. In yet another embodiment, an anti-B7-H4 antibody or its antigen-binding fragment, or a pharmaceutical composition, is administered to a patient diagnosed with cancer (e.g., a human patient) to deplete the patient's cancer cells that express B7-H4.

[0272] In further embodiments, an anti-B7-H4 antibody or its antigen-binding fragment, or a pharmaceutical composition, is administered to the patient as described above, and is further administered in combination with additional therapeutic agents, such as chemotherapeutic agents or immunostimulants, such as T-cell checkpoint inhibitors.

[0273] In exemplary embodiments, additional therapeutic agents are PD-1 antagonists, such as antagonistic PD-1 antibodies. Suitable PD-1 antibodies include, for example, OPDIVO (nivolumab), KEYTRUDA (pembrolizumab), or MEDI-0680 (AMP-514, WO2012 / 145493). Other suitable PD-1 antibodies include, for example, camrelizumab (SHR-1210), tisrelizumab (BGB-A317), or spartalizumab (NPVPDR001, NVS240118, PDR001). Additional therapeutic agents may also include pizilizumab (CT-011). A recombinant protein called AMP-224, which contains the extracellular domain of PD-L2 (B7-DC) fused to the Fc portion of IgG1, can also be used to antagonize the PD-1 receptor.

[0274] In another exemplary embodiment, the additional therapeutic agent is a PD-L1 antagonist, such as an antagonistic PD-L1 antibody. Suitable PD-L1 antibodies include, for example, TECENTRIQ (atezolizumab), durvalumab (MEDI4736), BMS-936559 (WO2007 / 005874), MSB0010718C (WO2013 / 79174), or rHigM12B7.

[0275] In yet another exemplary embodiment, the additional therapeutic agent is a GITR agonist, such as an antagonistic GITR antibody. Suitable GITR antibodies include, for example, TRX-518 (WO06 / 105021, WO09 / 009116), MK-4166 (WO11 / 028683), or the GITR antibody disclosed in WO2015 / 031667. In another embodiment, the additional therapeutic agent is the GITR antibody disclosed in WO2017 / 015623. In certain embodiments, the GITR antibody is selected from the following: a) an antibody comprising a GITR-binding domain (GITR-BD) having a CDR1 having the sequence of SEQ ID NO: 411, a CDR2 having the sequence of SEQ ID NO: 412, and a CDR3 having the sequence of SEQ ID NO: 413; b) an antibody comprising a GITR-BD having 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 having the sequence of SEQ ID NO: 411, a CDR2 having the sequence of SEQ ID NO: 412, and a CDR3 having the sequence of SEQ ID NO: 413, and (ii) a phosphorus (iii) the linker is a polypeptide, (iii) the hinge is a polypeptide derived from an immunoglobulin hinge region, and (iv) 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, where (i) GITR-BD has the amino acid sequence of SEQ ID NO: 414, (ii) the linker is a polypeptide, (iii) the hinge is a polypeptide derived from an immunoglobulin hinge region, and (iv) Fc is an immunoglobulin Fc polypeptide; and e) a tetravalent molecule comprising two copies of a polypeptide having the sequence of SEQ ID NO: 415. In any of the above embodiments of the tetravalent molecule, the hinge may have the sequence of SEQ ID NO: 416, 417, or 418. In any of the above embodiments of the tetravalent molecule, the linker may have an amino acid sequence selected from GG, GGG, and SEQ ID NOs: 419-425. In certain embodiments, the hinge has sequence number 417, and the linker has one of sequence numbers 419 to 423.

[0276] In yet another exemplary embodiment, the additional therapeutic agent is a CD80 extracellular domain (CD80 ECD), e.g., SEQ ID NO: 426; or a CD80 ECD fusion molecule comprising a CD80 ECD and a fusion partner as disclosed in WO2017 / 079117. In a particular embodiment, the CD80 ECD fusion molecule is a CD80 ECD-Fc fusion protein. In a particular embodiment, the CD80 ECD-Fc fusion protein has the sequence of SEQ ID NO: 427 or 428.

[0277] 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 particular embodiment, the anti-CSF1R antibody is selected from: a) an antibody comprising a heavy chain having the sequence of SEQ ID NO: 429 and a light chain having the sequence of SEQ ID NO: 430; b) an antibody comprising a heavy chain having a heavy chain (HC) complementarity determining region 1 (CDR1) having the sequence of SEQ ID NO: 431, an HC CDR2 having the sequence of SEQ ID NO: 432, and an HC CDR3 having the sequence of SEQ ID NO: 433, and a light chain having a light chain (LC) CDR1 having the sequence of SEQ ID NO: 434, an LC CDR2 having the sequence of SEQ ID NO: 435, and an LC CDR3 having the sequence of SEQ ID NO: 436; and c) an antibody comprising a heavy chain having the sequence of SEQ ID NO: 437 and a light chain having the sequence of SEQ ID NO: 438.

[0278] While the patients are typically human, non-human mammals, including transgenic mammals, can also be treated.

[0279] In some embodiments, the present invention relates to antibodies or antigen-binding fragments thereof or pharmaceutical compositions provided herein for use as pharmaceuticals. In some embodiments, the present invention relates to antibodies or antigen-binding fragments thereof or pharmaceutical compositions provided herein for use in methods for treating cancer. In some embodiments, the present invention relates to antibodies or antigen-binding fragments thereof or pharmaceutical compositions provided herein for use in methods for treating cancer in a subject, comprising administering an effective amount of the antibody or antigen-binding fragments thereof or pharmaceutical compositions provided herein to the subject.

[0280] 1.4.1.1 Route of administration and dosage The antibodies or antigen-binding fragments or compositions described herein can be delivered to a subject by various routes, including parenteral, subcutaneous, intravenous, intradermal, transdermal, intranasal, intratumoral, and intratumoral lymph node administration. In one embodiment, the antibody or antigen-binding fragment or composition is administered via an intravenous route.

[0281] The amount of antibody, its antigen-binding fragment, or composition effective in treating a disease condition will depend on the nature of the disease. The precise dose to be used in the composition will also depend on the route of administration and the severity of the disease.

[0282] 1.4.2 Use of Detection and Diagnostics Using the anti-B7-H4 antibody or its antigen-binding fragment described herein (see, for example, Section 5.2), B7-H4 protein levels in biological samples can be analyzed using classical methods known to those skilled in the art, including immunoassays such as enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, and Western blotting. Suitable antibody assay labels are known in the art and include enzymatic labels such as glucose oxidase; radioisotopes such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (121In), and technetium (99Tc); luminescence labels such as luminol; and fluorescent labels such as fluorescein and rhodamine, as well as biotin. Such labels can be used to label the antibody or its antigen-binding fragment described herein. Alternatively, a secondary antibody or its antigen-binding fragment that recognizes the anti-B7-H4 antibody or its antigen-binding fragment described herein may be labeled and used in combination with the anti-B7-H4 antibody or its antigen-binding fragment to detect B7-H4 protein levels.

[0283] Assays on B7-H4 protein expression levels are intended to involve qualitatively or quantitatively measuring or estimating the level of B7-H4 protein in a first biological sample, either directly (e.g., by measuring or estimating absolute protein levels) or relatively (e.g., by comparing it to disease-related protein levels in a second biological sample). The B7-H4 polypeptide expression level in the first biological sample can be measured or estimated and compared to a standard B7-H4 protein level obtained from a second biological sample taken from a population of healthy individuals, or determined by averaging the levels of a population of healthy individuals. As is understood in the art, once a “standard” B7-H4 polypeptide level is determined, it can be repeatedly used as a reference standard.

[0284] As used herein, the term “biological sample” refers to a biological sample obtained from a subject, cell line, tissue, or other cell source potentially expressing B7-H4. Methods for tissue biopsy and fluid collection from animals (e.g., humans) are well known in the art. Biological samples include peripheral mononuclear blood cells. Biological samples may also be blood samples, in which case circulating tumor cells (i.e., “CTCs”) may express and detect B7-H4.

[0285] The anti-B7-H4 antibodies described herein are well known and standard to those skilled in the art and can be used for prognostic, diagnostic, monitoring, and screening applications, including in vitro and in vivo applications, based on this specification. Prognostic, diagnostic, monitoring, and screening assays and kits for in vitro assessment and evaluation of immune system status and / or immune response may be used to predict, diagnose, and monitor patient samples, including those from patients known or suspected to have immune system dysfunction or cancer. This type of prognostic and diagnostic monitoring and evaluation has already been practiced using antibodies against the HER2 protein in breast cancer (HercepTest™, Dako), and this assay is also used to evaluate patients undergoing antibody therapy with Herceptin®. In vivo applications include targeted cell therapy, immune system modification, and radiographic imaging of immune responses.

[0286] The anti-B7-H4 antibodies and their antigen-binding fragments described herein may be made to possess detectable or functional labels. When fluorescent labeling is used, specific binding members may be identified and quantified using currently available microscopy and / or fluorescent cell sorting (FACS) methods known in the art. The anti-B7-H4 antibodies or their antigen-binding fragments described herein may be made to possess fluorescent labeling. Exemplary fluorescent labels include, for example, reactive and conjugated probes such as aminocoumarin, fluorescein, and Texas Red, Alexa Fluor dyes, Cy dyes, and DyLight dyes. Anti-B7-H4 antibodies can harbor radiolabels 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 radiolabeling is used, the 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. If the label is an enzyme, detection may be achieved by any of the currently available colorimetric, spectrophotometric, fluorescence spectrophotometric, amperometric, or gasometric methods known in the art. This can be achieved by contacting a sample or control sample with an anti-B7-H4 antibody or its antigen-binding fragment under conditions that allow for the formation of a complex between the antibody or its antigen-binding fragment and B7-H4. All complexes formed between the antibody or its antigen-binding fragment and B7-H4 are detected and compared in the sample and control. Based on the specific binding of the antibody or its antigen-binding fragment described herein to B7-H4, B7-H4 expression on the cell surface can be specifically detected using the antibody or its antigen-binding fragment. The antibody or its antigen-binding fragment described herein can also be used to purify B7-H4 via immunoaffinity purification.

[0287] This specification also includes assay systems that can be prepared, for example, in the form of test kits for the quantitative analysis of the degree of B7-H4 presence. The system, or test kit, may comprise a labeled component, such as a labeled antibody or antigen-binding fragment, and one or more additional immunochemical reagents. For details of the kits, please refer to Section 5.6 below.

[0288] In some embodiments, this specification provides a method for in vitro detection of B7-H4 in a sample, comprising contacting the sample with an antibody or its antigen-binding fragment. In some embodiments, this specification provides the use of an antibody or its antigen-binding fragment provided herein for in vitro detection of B7-H4 in a sample. In one embodiment, this specification provides an antibody or its antigen-binding fragment or pharmaceutical composition provided herein for use in the detection of B7-H4 in a subject or in a sample taken from a subject. In one embodiment, this specification provides an antibody or its antigen-binding fragment or pharmaceutical composition provided herein for use as a diagnostic agent. In one preferred embodiment, the antibody includes a detectable label. In one preferred embodiment, B7-H4 is human B7-H4. In one preferred embodiment, the subject is human.

[0289] 1.5 Kit This specification provides kits comprising one or more antibodies or antigen-binding fragments thereof or complexes thereof as described herein. In certain embodiments, this specification provides pharmaceutical packs or kits comprising one or more containers filled with one or more components of the pharmaceutical compositions described herein, such as one or more antibodies or antigen-binding fragments thereof provided herein. Such containers may optionally be accompanied by a notice in the form prescribed by a government agency that regulates the manufacture, use, or sale of pharmaceutical or biological products, the notice reflecting the agency's approval for manufacture, use, or sale for human administration.

[0290] This specification also provides kits that can be used in diagnostic methods. In one embodiment, the kit comprises an antibody or its antigen-binding fragment, preferably a purified antibody or its antigen-binding fragment, in one or more containers. In a particular embodiment, the kit described herein comprises a substantially isolated B7-H4 antigen (e.g., human B7-H4) that can be used as a control. In another particular embodiment, the kit described herein further comprises a control antibody or its antigen-binding fragment that does not react with the B7-H4 antigen. In yet another particular embodiment, the kit described herein comprises one or more elements for detecting the binding of the antibody or its antigen-binding fragment to the B7-H4 antigen (for example, the antibody or its antigen-binding fragment can be bound to a detectable substrate, e.g., a fluorescent compound, an enzyme substrate, a radioactive compound or a luminescent compound, or a second antibody or its antigen-binding fragment that recognizes the first antibody or its antigen-binding fragment can be bound to a detectable substrate). In a particular embodiment, the kit provided herein may comprise 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 means of the kit includes a solid support to which the B7-H4 antigen is attached. Such a kit may also include a non-attached reporter-labeled anti-human antibody or its antigen-binding fragment, or an anti-mouse / rat antibody or its antigen-binding fragment. In this embodiment, the binding of the antibody or its antigen-binding fragment to the B7-H4 antigen can be detected by the binding of the reporter-labeled antibody or its antigen-binding fragment.

[0291] The following examples are provided as illustrations only and are not limiting. [Examples]

[0292] The examples in this section (i.e., Section 6) are provided as illustrations and are not limiting.

[0293] Example 1: Evaluation of the incidence of B7-H4 expression in multiple indications The presence of B7-H4 was detected in stored samples, a mixture of whole sections, and tumor microarrays using the B7-H4 mouse monoclonal antibody A57.1 (ATCC catalog number PTA-5180). Samples were treated with the primary antibody and detected using a polymer detection system connected to a DAB (Ventana Medical Systems).

[0294] B7-H4 was readily detected in the membranes and cytosols of tumor tissue collected from patients with various cancers, including invasive ductal carcinoma, trinegative breast cancer, ovarian cancer, non-small cell lung cancer, and endometrial cancer (Figure 1). Furthermore, it was frequently observed in the indications listed in Table 11. [Table 11]

[0295] 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, for example, 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).

[0296] In subsequent experiments, the B7-H4 occurrence rate in various tumor types was evaluated by immunohistochemistry (IHC) (Figure 1B). The following tumor types and disease stages were examined: endometrium, invasive ductal carcinoma (IDC), trinegative breast cancer (TN), trinegative breast cancer (late stage), ovarian cancer, bladder cancer (early stage), non-small cell lung cancer (squamous cell, early stage), non-small cell lung cancer (squamous cell, late stage), head and neck cancer, bladder cancer (late stage), small cell lung cancer, cholangiocarcinoma, non-small cell lung cancer (adenocarcinoma, adenocarcinoma), thyroid cancer, pancreatic cancer, gastric cancer, melanoma, glioblastoma or glioblastoma multiforme (GBM), and Merkel cell carcinoma. B7-H4 surface protein expression on tumor cells was evaluated in tumor samples obtained from various available tumor types and disease stages (early (I / II), late (III / IV)). IHC intensity was assessed using antibody A57.1 on a scale of 0 to 3, and samples were assigned a single IHC score based on the highest intensity observed in at least 10% of all tumor cells across the entire section. A total of 50 to 100 samples were evaluated for each tumor type.

[0297] Example 2: Generation of novel antibodies against human B7-H4 Using an in vitro yeast display system, B7-H4 specific antibodies were isolated from a full-length human IgG1 naive antibody library. The libraries were designed to mimic the immune system; they did not contain predefined heavy-light chain pairs. The libraries were subjected to multiple rounds of positive and negative selection strategies using B7-H4 protein to enrich IgG that cross-reacted with human, cynomolgus monkey, and mouse B7-H4 targets. After four rounds of selection, the resulting IgG were sequenced to produce unique antibodies, and both strong binding affinity and monomer binding affinity to recombinant B7-H4 ectodomain were evaluated for epitope binning and target-specific cell binding.

[0298] A more detailed description of the selection, affinity maturation, and analytical methods used to generate and characterize the B7-H4 antibody is provided below.

[0299] Materials and methods Antigens were biotinylated using Pierce's EZ-Link Sulfo-NHS-Biotinylation kit. 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 isolation columns were purchased from Miltenyi Biotec. Goat anti-human IgG-PE (Human-PE) was obtained from Southern Biotech.

[0300] Naive Discovery Eight naive human synthetic yeast libraries, each with approximately 109 antibodies, were grown 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). In the first two rounds of selection, magnetic bead sorting using the Miltenyi MACS system was performed as previously described (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 10¹⁰ cells / library) were incubated in wash buffer (phosphate-buffered saline (PBS) / 0.1% bovine serum albumin (BSA)) at 30°C for 30 minutes in the presence of 10 ml of 10 nM biotinylated Fc fusion antigen. After washing once with 40 ml of ice-cold wash buffer, the cell pellet was resuspended in 20 ml of wash buffer, Streptavidin MicroBeads (500 μl) were added to the yeast, and incubated at 4°C for 15 minutes. The yeast was then pelletized, resuspended in 20 ml of wash buffer, and Miltenyi The LS column was loaded. After loading 20 mL, the column was washed three times with 3 mL of washing buffer. The column was then removed from the magnetic field, and the yeast was eluted with 5 mL of growth medium and grown overnight. Selection for the next round was performed using flow cytometry.Approximately 2 × 10⁷ yeast cells were pelleted, washed three times with washing buffer, and incubated at 30°C with biotinylated Fc fusion antigens (10–1 nM) at reduced concentrations under equilibrium conditions, with 10 nM biotinylated Fc fusion antigens of different species to obtain cross-reactivity, or with a multispecificity removal reagent (PSR) to remove nonspecific antibodies from selection. For PSR removal, 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)). The yeast was then washed twice with washing buffer and stained with LC-FITC (1:100 dilution) and SA-633 (1:500 dilution) or EAPE (1:50 dilution) secondary reagent at 4°C for 15 minutes. After washing twice with wash buffer, the cell pellet was resuspended in 0.3 mL of wash buffer and transferred to a sorting tube capped with a strainer. Sorting was performed using a FACS ARIA sorter (BD Biosciences) to determine the sort gate and select antibodies with the desired characteristics. The selection round was repeated until a population with all the desired characteristics was obtained. After the final round of sorting, the yeast was plated and individual colonies were picked for characterization.

[0301] Antibody optimization Antibody optimization was performed via light chain batch shuffling, followed by introducing diversity into the heavy chain and light chain variable regions as described below. Several combinations of these approaches were used for each antibody.

[0302] Light chain batch shuffling: Heavy chain plasmids derived from naive selection output were extracted from yeast using the smash-and-grab method, grown, and subsequently purified from E. coli to transform into a light chain library with a diversity of 5 × 10⁶. Selection was performed using the same conditions as in naive discovery, by one round of MACS and four rounds of FACS.

[0303] Selection of CDRH1 and CDRH2: The single antibody CDRH3 was recombined into a pre-existing library containing 1 × 10⁸ diverse CDRH1 and CDRH2 variants, and selection was performed by one round of MACS and four rounds of FACS, as described in Naive Discovery. In each FACS round, the PSR binding, species cross-reactivity, and affinity pressure of the library were examined, and sorting was performed to obtain a population with the desired characteristics. In these selections, affinity pressure was applied using reduced concentrations of biotinylated HIS-B7-H4 antigen (100–1 nM) under equilibrium conditions at 30°C.

[0304] VH Mut Selection: Mutations were introduced into the heavy chain variable region (VH) by error-prone PCR. The library was then prepared by transforming yeast already containing the parental light chain plasmid with this mutant VH and heavy chain expression vector. Selection was performed using two rounds of FACS sorting, similar to the previous cycle. In each FACS round, PSR binding and affinity pressures of the library were examined, and sorting was performed to obtain a population with the desired characteristics. For the selection of CDRH1 and CDRH2, affinity pressures for these selections were performed as described above.

[0305] Selection of CDRL1 and CDRL2: CDRL3 monoantibody was recombined into a pre-built library containing approximately 5 × 10⁵ diverse CDRL1 and CDRL2 variants, and selection was performed using three rounds of FACS sorting, as in the previous cycle. In each FACS round, the PSR binding and affinity pressures of the library were examined, and sorting was performed to obtain a population with the desired characteristics. In the selection of CDRH1 and CDRH2, affinity pressures of these selections were performed as described above.

[0306] Antibody production and purification Yeast clones were grown to saturation, and then expression was induced at 30°C for 48 hours with shaking. After expression induction, the yeast cells were pelleted, and the supernatant was collected for purification. IgG was purified using a Protein A column and eluted with acetic acid at pH 2.0. Fab fragments were generated by papain digestion and purified using KappaSelect (GE Healthcare LifeSciences).

[0307] ForteBio K D measurement ForteBio affinity measurements were performed as generally previously described on Octet RED384 (see Estep et al, High throughput solution-based measurement of antibody-antigen affinity and epitope binning. Mabs 5(2), 270-278 (2013)). Briefly, ForteBio affinity measurements were performed by loading IgG online onto an AHQ sensor. The sensor was equilibrated offline in assay buffer for 30 minutes and monitored online for 60 seconds to establish a baseline. The IgG-loaded sensor was exposed to 100 nM antigen for 3 minutes, then transferred to assay buffer for 3 minutes for dissociation rate measurement. For monovalent affinity assessment, Fab was used instead of IgG. In this assessment, unbiotinylated Fc fusion antigen was loaded online onto an AHQ sensor. The sensor was equilibrated offline in assay buffer for 30 minutes and monitored online for 60 seconds to establish a baseline. The antigen-loaded sensor was exposed to 100 nM Fab for 3 minutes, and then transferred to assay buffer for 3 minutes for dissociation rate measurement. All reaction rates were analyzed using a 1:1 binding model.

[0308] ForteBio Epitope Binning / Ligand Blocking Epitope binning / ligand blocking was performed using a standard sandwich-type cross-blocking assay. A control anti-target IgG was loaded onto an AHQ sensor, and unoccupied Fc binding sites on the sensor were blocked with an unrelated human IgG1 antibody. The sensor was then exposed to 100 nM of the target antigen, followed by exposure to a second anti-target antibody or ligand. Additional binding by the secondary antibody or ligand after antigen binding indicates an unoccupied epitope (non-competitive), while the absence of binding indicates epitope blocking (competitive or ligand blocking).

[0309] Four distinct portions of the B7-H4 extradomain were identified by SPR using four non-competitive antibodies ("Binning antibodies" 1, 2, 3, and 4). All antibodies generated during this campaign were evaluated for competitive binding to the B7-H4 extradomain with these four binning antibodies. If a B7-H4 antibody competed with one of the four binning antibodies (e.g., Binning antibody 1), it was determined that the B7-H4 antibody was located in that BIN (e.g., BIN1). If a B7-H4 antibody competed with two of the four binning antibodies (e.g., Binning antibodies 2 and 3), it was determined that the B7-H4 antibody was located in both of their BINs (e.g., BIN2 / 3).

[0310] IgG antibodies were classified into at least four binding bins, and >90% of antibodies bound to human / cynomolgus monkey or mouse B7-H4 with affinity responses ranging from >0.1 nm to <100 nM. Approximately 75% of recombinant protein-binding molecules also bind to B7-H4 on cells.

[0311] Cell binding analysis 100,000 cells overexpressing the antigen were washed with washing buffer and incubated with 100 μl of 100 nM IgG at room temperature for 5 minutes. The cells were then washed twice with washing buffer and incubated on ice for 15 minutes with 100 μl of 1:100 anti-human IgGPE. The cells were then washed twice with washing buffer and analyzed using a FACS Canto II analyzer (BD Biosciences).

[0312] PSR binding assay The PSR assay was performed as previously described (see Xu Y, et al. (2013) Addressing polyspecificity of antibody selected from an in vitro yeast presentation system: A FACS-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 concentrated membrane fraction was biotinylated using NHS-LCBiotin (Pierce, Thermo Fisher). This multispecificity reagent was incubated with IgG-presenting yeast and then washed. Next, a secondary labeling mix (Extravidin-R-PE, anti-human LC-FITC, and propidium iodide) was added to the mixture. The samples were analyzed using a FACSCanto II analyzer (BD Biosciences) with an HTS sample injector. Flow cytometry data were analyzed for mean fluorescence intensity (MFI) of the R-PE channel to evaluate nonspecific binding. Based on three reference antibodies exhibiting low, medium, and high PSR MFI values, the MFI values ​​were normalized to a range of 0-1.

[0313] Dynamic scanning fluorescence analysis Add 10 μL of 20×Sypro Orange to 20 μL of 0.2-1 mg / mL mAb or Fab solution. RT-PCR instrument (BioRad CFX96 RT Using PCR, the temperature of the sample plate is increased in 0.5C increments from 40 to 95C, and equilibrated for 2 minutes at each temperature. Tm is extracted using the negative first derivative of the raw data.

[0314] AC-SINS The AC-SINS assay 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-type 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 using Molecular Devices SpectraMax M2 and SoftMax. Wavelength shifts were measured using Pro6 software. Self-interacting clones exhibit a larger wavelength shift compared to PBS samples.

[0315] Example 3: Structural characterization of human monoclonal antibodies against B7-H4 The B7-H4 antibody is of the human IgG1 / κ isotype. The variable heavy chain (VH) region contains the alleles of the germline genes VH1, VH3, and VH4, and the variable light chain (VL) of the germline gene alleles, including Vk1 and Vk3. Several subsets of the antibody share high identity ranging from 72 to 100% within the respective CDR regions of VH and VL. Antibodies sharing the same germline alleles have high identity ranging from 88 to 100% in the VH and VL framework (FR) regions. The sequences of the B7-H4 antibody are shown in Tables 1 to 10.

[0316] Example 4: Characterization of monoclonal antibodies that bind to B7-H4 protein The binding affinity of the anti-B7-H4 antibody to the B7-H4 extracellular domain (ECD) was measured by biolayer interferometry (BLI). Details of the ForteBio affinity assay are described in Example 2 above. Briefly, recombinant human B7-H4-human IgG1 (SEQ ID NO: 409) protein was immobilized on a protein A chip, followed by isotype control hIgG1 to saturate the remaining binding sites on the capture chip. Then, the binding of the anti-B7-H4 antibody was evaluated. Furthermore, antibody binding to cynomolgus monkey (SEQ ID NO: 2) and mouse B7-H4 (SEQ ID NO: 3) was evaluated using the same protocol. The results are summarized in Table 12. B7-H4-Human IgG1: MASLGQILFWSIISIIIILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLT DAGTYKCYIITSKGKGNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTVVWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTESEIK RRSHLQLLNSKASGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 409) [Table 12-1] [Table 12-2]

[0317] Furthermore, the binding affinity of selected anti-B7-H4 antibodies to the N-terminal domain of human B7-H4 (B7-H4 IgV-human IgG1; SEQ ID NO: 410) was measured by surface plasmon resonance (SPR). Briefly, anti-human Fab antibodies were immobilized on the surface of a carboxyl-derivativeized SPR chip, and anti-B7-H4 antibodies were captured on the resulting surface at 5 ug / ml for 30 seconds. Then, various concentrations of B7-H4 IgV-human IgG1 (0 nM, 3.7 nM, 11.1 nM, 33.3 nM, 100 nM, and 300 nM) were flowed onto the surface, binding to the anti-B7-H4 antibodies during the binding phase, followed by washing with buffer during the dissociation phase. The data were fitted using a 1:1 binding model, and the results are summarized in Table 13. B7-H4 IgV-Human IgG1: MASLGQILFWSIISIIIILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITSKGKGNANLEYKTGAFSGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 410) [Table 13]

[0318] Therefore, multiple assays indicate that the antibody binds to B7-H4.

[0319] Example 5: Epitope Mapping The binding bins of the anti-B7-H4 antibody were measured by biolayer interferometry (BLI). The epitope mapping assay is described in detail in Example 2 above. Briefly, the first anti-B7-H4 antibody was captured on a protein A chip, followed by a control human IgG1 antibody, saturating additional binding sites on the chip. The sensor chip was then exposed to the antigen (B7-H4-human IgG1) (SEQ ID NO: 409), followed by the second anti-B7-H4 antibody, and its binding was evaluated. The epitope bins were determined by comparing the binding results with those obtained after using a non-B7-H4 antibody as the first antibody. The results are summarized in Table 12.

[0320] Example 6: Characterization of monoclonal antibodies that bind to B7-H4 expressed on the surface of cells HEK293T cell lines were transfused to express full-length human, cynomolgus monkey, mouse, or rat B7-H4. Furthermore, the ability of antibodies to bind B7-H4 expressed on the cell surface was evaluated using the SK-BR-3 breast cancer cell line, which expresses endogenous human B7-H4. (1 × 10⁻⁶) 5 Individual parental HEK293T cell lines or transfected HEK293T cell lines were incubated with 100 nM B7-H4 antibody. After incubation, cells were pelleted, washed, incubated with secondary labeled antibody, and samples were obtained using a flow cytometer. The binding ratio (FOP) to the parental cell line was calculated as follows: MFI transfected HEK293T cells / MFI parental HEK293T cells. FOP values ​​greater than 10 were considered to indicate specific binding.

[0321] To evaluate the cell binding ability of B7-H4 antibodies, 1 × 10⁵ SK-BR-3 cells or 293 cells transfected to express cynomolgus monkey, mouse, or rat B7-H4 were incubated with a titration-dose of B7-H4 antibody. After incubation, cells were pelleted, washed, incubated with a secondary labeled antibody, and samples were obtained using a flow cytometer. Data were analyzed using FlowJo software, and MFI was plotted against antibody concentration. EC50 cell binding ability was calculated using a nonlinear regression curve approximation method (GraphPad Prism).

[0322] All B7-H4 antibodies showed binding to HEK293T cells expressing human B7-H4, cynomolgus monkey B7-H4, or mouse B7-H4. The antibodies also showed potent dose-dependent binding to SK-BR-3 cells endogenously expressing human B7-H4 on their cell surface, or to HEK293T cell lines transfected to express cynomolgus monkey, mouse, or rat B7-H4 on their cell surface. These data demonstrate that the majority of B7-H4 antibodies fully cross-react with cynomolgus monkey, mouse, and rat B7-H4 (Figures 2A and 2B, Table 14).

[0323] [Table 14-1] [Table 14-2]

[0324] Example 7: Characterization of monoclonal antibody T cell checkpoint blocking activity To characterize the T cell checkpoint blocking activity of the B7-H4 antibody, primary human T cells were enriched from peripheral blood mononuclear cells (PBMCs) using the EasySep® Human T Cell Enrichment Kit, following the manufacturer's instructions. The enriched T cells were then treated with anti-CD3 / anti-CD28 In the presence of Dynabeads, 2 × 10 5Cells / mL were incubated at 37°C at a ratio of 1 bead per cell. After 6 days, the beads were removed magnetically, the T cells were washed, and 1 × 10⁶ cells were incubated at 37°C in the presence of 10 U / mL IL-2. 6 The cells were incubated at a concentration of cells / mL. After 4 days, the T cells were washed and 1 × 10⁶ cells were removed. 6 Cells / mL, 2 × 10 in the presence of 10 ug / mL antibody or antibody dose escalation. 6 The cells were incubated with artificial antigen-presenting cells (aAPCs) at 37°C at a rate of cells / mL. aAPCs are HEK293 T cell lines transfected to co-express the scFv form of the anti-human CD3 clone OKT3 and full-length human B7-H4 on the cell surface. aAPCs were treated with mitomycin C at 37°C for 1 hour and thoroughly washed before being added to the T cell co-culture. After 72 hours of co-culture of T cells, aAPCs, and B7-H4 antibody, the plates were centrifuged, the supernatant was collected, and IFNγ production was evaluated by ELISA. Cells were harvested, stained, and proliferation was evaluated by FACS (specifically, cells in each well were incubated in the presence of antibodies against CD4 and CD8). The cells were then washed, fixed, permeabilized, and incubated in the presence of Edu-Click reagent according to the manufacturer's instructions. After washing, the number of growing CD4+, CD8+, or total T cells was measured using flow cytometry. Data were analyzed using FlowJo software. For samples treated with a single concentration of antibody, data were calculated and reported as an increase ratio compared to a control human IgG-treated sample. For samples treated with dose-escalating antibodies, IFNγ production was plotted against antibody concentration, and EC50 potency was calculated using a nonlinear regression curve approximation method (GraphPad Prism).

[0325] All BIN2 / 3 and BIN3 antibodies are IFNγ-producing and / or CD4 + CD8 +Alternatively, T cell checkpoint blocking activity was reproducibly induced, as measured by an increase in overall T cell proliferation. Figure 3A shows that antibody 15441 increases T cell proliferation by at least 3% and CD4+ and CD8+ T cell proliferation by at least 2%. Figure 3A also shows that antibody 15461 increases T cell proliferation by at least 21%, CD4+ T cell proliferation by at least 9%, and CD8+ T cell proliferation by at least 11%. Figures 3B–3D show other dose-dependent T cell checkpoint blocking activities. [Table 15] [Table 16-1] [Table 16-2]

[0326] Example 8: Production of afucosylated and fucosylated monoclonal antibodies Antibodies with an Fc region in which the fucose content of the glycan portion is reduced may show higher ADCC activity compared to fully fucosylated antibodies (Niwa R et al., Clinical Cancer Research 11(6):2327-36(2005)). B7-H4 antibodies were used to produce normal fucosylated antibodies in CHO-x cells (Yamane-Ohnuki N, et al. Biotechnology and It was generated in Bioengineering 87(5):614-22(2004) and in the CHO cell line (CHO-y cells) (ibid.) to produce afucosylated antibodies.

[0327] Example 9: Characterization of binding of afucosylated and fucosylated monoclonal antibodies Fucosylated and afucosylated anti-B7-H4 antibodies were characterized by SPR according to the protocol described in Example 4. The antibodies showed similar binding to the human B7-H4 protein, and therefore glycosylation did not affect binding (Table 17). [Table 17]

[0328] Fucosylated and afucosylated anti-B7-H4 antibodies were also characterized by flow cytometry. In these experiments, HEK293T cell lines were transfused to express full-length cynomolgus monkey, mouse, or rat B7-H4. Furthermore, the cell-binding ability of B7-H4 antibodies was evaluated using endogenous human B7-H4 expressing the SK-BR-3 breast cancer cell line. 1 × 10⁵ SK-BR-3 or 293 cells transfused to express full-length human, cynomolgus monkey, mouse, or rat B7-H4 were incubated in the presence of a titration-dose of B7-H4 antibody. After incubation, cells were pelleted, washed, incubated in the presence of a secondary labeled antibody, and measured by flow cytometry. Data were analyzed using FlowJo software. MFI was plotted against antibody concentration, and EC50 cell-binding ability was calculated using a nonlinear regression curve approximation method (GraphPad Prism).

[0329] The B7-H4 antibody showed potent dose-dependent binding to SK-BR-3 cells that endogenously express B7-H4 on their cell surface, or to 293 cell lines transfected to express cynomolgus monkey, mouse, or rat B7-H4 on their cell surface. Binding ability and interspecies cross-reactivity were not affected by whether the antibody was fucosylated or afucosylated (Figures 4A-4D and Table 17).

[0330] Example 10: Binding of afucosylated and fucosylated monoclonal antibodies to the human Fcγ receptor IIIa (FcγRIIIa) V158 allele. Antibody 20502 was produced as both fucosylated (Ab-F) and afucosylated (Ab-A) and its binding affinity of the Fc region to FcgRIIIa(V158) was tested by surface plasmon resonance (SPR). Briefly, protein A was covalently bonded to a dextran tip using an amine coupling kit containing 100 mM ethylenediamine in 100 mM sodium borate buffer at pH 8.0 as a blocking reagent. Ab-A or Ab-F were captured at two densities in separate flow cells, and the protein A derivatization flow was used as a reference control. FcγRIIIA(V158) was diluted in HBS-P+ running buffer and injected in two-run cycles at six concentrations (0 nM, 1.37 nM, 12.3 nM, 37 nM, 111 nM, 333 nM, and 1000 nM). The binding constant, dissociation constant, and affinity of the Ab-A bond are determined by the Biacore T200 Evaluation. The calculations were performed using the Software 1:1 binding model. The affinity constants for Ab-A and Ab-F binding were measured using the Biacore T200 Evaluation Software steady-state affinity model. The afucosylated B7-H4 antibody (Ab-A) showed a 140-fold higher affinity for the Fcγ receptor IIIA (V158) than the same antibody with fucosylated Fc (Ab-F) (Figures 5A and 5B, Table 18). [Table 18]

[0331] Example 11: T cell checkpoint blocking activity of afucosylated and fucosylated monoclonal antibodies Based on the manufacturer's instructions, primary human T cells were enriched from PBMCs using the EasySep® Human T Cell Enrichment Kit. The enriched T cells were divided into 2 × 10⁶ cells. 5 Cells / mL were incubated at 37°C in the presence of anti-CD3 / anti-CD28 Dynabeads at a ratio of 1 bead per cell. After 6 days, the beads were removed magnetically, and the T cells were washed. 6The cells were incubated at 37°C in the presence of 10 U / mL IL-2 at a concentration of 10 U / mL / mL. After 4 days, the T cells were washed and 2 × 10⁶ cells were collected. 6 Along with artificial antigen-presenting cells (aAPCs) at a concentration of cells / mL, 1 × 10 6 Cells / mL were incubated at 37°C in the presence of dose-escalating B7-H4 antibody. aAPC was treated with mitomycin C at 37°C for 1 hour, thoroughly washed, and then added to co-culture with T cells. After 72 hours of co-culture of T cells, aAPC, and B7-H4 antibody, the plates were centrifuged, the supernatant was collected, and IFNγ production was evaluated by ELISA. IFNγ production was plotted against antibody concentration, and EC50 potency was calculated using a nonlinear regression curve approximation method (GraphPad Prism).

[0332] The B7-H4 antibody showed potent T-cell checkpoint blocking activity, as measured by increased IFNγ production. Furthermore, there was no clear difference in efficacy between the afucosylated antibody and the fucosylated antibody (Figure 6A and Table 19). [Table 19]

[0333] Next, primary human T cells were enriched from HLA-A2+ donor PBMCs using a human pan-T cell isolation kit, following the manufacturer's instructions. First, 5 × 10⁶ enriched pan-T cells were activated for 48 hours in the presence of 1.5 × 10⁷ anti-CD3 / anti-CD28 Dynabeads, 100 ng / mL IL-2, and 15 ng / mL IL-7 to generate T cells expressing the MART-1 TCR. Then, 5 × 10⁶ activated T cells were transduced with MART-1 TCR lentiviral particles in the presence of 200 ng / mL IL-2, 30 ng / mL IL-7, and 10 ug / mL polyblen. 24 hours after transduction, MART-1 TCR+ pan-T cells were enriched with 33.3 ng / mL IL-2 and 5 ng / mL Cells were grown in the presence of IL-7 for 15 days. To generate target cell lines expressing HLA-A2, human breast cancer cell lines MDA-MB-468 and SK-BR-3 expressing endogenous B7-H4 were transduced with HLA-A2 lentiviral particles for 48 hours. Furthermore, B7-H4 was knocked out of the HLA-A2+ SK-BR-3 cell line. MART-1 TCR+ pan-T cells were co-cultured in a 1:1 E:T ratio in the presence of various target cell lines, 500 μg / mL of MART-1 peptide, and 67 nM of B7-H4 antibody 20502 (afucosylated) or a human isotype control. After 24 hours of co-incubation, plates were centrifuged, supernatant was collected, and IL-2 production was evaluated using AlphaLisa according to the manufacturer's instructions.

[0334] As shown in Figure 6B, IL-2 production was reduced in wells containing SK-BR-3 B7-H4+ cells compared to SK-BR-3 KO cells, indicating that B7-H4 exhibits T cell checkpoint ligand activity when endogenously expressed at physiological levels in this assay. Furthermore, the B7-H4 antibody also demonstrated T cell checkpoint blocking activity, as an increase in IL-2 production was measured compared to isotype control-treated cells (Figure 6B). Therefore, this data confirms the T cell checkpoint blocking activity of B7-H4 antibody 20502 (afcosylated) and also demonstrates that B7-H4 antibody 20502 exhibits T cell checkpoint blocking activity in cells that endogenously express B7-H4.

[0335] Example 12: ADCC activity of afucosylated and fucosylated monoclonal antibodies The ADCC activity against B7-H4-expressing target cell lines was evaluated using B7-H4 antibodies. Specifically, primary human PBMC cells were subjected to 1 × 10⁶ tests. 6The cells were activated at 37°C with 200 IU / mL IL-2 at a concentration of cells / mL. The following day, the cells were washed and incubated with calcein-AM labeled SK-BR-3 target cells at an effector:target ratio of 40:1. Four hours after incubation, the lysis of target cells was quantified using a fluorometer. Triton / X-treated samples were used as the maximum lysis control, while samples treated only with culture medium were used as the background lysis control. The percentage of specific lysis was calculated as follows: [1 - ((sample - culture medium control) / (maximum lysis - culture medium control))] × 100. The percentage of specific lysis was plotted against antibody concentration, and the EC50 potency was calculated using a nonlinear regression curve approximation method (GraphPad Prism).

[0336] The B7-H4 antibody showed potent dose-dependent ADCC activity against the endogenous B7-H4-expressing mammary cell line SK-BR-3. Furthermore, the afucosylated antibody showed significantly potent ADCC activity compared to the fucosylated antibody (Figure 7 and Table 20). [Table 20]

[0337] Example 13: Correlation between ADCC activity and receptor density The density of B7-H4 was quantified on the surface of SK-BR-3, HCC1569, ZR-75-1, MDA-MB-48, and HCC1964 cells by FACS according to the manufacturer's specifications. Specifically, 1 × 10⁻⁶ 5Each cell was incubated on ice for 25 minutes in the presence of 15 ug / mL B7-H4 antibody. In parallel, one drop of Quantum® Simply Cellular (QSC) microspheres (pre-coated with a high concentration of anti-mouse IgG capture antibody) was also incubated on ice for 25 minutes in the presence of 15 ug / mL B7-H4 antibody. After incubation, the cells and QSC microspheres were pelleted, washed, and samples were acquired on a flow cytometer. Data were analyzed using FlowJo software. Average fluorescence intensity (MFI) was calculated and entered into a QuickCal® spreadsheet. Regressions relating the fluorescence channel values ​​of each bead to pre-assigned antibody-binding capacity (ABC) values ​​are automatically calculated. ABC values ​​are assigned when the MFI values ​​of labeled cells are added to the template.

[0338] ADCC activity of B7-H4 antibodies against B7-H4-expressing target cell lines with different levels of B7-H4 cell surface density was evaluated. Specifically, 1 × 10⁴ SK-BR-3, HCC1569, ZR-75-1, MDA-MB-468, or HCC1964 target cells were co-incubated at 4°C with dose-escalating B7-H4 antibodies. After 25 minutes, single-use vials of Promega's Jurkat-human CD16 reporter cells were thawed, and 7.5 × 10⁴ cells were added to the target cell / B7-H4 antibody mixture and incubated at 37°C. After 24 hours, the samples were brought to room temperature (RT) and incubated in the presence of Bio-Glo buffer. Substrate and luminescence were quantified using an EnVision multi-label reader. Data were plotted as luminescence against antibody concentration, and the potency of EC50 was calculated using a nonlinear regression curve approximation method (GraphPad Prism).

[0339] The ADCC activity of B7-H4 antibodies was dependent on the B7-H4 cell surface density: as the number of cell surface molecules decreased, the amount of maximum ADCC activity also decreased. Furthermore, afucosylated antibodies showed higher ADCC activity compared to fucosylated antibodies, particularly against target cells with low levels of B7-H4 cell surface density (Figure 8).

[0340] Example 14: In vivo antitumor effect Seven-week-old female BALB / c mice were purchased from Charles River Laboratories (Hollister, CA) and acclimatized for up to three weeks before the start of the experiment. The mouse colorectal cancer cell line CT26 was modified to express a chimeric protein consisting of a mouse B7-H4 extracellular domain with a mouse B7H3 transmembrane domain. These tumor cells were placed subcutaneously in the right flank of mice at a rate of 1.0 × 10⁶ 6 Cells were transplanted at a dose of 200 μl per mouse. Prior to inoculation, cells were subcultured at least three times in RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 2 mM L-glutamine. Cells were grown at 37°C under a humidified atmosphere of 5% CO2. Cells were harvested when they reached 80-85% confluence and were measured at a rate of 5 × 10⁶ cells per milliliter. 6 The cells were resuspended in a 1:1 mixture of serum-free RPMI1640 and Matrigel.

[0341] After cell transplantation for tumor growth, mice were monitored twice a week. Tumor measurements involved measuring the length and width of each tumor using calipers, and calculating the volume according to the formula: Tumor volume (mm3) = (width (mm) × length (mm2)) / 2. On the first day of treatment, all tumors were measured, outliers were excluded, and mice were randomly assigned to the treatment group. Antibodies including 20502 (Figure 9A) and 22213 (Figure 9B) were used for anti-B7-H4 treatment. As a control, mice were administered polyclonal human IgG (Bio X Cell, BE0092) or mouse IgG2a (Bio X Cell, BE0085). Antibodies were administered intravenously (iv) twice a week starting on day 4 or 5 after inoculation for four doses.

[0342] Tumors were measured at least twice a week until the tumor volume exceeded 10% of the animal's body weight, or approximately 2000 mm³. Changes in tumor size are shown by graphing individual tumors compared to the day the animals were inoculated with CT26 cells. P-values ​​were calculated using independent two-group two-sided t-test analysis of tumor volume calculated on each day of the study. Treatment with 20502 or 22213 significantly reduced tumor growth compared to human IgG controls when administered at doses of 10–20 mg / kg (p<0.05) (Figures 9A and 9B).

[0343] Similar experiments were conducted using mice transplanted with 4T1 cells (mouse mammary cancer cell line) or B16 (mouse melanoma cell line). These mice were treated with either a 20 mg / kg mouse surrogate of 20502 called 20502-mouse IgG2a-F, which contains the 20502 variable region fused to fucosylated mouse IgG2a, or a mouse IgG control antibody. Treatment with 20502-mouse IgG2a-F resulted in a significant reduction in tumor growth compared to the mouse IgG control in both the 4T1 mammary cancer model and the B16 melanoma model (Figure 10).

[0344] Additional experiments were also conducted using afcosylated 20502 in the MX-1 human breast cancer xenograft model. Female NSG (NOD-scid, IL2R gammanull) mice were purchased from Jackson Laboratory (Bar-Haber, ME) and adapted one week before the start of the study. The human breast cancer cell line MX-1 has been previously shown to endogenously express the cell surface B7H4 protein. These tumor cells were transplanted subcutaneously into the right flank of mice at a rate of 1.0 × 10⁶ cells / 100 μl / mouse in a 1:1 mixture of serum-free RPMI1640 and 5 × 10⁶ cells per milliliter of Matrigel.

[0345] After cell transplantation for tumor growth, mice were monitored twice weekly. Tumor measurements involved measuring the length and width of each tumor using calipers, and calculating the volume according to the formula: Tumor volume (mm³) = (width (mm) × length (mm)²) / 2. On day 7 post-inoculation, all tumors were measured, outliers were excluded, and mice were randomly assigned to the treatment group. All animals were administered a DNA construct that induces constitutive human IL-15 expression intracirculatoryly for the remainder of the study. On day 9, half of the mice received intravenous (iv) injection of 20 × 10⁶ human peripheral blood mononuclear cells (PBMCs) obtained from StemCell Technologies (Takwira, WA). From day 11, mice were administered afucosylated 20502 (20 mg / kg) or saline as a negative control. The therapeutic agent was administered intravenously (iv) twice weekly for four doses.

[0346] Tumors were measured at least twice a week until tumor volume exceeded 10% of the animal's body weight, or until the animal showed a decrease of 15% or more from its initial body weight. P-values ​​were calculated by comparing the mean tumor volume of all treatment groups at day 28 using one-way ANOVA. As shown in Figure 11, treatment with afcosylated 20502 significantly reduced tumor growth compared to saline controls only when mice were pre-injected with human PBMCs (p<0.05).

[0347] Example 15: In vivo antitumor efficacy when combined with anti-PD-1 antibody. method Eight-week-old female BALB / c mice were purchased from Charles River Laboratories (Hollister, CA) and acclimatized for up to two weeks before the start of the study. The mouse mammary cancer cell line 4T1 was modified to express a chimeric protein containing the extracellular domain of mouse B7-H4 and the transmembrane domain of mouse B7H3. Tumor cells were orthotopically transplanted into the mammary fat pads of mice at a rate of 0.5 × 10⁵ cells / 50 μl / mouse. Prior to inoculation, the cells were subcultured at least three times in RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS). The cells were grown at 37°C under a humidified atmosphere of 5% CO₂. When the cells reached 80–85% confluence, they were harvested, resuspended in serum-free RPMI1640, and inoculated into the mammary fat pads of the ventral abdomen of each mouse.

[0348] After cell transplantation for tumor growth, mice were monitored twice a week. The length and width of each tumor were measured using calipers, and the volume was calculated according to the formula: Tumor volume (mm³) = (width (mm) × length (mm)²) / 2. On the first day of treatment, all tumors were measured, outliers were excluded, and mice were randomly assigned to the treatment group. For anti-B7-H4 treatment, a mouse substitute for 20502 called 20502-mouse IgG2a-F, which contains the 20502 variable region fused to fucosylated mouse IgG2a, was used. As a control, mice were administered mouse IgG2a (anti-HEL). 20502-mouse IgG2a-F or mouse IgG2a was administered intravenously (iv) twice a week for four doses, starting from day 11 after inoculation. Anti-PD-1 (a modified version of RMP1-14 (Bio X Cell) containing the Fc silent mouse IgG2a domain) was administered intraperitoneally (ip) twice a week for three doses, starting 11 days after inoculation. The tumor volume was 10% of the animal's body weight, or approximately 2000 mm³. 3 We continued to measure the tumor at least twice a week until it exceeded a certain level.

[0349] result Changes in tumor size, mean tumor volume, and survival rates are shown in Figures 12A, 12B, and 12C, respectively. Treatment with either 20502-mouse IgG2a-F or anti-PD-1 significantly reduced tumor growth compared to mouse IgG2a controls (p<0.05). Co-administration of 20502-mouse IgG2a-F and anti-PD-1 significantly enhanced tumor growth inhibition compared to either monotherapy (p<0.05). Furthermore, combination therapy resulted in complete tumor regression in 5 out of 12 mice. P-values ​​were calculated using one-way ANOVA of tumor volume calculated on each day of the study, with multiple comparisons between groups.

[0350] Example 16: Anti-B7-H4 antibody increases NK cell and T cell infiltration and upregulates PD-L1. Eight-week-old female BALB / c mice were purchased from Charles River Laboratories (Hollister, CA), and 4T1+ mouse B7-H4 / H3 cells were orthotopically transplanted into the mammary fat pad at a rate of 0.5 × 10⁵ cells / 50 μl / mouse. On the first day of treatment, all tumors were measured, outliers were excluded, and mice were randomly assigned to the treatment group. Mice were administered human IgG1 (anti-HEL) or afucosylated 20502 at a dose of 20 mg / kg by intravenous (iv) injection twice (on days 11 and 14 after inoculation).

[0351] Twenty-four hours after the second dose, the mice were euthanized and perfused with phosphate-buffered saline (PBS). The tumors were then extracted, fixed in 10% formalin for 5 hours, rinsed with PBS, and exposed to 30% sucrose solution for at least 24 hours. In a -15°C chamber (cryostat), the tumors were embedded in Tissue-Tek® OCT compound and sectioned into 20 μm sections. Slides with mounted tissues were rinsed with 0.3% Triton in PBS, blocked with 5% normal goat serum, and stained overnight with primary antibodies (anti-NKp46, anti-CD3, or anti-PD-L1; 1:500). Two serial sections from each tumor were stained.

[0352] Following an overnight incubation, the slides were rinsed with 0.3% Triton, then incubated for 3 hours in a dark, humid chamber in the presence of a secondary antibody (1:400), and rinsed. The tissue was then fixed with 1% paraformaldehyde and rinsed with PBS. The coverslips were mounted using Vectashield® containing DAPI, sealed with Cytoseal®, and dried in a dark chamber. Images were acquired manually using a fluorescence microscope and camera. As shown in Figure 13, treatment with afucosylated 20502 resulted in an increase in natural killer (NK) cells (measured with anti-NKp46 antibody), an increase in CD3+ T cells at the tumor periphery, and a slight increase in CD3+ T cells in the tumor center. Increased PD-L1 expression was also observed.

[0353] Example 17: Anti-B7-H4 antibody significantly reduces in vivo tumor growth in 4T1- and B16- mouse B7-H4 / H3 models in a dose-dependent manner. Six-to-eight-week-old female BALB / c or C57Bl / 6 mice were purchased from Charles River Laboratories (Hollister, CA) and acclimatized up to three weeks before the start of the study. Mouse mammary cancer cell line 4T1 and melanoma cell line B16-F10 were modified to express a chimeric protein consisting of the extracellular domain of mouse B7-H4 and the transmembrane domain of mouse B7-H3. These tumor cells were 0.05 × 10⁶ for 4T1+ mouse B7-H4 / H3. 6 Cells / 50 μl / mouse orthotopically into the mammary gland fat pad, or 0.5 × 10 for B16+ mice B7-H4 / H3. 6 Cells were transplanted subcutaneously into the right flank of mice at a dose of 100 μl per mouse. Prior to inoculation, the cells were subcultured at least three times in RPMI-1640 or DMEM medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 2 mM L-glutamine. The cells were grown at 37°C under a humidified atmosphere of 5% CO2.

[0354] Mice were monitored twice a week after cell transplantation to promote tumor growth. Tumor measurements involved measuring the length and width of each tumor using calipers, and calculating the volume according to the formula: Tumor volume (mm³) = (width (mm) × length (mm)²) / 2. On the first day of treatment, all tumors were measured, outliers were excluded, and mice were randomly assigned to treatment groups. Mice were administered four times by intravenous (iv) injection of 20502-mouse IgG2a-F antibody (anti-B7-H4, mouse IgG2a, fucosylated (also called "cmFPA150-F") or mouse IgG2a control antibody on day 11 (4T1+mouse B7-H4 / H3) or day 6 (B16+mouse B7-H4 / H3) after inoculation, except for the 20 mg / kg group, which was administered twice a week continuously until the end of the study.

[0355] Tumors were measured at least twice a week until the tumor volume exceeded 10% of the animal's body weight, or approximately 2000 mm³. Changes in tumor size are shown by graphing the mean tumor volume relative to the day of inoculation. Treatment with 20502-mouse IgG2a-F significantly reduced tumor growth compared to mouse IgG2a controls when administered at doses of 1 mg / kg or higher in 4T1+ mouse B7-H4 / H3 models (see Figures 14A and 14B) and at doses of 3 mg / kg or higher in B16+ mouse B7-H4 / H3 models (see Figures 15A and 15B) (p<0.05). Statistical significance was calculated using one-way ANOVA comparing all treatment groups with mouse IgG2a.

[0356] Figures 14A and 14B show that the 20502-mouse IgG2a-F antibody significantly reduces the proliferation of 4T1 cells expressing B7-H4 / H3. BALB / c mice were orthotopically inoculated with 4T1 mammary cancer cells modified to express mouse B7-H4 ECD fused to B7-H3™. From day 11 after inoculation, the mice were administered the 20502-mouse IgG2a-F antibody (anti-B7-H4, mouse IgG2a, fucosylated) twice a week. The 20502-mouse IgG2a-F antibody showed a dose-dependent reduction in tumor growth, and one-way ANOVA at day 30 showed significant inhibition 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).

[0357] Figures 15A and 15B show that the 20502-mouse IgG2a-F antibody significantly reduces the proliferation of B16 cells expressing B7-H4 / H3. C57Bl / 6 mice were subcutaneously inoculated with B16-F10 melanoma cells modified to express mouse B7-H4 ECD fused to B7-H3™. From day 6 after inoculation, the mice were administered the 20502-mouse IgG2a-F antibody (anti-B7-H4, mouse IgG2a, fucosylated) twice a week. The 20502-mouse IgG2a-F antibody showed a dose-dependent reduction in tumor growth (Figure 15A), and one-way ANOVA on day 23 showed significant inhibition 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) (Figure 15B).

[0358] Therefore, the 20502-mouse IgG2a-F antibody significantly reduced tumor size in a dose-dependent manner in two different cancer models. These data in breast cancer and melanoma cell lines suggest that the 20502-mouse IgG2a-F antibody can be used to treat cancer patients. Sequence List [Table 21-1] [Table 21-2] [Table 21-3] [Table 21-4]

[0359] The present invention is not limited in scope by the specific embodiments described herein. In fact, various modifications of the present invention will be apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to be included within the scope of the accompanying claims.

[0360] All references cited herein (e.g., publications or patents or patent applications) are incorporated herein by reference in whole for any purpose, just as each individual reference (e.g., publication or patent or patent application) is incorporated by reference in whole for any purpose.

[0361] Other embodiments are within the scope of the attached claims. In certain embodiments, for example, the following items are provided: (Item 1) (a) Sequence numbers 458-463, respectively; (b) Sequence numbers 5-10, respectively; (c) Sequence numbers 15-20, respectively; (d) Sequence numbers 25-30, respectively; (e) Sequence numbers 35-40, respectively; (f) Sequence numbers 45-50, respectively; (g) Sequence IDs 55-60, respectively; (h) Sequence numbers 65-70, respectively; (i) Sequence numbers 75-80, respectively; (j) Sequence numbers 85-90, respectively; (k) Sequence numbers 95-100, respectively; (l) Sequence numbers 105-110, respectively; (m) Sequence numbers 115-120, respectively; (n) Sequence numbers 125-130, respectively; (o) Sequence numbers 135-140, respectively; (p) Sequence numbers 145-150, respectively; (q) Sequence numbers 155-160, respectively; (r) Sequence numbers 165-170, respectively; (s) Sequence numbers 175-180, respectively; (t) Sequence IDs 185-190 and (u) An isolated antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, comprising 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, selected from the group consisting of SEQ ID NOs. 195-200. (Item 2) The antibody or antigen-binding fragment according to item 1, wherein the antibody or antigen-binding fragment contains a VH having the amino acid sequence of SEQ ID NOs: 11, 21, 31, 41, 464, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, or 201. (Item 3) The antibody or antigen-binding fragment according to item 1 or item 2, wherein the antibody or antigen-binding fragment comprises a VL having the amino acid sequence of SEQ ID NOs. 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182, 192, or 202. (Item 4) An isolated antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region has the amino acid sequence of SEQ ID NOs: 11, 21, 31, 41, 464, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, or 201. (Item 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, and the light chain variable region has the amino acid sequence of SEQ ID NOs: 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182, 192, or 202. (Item 6) (a) Sequence IDs 464 and 42, respectively; (b) Sequence IDs 11 and 12, respectively; (c) Sequence IDs 21 and 22, respectively; (d) Sequence IDs 31 and 32, respectively; (e) Sequence IDs 41 and 42, respectively; (f) Sequence IDs 51 and 52, respectively; (g) Sequence IDs 61 and 62, respectively; (h) Sequence IDs 71 and 72, respectively; (i) Sequence IDs 81 and 82, respectively; (j) Sequence IDs 91 and 92, respectively; (k) Sequence IDs 101 and 102, respectively; (l) Sequence IDs 111 and 112, respectively; (m) Sequence IDs 121 and 122, respectively; (n) Sequence IDs 131 and 132, respectively; (o) Sequence IDs 141 and 142, respectively; (p) Sequence IDs 151 and 152, respectively; (q) Sequence IDs 161 and 162, respectively; (r) Sequence IDs 171 and 172, respectively; (s) Sequence IDs 181 and 182, respectively; (t) Sequence IDs 191 and 192 respectively; or (u) An isolated antibody or its antigen-binding fragment that specifically binds to human B7-H4, comprising a heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs. 201 and 202, respectively. (Item 7) The antibody or antigen-binding fragment according to any one of items 1 to 6, further comprising a heavy chain constant region. (Item 8) The antibody or antigen-binding fragment according to item 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. (Item 9) The antibody or antigen-binding fragment according to any one of items 1 to 8, wherein the antibody or antigen-binding fragment further comprises a light chain constant region. (Item 10) The antibody or antigen-binding fragment according to item 9, wherein the light chain constant region is selected from the group consisting of human immunoglobulin IgGκ and IgGλ light chain constant regions. (Item 11) The antibody or antigen-binding fragment according to any one of items 1 to 6, further comprising 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 the light chain constant region is a human IgGκ light chain constant region. (Item 12) The antibody or antigen-binding fragment according to any one of items 1 to 6, wherein the antibody or antigen-binding fragment comprises a heavy chain having the amino acid sequence of SEQ ID NOs: 13, 23, 33, 43, 469, 53, 63, 73, 83, 93, 103, 113, 123, 133, 143, 153, 163, 173, 183, 193, or 203. (Item 13) The antibody or antigen-binding fragment according to any one of items 1 to 6 or 12, wherein the antibody or antigen-binding fragment comprises a light chain having the amino acid sequence of SEQ ID NOs: 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, 114, 124, 134, 144, 154, 164, 174, 184, 194, or 204. (Item 14) The antibody or its antigen-binding fragment: (a) Sequence IDs 469 and 44, respectively; (b) Sequence IDs 13 and 14, respectively; (c) Sequence IDs 23 and 24, respectively; (d) Sequence IDs 33 and 34, respectively; (e) Sequence IDs 43 and 44, respectively; (f) Sequence IDs 53 and 54, respectively; (g) Sequence IDs 63 and 64, respectively; (h) Sequence IDs 73 and 74, respectively; (i) Sequence IDs 83 and 84, respectively; (j) Sequence IDs 93 and 94, respectively; (k) Sequence IDs 103 and 104, respectively; (l) Sequence IDs 113 and 114, respectively; (m) Sequence IDs 123 and 124, respectively; (n) Sequence IDs 133 and 134, respectively; (o) Sequence IDs 143 and 144, respectively; (p) Sequence IDs 153 and 154, respectively; (q) Sequence numbers 163 and 164, respectively; (r) Sequence IDs 173 and 174, respectively; (s) Sequence IDs 183 and 184, respectively; (t) Sequence IDs 193 and 194 respectively; or (u) An antibody or antigen-binding fragment according to any one of items 1 to 6, comprising a heavy chain and a light chain having the amino acid sequences of sequence numbers 203 and 204, respectively. (Item 15) An isolated antibody or its antigen-binding fragment that specifically binds to human B7-H4, wherein the antibody or its antigen-binding fragment is 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, 20496, and is classified as VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL The antibody or its antigen-binding fragment, comprising CDR3. (Item 16) The antibody or antigen-binding fragment described in item 15, wherein the CDR is a Kabat-defined CDR, a Chothia-defined CDR, or an AbM-defined CDR. (Item 17) An isolated antibody or its antigen-binding fragment that specifically binds to human B7-H4, wherein the antibody or its antigen-binding fragment comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 as defined by Chothia or AbM in 20502.1. (Item 18) Isolated antibodies or antigen-binding fragments thereof that specifically bind to human B7-H4, each having 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, respectively, corresponding to sequence numbers 458-463. (Item 19) (i) The antibody or antigen-binding fragment according to item 18, wherein the antibody or antigen-binding fragment comprises a variable heavy chain region having the amino acid sequence of SEQ ID NO: 464 and a variable light chain region having the amino acid sequence of SEQ ID NO: 42, or (ii) The antibody or antigen-binding fragment according to item 18, wherein the antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 469 and a light chain having the amino acid sequence of SEQ ID NO: 44. (Item 20) Isolated antibodies or antigen-binding fragments thereof that specifically bind to human B7-H4, each having 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, respectively, corresponding to sequence numbers 35-40. (Item 21) (i) The antibody or antigen-binding fragment thereof comprises a variable heavy chain region having the amino acid sequence of SEQ ID NO: 41 and a variable light chain region having the amino acid sequence of SEQ ID NO: 42, or (ii) The antibody or antigen-binding fragment thereof according to item 20, wherein the antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 43 and a light chain having the amino acid sequence of SEQ ID NO: 44. (Item 22) Isolated antibodies or antigen-binding fragments thereof that specifically bind to human B7-H4, each having 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, respectively, corresponding to sequence numbers 65-70. (Item 23) (i) The antibody or antigen-binding fragment thereof comprises a variable heavy chain region having the amino acid sequence of SEQ ID NO: 71 and a variable light chain region having the amino acid sequence of SEQ ID NO: 72, or (ii) The antibody or antigen-binding fragment thereof according to item 22, wherein the antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 73 and a light chain having the amino acid sequence of SEQ ID NO: 74. (Item 24) An isolated antibody or antigen-binding fragment that binds to the same human B7-H4 epitope as the antibody or antigen-binding fragment described in any one of items 1 to 23. (Item 25) The antibody or antigen-binding fragment according to any one of items 1 to 24, wherein the antibody or antigen-binding fragment is a human antibody or antigen-binding fragment. (Item 26) The antibody or antigen-binding fragment according to any one of items 1, 15-20, 22, or 24, wherein the antibody or antigen-binding fragment is a mouse, humanized, or chimeric antibody or antigen-binding fragment thereof. (Item 27) The antibody or antigen-binding fragment described in any one of items 1 to 26, wherein the antibody or antigen-binding fragment induces T cell proliferation. (Item 28) The antibody or antigen-binding fragment according to item 27, wherein the antibody or antigen-binding fragment increases T cell proliferation by at least 21% compared to treatment with a control antibody. (Item 29) The antibody or antigen-binding fragment described in item 27, wherein the antibody or antigen-binding fragment increases T cell proliferation by approximately 5% to approximately 35% compared to treatment with a control antibody. (Item 30) The antibody or antigen-binding fragment described in any one of items 1 to 29, wherein the antibody or antigen-binding fragment induces CD4+ T cell proliferation. (Item 31) The antibody or antigen-binding fragment according to item 30, wherein the antibody or antigen-binding fragment increases CD4+ T cell proliferation by at least 9% compared to treatment with a control antibody. (Item 32) The antibody or antigen-binding fragment described in item 30, wherein the antibody or antigen-binding fragment increases CD4+ T cell proliferation by approximately 5% to approximately 15% compared to treatment with a control antibody. (Item 33) The antibody or antigen-binding fragment described in any one of items 1 to 32, wherein the antibody or antigen-binding fragment induces CD8+ T cell proliferation. (Item 34) The antibody or antigen-binding fragment according to item 33, wherein the antibody or antigen-binding fragment increases CD8+ T cell proliferation by at least 11% compared to treatment with a control antibody. (Item 35) The antibody or antigen-binding fragment described in item 34, wherein the antibody or antigen-binding fragment increases CD8+ T cell proliferation by approximately 5% to approximately 15% compared to treatment with a control antibody. (Item 36) The antibody or antigen-binding fragment described in any one of items 1 to 35, wherein the antibody or antigen-binding fragment induces interferon-gamma (IFNγ) production. (Item 37) The antibody or antigen-binding fragment according to item 36, wherein the antibody or antigen-binding fragment can increase IFNγ production by at least twofold, at least threefold, at least fourfold, and at least fivefold, at least sixfold, at least sevenfold, at least eightfold, about twofold to about tenfold, or about threefold to about tenfold. (Item 38) The antibody or antigen-binding fragment described in any one of items 1 to 37, wherein the antibody or antigen-binding fragment can induce antibody-dependent cell-mediated cytotoxicity (ADCC) in B7-H4 expressing cells. (Item 39) The antibody or antigen-binding fragment described in item 38, wherein the antibody or antigen-binding fragment induces specific lysis in at least 20%, at least 30%, at least 40%, about 20% to about 50%, or about 30% to about 50% of B7-H4 expressing cells. (Item 40) The antibody or antigen-binding fragment described in any one of items 1 to 39 inhibits tumor growth in a mouse CT26 colorectal cancer model, a mouse mammary cancer 4T1 model, or a melanoma cell line B16-mouse B7-H4 / H3 model. (Item 41) The antibody or antigen-binding fragment according to item 40, wherein the antibody or antigen-binding fragment 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. (Item 42) An antibody or antigen-binding fragment according to any one of items 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 inhibition of ADCC activity, and / or the inhibition of tumor growth is dose-dependent. (Item 43) The antibody or antigen-binding fragment described in any one of items 1 to 42, wherein the antibody or antigen-binding fragment binds to cynomolgus monkey B7-H4. (Item 44) The antibody or antigen-binding fragment thereof, as described in any one of items 1 to 43, wherein the antibody or antigen-binding fragment thereof binds to rat B7-H4. (Item 45) The antibody or antigen-binding fragment described in any one of items 1 to 44, wherein the antibody or antigen-binding fragment binds to mouse B7-H4. (Item 46) The antibody or antigen-binding fragment described in any one of items 1 to 45, wherein the antibody or antigen-binding fragment binds to the IgV domain of human B7-H4. (Item 47) The antibody or antigen-binding fragment described in any one of items 1 to 46, wherein the antibody or antigen-binding fragment is afucosylated. (Item 48) A full-length antibody, or an antigen-binding fragment thereof, as described in any one of items 1 to 47. (Item 49) An antigen-binding fragment, which is an antibody or antigen-binding fragment as described in any one of items 1 to 47. (Item 50) The antigen-binding fragments include Fab, Fab', F(ab')2, single-chain Fv(scFv), disulfide-bonded Fv, V-NAR domain, IgNar, intracellular antibody, IgGΔCH2, minibody, F(ab')3, tetrabody, triabody, diabody, single-domain antibody, DVD-Ig, Fcab, and mAb. 2 Antigen-binding fragments as described in item 49, including (scFv)2 or scFv-Fc. (Item 51) An antibody or antigen-binding fragment according to any one of items 1 to 50, further comprising a detectable label. (Item 52) An isolated polynucleotide comprising a nucleic acid molecule encoding the heavy chain variable region or heavy chain of an antibody or antigen-binding fragment described in any one of items 1 to 51. (Item 53) The isolated polynucleotides described in item 52, wherein the nucleic acid molecule encodes the VH of SEQ ID NOs: 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 NOs: 13, 23, 33, 43, 469, 53, 63, 73, 83, 93, 103, 113, 123, 133, 143, 153, 163, 173, 183, 193, or 203. (Item 54) The isolated polynucleotide described in item 52, wherein the nucleic acid molecule has the sequence of SEQ ID NOs: 213, 223, 233, 243, 470, 253, 263, 273, 283, 283, 293, 303, 313, 323, 333, 343, 353, 363, 373, 383, 393, or 403. (Item 55) The isolated polynucleotide described in item 52, wherein the nucleic acid molecule has (i) the sequence of SEQ ID NOs: 213, 223, 233, 243, 470, 253, 263, 273, 283, 293, 303, 313, 323, 333, 343, 353, 363, 373, 383, 393, or 403, and (ii) the sequence of SEQ ID NO: 408. (Item 56) An isolated polynucleotide comprising a light chain variable region or a nucleic acid molecule encoding a light chain of an antibody or antigen-binding fragment described in any one of items 1 to 51. (Item 57) The isolated polynucleotide described in item 56, wherein the nucleic acid molecule encodes the VL of SEQ ID NOs: 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 NOs: 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, 114, 124, 134, 144, 154, 164, 174, 184, 194, or 204. (Item 58) The isolated polynucleotide described in item 56, wherein the nucleic acid molecule has the sequence of SEQ ID NOs: 214, 224, 234, 244, 254, 264, 274, 284, 294, 304, 314, 324, 334, 344, 354, 364, 374, 384, 394, or 404. (Item 59) The isolated polynucleotide described in item 56, wherein the nucleic acid molecule has (i) the sequence of SEQ ID NOs: 214, 224, 234, 244, 254, 264, 274, 284, 294, 304, 314, 324, 334, 344, 354, 364, 374, 384, 394, or 404, and (ii) the sequence of SEQ ID NO: 406. (Item 60) An isolated polynucleotide comprising a heavy chain variable region or heavy chain of an antibody or antigen-binding fragment described in any one of items 1 to 51, and a nucleic acid molecule encoding a light chain variable region or light chain of an antibody or antigen-binding fragment described in any one of items 1 to 51. (Item 61) An isolated vector containing a polynucleotide as described in any one of items 52-60. (Item 62) A host cell comprising a polynucleotide as described in any one of items 52-60, a vector as described in item 61, or a first vector containing a polynucleotide as described in any one of items 52-55 and a second vector containing a polynucleotide as described in any one of items 56-59. (Item 63) Host cells as described in item 62, selected from the group consisting of E. coli, Pseudomonas, Bacillus, Streptomyces, yeast, CHO, YB / 20, NS0, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, HepG2, SP2 / 0, R1.1, BW, LM, COS1, COS7, BSC1, BSC40, BMT10 cells, plant cells, insect cells, and human cells in tissue culture. (Item 64) The host cells described in item 62 or 63, wherein the host cells lack the functional α-1,6-fucosyltransferase gene (FUT8). (Item 65) A method for producing an antibody or antigen-binding fragment that binds to human B7-H4, comprising culturing a host cell described in any one of items 62 to 64, thereby expressing the nucleic acid molecule, and producing the antibody or its antigen-binding fragment. (Item 66) An isolated antibody or its antigen-binding fragment that specifically binds to human B7-H4 and is encoded by a polynucleotide described in any one of items 52-60. (Item 67) A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described in any one of items 1 to 51 or 66, and a pharmaceutically acceptable excipient. (Item 68) (i) an antibody or antigen-binding fragment as described in any one of items 1 to 51 or 66, and (ii) a pharmaceutically acceptable excipient, wherein at least 95% of the antibody or antigen-binding fragment in the composition is afcosylated. (Item 69) (i) an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4 and has 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 to 460, respectively; and (ii) a pharmaceutically acceptable excipient, wherein at least 95% of the antibody or antigen-binding fragment in the composition is afcosylated. (Item 70) (i) The antibody or its antigen-binding fragment comprises a variable heavy chain region having the amino acid sequence of SEQ ID NO: 464 and a variable light chain region having the amino acid sequence of SEQ ID NO: 42, or (ii) The antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 469 and a light chain having the amino acid sequence of SEQ ID NO: 44, according to item 69. (Item 71) (i) an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4 and has 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; and (ii) a pharmaceutically acceptable excipient, wherein at least 95% of the antibody or antigen-binding fragment in the composition is afcosylated. (Item 72) (i) The antibody or its antigen-binding fragment comprises a variable heavy chain region having the amino acid sequence of SEQ ID NO: 41 and a variable light chain region having the amino acid sequence of SEQ ID NO: 42, or (ii) The antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 43 and a light chain having the amino acid sequence of SEQ ID NO: 44, according to item 71. (Item 73) (i) an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4 and has 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 to 70, respectively; and (ii) a pharmaceutically acceptable excipient, wherein at least 95% of the antibody or antigen-binding fragment in the composition is afcosylated. (Item 74) (i) The antibody or its antigen-binding fragment comprises a variable heavy chain region having the amino acid sequence of SEQ ID NO: 71 and a variable light chain region having the amino acid sequence of SEQ ID NO: 72, or (ii) The antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 73 and a light chain having the amino acid sequence of SEQ ID NO: 74, according to item 73. (Item 75) A pharmaceutical composition according to any one of items 67 to 74, wherein fucosylation is undetectable in the composition. (Item 76) A pharmaceutical composition according to any one of items 67 to 75, further comprising an anti-PD-1 antibody or an antigen-binding fragment thereof. (Item 77) The pharmaceutical composition according to item 76, wherein the anti-PD-1 antibody or its antigen-binding fragment is nivolumab or pembrolizumab. (Item 78) The pharmaceutical composition according to item 76, wherein the anti-PD-1 antibody or its antigen-binding fragment is AMP-514, camrelizumab, tislerizumab, and spartalizumab. (Item 79) A pharmaceutical composition according to any one of items 67 to 75, further comprising an anti-PD-L1 antibody or an antigen-binding fragment thereof. (Item 80) A method for inducing T cell proliferation, comprising contacting T cells with an antibody or its antigen-binding fragment described in any one of items 1 to 51 or 66, or a pharmaceutical composition described in any one of items 67 to 79. (Item 81) The method according to item 80, which reduces T cell proliferation by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80%. (Item 82) A method for inducing CD4+ T cell proliferation, comprising contacting CD4+ T cells with an antibody or its antigen-binding fragment described in any one of items 1 to 51 or 66, or a pharmaceutical composition described in items 67 to 79. (Item 83) The method according to item 82, which reduces CD4+ T cell proliferation by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80%. (Item 84) A method for inducing CD8+ T cell proliferation, comprising contacting CD8+ T cells with an antibody or antigen-binding fragment thereof as described in any one of items 1 to 51 or 66, or a pharmaceutical composition as described in any one of items 67 to 79. (Item 85) The method according to item 84, which reduces CD8+ T cell proliferation by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80%. (Item 86) A method for inducing interferon-γ production, comprising contacting T cells with an antibody or its antigen-binding fragment described in any one of items 1 to 51 or 66, or a pharmaceutical composition described in any one of items 67 to 79. (Item 87) The method according to item 86, which increases interferon-gamma production by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80%. (Item 88) A method for killing cells expressing B7-H4, comprising contacting the cells with an antibody or antigen-binding fragment thereof as described in any one of items 1 to 51 or 66, or a pharmaceutical composition as described in any one of items 67 to 79. (Item 89) A method for depleting B7-H4 expressing cells from a cell population, comprising contacting the cell population with an antibody or its antigen-binding fragment described in any one of items 1 to 51 or 66, or a pharmaceutical composition described in any one of items 67 to 79. (Item 90) The method described in item 88 or 89, wherein the killing or depletion is carried out via ADCC. (Item 91) The method according to any one of items 80 to 90, further comprising contacting the T cells, the CD4+ T cells, the CD8+ T cells, the cells, or the cell population with an anti-PD-1 antibody or its antigen-binding fragment. (Item 92) The method according to item 91, comprising contacting an antibody or its antigen-binding fragment as described in any one of items 1 to 51 or 66, or a pharmaceutical composition as described in any one of items 67 to 78, and simultaneously contacting an anti-PD-1 antibody or its antigen-binding fragment. (Item 93) The method according to item 91, comprising contacting an antibody or its antigen-binding fragment as described in any one of items 1 to 51 or 66, or a pharmaceutical composition as described in any one of items 67 to 78, and then contacting an anti-PD-1 antibody or its antigen-binding fragment in succession. (Item 94) The method according to any one of items 80 to 90, further comprising contacting the T cells, the CD4+ T cells, the CD8+ T cells, the cells, or the cell population with an anti-PD-L1 antibody or its antigen-binding fragment. (Item 95) The method according to item 94, comprising simultaneously contacting an antibody or its antigen-binding fragment described in any one of items 1 to 51 or 66, or a pharmaceutical composition described in any one of items 67 to 75, and contacting an anti-PD-L1 antibody or its antigen-binding fragment. (Item 96) The method according to item 94, comprising contacting an antibody or its antigen-binding fragment described in any one of items 1 to 51 or 66, or a pharmaceutical composition described in any one of items 67 to 75, and then contacting an anti-PD-L1 antibody or its antigen-binding fragment in succession. (Item 97) The method according to any one of items 80 to 96, wherein the contact is in vitro. (Item 98) The method according to any one of items 80 to 96, wherein the contact is within the body of the test subject. (Item 99) A method for treating cancer expressing B7-H4 in a subject, comprising administering to a subject an effective amount of an antibody or its antigen-binding fragment described in any one of items 1 to 51 or 66, or a pharmaceutical composition described in any one of items 67 to 79. (Item 100) The method according to item 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. (Item 101) The method according to item 100, wherein the breast cancer is a trine-negative breast cancer, or the non-small cell lung cancer is a squamous cell carcinoma. (Item 102) The method according to item 100, wherein the non-small cell lung cancer is adenocarcinoma. (Item 103) The method according to item 99, wherein the cancer is selected from the group consisting of head and neck cancer, small cell lung cancer, gastric cancer, and melanoma. (Item 104) The method according to item 100, wherein the cancer is ovarian cancer and serous adenocarcinoma. (Item 105) The method according to item 101, wherein the cancer is breast cancer and ductal carcinoma. (Item 106) The method according to any one of items 99 to 105, wherein the cancer has an insufficient response to a PD-1 inhibitor. (Item 107) The method according to any one of items 99 to 106, wherein the cancer has an insufficient response to a PD-L1 inhibitor. (Item 108) The method according to any one of items 99 to 107, wherein the cancer expresses a low level of PD-L1. (Item 109) The method according to any one of items 99 to 108, wherein the subject is a human. (Item 110) The method according to any one of items 99 to 109, further comprising administering an anti-PD-1 antibody or an antigen-binding fragment thereof to the subject. (Item 111) The method according to item 110, comprising administering an antibody or its antigen-binding fragment as described in any one of items 1 to 51 or 66, or a pharmaceutical composition as described in any one of items 67 to 78, and simultaneously administering an anti-PD-1 antibody or its antigen-binding fragment. (Item 112) The method according to item 110, comprising administering an antibody or its antigen-binding fragment as described in any one of items 1 to 51 or 66, or a pharmaceutical composition as described in any one of items 67 to 78, followed by the administration of an anti-PD-1 antibody or its antigen-binding fragment. (Item 113) The method according to item 112, wherein the administration of the anti-PD-1 antibody or its antigen-binding fragment is performed after the administration of the antibody or its antigen-binding fragment according to any one of items 1 to 51 or 66, or the pharmaceutical composition according to any one of items 67 to 78. (Item 114) The method according to any one of items 91-93, 97, 98 and 110-113, wherein the anti-PD-1 antibody or its antigen-binding fragment is nivolumab or pembrolizumab. (Item 115) The method according to any one of items 91-93, 97, 98 and 110-113, wherein the anti-PD-1 antibody or its antigen-binding fragment is AMP-514, camrelizumab, tislerizumab, or spartalizumab. (Item 116) A method for detecting B7-H4 in a sample, comprising contacting the sample with an antibody or an antigen-binding fragment thereof described in any one of items 1 to 51 or 66. (Item 117) The method described in item 116, wherein the sample is obtained from the cancer of a human subject. (Item 118) A kit comprising an antibody or its antigen-binding fragment as described in any one of items 1-51 or 66, or a pharmaceutical composition as described in any one of items 67-79, and a) a detection reagent, b) B7-H4 antigen, c) a notice reflecting approval for use or sale for human administration, or d) a combination thereof. (Item 119) An isolated antibody or antigen-binding fragment thereof as described in any one of items 1 to 51 or 66, wherein the antibody or antigen-binding fragment inhibits the T cell checkpoint blocking activity of B7-H4. (Item 120) The isolated antibody or its antigen-binding fragment as described in item 119, wherein the T cell checkpoint blocking activity is measured by increased IL-2 production compared to control cells. (Item 121) (i) an antibody or antigen-binding fragment as described in any one of items 1 to 51 or 66, and (ii) a pharmaceutically acceptable excipient, wherein the antibody or antigen-binding fragment inhibits the T-cell checkpoint blocking activity of B7-H4. (Item 122) The pharmaceutical composition according to item 121, wherein the T cell checkpoint blocking activity is measured by increased IL-2 production compared to control cells.

Claims

[Claim 1] The invention as shown in the drawings.

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