B7-h4 antibody-drug conjugates and methods of use thereof

Abstract

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Description

B7-H4 ANTIBODY-DRUG CONJUGATES AND METHODS OF USE THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present invention claims the benefit of priority to United States Provisional Application No. 63 / 608,714, filed on December 11, 2023, the contents of which are incorporated herein by reference.FIELD OF THE INVENTION

[0002] he invention is generally related to the field of antibody-drug conjugates, and more particularly to compositions and methods for targeting tumor cells expressing B7- H4 for elimination with cytotoxin payloads attached to the antibody molecule.BACKGROUND OF THE INVENTION

[0003] The B7 family plays an important role in both positive and negative regulation of immune responses by engaging a variety of receptors on lymphocytes (Rahbar, R. et al., Cancer Immunol Res, 3(2): 184-195(2015)). B7-H4 (also referred to as VTCN1 or B7x or B7S1) is a member of the B7 family and inhibits T-cell function. It is also upregulated on a variety of tumors and has been proposed to promote tumor growth. High B7-H4 expression is found in numerous tumor tissues providing a correlation of the level of expression on tumor cells with adverse clinical and pathologic features, including tumor aggressiveness.

[0004] The biological activity of B7-H4 has been associated with decreased inflammatory CD4+T-cell responses and a correlation between B7-H4-expressing tumor- associated macrophages and FoxP3+ regulatory T cells (Tregs) within the tumor microenvironment. Since B7-H4 is expressed on tumor cells and tumor-associated macrophages in various cancer types, therapeutic blockade of B7-H4 could favorably alter the tumor microenvironment allowing for antigen-specific clearance of tumor cells (Podojil, JR. and Miller, SD, Immunol Rev, 276(l):40-51 (2017)).

[0005] cDNA encoding the human B7-H4 protein was identified and cloned from placental cDNA (Sica, G. L. et al. (2003) "B7-H4, A Molecule of The B7 Family, Negatively Regulates T Cell Immunity," Immunity 18:849-861; Zang, X. et al. (2003) B7x: A Widely Expressed B7 Family Member That Inhibits T Cell Activation," Proc. Natl. Acad. Sci. (USA) 100: 10388-10392). B7-H4 is discussed in U.S. Pat. Nos. 7,931,896; 7,875,702; 7,847,081; and 7,622,565.

[0006] Anti-B7-H4 antibodies are disclosed in U.S. Pat. Nos. 9,574,000; 7,888,477; 7,737,255; 7,619,068; and 6,962,980.

[0007] Human B7-H4 protein possesses 282 amino acid residues, which have been categorized as including an amino terminal extracellular domain, a large hydrophobic transmembrane domain and a very short intracellular domain (consisting of only 2 amino acid residues). Like other B7 family members, B7-H4 possesses a pair of Ig-like regions in its extracellular domain. The B7-H4 protein has an overall structure of a type I transmembrane protein. The protein has minimal (about 25%) homology with other B7 family members (Zang, X. et al. (2003) “B7x: A Widely Expressed B7 Family Member That Inhibits T Cell Activation," Proc. Natl. Acad. Sci. (USA) 100: 10388-10392).

[0008] B7-H4 is also expressed on tumor-associated macrophages (TAMs). TAMs inhibit anti -tumor immune responses through the release of humoral mediators and also protect tumors from immune recognition by hampering cell-mediated immune responses through the cell-surface expression of inhibitory molecules such as B7-H4. TAMs derive from resident macrophages or from monocytes recruited by the tumor microenvironment and polarized at the tumor site. Tumor infiltration with TAMs has been associated with poor patient survival and targeting TAMs represents a promising strategy against cancer. Several approaches have already been developed, including depletion with clodronate liposomes; tumor recruitment inhibition by CFSR-1 and CCL2 targeting; and “reeducation” through activation via anti-CD40 mAbs, or HRG plasma protein, or mannose receptor (Dangai, D. et al., Cancer Res, 73(15): 4820-4829 (2013)).

[0009] Since B7-H4 is expressed on tumor cells and TAMs in various cancer types, directing therapeutics against B7-H4 could have tremendous synergistic outcomes in favorably altering the tumor micro-environment and eliminating cancer cells. Thus, there is a need for new immunomodulators of B7-H4.

[0010] Therefore, it is an object of the invention to provide compositions that target B7-H4 expressed on tumor cells and deliver cytotoxic payloads to destroy the tumor cells. Such compositions are useful for the treatment of cancer.SUMMARY OF THE INVENTION

[0011] Compositions and methods of their use for targeting B7-H4 expressing cells and delivering cytotoxic payloads are provided. Such compositions are useful for the treatment of cancers. In one embodiment the compositions are antibodies that specifically bind to B7-H4 and are conjugated to cytotoxic payloads via a linker. Thecompounds can be antibodies or antigen binding fragments thereof, fusion proteins, aptamers, or agents that specifically bind B7-H4.

[0012] One embodiment provides a method for treating infection in a subject in need thereof by administering an effective amount of a composition that targets B7-H4 expressed on the surface of a cell and delivers a cytotoxic payload to the cell.

[0013] In particular embodiments the agent is a B7-H4 fusion protein, for example a fusion protein that includes an extracellular domain of B7-H4 or functional variant thereof linked to an immunoglobulin domain.

[0014] In some embodiments, the cancer is characterized by increased expression of B7-H4. In particular embodiments, the cancer is an ovarian, breast, lung, thyroid, gastrointestinal cancer, acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), endometrial cancer, brain cancer, head and neck cancer, pancreatic cancer, cholangiocarcinomas and bladder cancer. The agent can be administered contemporaneously or in combination with a vaccine or a component thereof.

[0015] Any of the disclosed methods can include administering to the subject a composition that targets B7-H4 expressed on the surface of a cell and delivers a cytotoxic payload to the cell.

[0016] One embodiment provides a method for assessing or predicting the efficacy of a treatment using an anti-B7-H4 binding moiety by assaying the cells of a subject in need of treatment to determine whether the cells express B7-H4, binding partners of B7- H4, or both. Exemplary cells to be assayed include but are not limited to cancer cells obtained from the subject. Exemplary cancer cells, include but are not limited to, ovarian, breast, lung, thyroid, gastrointestinal cancer, acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), endometrial cancer, brain cancer, head and neck cancer, pancreatic cancer, cholangiocarcinomas (BTC) and bladder cancer.

[0017] One embodiment provides an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof that has a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:3.

[0018] One embodiment provides a nucleic acid that encodes a light chain according to SEQ ID NO:3. Another embodiment provides a nucleic acid having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:7.

[0019] One embodiment provides an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof that has a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:8.

[0020] One embodiment provides a nucleic acid encoding heavy chain SEQ ID NO:8. Another embodiment provides a nucleic acid having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 12.

[0021] One embodiment provides an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof that has a light chain containing CDRs according to SEQ ID Nos: 4, 5, and 6.

[0022] Another embodiment provides an anti-B7-H4 antibody-drug conjugate or an antigen binding fragment thereof having a heavy chain containing CDRs according to SEQ ID Nos: 9, 10, and 11.

[0023] In one embodiment, an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof has a light chain containing CDRs according to SEQ ID Nos: 4, 5, and 6 and a heavy chain containing CDRs according to SEQ ID Nos: 9, 10, and 11.

[0024] One embodiment provides an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof having a light chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 3 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:8.

[0025] Another embodiment provides an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof that has a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 13.

[0026] In one embodiment, the antibody-drug conjugate light chain contains CDRs with amino acid sequences according to SEQ ID Nos: 14, 15, and 16.

[0027] One embodiment provides an antibody-drug conjugate heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:29.

[0028] Another embodiment provides an antibody-drug conjugate heavy chain containing CDRs with amino acid sequences according to SEQ ID NOs: 9, 11, and 30.

[0029] One embodiment provides an anti-B7-H4 antibody-drug conjugate, or antigen binding fragment thereof containing light chain CDRs according to SEQ ID Nos: 14, 15, and 16 and heavy chain CDRs according to SEQ ID Nos: 9, 11, and 30.

[0030] One embodiment provides an anti-B7-H4 antibody-drug conjugate, or antigen binding fragment thereof containing a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 13 and a heavychain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:29.

[0031] One embodiment provides an anti-B7-H4 antibody-drug conjugate light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:46.

[0032] Another embodiment provides an anti-B7-H4 antibody-drug conjugate light chain containing CDRs with amino acid sequences according to SEQ ID Nos: 14, 16, and 47.

[0033] One embodiment provides an antibody-drug conjugate heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:49.

[0034] Another embodiment provides an antibody-drug conjugate heavy chain containing CDRs with amino acid sequences according to SEQ ID Nos: 9, 11, and 47.

[0035] One embodiment provides an antibody-drug conjugate, or antigen binding fragment thereof containing light chain CDRs according to SEQ ID Nos: 14, 16, and 47, and heavy chain CDRs according to SEQ ID Nos: 9, 11, and 50.

[0036] One embodiment provides an anti-B7-H4 antibody-drug conjugate, or an antigen binding fragment thereof containing a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:46 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:49.

[0037] One embodiment provides an antibody-drug conjugate light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:52.

[0038] In another embodiment, an antibody-drug conjugate light chain contains CDRs with amino acid sequences according to SEQ ID Nos: 53, 54, and 55.

[0039] One embodiment provides an antibody-drug conjugate heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:65.

[0040] Another embodiment provides an antibody-drug conjugate heavy chain containing CDRs with amino acid sequences according to SEQ ID Nos: 66, 67, and 68.

[0041] One embodiment provides an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof containing light chain CDRs according to SEQ ID Nos: 53, 54, and 55, and heavy chain CDRs according to SEQ ID Nos: 66, 67, and 68.

[0042] One embodiment provides an antibody-drug conjugate, or antigen binding fragment thereof, containing a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 52 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:65.

[0043] One embodiment provides an antibody-drug conjugate light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:85.

[0044] Another embodiment provides an antibody-drug conjugate light chain containing CDRs with amino acid sequences according to SEQ ID Nos: 4, 86, and 87.

[0045] One embodiment provides an antibody-drug conjugate heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:89.

[0046] One embodiment provides an antibody-drug conjugate heavy chain containing CDRs with amino acid sequences according to SEQ ID Nos: 9, 90, and 91.

[0047] Another embodiment provides an anti-B7-H4 antibody-drug conjugate, or antigen binding fragment thereof, containing light chain CDRs according to SEQ ID Nos: 4, 86, and 87 and heavy chain CDRs according to SEQ ID Nos: 9, 90, and 91.

[0048] One embodiment provides an anti-B7-H4 antibody-drug conjugate, or antigen binding fragment thereof, containing a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:85 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 89.

[0049] One embodiment provides an antibody-drug conjugate light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:93.

[0050] Another embodiment provides an antibody-drug conjugate light chain containing CDRs with amino acid sequences according to SEQ ID Nos: 94, 95, and 96.

[0051] One embodiment provides an antibody-drug conjugate heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:98.

[0052] Another embodiment provides an antibody-drug conjugate heavy chain containing CDRs with amino acid sequences according to SEQ ID Nos: 9, 99, and 100.

[0053] One embodiment provides an anti-B7-H4 antibody-drug conjugate, or antigen binding fragment thereof, having light chain CDRs according to SEQ ID Nos: 94, 95, and 96, and heavy chain CDRs according to SEQ ID Nos: 9, 99, and 100.

[0054] One embodiment provides an anti-B7-H4 antibody-drug conjugate, or antigen binding fragment thereof, having a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:93 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:98.

[0055] One embodiment provides an antibody-drug conjugate light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 102.

[0056] Another embodiment provides an antibody-drug conjugate light chain containing CDRs with amino acid sequences according to SEQ ID Nos: 103, 104, and 105.

[0057] One embodiment provides an antibody-drug conjugate heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 107.

[0058] Another embodiment provides an antibody-drug conjugate heavy chain containing CDRs with amino acid sequences according to SEQ ID Nos: 9, 108, and 109.

[0059] In one embodiment an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof, contains light chain CDRs according to SEQ ID Nos: 103, 104, and 105 and heavy chain CDRs according to SEQ ID Nos: 9, 108, and 109.

[0060] One embodiment provides an anti-B7-H4 antibody-drug conjugate, or antigen binding fragment thereof, containing a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 102 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 107.

[0061] One embodiment provides an antibody-drug conjugate light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 111.

[0062] Another embodiment provides an antibody-drug conjugate light chain containing CDRs with amino acid sequences according to SEQ ID Nos: 95, 112, and 113.

[0063] One embodiment provides an antibody-drug conjugate heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:115.

[0064] One embodiment provides an antibody heavy chain containing CDRs with amino acid sequences according to SEQ ID Nos: 116, 117, and 118.

[0065] In one embodiment, the anti-B7-H4 antibody-drug conjugate, or antigen binding fragment thereof, contains light chain CDRs according to SEQ ID Nos: 95, 112, and 113, and heavy chain CDRs according to SEQ ID Nos: 116, 117, and 118.

[0066] One embodiment provides an anti-B7-H4 antibody-drug conjugate, or antigen binding fragment thereof, containing a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 111 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 115.

[0067] One embodiment provides an antibody-drug conjugate light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 120.

[0068] Another embodiment provides an antibody-drug conjugate light chain containing CDRs with amino acid sequences according to SEQ ID Nos: 54, 55, and 121.

[0069] One embodiment provides an antibody-drug conjugate heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 123.

[0070] Another embodiment provides an antibody-drug conjugate heavy chain containing CDRs with amino acid sequences according to SEQ ID Nos: 66, 124, and 125.

[0071] One embodiment provides an anti-B7-H4 antibody-drug conjugate, or antigen binding fragment thereof, containing light chain CDRs according to SEQ ID Nos: 54, 55, and 121 and heavy chain CDRs according to SEQ ID Nos: 66, 124, and 125.

[0072] One embodiment provides an anti-B7-H4 antibody-drug conjugate, or an antigen binding fragment thereof, containing a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 120 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 123.

[0073] One embodiment provides an anti-B7-H4 antibody-drug conjugate heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 128

[0074] Another embodiment provides an anti-B7-H4 antibody-drug conjugate heavy chain containing CDRs with amino acid sequences according to SEQ ID Nos: 9, 129, and 130.

[0075] In one embodiment, an anti-B7-H4 antibody-drug conjugate, or antigen binding fragment thereof, contains light chain CDRs according to SEQ ID Nos: 14, 15, and 16 and heavy chain CDRs according to SEQ ID Nos: 9, 129, 130.

[0076] One embodiment provides an anti-B7-H4 antibody-drug conjugate, or antigen binding fragment thereof, containing a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 13 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 128.

[0077] One embodiment provides an anti-B7-H4 antibody-drug conjugate, or an antigen binding fragment thereof having a light chain with an amino acid sequence according to any one of SEQ ID NO:3, 13, 46, 52, 85, 93, 102, 111, or 120.

[0078] Another embodiment provides an anti-B7-H4 antibody-drug conjugate, or an antigen binding fragment thereof having a heavy chain with an amino acid sequence according to any one of SEQ ID NO:8, 29, 49, 65, 89, 98, 107, 115, 123, or 128.

[0079] One embodiment provides an anti-B7-H4 antibody-drug conjugate, or an antigen binding fragment thereof having a light chain with an amino acid sequence according to any one of SEQ ID NOs: 3, 13, 46, 52, 85, 93, 102, 111, or 120, and a heavy chain with an amino acid sequence according to any one of SEQ ID Nos: 8, 29, 49, 65, 89, 98, 107, 115, 123, or 128..

[0080] Also provided is an anti-B7-H4 antibody-drug conjugate, or antigen binding fragment thereof having three light chain CDRs with amino acid sequences that are selected from the group consisting of SEQ ID Nos: 4, 5, 6, 14, 15, 16, 47, 53, 54, 55, 86, 87, 94, 95, 96, 103, 104, 105, 112, 113, or 121.

[0081] One embodiment provides an anti-B7-H4 antibody-drug conjugate, or antigen binding fragment thereof having three heavy chain CDRs with amino acid sequences that are selected from the group consisting of SEQ ID Nos: 9, 10, 11, 30, 50, 66, 67, 68, 90, 91, 99, 100, 108, 109, 116, 117, 118, 124, 125, 129, or 130.

[0082] One embodiment provides an anti-B7-H4 antibody-drug conjugate, or antigen binding fragment thereof having three light chain CDRs with amino acid sequences that are selected from the group consisting of SEQ ID NOs: 4, 5, 6, 14, 15, 16, 47, 53, 54, 55, 86, 87, 94, 95, 96, 103, 104, 105, 112, 113, and 121, and three heavy chainCDRs with amino acid sequences that are selected from the group consisting of SEQ ID NOs: 9, 10, 11, 30, 50, 66, 67, 68, 90, 91, 99, 100, 108, 109, 116, 117, 118, 124, 125, 129, and 130.

[0083] Another embodiment provides an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof having a light chain variable domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 19, 20, 21, 22, or 23, and a heavy chain variable domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs:34, 35, 36, or 37.

[0084] One embodiment provides an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof having a light chain having an amino acid sequence according to any one of SEQ ID NOs: 24, 25, 26, 27, or 28, and a heavy chain having an amino acid sequence according to any one of SEQ ID NOs: 38, 39, 40, or 41.

[0085] Another embodiment provides an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof having a light chain having an amino acid sequence according to any one of SEQ ID NOs: 24, 25, 26, 27, or 28, and a heavy chain having an amino acid sequence according to any one of SEQ ID NOs: 42, 43, 44, or 45.

[0086] Also provided is an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof having a light chain variable domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs:58, 59, 60, or 61, and a heavy chain variable domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 70, 71, 72, 73, or 74.

[0087] One embodiment provides an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof having a light chain having an amino acid sequence according to any one of SEQ ID NOs: 62, 63, or 64, and a heavy chain having an amino acid sequence according to any one of SEQ ID NOs: 75, 76, 77, 78, or 79.

[0088] One embodiment provides an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof having a light chain having an amino acid sequence according to any one of SEQ ID NOs: 62, 63, or 64, and a heavy chain having an amino acid sequence according to any one of SEQ ID NOs: 80, 81, 82, 83, or 84.

[0089] Another embodiment provides an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof having six complementarity determining regions (CDRs), wherein the CDRs include the three light chain CDRs of a polypeptide selectedfrom the group consisting of SEQ ID NO:4, 5, 6, 14, 15, 16, 49, 55, 56, 57, 88, 89, 96, 97, 97, 105, 106, 107, 114, 115, or 123, or a variant thereof comprising at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO4, 5, 6, 14, 15, 16, 49, 55, 56, 57, 88, 89, 96, 97, 97, 105, 106, 107, 114, 115, or 123, and the three heavy chain CDRs of a polypeptide selected from the group consisting of SEQ ID NO:9, 10, 11, 31, 52, 68, 69, 70, 92, 93, 101, 102, 118, 119, 120, 126, 127, 131, or 132, or a variant thereof comprising at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 9, 10, 11, 31, 52, 68, 69, 70, 92, 93, 101, 102, 118, 119, 120, 126, 127, 131, or 132, and wherein the antibody-drug conjugate or antigen binding fragment thereof binds to B7-H4.

[0090] Another embodiment provides an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof having two light chains and two heavy chains, wherein the two light chains include a polypeptide selected from the group consisting of SEQ ID NO: 24, 25, 26, 27, 28, 62, 63, or 64, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 24, 25, 26, 27, 28, 62, 63, or 64, and the two heavy chains include a polypeptide selected from the group consisting of SEQ ID NO: 38, 39, 40, 41, 75, 76, 77, 78, or 79, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 38, 39, 40, 41, 75, 76, 77, 78, or 79, and wherein the antibody or antigen binding fragment thereof binds to B7-H4.

[0091] One embodiment provides an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof having two light chains and two heavy chains, wherein the two light chains include a polypeptide selected from the group consisting of SEQ ID NO: 24, 25, 26, 27, 28, 62, 63, or 64, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 24, 25, 26, 27, 28, 62, 63, or 64, and the two heavy chains include a polypeptide selected from the group consisting of SEQ ID NO: 42, 43, 44, 45, 80, 81, 82, 83, or 84, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 42, 43, 44, 45, 80, 81, 82, 83, or 84, and wherein the antibody or antigen binding fragment thereof binds to B7-H4.

[0092] One embodiment provides an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof having two light chains and two heavy chains, wherein the two light chains include a polypeptide selected from the group consisting of SEQ ID NO: 24, 25, 26, 27, or 28, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%,95%, 99%, or more sequence identity to SEQ ID NO: 24, 25, 26, 27, or 28, and the two heavy chains include a polypeptide selected from the group consisting of SEQ ID NO: 38, 39, 40, or 41, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 38, 39, 40, or 41, and wherein the antibody or antigen binding fragment thereof binds to B7-H4.

[0093] Another embodiment provides an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof having two light chains and two heavy chains, wherein the two light chains include a polypeptide selected from the group consisting of SEQ ID NO: 24, 25, 26, 27, or 28, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 24, 25, 26, 27, or 28, and the two heavy chains include a polypeptide selected from the group consisting of SEQ ID NO: 42, 43, 44, or 45, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 42, 43, 44, or 45, and wherein the antibody-drug conjugate or antigen binding fragment thereof binds to B7-H4.

[0094] One embodiment provides an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof having two light chains and two heavy chains, wherein the two light chains include a polypeptide selected from the group consisting of SEQ ID NO: 62, 63, or 64, or a variant thereof comprising at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 62, 63, or 64, and the two heavy chains include a polypeptide selected from the group consisting of SEQ ID NO: 75, 76, 77, 78, or 79, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 75, 76, 77, 78, or 79, and wherein the antibodydrug conjugate or antigen binding fragment thereof binds to B7-H4.

[0095] Another embodiment provides an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof having two light chains and two heavy chains, wherein the two light chains include a polypeptide selected from the group consisting of SEQ ID NO: 62, 63, or 64, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 62, 63, or 64, and the two heavy chains include a polypeptide selected from the group consisting of SEQ ID NO: 80, 81, 82, 83, or 84, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 80, 81, 82, 83, or 84, and wherein the antibody-drug conjugate or antigen binding fragment thereof binds to B7-H4.

[0096] On other embodiment provides an anti-B7-H4 antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-B7-H4 antibody comprising alight chain having an amino acid sequence according to SEQ ID NOs: 63, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 63 and a heavy chain having an amino acid sequence according to SEQ ID NOs: 78, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO:78, a linker and a cytotoxic payload, wherein the linker and payload comprises a structure represented by Compound 1.

[0097] Another embodiment provides an anti-B7-H4 antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-B7-H4 antibody comprising a light chain having an amino acid sequence according to SEQ ID NOs: 132, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 132 and a heavy chain having an amino acid sequence according to SEQ ID NOs: 133, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 133, a linker and a cytotoxic payload, wherein the linker and payload comprises a structure represented by Compound 1.BREIF DESCRIPTION OF THE DRAWINGS

[0098] Figures 1A-B. Anti-tumor activity of B7-H4 antibody drug conjugate (ADC) in a PDX model of triple negative breast cancer. 1 A) Mean tumor progression following 3 weekly doses of vehicle control, 3 weekly doses of B7-H4 ADC (1.5 mg / kg), or a single dose of 4.5 mg / kg B7-H4 ADC. IB) Growth of individual tumor in each of the 3 treatment groups. Mean tumor volume at endpoint was <13mm3for the 4.5 mg / kg treatment group and ~40mm3for the 1.5 mg / kg treatment group.

[0099] Figure 2. Anti-tumor activity of B7-H4 ADC in an orthotopic xenograft model of HER2+ breast cancer (HCC1569). Mean tumor progression is shown following no treatment or following two doses of B7-H4 ADC at 1.5, 3, or 6 mg / kg (Q21D, Days 14 and 35 post-inoculation, indicated by dashed lines).

[0100] Figure 3. Anti-tumor activity of B7-H4 ADC in a transgenic xenograft model of colon cancer (HT29-hB7-H4). Growth of individual tumors is shown following no treatment or following a single dose administration (on Day 8 post-inoculation) of B7- H4 ADC at 1, 3, or 6 mg / kg. 6 of 7 mice in each of the 3 and 6 mg / kg treatment groups achieved complete response (CRs) with no measurable tumor for >3 weeks of consecutive measurements.

[0101] Figure 4. Anti-tumor activity of B7-H4 ADC in xenograft models of colon cancer. Four tumor models were analyzed in parallel. These four tumor models werecomposed (at the time of inoculation, “Day 0”) of a) 100% HT29 cells (B7-H4-negative), b) 25% (transgenic) HT29-hB7-H4 cells mixed with 75% HT29 cells, c) 75% HT29-hB7- H4 cells mixed with 25% HT29 cells, or d) 100% HT29-hB7-H4 cells. Mean tumor progression is shown for each tumor type, comparing an untreated control group to a group treated with a single 3 mg / kg dose of B7-H4 ADC (Day 14 post-inoculation, indicated by a dashed line). All four control groups displayed highly similar growth rates. B7-H4 ADC treatment had no measurable effect on B7-H4-negative HT29 tumors. 100% of tumors composed of 100% HT29-hB7H4 showed significant regression below baseline measurement. Similarly, 100% of mixed tumors composed of -75% B7-H4+ tumor cells showed significant regression below baseline measurement. All the tumors composed of -25% B7-H4+ cells that were treated with B7-H4 ADC achieved a partial response (>30% tumor reduction) with a mean regression of 50% below baseline measurement. Combined results suggest a strong bystander effect for B7-H4 ADC in tumor with heterogenous B7-H4 expression.

[0102] Figures 5A-B. Anti-tumor activity of B7-H4 ADC in a xenograft model of transgenic ovarian cancer (OVCAR3-B7-H4-OE). 5A) Mean tumor progression is shown following no treatment or following a single dose administration of B7-H4 ADC 6 mg / kg. 5B) Regression of tumors following a single dose administration of 1 or 3 mg / kg B7-H4 ADC. 100% complete responses (CR’s) were achieved in all treated tumors at 1 or 3 mg / kg, with no detectable tumor for 2 or more successive measurements.

[0103] Figures 6A-B. Anti-tumor activity of B7-H4 ADC in a xenograft model of ER / PR+ breast cancer (ZR-75-1). 6A) IHC stain for B7-H4 protein expression in a xenograft ZR-75-1 tumor in mice. 6B) Tumor progression in sham control mice or following 3 x Q7D doses of 1, 3, or 6 mg / kg B7-H4 ADC (on Days 1, 8, and 15 of treatment, as indicated by dashed lines). Tumor growth inhibition of >80% was achieved at all dose levels.

[0104] Figure 7. Comparison of the relative binding of 3 anti-B7-H4 antibodies to recombinant human B7-H4 protein using an ELISA analysis. Relative binding is plotted for B7-H4 MAB (the parent antibody for B7-H4 ADC containing an identical variable domain), B7-H4 ADC (the antibody intermediate for B7-H4 ADC), and clone AZ-E02, an anti-human B7-H4 antibody published by AstraZeneca. Similar binding is seen for B7-H4 MAB and B7-H4 ADC, and comparable or superior binding of B7-H4 ADC compared to AZ-E02 is observed over a concentration gradient of antibody.

[0105] Figure 8. Comparison of the relative binding of 3 anti-B7-H4 antibodies to cancer cell lines. Flow cytometry analysis was performed to measure binding to live cancer cells. The Mean Fluorescence Intensity (MFI) is plotted for B7-H4 MAB (the parent antibody for B7-H4 ADC containing an identical variable domain), B7-H4 ADC (the antibody intermediate for B7-H4 ADC ADC), and clone AZ-E02, an anti-human B7- H4 antibody published by AstraZeneca. Similar binding is seen for B7-H4 MAB and B7- H4 ADC, and comparable or superior binding of B7-H4 ADC compared to AZ-E02 is seen for each cell line. None of the antibodies show significant binding to B7-H4 negative cells.

[0106] Figure 9. Comparison of the kinetics internalization of two pHRODO- labeled anti-B7H4 antibodies, B7-H4 ADC and AZ-E02 in A) a B7-H4-positive cell line (OVCAR3 ovarian cancer cells transduced to express a human B7-H4 transgene) or B) a B7-H4-negative cell line (A549 lung cancer cells). The pHrodo dye is pH sensitive, resulting in fluorescence only under acidic conditions present in intracellular lysosomes. Both antibodies show similar levels and kinetics of uptake into intracellular lysosomes of B7-H4+ cells. Neither antibody was significantly internalized in the absence of B7-H4 target expression in A549 cells.

[0107] Figure 10. Representative experiment demonstrating B7-H4 target-dependent cytotoxicity of B7-H4 ADC on SK-BR-3 (wildtype [WT], B7-H4-positive) vs. a B7-H4- negative knockout (KO) derivative cell lines, in vitro. Sensitivity of the cell line to free MMAE is shown for reference.

[0108] Figures 11 A-B. Antibody-dependent cellular cytotoxicity analysis ofLNCB74. An ADCC assay was performed on SKBR3 (B7-H4+ HER2+) tumor cells cocultured with human PBMCs. 11 A) Donor 1 and 1 IB) Donor 2.

[0109] Figures 12A-E. Comparing internalization of LNCB74 and other anti-B7-H4 antibodies. 12A) Flow cytometric measurement of LNCB74 antibody intermediate binding to B7-H4-negative and positive cell lines. Kinetics of internalization of LNCB74 antibody intermediate and comparator B7-H4 antibodies using Fc-glycan-conjugated- pHrodo-labeled antibodies on 12B) SKBR3 cells, 12C) SKBR3 knockout cells, 12D) OVCAR3 cells, 12E) HCC1569 cells. Relative internalization was measured using an Opera Phenix confocal imaging system.

[0110] Figure 13. Cytotoxicity assays of LNCB74 in tumor cells from various indications. Dose-dependent killing of B7-H4+ SKBR3 breast cancer cells by LNCB74 invitro. Low toxicity to a B7-H4- knockout derivative, and sensitivity of SKBR3 cells to free MMAE toxin are shown for reference.

[0111] Figures 14A-E. In vivo anti-tumor toxicity. 14A) In vitro surface expression of B7-H4 on cell lines used in xenograft tumor models. 14B) In vivo anti-tumor effect on transgenic HT-29 cells overexpressing human B7-H4. Anti -tumor effect in the HCC1569 (14C) and ZR-75-1 (14D) orthotopic breast cancer tumor models. 14E) Anti-tumor effect in the OVCAR3 ovarian model. Dashed line indicates day of dosing.

[0112] Figures 1 A-B5. Analysis of bystander activity in lower P-value tumors. 15A) Immunofluorescent staining of cryosections from xenograft mouse tumors (collected on treatment ‘Day O’). Tumor models were generated with 100%, -75%, -25%, or 0% B7- H4 positive cells by mixing transgenic HT29-hB7-H4 cells with B7-H4-negative HT29 ‘wildtype’ cells. 15B) Dose response for LNCB74 anti -tumor activity using an HT29- hB7-H4 transgenic tumor model demonstrating an anti-tumor / bystander effect of a single dose of 3 mg / kg LNCB74 in a ‘mixed’ models composed of 100%, 75%, or 25% HT29 B7-H4+ cells.

[0113] Figures 16A-C. Activity of LNCB74 in three B7-H4-positive PDX models. 16A) Durable regression was observed in TNBC model CTG-0012 following fractionated dosing of 1.5 mg / kg weekly x 3 as well as with a single bolus of 4.5 mg / kg. 16B) In HR+BC model CTG-2328, a single dose of 3 mg / kg or 4.5 mg / kg dose was effective. 16C) In TNBC model CTG-0670, a single dose of 3 mg / kg resulted in control of tumor growth. H4C image above each graph is the IHC demonstrating the presence of B7-H4 expression within the model.

[0114] Figures 17A-G. Comparison of anti-B7-H4 ADCs (DAR4). 17A) Schematic of the structural differences between LNCB74 and a similar Anti-B7-H4 ADC with a Val-Cit conjugated to MMAE. 17B) In an HCC1569 (HER+BC) model, after a single dose LNCB74 outperformed the comparator ADC in all dose comparisons tested. 17C) In an OVCAR3 ovarian cancer model, LNCB74 also provided more robust and durable tumor control after a single dose when compared to the comparator anti-B7-H4 ADC with Val-Cit linker, despite both having a DAR4 MMAE payload.

[0115] Figure 18. PK analysis (LC / MS / MS) of LNCB74 ADC and Total Antibody in rats following single dose administration at a dose of 3 mg / kg, i.v.

[0116] A panel of B7-H4-expressing cancer cell lines were assayed for sensitivity. EC50 values for B7-H4 ADC cytotoxicity are listed in Table 1. Where possible, values are shown for B7-H4-negative isogenic cell lines. Serial dilutions of MMAE or B7-H4ADC were prepared in complete media ranging from 666 nM (100 pg / mL) to 1.76 pM (256 pg / mL). Cells were plated in 96-well plates and incubated in the presence of test articles at 37°C. After 120 h, cell viability was measured using a CellTiter-Glo kit (Promega), according to the manufacturer’s instructions.DETAILED DESCRIPTION OF THE INVENTIONI. Definitions

[0117] As used herein, a molecule is said to be able to “immunospecifically bind” a second molecule if such binding exhibits the specificity and affinity of an antibody to its cognate antigen. Antibodies are said to be capable of immunospecifically binding to a target region or conformation (“epitope”) of an antigen if such binding involves the antigen recognition site of the immunoglobulin molecule. An antibody that immunospecifically binds to a particular antigen may bind to other antigens with lower affinity if the other antigen has some sequence or conformational similarity that is recognized by the antigen recognition site as determined by, e.g., immunoassays, BIACORE® assays, or other assays known in the art, but would not bind to a totally unrelated antigen. In some embodiments, however, antibodies (and their antigen binding fragments) will not cross-react with other antigens. Antibodies may also bind to other molecules in a way that is not immunospecific, such as to FcR receptors, by virtue of binding domains in other regions / domains of the molecule that do not involve the antigen recognition site, such as the Fc region.

[0118] As used herein, a molecule is said to “bind” a second molecule if such binding exhibits the specificity and affinity of a receptor to its cognate binding ligand. A molecule can be capable of binding to more than one other molecule.

[0119] As used herein, the term “antibody” is intended to denote an immunoglobulin molecule that possesses a “variable region” antigen recognition site and include antigen-binding fragments of antibodies. The term “variable region” is intended to distinguish such domain of the immunoglobulin from domains that are broadly shared by antibodies (such as an antibody Fc domain). The variable region includes a “hypervariable region” whose residues are responsible for antigen binding. The hypervariable region includes amino acid residues from a “Complementarity Determining Region” or “CDR” (z.e., typically at approximately residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and at approximately residues 27-35 (Hl), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequencesof Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or those residues from a “hypervariable loop” (i.e., residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26- 32 (Hl), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk, 1987 , J. Mol. Biol. 196:901-917). “Framework Region” or “FR” residues are those variable domain residues other than the hypervariable region residues as herein defined. The term antibody includes monoclonal antibodies, multi-specific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camelized antibodies (See e.g., Muyldermans et al., 2001, Trends Biochem. Sci. 26:230; Nuttall el al., 2000, Cur. Pharm. Biotech. 1 :253; Reichmann and Muyldermans, 1999, J. Immunol. Meth. 231 :25; International Publication Nos. WO 94 / 04678 and WO 94 / 25591; U.S. Patent No. 6,005,079), single-chain Fvs (scFv) (see, e.g., see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer- Verlag, New York, pp. 269-315 (1994)), single chain antibodies, disulfide-linked Fvs (sdFv), intrabodies, diabodies, triabodies, tetrabodies, Bis-scFv, minibodies, Fab2, Fab3and anti -idiotypic (anti-Id) antibodies (including, e.g., anti-Id and anti-anti-Id antibodies to antibodies). In particular, such antibodies include immunoglobulin molecules of any type e.g., IgG, IgE, IgM, IgD, IgA and IgY), class e.g., IgGi, IgG?, IgGs, IgG4, IgAi and IgA2) or subclass.

[0120] As used herein, the term “antigen binding fragment” of an antibody refers to one or more portions of an antibody that contain the antibody’s Complementarity Determining Regions (“CDRs”) and optionally the framework residues that include the antibody’s “variable region” antigen recognition site and exhibit an ability to immunospecifically bind antigen. Such fragments include Fab', F(ab')2, Fv, single chain (ScFv), and mutants thereof, naturally occurring variants, and fusion proteins including the antibody’s “variable region” antigen recognition site and a heterologous protein e.g., a toxin, an antigen recognition site for a different antigen, an enzyme, a receptor or receptor ligand, etcf

[0121] As used herein, the term “fragment” refers to a peptide or polypeptide including an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least 80contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues.

[0122] As used herein the term “modulate” relates to a capacity to alter an effect, result, or activity (e.g., signal transduction). Such modulation can be agonistic or antagonistic. Antagonistic modulation can be partial (z.e., attenuating, but not abolishing) or it can completely abolish such activity (e.g., neutralizing). Modulation can include internalization of a receptor following binding of an antibody or a reduction in expression of a receptor on the target cell. Agonistic modulation can enhance or otherwise increase or enhance an activity (e.g., signal transduction). In a still further embodiment, such modulation can alter the nature of the interaction between a ligand and its cognate receptor so as to alter the nature of the elicited signal transduction. For example, the molecules can, by binding to the ligand or receptor, alter the ability of such molecules to bind to other ligands or receptors and thereby alter their overall activity. In some embodiments, such modulation will provide at least a 10% change in a measurable immune system activity, at least a 50% change in such activity, or at least a 2-fold, 5-fold, 10-fold, or at least a 100-fold change in such activity.

[0123] The term “substantially,” as used in the context of binding or exhibited effect, is intended to denote that the observed effect is physiologically or therapeutically relevant. Thus, for example, a molecule is able to substantially block an activity of a ligand or receptor if the extent of blockage is physiologically or therapeutically relevant (for example if such extent is greater than 60% complete, greater than 70% complete, greater than 75% complete, greater than 80% complete, greater than 85% complete, greater than 90% complete, greater than 95% complete, or greater than 97% complete). Similarly, a molecule is said to have substantially the same immunospecificity and / or characteristic as another molecule, if such immunospecificities and characteristics are greater than 60% identical, greater than 70% identical, greater than 75% identical, greater than 80% identical, greater than 85% identical, greater than 90% identical, greater than 95% identical, or greater than 97% identical).

[0124] As used herein, the “co-stimulatory” signals encompass positive costimulatory signals (e.g., signals that result in enhancing an activity) and negative costimulatory signals (e.g., signals that result in inhibiting an activity).

[0125] The term “derivative” refers to an antibody or antigen-binding fragment thereof that immunospecifically binds to the same target of a parent or reference antibody but which differs in amino acid sequence from the parent or reference antibody or antigen binding fragment thereof by including one, two, three, four, five or more amino acid substitutions, additions, deletions or modifications relative to the parent or reference antibody or antigen binding fragment thereof. In some embodiments such derivatives will have substantially the same immunospecificity and / or characteristics, or the same immunospecificity and characteristics as the parent or reference antibody or antigen binding fragment thereof. The amino acid substitutions or additions of such derivatives can include naturally occurring (z.e., DNA-encoded) or non-naturally occurring amino acid residues. The term “derivative” encompasses, for example, chimeric or humanized variants, as well as variants having altered CHI, hinge, CH2, CH3 or CH4 regions, so as to form, for example antibodies, etc., having variant Fc regions that exhibit enhanced or impaired effector or binding characteristics.

[0126] As used herein, a “chimeric antibody” is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules such as antibodies having a variable region derived from a non-human antibody and a human immunoglobulin constant region.

[0127] As used herein, the term “humanized antibody” refers to an immunoglobulin including a human framework region and one or more CDR’s from a non-human (usually a mouse or rat) immunoglobulin. The non-human immunoglobulin providing the CDR's is called the “donor” and the human immunoglobulin providing the framework is called the “acceptor.” Constant regions need not be present, but if they are, they should be substantially identical to human immunoglobulin constant regions, i.e., at least about 85- 99%, or about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDR’s, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A humanized antibody is an antibody including a humanized light chain and a humanized heavy chain immunoglobulin. For example, a humanized antibody would not encompass a typical chimeric antibody, because, e.g., the entire variable region of a chimeric antibody is non-human.

[0128] The term “endogenous concentration” refers to the level at which a molecule is natively expressed (i.e., in the absence of expression vectors or recombinant promoters) by a cell (which cell can be a normal cell, a cancer cell or an infected cell).

[0129] As used herein, the terms “treat,” “treating,” “treatment” and “therapeutic use” refer to the elimination, reduction or amelioration of one or more symptoms of a disease or disorder. As used herein, a “therapeutically effective amount” refers to that amount of a therapeutic agent sufficient to mediate a clinically relevant elimination, reduction or amelioration of such symptoms. An effect is clinically relevant if its magnitude is sufficient to impact the health or prognosis of a recipient subject. A therapeutically effective amount may refer to the amount of therapeutic agent sufficient to delay or minimize the onset of disease, e.g., delay or minimize the spread of cancer. A therapeutically effective amount may also refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease.

[0130] As used herein, the term “prophylactic agent” refers to an agent that can be used in the prevention of a disorder or disease prior to the detection of any symptoms of such disorder or disease. A “prophylactically effective” amount is the amount of prophylactic agent sufficient to mediate such protection. A prophylactically effective amount may also refer to the amount of the prophylactic agent that provides a prophylactic benefit in the prevention of disease.

[0131] As used herein, the term “cancer” refers to a neoplasm or tumor resulting from abnormal uncontrolled growth of cells. As used herein, cancer explicitly includes leukemias and lymphoma. The term “cancer” refers to a disease involving cells that have the potential to metastasize to distal sites and exhibit phenotypic traits that differ from those of non-cancer cells, for example, formation of colonies in a three-dimensional substrate such as soft agar or the formation of tubular networks or web-like matrices in a three-dimensional basement membrane or extracellular matrix preparation. Non-cancer cells do not form colonies in soft agar and form distinct sphere-like structures in three- dimensional basement membrane or extracellular matrix preparations.

[0132] As used herein, an “immune cell” refers to any cell from the hemopoietic origin including, but not limited to, T cells, B cells, monocytes, dendritic cells, and macrophages.

[0133] As used herein, “inflammatory molecules” refer to molecules that result in inflammatory responses including, but not limited to, cytokines and metalloproteases such as including, but not limited to, IL-ip, TNF-a, TGF-beta, IFN-y, IL-18, IL-17, IL-6, IL- 23, IL-22, IL-21, and MMPs.

[0134] As used herein, “valency” refers to the number of binding sites available per molecule.

[0135] As used herein, the terms “immunologic,” “immunological” or “immune” response is the development of a beneficial humoral (antibody mediated) and / or a cellular (mediated by antigen-specific T cells or their secretion products) response directed against a peptide in a recipient patient. Such a response can be an active response induced by administration of immunogen or a passive response induced by administration of antibody or primed T-cells. A cellular immune response is elicited by the presentation of polypeptide epitopes in association with Class I or Class II MHC molecules to activate antigen specific CD4+T helper cells and / or CD8+cytotoxic T cells. The response may also involve activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia cells, eosinophils, activation or recruitment of neutrophils or other components of innate immunity. The presence of a cell-mediated immunological response can be determined by proliferation assays (CD4+T cells) or CTL (cytotoxic T lymphocyte) assays. The relative contributions of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be distinguished by separately isolating antibodies and T-cells from an immunized syngeneic animal and measuring protective or therapeutic effect in a second subject.

[0136] An “immunogenic agent” or “immunogen” is capable of inducing an immunological response against itself on administration to a mammal, optionally in conjunction with an adjuvant.

[0137] As used herein, the terms “individual,” “host,” “subject,” and “patient” are used interchangeably herein, and refer to a mammal, including, but not limited to, humans, rodents, such as mice and rats, and other laboratory animals.

[0138] As used herein, the term “polypeptide” refers to a chain of amino acids of any length, regardless of modification (e.g., phosphorylation or glycosylation). The term polypeptide includes proteins and fragments thereof. The polypeptides can be “exogenous,” meaning that they are “heterologous,” i.e., foreign to the host cell being utilized, such as human polypeptide produced by a bacterial cell. Polypeptides are disclosed herein as amino acid residue sequences. Those sequences are written left to right in the direction from the amino to the carboxy terminus. In accordance with standard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gin, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (He, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P),Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Vai, V).

[0139] As used herein, the term “variant” refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and / or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.

[0140] Modifications and changes can be made in the structure of the polypeptides of the disclosure and still obtain a molecule having similar characteristics as the polypeptide (e.g., a conservative amino acid substitution). For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity. Because it is the interactive capacity and nature of a polypeptide that defines that polypeptide’s biological functional activity, certain amino acid sequence substitutions can be made in a polypeptide sequence and nevertheless obtain a polypeptide with like properties.

[0141] In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a polypeptide is generally understood in the art. It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still result in a polypeptide with similar biological activity. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. Those indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0142] It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant polypeptide, which in turn defines the interaction of the polypeptide with other molecules, such as enzymes, substrates,receptors, antibodies, antigens, and cofactors. It is known in the art that an amino acid can be substituted by another amino acid having a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, the substitution of amino acids whose hydropathic indices are within ± 2 is preferred, those within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred.

[0143] Substitution of amino acids can also be made on the basis of hydrophilicity, particularly where the biological functional equivalent polypeptide or peptide thereby created is intended for use in immunological embodiments. The following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); proline (-0.5 + 1); threonine (-0.4); alanine (-0.5); histidine (-0.5); cysteine (- 1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biological equivalent, and in particular, an immunologically equivalent polypeptide. In such changes, the substitution of amino acids whose hydrophilicity values are within ± 2 is preferred, those within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred.

[0144] As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various foregoing characteristics into consideration are well known to those of skill in the art and include (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gin, His), (Asp: Glu, Cys, Ser), (Gin: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gin), (He: Leu, Vai), (Leu: He, Vai), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Trp: Tyr), (Tyr: Trp, Phe), and (Vai: lie, Leu). Embodiments of this disclosure thus contemplate functional or biological equivalents of a polypeptide as set forth above. In particular, embodiments of the polypeptides can include variants having about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the polypeptide of interest.

[0145] The term “percent (%) sequence identity” is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical with the nucleotides or amino acids in a reference nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.

[0146] For purposes herein, the % sequence identity of a given nucleotides or amino acids sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given sequence C that has or comprises a certain % sequence identity to, with, or against a given sequence D) is calculated as follows:

[0147] 100 times the fraction W / Z,

[0148] where W is the number of nucleotides or amino acids scored as identical matches by the sequence alignment program in that program’s alignment of C and D, and where Z is the total number of nucleotides or amino acids in D. It will be appreciated that where the length of sequence C is not equal to the length of sequence D, the % sequence identity of C to D will not equal the % sequence identity of D to C.

[0149] As used herein, the term “pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water and emulsions such as an oil / water or water / oil emulsion, and various types of wetting agents.

[0150] IL Compositions

[0151] The high levels of B7-H4 expression found in numerous tumor tissues, for example, human ovarian cancers, points to a key role for B7-H4 in mediating immune suppression. Also, Tumor-Associated Macrophages (TAMs) expressing B7-H4 have been found to suppress tumor-associated antigen-specific T cell immunity (Kryczek, I. et al. (2006) "B7-H4 Expression Identifies A Novel Suppressive Macrophage Population In Human Ovarian Carcinoma," J. Exp. Med. 203(4):871-881). The intensity of B7-H4 expression in TAMs correlates significantly with Treg cell numbers in the tumor. Furthermore, B7-H4 expressed on TAMs, is associated with poor patient outcome (Kryczek, I. et al. (2006) "B7-H4 Expression Identifies A Novel Suppressive Macrophage Population In Human Ovarian Carcinoma," J. Exp. Med. 203(4):871-881). Additionally, B7-H4 can be expressed on myeloid derived suppressor cells (MDSCs) where it may exert an immune suppressive effect in viral infection (Garg, A et al. (2017) “Human Immunodeficiency Virus Type-1 Myeloid Derived Suppressor Cells InhibitCytomegalovirus Inflammation through Interleukin-27 and B7-H4” Sci. Rep. Mar 24;7:44485). While in a study of uterine cancer, B7-H4 expression in the tumor microenvironment was associated with increased infiltration of MDSCs (Vanderstraeten, A. et al. (2014) “Mapping the immunosuppressive environment in uterine tumors: implications for immunotherapy,” Cancer Immunol. Immunother. Jun;63(6):545-57). Therefore, B7-H4 may be expressed on tumor cells, TAM and / or MDSCs where it may exert immune suppressive signaling in cancer.

[0152] Neutrophils are a major component of the host innate defense against infection and also contribute to autoimmune pathogenesis and chronic inflammation. During infection, neutrophils rapidly migrate to sites of inflammation, become activated, and initiate a cascade of defense mechanisms including phagocytosis, killing, and degradation of microorganisms by antimicrobial and proteolytic proteins, along with the generation of reactive oxygen species. Neutrophils also participate in tissue breakdown, remodeling, wound healing, and modulation of other inflammatory and adaptive immune components. Due to their short life span, neutrophils have to be resupplied continuously during infection and inflammation by expansion from myeloid progenitor cells in the bone marrow. In vitro B7-H4 inhibits the growth of bone marrow-derived neutrophil progenitors, suggesting an inhibitory function of B7-H4 in neutrophil expansion (Zhu, G. et al., Blood, 113: 1759-1767 (2009)). Thus, compositions that modulate B7-H4 signal transduction are provided.A. B7-H4 Sequences

[0153] One embodiment provides B7-H4 polypeptides. The polypeptides can include an amino acid sequence of full-length B7-H4, or a fragment or variant thereof, or a fusion protein thereof.

[0154] One embodiment provides human B7-H4 proteins or polypeptides thereof Sequences for human B7-H4 are known in the art. For example, a consensus sequence for B7-H4 isMASLGQILFW SIISIIIILA GAIALIIGFG ISGRHSITVT TVASAGNIGE DGILSCTFEP61 DIKLSDIVIQ WLKEGVLGLV HEFKEGKDEL SEQDEMFRGR TAVFADQVIV GNASLRLKNVQLTDAGTYKC YIITSKGKGN ANLEYKTGAF SMPEVNVDYN ASSETLRCEA PRWFPQPTVVWASQVDQGAN FSEVSNTSFE LNSENVTMKV VSVLYNVTIN NTYSCMIEND IAKATGDIKVTESEIKRRSH LQLLNSKASL CVSSFFAISW ALLPLSPYLM LK

[0155] (SEQ ID NO: 1, Q7Z7D3 (VTCN1 HUMAN)), where amino acids 1-27 are a signal sequence (underlined according to US Patent Publication No. 2016 / 0039905 which is incorporated by referenced in its entirety). Sica et al. indicated that amino acids 1-20 comprise the signal sequence. UniProtKB accession #Q7Z7D3 indicates that the extracellular domain includes amino acids 25-259.

[0156] Another embodiment provides mouse B7-H4 proteins and polypeptides. Sequences for mouse B7-H4 are known in the art. For example, a consensus sequence for mouse B7-H4 is MASLGQIIFWSIINIIIILAGAIALIIGFGISGKHFITVTTFTSAGNIGEDGTLSCTFEP DIKLNGIVIQWLKEGIKGLVHEFKEGKDDLSQQHEMFRGRTAVFADQVVVGNAS LRLKNVQLTDAGTYTCYIRTSKGKGNANLEYKTGAFSMPEINVDYNASSESLRC EAPRWFPQPTVAWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNT YSCMIENDIAKATGDIKVTDSEVKRRSQLQLLNSGPSPCVFSSAFVAGWALLSLS CCLMLR

[0157] (SEQ ID NO:2, Q7TSP5 (VTCN1 MOUSE) which is incorporated by reference in its entirety.)Antibody-Drug Conjugates

[0158] Antibody-drug conjugates of the invention are comprised of an anti-B7-H4 antibody, a cytotoxin and a linker. Antibodies are comprised of light and heavy chains. Each chain is comprised of a variable domain and a constant domain. The constant domain of the light chain can be kappa or lamba sequences as are well known in the art. The heavy chain constant domain is comprised of a first constant domain, a hinge and an Fc domain. Antibodies come in 5 classes, as determined by their sequences. The classes are IgA, IgD, IgE, IgG and IgM. The IgG antibodies comprise 4 subclasses, including IgGl, IgG2, IgG3 and IgG4. The antibody-drug conjugates of the invention are typically of the IgG class and more typically either IgGl or IgG4. If the IgGl classes are utilized, the constant domain sequence can be wild type or contain specific mutations to reduce the binding to Fc gamma receptors. These mutations are well known in the art, but include mutating the glycosylation site (e.g., N297Q), mutating the Fc gamma receptor binding site (L234A and L235A or L234F, L235E and P331S). If the IgG4 class is utilized, the constant domain sequence can be wild type or contain a specific mutation (S228P) to reduce chain exchange as is well known in the art.

[0159] The linker can be conjugated to the antibody by means well known in the art. For example, stochastic conjugation may be used to attach the linker to cysteine residueswithin the antibody. Other means include site specific conjugation, where a specific sequence is added to either the heavy chain or light chain and will serve as linker location. Examples include CAAX, as described in WO 2012 / 153193 and WO 2023 / 194800, wherein C is a cysteine residue, A is an aliphatic amino acid, and X is selected from glutamine, glutamate, serine, cysteine, methionine, alanine, and leucine. An exemplary CAAX sequence is CVIM. A preferred site-specific linker sequence is CVIM, which can be attached to the C-terminus of the light chain via a short spacer comprised of a series of 4, 5, 6, 7, 8, or more glycine residues. A preferred spacer is a series of 7 glycine residues.

[0160] Anti-B7-H4 antibodies, cytotoxins and linkers are well known in the art and are described below.1. Anti-B7-H4 Antibody Sequences a. B1A1 Sequences

[0161] One embodiment provides a murine monoclonal antibody is produced by hybridoma clone Bl Al contains two light chains and two heavy chains and specifically binds to B7-H4. i. Light Chain

[0162] One embodiment provides a murine monoclonal antibody or antigen binding fragment thereof that has a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence: DIVMTOSHKFMSTSVGDRVSITCKASODVRTAVAWYOOKPGOSPKLLIYSTSYR YTGVPDRFTGSGSGTEFTFTISSVQAEDLAVYYCOQYYVTPLTFGAGTKLELK (SEQ ID NO:3) that specifically binds to B7-H4.

[0163] The CDRs are of SEQ ID NO:3 are bolded and underlined and are:CDR1 KASQDVRTAVA (SEQ ID NO:4);CDR2 STSYRYT (SEQ ID NO: 5); and CDR3 QQYYVTPLT (SEQ ID NO:6).

[0164] Another embodiment provides a nucleic acid that encodes the light chain (SEQ ID NO:3).

[0165] An exemplary nucleic acid that encodes light chain (SEQ ID NO:3) is GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACA GGGTCAGTATCACCTGCAAGGCCAGTCAGGATGTGAGAACTGCTGTAGCCTG GTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTACTCGACATCCT ACCGGTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGAATTCACTTTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAATATTATGTTACTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA (SEQ ID N0:7). ii. Heavy Chain

[0166] One embodiment provides a murine monoclonal antibody or antigen binding fragment thereof that has a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence: EVOLOQSGTVLARPGASVKMSCKASGYTFTSYWMHWIKORPGOGLEWIGAIYP GNSDTKYNOKFKDKAKLTAVTSASTAYMELSSLTNEDSAVYYCTSTVRNVMD YWGQGTSVTVSS (SEQ ID NO: 8) and specifically binds to B7-H4.

[0167] The CDRs of SEQ ID NO: 8 are bolded and underlined and are:CDR1 SYWMH (SEQ ID NO: 9);CDR2 AIYPGNSDTKYNQKFKDK (SEQ ID NO: 10); andCDR3 TVRNVMDY (SEQ ID NO: 11).

[0168] Another embodiment provides a nucleic acid encoding heavy chain (SEQ ID NO:8).

[0169] An exemplary nucleic acid that encodes heavy chain (SEQ ID NO:8) is GAGGTTCAGCTCCAGCAGTCTGGGACTGTGCTGGCAAGGCCTGGGGCTTCAG TGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACCAGCTACTGGATGCAC TGGATAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGCGCTATTTATC CTGGAAATAGTGATACTAAATACAACCAGAAGTTCAAGGACAAGGCCAAACT GACTGCAGTCACATCTGCCAGCACTGCCTACATGGAGCTCAGCAGCCTGACA AATGAGGACTCTGCGGTCTATTACTGTACATCTACGGTACGGAATGTTATGGA CTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 12).

[0170] One embodiment provides an antibody, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain having CDRs according to SEQ ID Nos: 4, 5, and 6 and specifically binds to B7-H4.

[0171] One embodiment provides an antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having a heavy chain having CDRs according to SEQ ID Nos: 9, 10, and 11 and specifically binds to B7-H4.

[0172] One embodiment provides an antibody, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain having CDRs according to SEQ ID Nos: 4, 5, and 6 and a heavy chain having CDRs according to SEQ ID Nos: 9, 10, and 11 and specifically binds to B7-H4.

[0173] One embodiment provides an antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having a light chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:3 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:8 and specifically binds to B7-H4. b. B1H1 Sequences

[0174] In one embodiment a murine monoclonal antibody is produced by hybridoma clone B1H1 and contains two light chains and two heavy chains. i. Light Chain

[0175] One embodiment provides a murine monoclonal antibody or antigen binding fragment thereof that has a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:

[0176] EIQMTQSPSSMSASLGDRITITCOATQDIVKSLNWYQOKPGKPPSFLIYYTAQLAEGVPSRFSGSGSGSDYSLTISNLESEDFADYYCLQFYEFPPTFGGGTK LEIK (SEQ ID NO: 13).

[0177] The CDRs are of SEQ ID NO: 13 are bolded and underlined and are:CDR1 QATQDIVKSLN (SEQ ID NO: 14);CDR2 YTAQLAE (SEQ ID NO: 15); andCDR3 LQFYEFPPT (SEQ ID NO: 16).

[0178] Another embodiment provides a nucleic acid that encodes the light chain(SEQ ID NO: 13).

[0179] An exemplary nucleic acid that encodes the light chain (SEQ ID NO: 13) is

[0180] GAAATCCAGATGACCCAGTCTCCATCCTCTATGTCTGCATCTCTGG GAGACAGAATAACCATCACTTGCCAGGCAACTCAAGACATTGTTAAGAGTTT AAACTGGTATCAACAAAAACCAGGGAAACCCCCTTCATTCCTGATCTATTATA CAGCTCAACTGGCAGAAGGGGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGGTCAGACTATTCTCTGACAATCAGCAACCTGGAGTCTGAAGATTTTGCAGACT ATTACTGTCTACAGTTTTATGAGTTTCCTCCGACGTTCGGTGGAGGCACCAAG CTGGAAATCAAA (SEQ ID NO: 17).

[0181] One embodiment provides a monoclonal antibody or antigen binding fragment thereof that has a light chain constant domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGG GGGGGCVIM (SEQ ID NO: 18) and specifically binds to B7-H4. ii. Humanized Light Chain

[0182] One embodiment provides an anti-B7-H4 monoclonal antibody or antigen binding fragment thereof that has a humanized light chain variable domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with one of the following amino acid sequences:Humanized B1H1 VL1:DIQMTQSPSSLSASVGDRVTITCQATQDIVKSLNWYQQKPGKPPKFLIYYTAQLA EGVPSRFSGSGSGTDYTLTISSLQSEDFATYYCLQFYEFPPTFGGGTKVEIK (SEQ ID NO: 19)Humanized B1H1 VL2:DIQMTQSPSSLSASVGDRVTITCQATQDIVKSLNWYQQKPGKPPKFLIYYTAQLA EGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQFYEFPPTFGGGTKVEIK (SEQ ID NO:20)Humanized B 1H1 VL3 :DIQMTQSPSSLSASVGDRVTITCQATQDIVKSLNWYQQKPGKAPKFLIYYTAQLA EGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQFYEFPPTFGGGTKVEIK (SEQ ID NO:21)Humanized B1H1 VL4:DIQMTQSPSSLSASVGDRVTITCQATQDIVKSLNWYQQKPGKPPKFLIYYTAQLA EGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQFYEFPPTFGGGTKVEIK (SEQ ID NO:22)Humanized B1H1 VL5:DIQMTQSPSSLSASVGDRVTITCQATQDIVKSLNWYQQKPGKPPKFAIYYTAQLA EGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQFYEFPPTFGGGTKVEIK (SEQ ID NO:23)One embodiment provides an anti-B7-H4 monoclonal antibody or antigen binding fragment thereof that has a humanized light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with one of the following amino acid sequences:Full Length B1H1 Light chain Variant 1:DIQMTQSPSSLSASVGDRVTITCQATQDIVKSLNWYQQKPGKPPKFLIYYTAQLA EGVPSRFSGSGSGTDYTLTISSLQSEDFATYYCLQFYEFPPTFGGGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGGG GCVIM (SEQ ID NO:24)Full Length B1H1 Light chain Variant 2:DIQMTQSPSSLSASVGDRVTITCQATQDIVKSLNWYQQKPGKPPKFLIYYTAQLAEGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQFYEFPPTFGGGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGGG GCVIM (SEQ ID NO:25)Full Length B1H1 Light chain Variant 3:DIQMTQSPSSLSASVGDRVTITCQATQDIVKSLNWYQQKPGKAPKFLIYYTAQLAEGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQFYEFPPTFGGGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGGG GCVIM (SEQ ID NO:26)Full Length B1H1 Light chain Variant 4:DIQMTQSPSSLSASVGDRVTITCQATQDIVKSLNWYQQKPGKPPKFLIYYTAQLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQFYEFPPTFGGGTKVEIKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGGGG C VIM (SEQ ID NO: 27)Full Length B1H1 Light chain Variant 5:DIQMTQSPSSLSASVGDRVTITCQATQDIVKSLNWYQQKPGKPPKFLIYYTAQLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQFYEFPPTFGGGTKVEIKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGGGG CVIM (SEQ ID NO:28) iii. Heavy Chain

[0183] One embodiment provides a murine monoclonal antibody or antigen binding fragment thereof that has a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:EVQLQQSGTVLARPGASVKMSCKVSGYPFTSYWMHWVKQRPGQGLEWIGAIY PGKSDTEYNPNFKGKAKLTAVTSATTAYMELSSLTNEDSAVYYCTSTWTHYFD YWGQGTTLTVSS (SEQ ID NO:29) and specifically binds to B7-H4.

[0184] The CDRs are of SEQ ID NO:29 are bolded and underlined and are:CDR1 SYWMH (SEQ ID NO: 9);CDR2 AIYPGKSDTEYNPNFKG (SEQ ID NO: 30); andCDR3 TVRNVMDY (SEQ ID NO: 11).

[0185] Another embodiment provides a nucleic acid encoding heavy chain (SEQ ID NO:29).

[0186] An exemplary nucleic acid that encodes heavy chain (SEQ ID NO:29) is GAGGTTCAGCTCCAGCAGTCTGGGACTGTTCTGGCAAGGCCTGGGGCTTCAG TGAAGATGTCCTGCAAGGTTTCTGGCTACCCCTTTACCAGCTACTGGATGCAC TGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGCGCTATTTATC CTGGAAAAAGTGACACTGAATACAACCCGAACTTCAAGGGCAAGGCCAAACT GACTGCAGTCACATCTGCCACCACTGCCTACATGGAGCTCAGCAGCCTGACA AATGAGGACTCTGCGGTCTATTACTGTACAAGTACCTGGACCCACTACTTTGA CTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO:31).

[0187] One embodiment provides a murine monoclonal antibody or antigen binding fragment thereof that has a heavy chain constant domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI<ALPAPIEI<TI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK (SEQ ID NO:32) and specifically binds to B7-H4. SEQ ID NO:32 contains L234A and L235A (AlaAla) to reduce and / or eliminate binding of the antibody to IgG receptors.

[0188] The underlined and bolded amino acids represent amino acids that differ from the L234F, L235E, P331S (FES) mutant sequence shown below.

[0189] Another embodiment provides a mutant heavy chain constant domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPSEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK (SEQ ID NO:33)The underlined and bolded amino acids of SEQ ID NO:33 represent L234F, L235E,P331S (FES) amino acids that differ from the AlaAla sequence above. iv. Humanized Heavy Chain

[0190] One embodiment provides an anti-B7-H4 monoclonal antibody or antigen binding fragment thereof that has a humanized heavy chain variable domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with one of the following amino acid sequences:Humanized B1H1 VH1 :EVQLVQSGAEVKKPGASVKVSCKVSGYPFTSYWMHWVRQAPGQGLEWIGAIYP GKSDTEYAPKFQGRVTLTADTSTSTAYMELSSLRSEDTAVYYCTSTWTHYFDYW GQGTTVTVSS (SEQ ID NO:34)Humanized B1H1 VH2:QVQLVQSGAEVKKPGASVKVSCKVSGYPFTSYWMHWVRQAPGQGLEWMGAIY PGKSDTEYAQKFQGRVTLTADTSTSTAYMELSSLRSEDTAVYYCTSTWTHYFDY WGQGTTVTVSS (SEQ ID NO: 35)Humanized B 1H1 VH3 :QVQLVQSGAEVKKPGASVKVSCKVSGYPFTSYWMHWVRQAPGQGLEWMGAIY PGKSDTEYAQKFQGRVTLTADTSTSTAYMELSSLRSEDTATYYCTSTWTHYFDY WGQGTTVTVSS (SEQ ID NO: 36)Humanized B1H1 VH4:QVQLVQSGAEVKKPGASVKVSCKVSGYPFTSYYMHWVRQAPGQGLEWMGAIY PGKSDTEYAQKFQGRVTLTADTSTSTAYMELSSLRSEDTATYYCTSTWTHYFDY WGQGTTVTVSS (SEQ ID NO: 37)One embodiment provides an anti-B7-H4 monoclonal antibody or antigen binding fragment thereof that has a humanized heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with one of the following amino acid sequences:Heavy chain B1H1 Variant 1 (AlaAla):EVQLVQSGAEVKKPGASVKVSCKVSGYPFTSYWMHWVRQAPGQGLEWIGAIYP GKSDTEYAPKFQGRVTLTADTSTSTAYMELSSLRSEDTAVYYCTSTWTHYFDYW GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 38)Heavy chain B1H1 Variant 2 (AlaAla):QVQLVQSGAEVKKPGASVKVSCKVSGYPFTSYWMHWVRQAPGQGLEWMGAIY PGKSDTEYAQKFQGRVTLTADTSTSTAYMELSSLRSEDTAVYYCTSTWTHYFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK (SEQ ID NO: 39)Heavy Chain B1H1 Variant 3 (AlaAla):QVQLVQSGAEVKKPGASVKVSCKVSGYPFTSYWMHWVRQAPGQGLEWMGAIY PGKSDTEYAQKFQGRVTLTADTSTSTAYMELSSLRSEDTATYYCTSTWTHYFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK (SEQ ID NO:40)Heavy chain B1H1 Variant 4 (AlaAla):QVQLVQSGAEVKKPGASVKVSCKVSGYPFTSYYMHWVRQAPGQGLEWMGAIY PGKSDTEYAQKFQGRVTLTADTSTSTAYMELSSLRSEDTATYYCTSTWTHYFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK (SEQ ID N0:41)

[0191] In another embodiment, the monoclonal antibody or antigen binding fragment thereof has a humanized heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with one of the following amino acid sequences:Heavy chain B1H1 variant 1 (FES):EVQLVQSGAEVKKPGASVKVSCKVSGYPFTSYWMHWVRQAPGQGLEWIGAIYP GKSDTEYAPKFQGRVTLTADTSTSTAYMELSSLRSEDTAVYYCTSTWTHYFDYW GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPSEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK (SEQ ID NO:42);Heavy chain B1H1 variant 2 (FES):QVQLVQSGAEVKKPGASVKVSCKVSGYPFTSYWMHWVRQAPGQGLEWMGAIY PGKSDTEYAQKFQGRVTLTADTSTSTAYMELSSLRSEDTAVYYCTSTWTHYFDY W GQGT T VT VS S ASTKGPS VFPLAPS SKSTSGGT AALGCLVKD YFPEP VT VSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CT<VS NKALPAPSEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK (SEQ ID NO:43);Heavy chain B1H1 variant 3 (FES):QVQLVQSGAEVKKPGASVTCVSCKVSGYPFTSYWMHWVRQAPGQGLEWMGAIY PGKSDTEYAQKFQGRVTLTADTSTSTAYMELSSIJLSEDTATYYCTSTWTHYFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VS NKALPAPSEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK (SEQ ID NO:44); andHeavy chain B1H1 variant 4 (FES):QVQLVQSGAEVKKPGASVKVSCKVSGYPFTSYYMHWVRQAPGQGLEWMGAIY PGKSDTEYAQKFQGRVTLTADTSTSTAYMELSSLRSEDTATYYCTSTWTHYFDY WGQGT1 VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VS NKALPAPSEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK (SEQ ID NO:45).

[0192] One embodiment provides an anti-B7H4 antibody-drug conjugate, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain having CDRs according to SEQ ID Nos: 14, 15, and 16.

[0193] One embodiment provides an anti-B7H4 antibody-drug conjugate, preferably a monoclonal antibody, or an antigen binding fragment thereof having a heavy chain containing CDRs according to SEQ ID Nos: 9, 11, and 30.

[0194] One embodiment provides an anti-B7H4 antibody-drug conjugate, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain containing CDRs according to SEQ ID Nos: 14, 15, and 16 and a heavy chain containing CDRs according to SEQ ID Nos: 9, 11, and 30.

[0195] One embodiment provides an anti-B7H4 antibody-drug conjugate, preferably a monoclonal antibody, or an antigen binding fragment thereof having a light chain at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 13 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:29.

[0196] Another embodiment provides an anti-B7H4 antibody-drug conjugate preferably a monoclonal antibody, or an antigen binding fragment thereof having a light chain variable domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or100% sequence identity to any one of SEQ ID NOs: 19, 20, 21, 22, or 23, and a heavy chain variable domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs:34, 35, 36, or 37. .

[0197] One embodiment provides an anti-B7H4 antibody-drug conjugate or antigen binding fragment thereof having a light chain having an amino acid sequence according to any one of SEQ ID NOs: 24, 25, 26, 27, or 28, and a heavy chain having an amino acid sequence according to any one of SEQ ID NOs: 38, 39, 40, or 41.

[0198] Another embodiment provides an anti-B7H4 antibody-drug conjugate or antigen binding fragment thereof having a light chain having an amino acid sequence according to any one of SEQ ID NOs: 24, 25, 26, 27, or 28, and a heavy chain having an amino acid sequence according to any one of SEQ ID NOs: 42, 43, 44, or 45.

[0199] One embodiment provides an anti-B7H4 antibody-drug conjugate or antigen binding fragment thereof having two light chains and two heavy chains, wherein the two light chains include a polypeptide selected from the group consisting of SEQ ID NO: 24, 25, 26, 27, or 28, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 24, 25, 26, 27, or 28, and the two heavy chains include a polypeptide selected from the group consisting of SEQ ID NO: 38, 39, 40, or 41, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 38, 39, 40, or 41, and wherein the antibody or antigen binding fragment thereof binds to B7-H4.

[0200] Another embodiment provides an antibody-drug conjugate or antigen binding fragment thereof having two light chains and two heavy chains, wherein the two light chains include a polypeptide selected from the group consisting of SEQ ID NO: 24, 25, 26, 27, or 28, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 24, 25, 26, 27, or 28, and the two heavy chains include a polypeptide selected from the group consisting of SEQ ID NO: 42, 43, 44, or 45, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 42, 43, 44, or 45, and wherein the antibody or antigen binding fragment thereof binds to B7-H4. c. B1H3 Sequences

[0201] In one embodiment a murine monoclonal antibody is produced by hybridoma clone B1H3 and contains two light chains and two heavy chains and specifically binds B7-H4. i. Light Chain

[0202] One embodiment provides an anti-B7-H4 murine monoclonal antibody or antigen binding fragment thereof that has a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:EIQMTOSPSSMSASLGDTITITCOATODIVKSLNWYOOKPGKPPSFLIYYTTQLA EGVPSRFSGSGSGSDYSLTISNLDSEDFADYYCLQFYEFPPTFGGGTKLEIK (SEQ ID NO:46)

[0203] The CDRs are of SEQ ID NO:46 are bolded and underlined and are:CDR1 QATQDIVKSLN (SEQ ID NO: 14);CDR2 YYTTQLAE (SEQ ID NO:47); and CDR3 LQFYEFPPT (SEQ ID NO: 16).

[0204] Another embodiment provides a nucleic acid that encodes the light chain (SEQ ID NO: 46).

[0205] An exemplary nucleic acid that encodes light chain (SEQ ID NO:46) is GAAATCCAGATGACCCAGTCTCCATCCTCTATGTCTGCATCTCTGGGAGACAC AATAACCATCACTTGCCAGGCAACTCAAGACATTGTTAAGAGTTTAAACTGG TATCAACAAAAACCAGGGAAACCCCCTTCATTCCTGATCTATTATACAACTCAACTGGCAGAAGGGGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGGTCAGAC TATTCTCTGACAATCAGCAACCTGGACTCTGAAGATTTTGCAGACTATTACTG TCTACAGTTTTATGAGTTTCCTCCGACGTTCGGTGGAGGCACCAAGCTGGAAA TCAAA (SEQ ID NO: 48). ii. Heavy Chain

[0206] One embodiment provides an anti-B7-H4 murine monoclonal antibody or antigen binding fragment thereof that has a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:EVQLQQSGTVLARPGASVKMSCKASGYTFSSYWMHWVKQRPGQGLEWIGAIY PGKSDTSYNOKFQGKAKLTAVTSASTAFMELTSLTNEDSAVYYCTSTWTHYFD YWGQGTTLTVSS (SEQ ID NO: 49).

[0207] The CDRs are of SEQ ID NO:49 are bolded and underlined and are:CDR1 SYWMH (SEQ ID NO: 9);CDR2 AIYPGKSDTEYNPNFKG (SEQ ID NO: 50); andCDR3 TVRNVMDY (SEQ ID NO: 11).

[0208] Another embodiment provides a nucleic acid encoding heavy chain (SEQ ID NO:49).

[0209] An exemplary nucleic acid that encodes heavy chain (SEQ ID NO:49) is GAGGTTCAGCTCCAGCAGTCTGGGACTGTTCTGGCAAGGCCTGGGGCTTCAG TGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTTCCAGCTACTGGATGCAC TGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGCGCTATTTATC CTGGAAAAAGTGATACTAGCTACAACCAGAAGTTCCAGGGCAAGGCCAAACT GACTGCAGTCACATCTGCCAGCACTGCCTTCATGGAGCTCACCAGCCTGACA AATGAGGACTCTGCGGTCTATTACTGTACAAGTACCTGGACCCACTACTTTGA CTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO:51).

[0210] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain containing CDRs according to SEQ ID Nos: 14, 16, and 47.

[0211] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having a heavy chain containing CDRs according to SEQ ID Nos: 9, 11, and 50.

[0212] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain containing CDRs according to SEQ ID Nos: 14, 16, and 47, and a heavy chain containing CDRs according to SEQ ID Nos: 9, 11, and 50.

[0213] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having a light chain at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:46 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:49. d. B1H10 Sequences

[0214] In one embodiment an anti-B7H4 murine monoclonal antibody is produced by hybridoma clone B1H10 and contains two light chains and two heavy chains. i. Light Chain

[0215] One embodiment provides an anti-B7H4 murine monoclonal antibody or antigen binding fragment thereof that has a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:DIQMTQTTSSLSASLGDRVTISCRASODISNYLNWYQQKPDGTIKLLIYYTSRLH SGVPSRFSGSGSGSDYSLTISNLEOEDIATYFCOQGNTLPWTFGGGTKLEFK (SEQ ID NO: 52).

[0216] The CDRs are of SEQ ID NO:52 are bolded and underlined and are:CDR1 RASQDISNYLN (SEQ ID NO:53);CDR2 YTSRLHS (SEQ ID NO: 54); andCDR3 QQGNTLPWT (SEQ ID NO 55).

[0217] Another embodiment provides a nucleic acid that encodes the light chain(SEQ ID NO: 52).An exemplary nucleic acid that encodes light chain (SEQ ID NO: 52) is GATATCCAGATGACACAAACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAG AGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGCAATTATTTAAACTGG TATCAGCAGAAACCAGATGGAACTATTAAACTCCTGATCTATTACACATCAA GATTACATTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGATCAGA TTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTT GCCAACAGGGTAATACGCTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGA ATTCAAA (SEQ ID NO:56).

[0218] One embodiment provides an anti-B7H4 monoclonal antibody or antigen binding fragment thereof that has a light chain constant domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKTIKLLIYYTSRLHS GVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTLPWTFGQGTKLEIKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGGGG CVIM (SEQ ID NO: 57) ii. Humanized B1H10 Light Chain

[0219] In another embodiment, the anti-B7H4 monoclonal antibody or antigen binding fragment thereof has a humanized light chain variable domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with one of the following amino acid sequences:Humanized B1H10 VL1 :DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKTIKLLIYYTSRLHS GVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTLPWTFGQGTKLEIK (SEQ ID NO:58)Humanized B1H10 VL2: DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHS GVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTLPWTFGQGTKLEIK (SEQ ID NO:59)Humanized B1H10 VL3: DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHS GVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGQTLPWTFGQGTKLEIK (SEQ ID NO: 60)Humanized B1H10 VL4: DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHS GVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGSTLPWTFGQGTKLEIK (SEQ ID NO:61)One embodiment provides an anti-B7H4 monoclonal antibody or antigen binding fragment thereof that has a humanized light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with one of the following amino acid sequences:Humanized B 1H10 Light Chain Variant 1 :DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKTIKLLIYYT SRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTLPWTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGG GGGGGCVIM (SEQ ID NO: 134)Humanized B1H10 Light Chain Variant 2: DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHS GVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTLPWTFGQGTKLEIKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGGGG C VIM (SEQ ID NO: 62)Humanized B1H10 Light Chain Variant 3: DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHS GVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGQTLPWTFGQGTKLEIKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGGGG CVIM (SEQ ID NO:63)Humanized B1H10 Light Chain Variant 4:DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHS GVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGSTLPWTFGQGTKLEIKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGGGG CVIM (SEQ ID NO: 64) iii. Heavy Chain

[0220] One embodiment provides an anti-B7H4 murine monoclonal antibody or antigen binding fragment thereof that has a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:EVOLOQSGPELVKPGASVKMSCKASGYTFTDYYMNWVROSHGKSLEWIGRVNPSNGGTNYNQKFKGKATLTVDKSLSTAYMQLSSLTSEDSAVYYCARRHNYAD FWGQGTTLTVSS (SEQ ID NO:65).

[0221] The CDRs of SEQ ID NO:65 are bolded and underlined and are:CDR1 DYYMN (SEQ ID NO: 66);CDR2 RVNPSNGGTNYNQKFKG (SEQ ID NO: 67); andCDR3 RHNYADF (SEQ ID NO: 68).

[0222] Another embodiment provides a nucleic acid encoding heavy chain (SEQ ID NO:65).An exemplary nucleic acid that encodes heavy chain (SEQ ID NO:65) isGAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGTAAGGCTTCTGGATACACATTCACTGACTACTACATGAACTGGGTGAGGCAGAGTCATGGAAAGAGCCTTGAGTGGATTGGACGTGTTAATC CTAGCAATGGTGGTACTAACTACAACCAGAAATTCAAGGGCAAGGCCACATT GACAGTAGACAAATCCCTCAGCACAGCCTACATGCAGCTCAGCAGCCTGACA TCTGAGGACTCTGCGGTCTATTACTGTGCAAGACGACATAACTACGCAGACTT CTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO:69). iv. Humanized B1H10 Heavy Chain

[0223] One embodiment provides an anti-B7H4 antibody-drug conjugate or antigenbinding fragment thereof that has a humanized heavy chain variable domain varianthaving at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with one of the following amino acid sequences:Humanized B1H10 VH1:EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWIGRVN PSNGGTNYAQKFQGRVTLTVDKSTSTAYMELSSLRSEDTAVYYCARRHNYADF WGQGTTVTVSS (SEQ ID NO: 70)Humanized B1H10 VH2:QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRV NPANGGTNYAQKFQGRVTLTVDTSTSTAYMELSSLRSEDTAVYYCARRHNYAD FWGQGTTVTVSS (SEQ ID NO:71)Humanized B1H10 VH3:QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRV NPSSGGTNYAQKFQGRVTLTVDTSTSTAYMELSSLRSEDTAVYYCARRHNYADF WGQGTTVTVSS (SEQ ID NO: 72)Humanized B1H10 VH4:QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRV NPSNGGTNYAQKFQGRVTLTVDTSKSTAYMELSSLRSEDTAVYYCARRHNYAD FWGQGTTVTVSS (SEQ ID NO:73)Humanized B1H10 VH5:QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRV NPSSGGTNYAQKFQGRVTLTVDTSKSTAYMELSSLRSEDTAVYYCARRHNYADF WGQGTTVTVSS (SEQ ID NO: 74)

[0224] One embodiment provides an anti-B7H4 monoclonal antibody or antigen binding fragment thereof that has a humanized heavy chain variant having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the one of the following amino acid sequences:Humanized B1H10 Heavy Chain Variant 1 (AlaAla):EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWIGRVN PSNGGTNYAQKFQGRVTLTVDKSTSTAYMELSSLRSEDTAVYYCARRHNYADF WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK (SEQ ID NO: 75)Humanized B1H10 Heavy Chain Variant 2 (AlaAla):QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRV NPANGGTNYAQKFQGRVTLTVDTSTSTAYMELSSLRSEDTAVYYCARRHNYAD FWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK (SEQ ID NO: 76)Humanized B1H10 Heavy Chain Variant 3 (AlaAla):QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRV NPSSGGTNYAQKFQGRVTLTVDTSTSTAYMELSSLRSEDTAVYYCARRHNYADF WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK (SEQ ID NO: 77)Humanized B1H10 Heavy Chain Variant 4 (AlaAla):QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRV NPSNGGTNYAQKFQGRVTLTVDTSKSTAYMELSSLRSEDTAVYYCARRHNYAD FWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK (SEQ ID NO: 78)Humanized B1H10 Heavy Chain Variant 5 (AlaAla):QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRV NPSSGGTNYAQKFQGRVTLTVDTSKSTAYMELSSLRSEDTAVYYCARRHNYADF WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK (SEQ ID NO: 79)

[0225] One embodiment provides an anti-B7H4 monoclonal antibody or antigen binding fragment thereof that has a humanized mutant heavy chain variant having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with one of the following amino acid sequences:Humanized B1H10 Heavy Chain Variant 1 (FES):EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWIGRVN PSNGGTNYAQKFQGRVTLTVDKSTSTAYMELSSLRSEDTAVYYCARRHNYADF WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VS NKALPAPSEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK (SEQ ID NO: 80)Humanized B1H10 Heavy Chain Variant 2 (FES):QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRV NPANGGTNYAQKFQGRVTLTVDTSTSTAYMELSSLRSEDTAVYYCARRHNYAD FWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPSEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK (SEQ ID NO: 81)Humanized B1H10 Heavy Chain Variant 3 (FES):QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRV NPSSGGTNYAQKFQGRVTLTVDTSTSTAYMELSSLRSEDTAVYYCARRHNYADF WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VS NKALPAPSEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK (SEQ ID NO: 82)Humanized B1H10 Heavy Chain Variant 4 (FES):QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRV NPSNGGTNYAQKFQGRVTLTVDTSKSTAYMELSSLRSEDTAVYYCARRHNYAD FWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPSEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:83)Humanized B1H10 Heavy Chain Variant 5 (FES):QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRVNPSSGGTNYAQKFQGRVTLTVDTSKSTAYMELSSLRSEDTAVYYCARRHNYADF WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VS NKALPAPSEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK (SEQ ID NO: 84)

[0226] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain containing CDRs according to SEQ ID Nos: 53, 54, or 55.

[0227] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having a heavy chain containing CDRs according to SEQ ID Nos: 66, 67, or 68.

[0228] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain containing CDRs according to SEQ ID Nos: 53, 54, or 55, and a heavy chain containing CDRs according to SEQ ID Nos: 66, 67, or 68.

[0229] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having a light chain at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:52 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 65.

[0230] Another embodiment provides an anti-B7H4 antibody preferably a monoclonal antibody, or an antigen binding fragment thereof having a light chain variable domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs:58, 59, 60, or 61, and a heavy chain variable domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs:70, 71, 72, 73, or 74. .

[0231] One embodiment provides an anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence according to any one of SEQ ID NOs: 62, 63, or 64, and a heavy chain having an amino acid sequence according to any one of SEQ ID NOs: 75, 76, 77, 78, or 79.

[0232] Another embodiment provides an anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence according to any one of SEQ ID NOs: 62, 63, or 64, and a heavy chain having an amino acid sequence according to any one of SEQ ID NOs: 80, 81, 82, 83, or 84.

[0233] One embodiment provides an anti-B7H4 antibody or antigen binding fragment thereof having two light chains and two heavy chains, wherein the two light chains include a polypeptide selected from the group consisting of SEQ ID NO: 62, 63, or 64, or a variant thereof comprising at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 62, 63, or 64, and the two heavy chains include a polypeptide selected from the group consisting of SEQ ID NO: 75, 76, 77, 78, or 79, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 75, 76, 77, 78, or 79.

[0234] Another embodiment provides an anti-B7H4 antibody or antigen binding fragment thereof having two light chains and two heavy chains, wherein the two light chains include a polypeptide selected from the group consisting of SEQ ID NO: 62, 63, or 64, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 62, 63, or 64, and the two heavy chains include a polypeptide selected from the group consisting of SEQ ID NO: 80, 81, 82, 83, or 84, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 80, 81, 82, 83, or 84. e. B2E6 Sequences

[0235] In one embodiment an anti-B7H4 murine monoclonal antibody is produced by hybridoma clone B2E6 and contains two light chains and two heavy chains. i. Light Chain

[0236] One embodiment provides an anti-B7H4 murine monoclonal antibody or antigen binding fragment thereof that has a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:ETVMTQSHKIMSTSVGDRVTITCKASODVRTAVAWYQOKPGQSPKLLISSASYO YTGVPDRFTGSGSGTDFTFTISSLQAEDLAVYYCHQYYNTPLTFGAGTKLELR (SEQ ID NO: 85).

[0237] The CDRs are of SEQ ID NO:85 are bolded and underlined and are:CDR1 KASQDVRTAVA (SEQ ID NO:4);CDR2 SASYQYT (SEQ ID NO:86); andCDR3 HQYYNTPLT (SEQ ID NO:87).

[0238] Another embodiment provides a nucleic acid that encodes the light chain(SEQ ID NO: 85).

[0239] An exemplary nucleic acid that encodes light chain (SEQ ID NO: 85) is GAAACTGTGATGACCCAGTCTCACAAAATCATGTCCACTTCAGTAGGAGACA GGGTCACCATCACCTGCAAGGCCAGTCAGGATGTGAGAACTGCTGTGGCCTG GTATCAACAGAAACCAGGACAATCTCCTAAATTACTAATTTCCTCGGCATCCT ACCAATACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGA TTTCACTTTCACCATCAGCAGTTTGCAGGCTGAAGACCTGGCAGTTTATTACT GTCATCAGTATTATAATACTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAG CTGAGA (SEQ ID NO: 88). ii. Heavy Chain

[0240] One embodiment provides an anti-B7H4 murine monoclonal antibody or antigen binding fragment thereof that has a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:EVQLQQSGTVLARPGASVKMSCKASGYTFTSYWMHWVKQRPGQGLEWIGAIY PGKSDTTYNQKFEGKAKLTAVTSDSTAYMDLSSLTNEDSAVYYCTSSVRNAM DYWGOGTSVTVSS (SEQ ID NO: 89).

[0241] The CDRs are of SEQ ID NO:89 are bolded and underlined and are:CDR1 SYWMH (SEQ ID NO: 9);CDR2 AIYPGKSDTTYNQKFEG (SEQ ID NO: 90); andCDR3 SVRNAMDY (SEQ ID NO:91).

[0242] Another embodiment provides a nucleic acid encoding heavy chain (SEQ ID NO:89).An exemplary nucleic acid that encodes heavy chain (SEQ ID NO:89) is GAGGTTCAGCTCCAGCAGTCTGGGACTGTGCTGGCAAGGCCTGGGGCTTCAG TGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATGCAC TGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGCGCTATTTATCCTGGAAAAAGTGATACTACCTACAACCAGAAGTTCGAGGGCAAGGCCAAACT GACTGCAGTCACATCTGACAGCACAGCCTACATGGATCTCAGTAGCCTGACA AATGAGGACTCTGCGGTCTATTACTGTACATCTTCGGTTCGGAATGCTATGGA CTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO:92).

[0243] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain having CDRs according to SEQ ID Nos: 4, 86, and 87

[0244] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having a heavy chain having CDRs according to SEQ ID Nos: 9, 90, and 91.

[0245] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain containing CDRs according to SEQ ID Nos: 4, 86, and 87, and a heavy chain containing CDRs according to SEQ ID Nos: 9, 90, and 91.

[0246] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having a light chain at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:85 and aheavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 89. f. B4B3 Sequences

[0247] In one embodiment an anti-B7H4 murine monoclonal antibody is produced by hybridoma clone B4B3 and contains two light chains and two heavy chains. i. Light Chain

[0248] One embodiment provides an anti-B7H4 murine monoclonal antibody or antigen binding fragment thereof that has a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:DVLMTQTPLSLPVSLGGQASISCRSSQUVHSNGNTYLEWYLQKPGQSPKLLIYK VSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKL EIK (SEQ ID NO:93)

[0249] The CDRs of SEQ ID NO:93 are bolded and underlined and are:CDR1 RSSQIIVHSNGNTYLE (SEQ ID NO: 94);CDR2 KVSNRFS (SEQ ID NO: 95); andCDR3 FQGSHVPWT (SEQ ID NO: 96).

[0250] Another embodiment provides a nucleic acid that encodes the light chain (SEQ ID NO:93).

[0251] An exemplary nucleic acid that encodes light chain (SEQ ID NO:93) is GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGGTCA AGCCTCCATCTCTTGCAGATCTAGTCAGATCATTGTACATAGTAATGGAAACA CCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGG ATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTG GGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGTGGACGTTCGGTGGAGG CACCAAGCTGGAAATCAAA (SEQ ID NO: 97). ii. Heavy Chain

[0252] One embodiment provides an anti-B7H4 murine monoclonal antibody or antigen binding fragment thereof that has a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:QVQLQQPGAELVKPGASVKLSCKASGYTFISYWMHWVKQRPGQGLEWIGEIDP SDSYTYYNQKFKGKATLTVDKS S ST AYMQLS SLTSEDS AVYYCARRKTWDWY FD VWGAGTT VT VS S (SEQ ID NO:98).

[0253] The CDRs are of SEQ ID NO:98 are bolded and underlined and are:CDR1 SYWMH (SEQ ID NOV);CDR2 EIDPSDSYTYYNQKFKG (SEQ ID NO: 99); andCDR3 RKTWDWYFDV (SEQ ID NO: 100).

[0254] Another embodiment provides a nucleic acid encoding heavy chain (SEQ ID NO:98).

[0255] An exemplary nucleic acid that encodes heavy chain (SEQ ID NO:98) is CAGGTCCAGCTGCAGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAG TGAAGCTGTCCTGCAAGGCTTCTGGATACACCTTCATTAGCTACTGGATGCAC TGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATCGGAGAGATTGATC CTTCTGATAGTTATACTTACTACAATCAAAAGTTCAAGGGCAAGGCCACATTG ACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACAT CTGAGGACTCTGCGGTCTATTACTGTGCAAGAAGGAAAACCTGGGACTGGTA CTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO:101).

[0256] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain containing CDRs according to SEQ ID Nos: 94, 95, and 96.

[0257] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having a heavy chain containing CDRs according to SEQ ID Nos: 9, 99, and 100.

[0258] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain containing CDRs according to SEQ ID Nos: 94, 95, and 96, and a heavy chain containing CDRs according to SEQ ID Nos: 9, 99, and 100.

[0259] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having a light chain at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:93 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 98. g. B4E11 Sequences

[0260] In one embodiment an anti-B7H4 murine monoclonal antibody is produced by hybridoma clone B4E11 and contains two light chains and two heavy chains. i. Light Chain

[0261] One embodiment provides an anti-B7H4 murine monoclonal antibody or antigen binding fragment thereof that has a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:DIVMTOSHKFMSTSVGDRVTITCKASODVSTAVAWYOOKPGOSPKLLISSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCOQHYSTPTFGGGTKLEIR (SEQ ID NO: 102).

[0262] The CDRs of SEQ ID NO: 102 are bolded and underlined and are:CDR1 KASQDVSTAVA (SEQ ID NO: 103);CDR2 SASYRYT (SEQ ID NO: 104); andCDR3 QQHYSTPT (SEQ ID NO: 105).

[0263] Another embodiment provides a nucleic acid that encodes the light chain (SEQ ID NO: 102).

[0264] An exemplary nucleic acid that encodes light chain (SEQ ID NO: 102) isGACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACA GGGTCACTATCACCTGCAAGGCCAGTCAGGATGTGAGTACTGCTGTAGCCTG GTATCAACAGAAACCAGGACAGTCTCCTAAACTACTGATTTCCTCGGCATCCTACCGGTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGA TTTCACTTTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACT GTCAGCAACATTATAGTACTCCGACGTTCGGTGGAGGCACCAAGCTGGAAAT C AGA (SEQ ID NO: 106). ii. Heavy Chain

[0265] One embodiment provides an anti-B7H4 murine monoclonal antibody or antigen binding fragment thereof that has a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:EVQLQQSGTVLARPGASVKMSCKASGYTFTSYWMHWVKERPGQGLEWIGAIY PGDSDTRYNQKFKGRAKLTAVTSANTAYMELSSLTNDDSAVFYCTCTTAGVL DYWGQGTSVTVSS (SEQ ID NO: 107).

[0266] The CDRs are of SEQ ID NO: 107 are bolded and underlined and are:CDR1 SYWMH (SEQ ID NO: 9);CDR2 AIYPGDSDTRYNQKFKG (SEQ ID NO: 108); andCDR3 TTAGVLDY (SEQ ID NO: 109).

[0267] Another embodiment provides a nucleic acid encoding heavy chain (SEQ ID NO: 107).

[0268] An exemplary nucleic acid that encodes heavy chain (SEQ ID NO: 107) is GAGGTTCAGCTCCAGCAGTCTGGGACTGTGCTGGCAAGGCCTGGGGCTTCAG TGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACCAGCTACTGGATGCAC TGGGTAAAAGAGAGGCCTGGACAGGGTCTGGAATGGATTGGCGCTATTTATC CTGGAGATAGTGATACTAGGTATAATCAGAAGTTCAAGGGCAGGGCCAAACT GACTGCAGTCACATCTGCCAACACTGCCTACATGGAGCTCAGCAGCCTGACA AATGATGACTCTGCGGTCTTCTACTGTACATGTACTACGGCTGGTGTTTTGGA CTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 110).

[0269] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain containing CDRs according to SEQ ID Nos: 103, 104, and 105.

[0270] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having a heavy chain containing CDRs according to SEQ ID Nos: 9, 108, and 109.

[0271] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain containing CDRs according to SEQ ID Nos: 103, 104, and 105, and a heavy chain containing CDRs according to SEQ ID Nos: 9, 108, and 109.

[0272] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having a light chain at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 102 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 107. h. B6C8 Sequences

[0273] In one embodiment an anti-B7H4 murine monoclonal antibody is produced by hybridoma clone B6C8 and contains two light chains and two heavy chains. i. Light Chain

[0274] One embodiment provides an anti-B7H4 murine monoclonal antibody or antigen binding fragment thereof that has a light chain having at least 50%, 60%, 70%,80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:DVVMTQTPLSLPVSLGDQASISCTSSQSIVHGNGNTYLEWYLQKPGQSPKLLIY KVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTK LEIK(SEQ ID NO: 111).

[0275] The CDRs of SEQ ID NO: 111 are bolded and underlined and are:CDR1 TSSQSIVHGNGNTYLE (SEQ ID NO: 112);CDR2 KVSNRFS (SEQ ID NO: 95); andCDR3 FQGSHVPYT (SEQ ID NO: 113).

[0276] Another embodiment provides a nucleic acid that encodes the light chain (SEQ ID NO: 111).

[0277] An exemplary nucleic acid that encodes light chain (SEQ ID NO: 111) is

[0278] GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTG GAGATCAGGCCTCCATCTCTTGCACATCTAGTCAGAGCATTGTACATGGTAAT GGAAACACCTATTTAGAATGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGC TCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTG AGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGTACACGTTC GGAGGGGGGACCAAGCTGGAAATAAAA(SEQ ID NO: 114). ii. Heavy Chain

[0279] One embodiment provides an anti-B7H4 murine monoclonal antibody or antigen binding fragment thereof that has a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:QVQLQQPGAELVKPGASVKLSCKASGYSFTSYWMNWVKQRPGRGLEWIGRIHP SDSETHYNQKFKSKATLTVDKSSSTAYIQLSSLTSEDSAVYFCARYGLFYGNDG YAMDHWGQGTSVTVSS (SEQ ID NO: 115).

[0280] The CDRs of SEQ ID NO: 115 are bolded and underlined and are:CDR1 SYWMN (SEQ ID NO: 116);CDR2 RIHPSDSETHYNQKFKS (SEQ ID NO: 117); andCDR3 YGLFYGNDGYAMDH (SEQ ID NO: 118)

[0281] Another embodiment provides a nucleic acid encoding heavy chain (SEQ ID NO:115).

[0282] An exemplary nucleic acid that encodes heavy chain (SEQ ID NO: 115) is CAGGTCCAACTGCAGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAG TGAAGCTGTCCTGCAAGGCTTCTGGCTACTCTTTCACCAGCTACTGGATGAAC TGGGTGAAGCAGAGGCCTGGACGAGGCCTCGAGTGGATTGGAAGGATTCATC CTTCTGATAGTGAAACTCACTACAATCAAAAGTTCAAGAGCAAGGCCACACT GACTGTAGACAAATCCTCCAGCACAGCCTACATCCAACTCAGCAGCCTGACA TCTGAGGACTCTGCGGTCTATTTTTGTGCAAGATACGGGCTCTTCTATGGTAA CGACGGATATGCTATGGACCACTGGGGTCAAGGAACCTCAG (SEQ ID NO: 119).

[0283] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain containing CDRs according to SEQ ID Nos: 97, 116 and 117.

[0284] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having a heavy chain containing CDRs according to SEQ ID Nos: 118, 119, and 120.

[0285] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain containing CDRs according to SEQ ID Nos 97, 114, and 115, and a heavy chain containing CDRs according to SEQ ID Nos: 118, 119, and 120.

[0286] One embodiment provides an antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having a light chain at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 113 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 117. i. B9H1 Sequences

[0287] In one embodiment an anti-B7H4 murine monoclonal antibody is produced by hybridoma clone B9H1 and contains two light chains and two heavy chains. i. Light Chain

[0288] One embodiment provides an anti-B7H4 murine monoclonal antibody or antigen binding fragment thereof that has a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:DIQMTQTTSSLSASLGDRVTISCRASODISFYLNWYQQKPDGTVKLLIYYTSRLH SGVPSRFSGSGSGTDYSLTISNLEOEDIATYFCOQGNTLPWTFGGGTKLEIK (SEQ ID NO: 120).

[0289] The CDRs of SEQ ID NO: 120 are bolded and underlined and are:CDR1 RASQDISFYLN (SEQ ID NO: 121);CDR2 YTSRLHS (SEQ ID NO: 54); andCDR3 QQGNTLPWT (SEQ ID NO 55).

[0290] Another embodiment provides a nucleic acid that encodes the light chain(SEQ ID NO: 120).

[0291] An exemplary nucleic acid that encodes light chain (SEQ ID NO: 120) isGATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAG AGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGCTTTTATTTAAACTGGT ATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGAT TATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTG CCAACAGGGTAATACACTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAA ATCAAA (SEQ ID NO: 122). ii. Heavy Chain

[0292] One embodiment provides an anti-B7H4 murine monoclonal antibody or antigen binding fragment thereof that has a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:EVQLQQSGPELVKPGASVKMSCKASGYTFTDYYMNWVKQSHGKSLEWIGRVN PSNGGTSYNQKFKGKATLTVDKSLSAAYMQLNSLTSEDSAVYYCARRHNYPD YWGQGTTLTVSS (SEQ ID NO: 123).

[0293] The CDRs of SEQ ID NO: 123 are bolded and underlined and are:CDR1 DYYMN (SEQ ID NO: 66);CDR2 RVNPSNGGTSYNQKFKG (SEQ ID NO: 124); andCDR3 RHNYPDY (SEQ ID NO: 125).

[0294] Another embodiment provides a nucleic acid encoding heavy chain (SEQ IDNO: 125).

[0295] An exemplary nucleic acid that encodes heavy chain (SEQ ID NO: 125) isGAGGTCCAGCTGCAACAGTCTGGACCTGAACTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGTAAGGCTTCTGGATACACATTCACTGACTACTACATGAACTGGGTGAAGCAGAGTCATGGAAAGAGCCTTGAGTGGATTGGACGTGTTAATC CTAGCAATGGTGGTACTAGCTACAACCAGAAGTTCAAGGGCAAGGCCACATT GACAGTAGACAAATCCCTCAGCGCAGCCTATATGCAGCTCAACAGCCTGACA TCTGAGGACTCTGCGGTCTATTACTGTGCAAGAAGGCATAACTACCCTGACTA CTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO: 126).

[0296] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain containing CDRs according to SEQ ID Nos: 54, 55, and 121.

[0297] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having a heavy chain containing CDRs according to SEQ ID Nos: 66, 124, and 125.

[0298] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain containing CDRs according to SEQ ID Nos: 54, 55, and 121, and a heavy chain containing CDRs according to SEQ ID Nos: 66, 124, and 125.

[0299] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having a light chain at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 120 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 123. j. B10D7 Sequences

[0300] In one embodiment an anti-B7H4 murine monoclonal antibody is produced by hybridoma clone B10D7 and contains two light chains and two heavy chains. i. Light Chain

[0301] One embodiment provides an anti-B7H4 monoclonal antibody or antigen binding fragment thereof that has a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:EIQMTQSPSSMSASLGDRITITCOATODIVKSLNWYQOKPGKPPSFLIYYTAQLA EGVPSRFSGSGSGSDYSLTISNLESEDFADYYCLQFYEFPPTFGGGTKLEIK (SEQ ID NO: 13).

[0302] The CDRs of SEQ ID NO: 13 are bolded and underlined and are:CDR1 QATQDIVKSLN (SEQ ID NO: 14);CDR2 YTAQLAE (SEQ ID NO: 15); andCDR3 LQFYEFPPT (SEQ ID NO: 16).

[0303] Another embodiment provides a nucleic acid that encodes the light chain (SEQ ID NO: 13).

[0304] An exemplary nucleic acid that encodes light chain (SEQ ID NO: 13) is

[0305] GAAATCCAGATGACCCAGTCTCCATCCTCTATGTCTGCATCTCTGGGAGACAGAATAACCATCACTTGCCAGGCAACTCAAGACATTGTTAAGAGTTT AAACTGGTATCAACAAAAACCAGGGAAACCCCCTTCATTCCTGATCTATTATA CAGCTCAACTGGCAGAAGGGGTCCCGTCAAGGTTCAGTGGCAGTGGGTCTGG GTCAGACTATTCTCTGACAATCAGCAACCTGGAGTCTGAAGATTTTGCAGACTATTACTGTCTACAGTTTTATGAGTTTCCTCCGACGTTCGGTGGAGGCACCAAG CTGGAAATCAA (SEQ ID NO: 127). ii. Heavy Chain

[0306] One embodiment provides an anti-B7H4 murine monoclonal antibody or antigen binding fragment thereof that has a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:EVQLQQSGTVLARPGASVKMSCKASGYPFTSYWMHWVKQRPGQGLEWIGAIY PGNSDTRYNPNFKGKANLTAVTSATTAYMELSSLTNEESAVYYCTSTWTHYFD YWGQGTTLTVSS (SEQ ID NO: 128).

[0307] The CDRs of SEQ ID NO: 128 are bolded and underlined and are:CDR1 SYWMH (SEQ ID NO: 9);CDR2 AIYPGNSDTRYNPNFKG (SEQ ID NO: 129); andCDR3 TWTHYFDY (SEQ ID NO: 130).

[0308] Another embodiment provides a nucleic acid encoding heavy chain (SEQ ID NO: 128).

[0309] An exemplary nucleic acid that encodes heavy chain (SEQ ID NO: 130) is GAGGTTCAGCTCCAGCAGTCTGGGACTGTGCTGGCAAGGCCTGGGGCTTCAG TGAAGATGTCCTGCAAGGCTTCTGGCTACCCCTTTACCAGCTACTGGATGCAC TGGGTAAAGCAGAGGCCTGGACAGGGTCTGGAATGGATTGGCGCTATTTATCCTGGAAATAGTGATACTAGGTACAACCCGAATTTCAAGGGCAAGGCCAACCT GACTGCAGTCACATCTGCCACCACTGCCTACATGGAGCTCAGCAGCCTGACA AATGAGGAATCTGCGGTCTATTACTGTACAAGTACCTGGACCCACTACTTTGA CTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO: 131).

[0310] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain containing CDRs according to SEQ ID Nos: 14, 15, and 16.

[0311] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having a heavy chain containing CDRs according to SEQ ID Nos: 9, 129, and 130.

[0312] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain containing CDRs according to SEQ ID Nos: 14, 15, and 16 and a heavy chain containing CDRs according to SEQ ID Nos: 9, 129, and 130.

[0313] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having a light chain at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 13 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 128.2. LNCB74 Antibody Sequences

[0314] One embodiment provides LNCB74, a monoclonal antibody comprising two light chains and two heavy chains and specifically binds to and is internalized by B7-H4 expressing cells.

[0315] B7-H4 protein is highly expressed in multiple tumor indications, including breast, ovarian, and endometrial cancers. B7-H4 expression is low and limited in normal healthy human tissues, providing a potential broad therapeutic index for a B7-H4 targeting antibody drug conjugate (ADC).

[0316] LNCB74 is a B7-H4 antibody conjugated to the microtubule disrupting payload monomethyl auristatin E (MMAE) with a drug-to-antibody ratio of 4 (DAR4). The ADC employs a glucuronidase-cleavable, site-specific linkage conjugated to an engineered cysteine in the antibody light chain via LigaChem Biosciences’ ConjuAll™ technology to increase stability in circulation, improve selective release of payload in tumor cells, and reduce payload release in non-tumor cells. LNCB74 incorporates an Fc mutation to minimize binding and uptake of LNCB74 by Fc receptor expressing immune cells. The ConjuAll technology, with its selective cleavage and release within tumor cells, combined with mitigation of off-target uptake via reduced Fc interactions, is proposed to improve the safety profile and therapeutic index of LNCB74 compared to other B7-H4 targeted ADCs.a. Light Chain

[0317] One embodiment provides LNCB74, a monoclonal antibody or antigen binding fragment thereof that has a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:DIQMTQSPSSLSASVGDRVTITCRASODISNYLNWYQQKPGKAPKLLIYYTSRLH SGVPSRFSGSGSGTDYTLTISSLOPEDFATYFCOQGNTLPWTFGOGTKLEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGG GGCVIM (SEQ ID NO: 132).

[0318] The CDRs of SEQ ID NO: 132 are bolded and underlined and are:CDR1 RASQDISNYLN (SEQ ID NO:53);CDR2 YTSRLHS (SEQ ID NO: 54); andCDR3 QQGNTLPWT (SEQ ID NO 55). b. Heavy Chain

[0319] One embodiment provides LNCB74, a monoclonal antibody or antigen binding fragment thereof that has a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRV NPSNGGTNYAQKFOGRVTLTVDTSKSTAYMELSSLRSEDTAVYYCARRHNYA DFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFP AVLQS SGL YSLS S VVTVPS S SLGTQT YICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<C KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG (SEQ ID NO: 133).

[0320] The CDRs of SEQ ID NO: 132 are bolded and underlined and are:CDR1 DYYMN (SEQ ID NO: 66);CDR2 RVNPSNGGTNYAQKFQG (SEQ ID NO: 135; and CDR3 RHNYADF (SEQ ID NO:68).3. Comparator Antibody Sequences a. LNCB74 Sequences

[0321] One embodiment provides a comparator antibody comprising two light chains and two heavy chains. The Fc portion of this comparator is wild-type, unlike LNCB74 which contains a LAL A mutation which functions to reduce Fc-driven immune cell engagement. i. Light Chain

[0322] One embodiment provides a comparator antibody or antigen binding fragment thereof that has a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:EIVMTQSPATLSVSPGERATLSCRASOSVSSNLAWYQQKPGQAPRLLIYGASTRA TGIPARFSGSGSGTEFTLTISSLOSEDFAVYYCOOYHSFPFTFGGGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 134).

[0323] The CDRs of SEQ ID NO: 134 are bolded and underlined and are:CDR1 RASQSVSSNLA (SEQ ID NO: 135);CDR2 GASTRAT (SEQ ID NO: 136); and CDR3 QQYHSFPFT (SEQ ID NO: 137). ii. Heavy Chain

[0324] One embodiment provides a comparator antibody or antigen binding fragment thereof that has a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:QLQLQESGPGLVKPSETLSLTCTVSGGSIKSGSYYWGWIRQPPGKGLEWIGNIYY SGSTYYNPSLRSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGSYPNQFDP WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG (SEQ ID NO: 138).

[0325] The CDRs of SEQ ID NO: 138 are bolded and underlined and are: CDR1 GSIKSGSYYWG (SEQ ID NO: 139);CDR2 NIYYSGSTYYNPSLRS (SEQ ID NO: 140); and CDR3 AREGSYPNQFDP (SEQ ID NO: 141).2. Antibody Compositions

[0326] The disclosed B7-H4-binding molecules can be antibodies or antigen binding fragments thereof. The disclosed antibodies and antigen binding fragments thereof include whole immunoglobulin (i.e., an intact antibody) of any class, fragments thereof, and synthetic proteins containing at least the antigen binding variable domain of an antibody. In some embodiments, the disclosed molecule contains both an antibody light chain as well as at least the variable domain of an antibody heavy chain. In other embodiments, such molecules can further include one or more of the CHi, hinge, CEE, CH3, and CH4 regions of the heavy chain (especially, the CHi and hinge regions, or the CHi, hinge and CEE regions, or the CHi, hinge, CEE and CEE regions). The antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgGi, IgG?, IgGs and IgG4. In some embodiments, the constant domain is a complement fixing constant domain where it is desired that the antibody exhibit cytotoxic activity, and the class is typically IgGi. In other embodiments, where such cytotoxic activity is not desirable, the constant domain can be of the IgG? or IgG4class. The antibody can include sequences from more than one class or isotype, and selecting particular constant domains to optimize desired effector functions is within the ordinary skill in the art.

[0327] The variable domains differ in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen.However, the variability is not usually evenly distributed through the variable domains of antibodies. It is typically concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of the variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies.

[0328] Also disclosed are fragments of antibodies which have bioactivity. The fragments, whether attached to other sequences or not, include insertions, deletions,substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment.

[0329] Techniques can also be adapted for the production of single-chain antibodies specific to B7-H4. Methods for the production of single-chain antibodies are well known to those of skill in the art. A single chain antibody can be created by fusing together the variable domains of the heavy and light chains using a short peptide linker, thereby reconstituting an antigen binding site on a single molecule. Single-chain antibody variable fragments (scFvs) in which the C-terminus of one variable domain is tethered to the N- terminus of the other variable domain via a 15 to 25 amino acid peptide or linker have been developed without significantly disrupting antigen binding or specificity of the binding. The linker is chosen to permit the heavy chain and light chain to bind together in their proper conformational orientation.

[0330] Divalent single-chain variable fragments (di-scFvs) can be engineered by linking two scFvs. This can be done by producing a single peptide chain with two VH and two VL regions, yielding tandem scFvs. ScFvs can also be designed with linker peptides that are too short for the two variable regions to fold together (about five amino acids), forcing scFvs to dimerize. This type is known as diabodies. Diabodies have been shown to have dissociation constants up to 40-fold lower than corresponding scFvs, meaning that they have a much higher affinity to their target. Still shorter linkers (one or two amino acids) lead to the formation of trimers (triabodies or tribodies). Tetrabodies have also been produced. They exhibit an even higher affinity to their targets than diabodies.

[0331] One embodiment provides a monoclonal antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules. Monoclonal antibodies include “chimeric” antibodies in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity.

[0332] On embodiment provides antibodies and antigen binding fragments thereof the specifically bind to human B7-H4.

[0333] One embodiment provides antibodies produced by a hybridoma from the group consisting of B1A1, B1H1, B1H3, B1H10, B2E6, B4B2, B4E11, B6C8, B9H1, and B10D7. a. Antibodies

[0334] The immunomodulatory agent can be an antibody. Suitable antibodies are known in the art or can be prepared by one of skill in the art. Nucleic acid and polypeptide sequences for B7-H4 are known in the art, and exemplary protein sequences are provided above. The sequences can be used, as discussed in more detail below, by one of skill in the art to prepare an antibody or antigen binding fragment thereof specific for B7-H4. The antibody, or antigen binding fragment, therefore, can be an agonist or antagonist of B7-H4 signaling.

[0335] The activity (i.e., agonist or antagonist) of an antibody or antigen binding fragment thereof that is specific for B7-H4, can be determined using functional assays that are known in the art, and include the assays discussed below. Typically the assays include determining if the antibody or antigen binding fragment thereof increases (i.e., agonist) or decreases (i.e., antagonist) signaling through B7-H4. Because B7-H4 signal transduction results in a suppressive immune response, agonizing B7-H4 causes a suppressed or reduced immune response. Antagonizing B7-H4 signaling inhibits the immune suppressive response resulting in an overall increase in immune response.

[0336] In some embodiments, the disclosed antibodies and antigen binding fragments thereof immunospecifically bind to B7-H4. In some embodiments, the antibody binds to an extracellular domain of B7-H4.

[0337] For example, molecules are provided that can immunospecifically bind to B7-H4:(I) arrayed on the surface of a cell (especially a live cell);(II) arrayed on the surface of a cell (especially a live cell) at an endogenous concentration;(III) arrayed on the surface of a live cell, and modulates binding between B7- H4 and a ligand thereof;(IV) arrayed on the surface of a live cell, and reduces or inhibits immune suppression by B7-H4;(V) arrayed on the surface of a live cell, and induces or enhances immune suppression by B7-H4;(VI) arrayed on the surface of a live cell, wherein the cell is a tumor cell;(VII) combinations of I-IV and VI;(VIII) combinations of I-III and V-IV; and(IX) arrayed on the surface of a live myeloid or lymphoid derived cancer cells (AML or ALL) and enhances apoptosis and differentiation resulting in reduced selfrenewal of cancer stem cells.

[0338] In some embodiments, the molecules are capable of inducing antibody dependent cell cytotoxicity (ADCC), complement dependent cytotoxicity (CDC) or cellular apoptosis through other mechanisms, of B7-H4 expressing cell.

[0339] To prepare an antibody or antigen binding fragment thereof that specifically binds to B7-H4, purified proteins, polypeptides, fragments, fusions, or epitopes to B7-H4 or polypeptides expressed from nucleic acid sequences thereof, can be used. The antibodies or antigen binding fragments thereof can be prepared using any suitable methods known in the art such as those discussed in more detail below. i. Human and Humanized Antibodies

[0340] Many non-human antibodies (e.g., those derived from mice, rats, or rabbits) are naturally antigenic in humans, and thus can give rise to undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the methods serves to lessen the chance that an antibody administered to a human will evoke an undesirable immune response.

[0341] Transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production can be employed. For example, it has been described that the homozygous deletion of the antibody heavy chain joining region (J(H)) gene in chimeric, and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ -line mutant mice will result in the production of human antibodies upon antigen challenge.

[0342] Optionally, the antibodies are generated in other species and “humanized” for administration in humans. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab’, F(ab’)2, or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient antibody are replaced byresidues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non- human residues. Humanized antibodies may also contain residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will contain substantially all of at least one, and typically two, variable domains, in which all or substantially all, of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0343] Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule. Humanization can be essentially performed by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, a humanized form of a nonhuman antibody (or a fragment thereof) is a chimeric antibody or fragment, wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0344] The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important in order to reduce antigenicity. According to the “best-fit” method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody. Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies.

[0345] It is further important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, humanized antibodies can be prepared by a process of analysis of the parental sequences and various conceptual humanized products using three dimensional models of the parental and humanized sequences. Three dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the consensus and import sequence so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.

[0346] The antibody can be bound to a substrate or labeled with a detectable moiety or both bound and labeled. The detectable moieties contemplated with the present compositions include fluorescent, enzymatic and radioactive markers. ii. Single-Chain Antibodies

[0347] Methods for the production of single-chain antibodies are well known to those of skill in the art. A single chain antibody is created by fusing together the variable domains of the heavy and light chains using a short peptide linker, thereby reconstituting an antigen binding site on a single molecule. Single-chain antibody variable fragments (scFvs) in which the C-terminus of one variable domain is tethered to the N-terminus of the other variable domain via a 15 to 25 amino acid peptide or linker have been developed without significantly disrupting antigen binding or specificity of the binding. The linker is chosen to permit the heavy chain and light chain to bind together in their proper conformational orientation. These Fvs lack the constant regions (Fc) present in the heavy and light chains of the native antibody. iii. Monovalent Antibodies

[0348] In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly Fab fragments, can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Papain digestion of antibodies typically produces two identicalantigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment, called the F(ab’)2 fragment, which has two antigen combining sites and is still capable of cross-linking antigen.

[0349] The Fab fragments produced in the antibody digestion also contain the constant domains of the light chain and the first constant domain of the heavy chain. Fab’ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain domain including one or more cysteines from the antibody hinge region. The F(ab’)2 fragment is a bivalent fragment comprising two Fab’ fragments linked by a disulfide bridge at the hinge region. Fab’-SH is the designation herein for Fab’ in which the cysteine residue(s) of the constant domains bear a free thiol group. Antibody fragments originally were produced as pairs of Fab’ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known. iv. Hybrid Antibodies

[0350] The antibody can be a hybrid antibody. In hybrid antibodies, one heavy and light chain pair is homologous to that found in an antibody raised against one epitope, while the other heavy and light chain pair is homologous to a pair found in an antibody raised against another epitope. This results in the property of multi-functional valency, i.e., ability to bind at least two different epitopes simultaneously. Such hybrids can be formed by fusion of hybridomas producing the respective component antibodies, or by recombinant techniques. Such hybrids may, of course, also be formed using chimeric chains. v. Conjugates or Fusions of Antibody Fragments

[0351] The targeting function of the antibody can be used therapeutically by coupling the antibody or a fragment thereof with a therapeutic agent. Such coupling of the antibody or fragment (e.g., at least a portion of an immunoglobulin constant region (Fc)) with the therapeutic agent can be achieved by making an immunoconjugate or by making a fusion protein, comprising the antibody or antibody fragment and the therapeutic agent.

[0352] Such coupling of the antibody or fragment with the therapeutic agent can be achieved by making an immunoconjugate or by making a fusion protein, or by linking the antibody or fragment to a nucleic acid such as an siRNA, comprising the antibody or antibody fragment and the therapeutic agent.

[0353] In some embodiments, the antibody is modified to alter its half-life. In some embodiments, it is desirable to increase the half-life of the antibody so that it is present in the circulation or at the site of treatment for longer periods of time. For example, it may be desirable to maintain titers of the antibody in the circulation or in the location to be treated for extended periods of time. Antibodies can be engineered with Fc variants that extend half-life, e.g., using Xtend™ antibody half-life prolongation technology (Xencor, Monrovia, CA). In other embodiments, the half-life of the anti-DNA antibody is decreased to reduce potential side effects. The conjugates disclosed can be used for modifying a given biological response. The drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin. vi. Exemplary B7H4 Antibodies

[0354] Exemplary B7H4 antibodies or antigen binding fragments are disclosed herein. The antibodies can include one or more heavy chain and one or more light chain of mouse anti-human B7H4 antibody B1H1 or B1H10. In some embodiments, the B7H4 antibody includes some or all of the light chain CDRs, the entire light chain variable region, some or all of the heavy chain CDRs, the entire heavy chain variable region, or a combination thereof of any of mouse anti-human B7H4 antibody B1H1 or B1H10. Exemplary combinations are disclosed below.

[0355] An anti-B7H4 antibody or antigen binding fragment thereof having a light chain with an amino acid sequence according to SEQ ID NO:24 and a heavy chain having an amino acid sequence according to SEQ ID NO:38.

[0356] An anti-B7H4 antibody or antigen binding fragment thereof having a light chain with an amino acid sequence according to SEQ ID NO:24 and a heavy chain having an amino acid sequence according to SEQ ID NO:39.

[0357] An anti-B7H4 antibody or antigen binding fragment thereof having a light chain with an amino acid sequence according to SEQ ID NO:24 and a heavy chain having an amino acid sequence according to SEQ ID NO:40.

[0358] An anti-B7H4 antibody or antigen binding fragment thereof having a light chain with an amino acid sequence according to SEQ ID NO:24 and a heavy chain having an amino acid sequence according to SEQ ID NO:41.

[0359] An anti-B7H4 antibody or antigen binding fragment thereof having a light chain with an amino acid sequence according to SEQ ID NO:24 and a heavy chain having an amino acid sequence according to SEQ ID NO:42.

[0360] An anti-B7H4 antibody or antigen binding fragment thereof with a light chain having an amino acid sequence according to SEQ ID NO:24 and a heavy chain having an amino acid sequence according to SEQ ID NO:43.

[0361] An anti-B7H4 antibody or antigen binding fragment thereof with a light chain having an amino acid sequence according to SEQ ID NO:24 and a heavy chain having an amino acid sequence according to SEQ ID NO:44.

[0362] An anti-B7H4 antibody or antigen binding fragment thereof with a light chain having an amino acid sequence according to SEQ ID NO:24 and a heavy chain having an amino acid sequence according to SEQ ID NO:45.

[0363] An anti-B7H4 antibody or antigen binding fragment thereof with a light chain having an amino acid sequence according to SEQ ID NO:25 and a heavy chain having an amino acid sequence according to SEQ ID NO:38.

[0364] An anti-B7H4 antibody or antigen binding fragment thereof with a light chain having an amino acid sequence according to SEQ ID NO:25 and a heavy chain having an amino acid sequence according to SEQ ID NO:39.

[0365] An anti-B7H4 antibody or antigen binding fragment thereof with a light chain having an amino acid sequence according to SEQ ID NO:25 and a heavy chain having an amino acid sequence according to SEQ ID NO:40.

[0366] An anti-B7H4 antibody or antigen binding fragment thereof with a light chain having an amino acid sequence according to SEQ ID NO:25 and a heavy chain having an amino acid sequence according to SEQ ID NO:41.

[0367] An anti-B7H4 antibody or antigen binding fragment thereof with a light chain having an amino acid sequence according to SEQ ID NO:25 and a heavy chain having an amino acid sequence according to SEQ ID NO:42.

[0368] An anti-B7H4 antibody or antigen binding fragment thereof with a light chain having an amino acid sequence according to SEQ ID NO:25 and a heavy chain having an amino acid sequence according to SEQ ID NO:43.

[0369] An anti-B7H4 antibody or antigen binding fragment thereof with a light chain having an amino acid sequence according to SEQ ID NO:25 and a heavy chain having an amino acid sequence according to SEQ ID NO:44.

[0370] An anti-B7H4 antibody or antigen binding fragment thereof with a light chain having an amino acid sequence according to SEQ ID NO:25 and a heavy chain having an amino acid sequence according to SEQ ID NO:45.

[0371] An anti-B7H4 antibody or antigen binding fragment thereof with a light chain having an amino acid sequence according to SEQ ID NO:26 and a heavy chain having an amino acid sequence according to SEQ ID NO:38.

[0372] An anti-B7H4 antibody or antigen binding fragment thereof with a light chain having an amino acid sequence according to SEQ ID NO:26 and a heavy chain having an amino acid sequence according to SEQ ID NO:39.

[0373] An anti-B7H4 antibody or antigen binding fragment thereof with a light chain having an amino acid sequence according to SEQ ID NO:26 and a heavy chain having an amino acid sequence according to SEQ ID NO:40.

[0374] An anti-B7H4 antibody or antigen binding fragment thereof with a light chain having an amino acid sequence according to SEQ ID NO:26 and a heavy chain having an amino acid sequence according to SEQ ID NO:41.

[0375] An anti-B7H4 antibody or antigen binding fragment thereof with a light chain having an amino acid sequence according to SEQ ID NO:26 and a heavy chain having an amino acid sequence according to SEQ ID NO:42.

[0376] An anti-B7H4 antibody or antigen binding fragment thereof with a light chain having an amino acid sequence according to SEQ ID NO:26 and a heavy chain having an amino acid sequence according to SEQ ID NO:43.

[0377] An anti-B7H4 antibody or antigen binding fragment thereof with a light chain having an amino acid sequence according to SEQ ID NO:26 and a heavy chain having an amino acid sequence according to SEQ ID NO:44.

[0378] An anti-B7H4 antibody or antigen binding fragment thereof with a light chain having an amino acid sequence according to SEQ ID NO:26 and a heavy chain having an amino acid sequence according to SEQ ID NO:45.

[0379] An anti-B7H4 antibody or antigen binding fragment thereof with a light chain having an amino acid sequence according to SEQ ID NO:27 and a heavy chain having an amino acid sequence according to SEQ ID NO:38.

[0380] An anti-B7H4 antibody or antigen binding fragment thereof with a light chain having an amino acid sequence according to SEQ ID NO:27 and a heavy chain having an amino acid sequence according to SEQ ID NO:39.

[0381] An anti-B7H4 antibody or antigen binding fragment thereof with a light chain having an amino acid sequence according to SEQ ID NO:27 and a heavy chain having an amino acid sequence according to SEQ ID NO:40.

[0382] An anti-B7H4 antibody or antigen binding fragment thereof with a light chain having an amino acid sequence according to SEQ ID NO:27 and a heavy chain having an amino acid sequence according to SEQ ID NO:41.

[0383] An anti-B7H4 antibody or antigen binding fragment thereof with a light chain having an amino acid sequence according to SEQ ID NO:27 and a heavy chain having an amino acid sequence according to SEQ ID NO:42.

[0384] An anti-B7H4 antibody or antigen binding fragment thereof with a light chain having an amino acid sequence according to SEQ ID NO:27 and a heavy chain having an amino acid sequence according to SEQ ID NO:43.

[0385] An anti-B7H4 antibody or antigen binding fragment thereof with a light chain having an amino acid sequence according to SEQ ID NO:27 and a heavy chain having an amino acid sequence according to SEQ ID NO:44.

[0386] An anti-B7H4 antibody or antigen binding fragment thereof with a light chain having an amino acid sequence according to SEQ ID NO:27 and a heavy chain having an amino acid sequence according to SEQ ID NO:45.

[0387] An anti-B7H4 antibody or antigen binding fragment thereof with a light chain having an amino acid sequence according to SEQ ID NO:28 and a heavy chain having an amino acid sequence according to SEQ ID NO:38.

[0388] An anti-B7H4 antibody or antigen binding fragment thereof with a light chain having an amino acid sequence according to SEQ ID NO:28 and a heavy chain having an amino acid sequence according to SEQ ID NO:39.

[0389] An anti-B7H4 antibody or antigen binding fragment thereof with a light chain having an amino acid sequence according to SEQ ID NO:28 and a heavy chain having an amino acid sequence according to SEQ ID NO:40.

[0390] An anti-B7H4 antibody or antigen binding fragment thereof with a light chain having an amino acid sequence according to SEQ ID NO:28 and a heavy chain having an amino acid sequence according to SEQ ID NO:41.

[0391] An anti-B7H4 antibody or antigen binding fragment thereof with a light chain having an amino acid sequence according to SEQ ID NO:28 and a heavy chain having an amino acid sequence according to SEQ ID NO:42.

[0392] An anti-B7H4 antibody or antigen binding fragment thereof with a light chain having an amino acid sequence according to SEQ ID NO:28 and a heavy chain having an amino acid sequence according to SEQ ID NO:43.

[0393] An anti-B7H4 antibody or antigen binding fragment thereof with a light chain having an amino acid sequence according to SEQ ID NO:28 and a heavy chain having an amino acid sequence according to SEQ ID NO:44.

[0394] An anti-B7H4 antibody or antigen binding fragment thereof with a light chain having an amino acid sequence according to SEQ ID NO:28 and a heavy chain having an amino acid sequence according to SEQ ID NO:45.5. Proteins and Polypeptides a. Protein and Polypeptide Compositions

[0395] The immunomodulatory agent can be a protein, polypeptide, or fusion protein. For example, the immunomodulatory agent can be an isolated or recombinant protein or polypeptide, or functional fragment, variant, or fusion protein thereof of B7- H4.

[0396] The protein or polypeptide, or functional fragment, variant, or fusion protein thereof can be an agonist or an antagonist. For example, in some embodiments an antagonist of B7-H4 is a B7-H4 polypeptide or a fragment or fusion protein thereof that binds to a ligand of B7-H4. The polypeptide can be a soluble fragment, for example the extracellular domain of B7-H4, or a functional fragment thereof, or a fusion protein thereof. In some embodiments, a soluble ligand of B7-H4 may serve as an agonist, increasing signal transduction through B7-H4.

[0397] The activity (i.e., agonist or antagonist) of a protein or polypeptide of B7-H4, or any fragment, variant or fusion protein thereof can be determined using functional assays that are known in the art, and include the assays discussed below. Typically the assays include determining if the protein, polypeptide or fragment, variant or fusion protein thereof increases (i.e., agonist) or decreases (i.e., antagonist) signaling through the B7-H4 receptor. In some embodiments the assay includes determining if the protein, polypeptide or fragment, variant, or fusion protein thereof increases (i.e., agonist) or decreases (i.e., antagonist) the immune response associated with B7-H4. Typically the assays include determining if the protein, polypeptide or fragment, variant, or fusion protein thereof increases (i.e., agonist) or decreases (i.e., antagonist) signaling through B7-H4. In some embodiments the assay includes determining if the protein, polypeptide or fragment, variant, or fusion protein thereof decreases (i.e., agonist) or increases (i.e.,antagonist) an immune response negatively regulated by B7-H4. In some embodiments the assay includes determining if the protein, polypeptide or fragment, variant, or fusion protein thereof increases (i.e., antagonist) the apoptosis and differentiation of acute myeloid leukemia cells and acute lymphoblastic leukemia cells resulting in reduced selfrenewal capacity of AML and ALL stem cells.

[0398] Nucleic acid and polypeptide sequences for B7-H4 are known in the art and exemplary protein and peptide sequences are provided above. The sequences can be used, as discussed in more detail below, by one of skill in the art to prepare any protein or polypeptide of B7-H4, or any fragment, variant, or fusion protein thereof. Generally, the proteins, polypeptides, fragments, variants, and fusions thereof of B7-H4 are expressed from nucleic acids that include sequences that encode a signal sequence. The signal sequence is generally cleaved from the immature polypeptide to produce the mature polypeptide lacking the signal sequence. The signal sequence can be replaced by the signal sequence of another polypeptide using standard molecule biology techniques to affect the expression levels, secretion, solubility, or other property of the polypeptide B7- H4 proteins with and without a signal sequence are disclosed. It is understood that in some cases, the mature protein as it is known or described in the art, i.e., the protein sequence without the signal sequence, is a putative mature protein. During normal cell expression, a signal sequence can be removed by a cellular peptidase to yield a mature protein. The sequence of the mature protein can be determined or confirmed using methods that are known in the art. i. Fragments

[0399] As used herein, a fragment of B7-H4 refers to any subset of the polypeptide that is at least one amino acid shorter than full length protein. Useful fragments include those that retain the ability to bind to their natural ligand or ligands. A polypeptide that is a fragment of any full-length B7-H4 typically has at least 20 percent, 30 percent, 40 percent, 50 percent, 60 percent, 70 percent, 80 percent, 90 percent, 95 percent, 98 percent, 99 percent, 100 percent, or even more than 100 percent of the ability to bind its natural ligand respectively as compared to the full-length protein.

[0400] Fragments of B7-H4 include cell free fragments. Cell free polypeptides can be fragments of full-length, transmembrane, polypeptides that may be shed, secreted or otherwise extracted from the producing cells. Cell free fragments of polypeptides can include some or all of the extracellular domain of the polypeptide and lack some or all of the intracellular and / or transmembrane domains of the full-length protein. In oneembodiment, polypeptide fragments include the entire extracellular domain of the full- length protein. In other embodiments, the cell free fragments of the polypeptides include fragments of the extracellular domain that retain biological activity of full-length protein. The extracellular domain can include 1, 2, 3, 4, or 5 contiguous amino acids from the transmembrane domain, and / or 1, 2, 3, 4, or 5 contiguous amino acids from the signal sequence. Alternatively, the extracellular domain can have 1, 2, 3, 4, 5 or more amino acids removed from the C-terminus, N-terminus, or both. In some embodiments the extracellular domain is the only functional domain of the fragment (e.g., the ligand binding domain). ii. Variants

[0401] Variants of B7-H4, and fragments thereof are also provided. In some embodiments, the variant is at least 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, or 99 percent identical to any one of SEQ ID NO: 1. Useful variants include those that increase biological activity, as indicated by any of the assays described herein, or that increase half-life or stability of the protein. The protein and polypeptides of B7-H4, and fragments, variants, and fusion proteins thereof can be engineered to increase biological activity. For example, in some embodiments, a B7-H4 polypeptide, protein, or fragment, variant or fusion thereof has been modified with at least one amino acid substitution, deletion, or insertion that increases a function thereof.

[0402] Finally, variant polypeptides can be engineered to have an increased half-life relative to wild type. These variants typically are modified to resist enzymatic degradation. Exemplary modifications include modified amino acid residues and modified peptide bonds that resist enzymatic degradation. Various modifications to achieve this are known in the art. The variants can be modified to adjust for effects of affinity for the receptor on the half-life of proteins, polypeptides, fragments, or fusions thereof at serum and endosomal pH. iii. Fusion Proteins

[0403] Fusion polypeptides have a first fusion partner including all or a part of a polypeptide B7-H4 fused to a second polypeptide directly or via a linker peptide sequence that is fused to the second polypeptide. The fusion proteins optionally contain a domain that functions to dimerize or multimerize two or more fusion proteins. The peptide / polypeptide linker domain can either be a separate domain, or alternatively can be contained within one of the other domains (first polypeptide or second polypeptide) of the fusion protein. Similarly, the domain that functions to dimerize or multimerize the fusionproteins can either be a separate domain, or alternatively can be contained within one of the other domains (first polypeptide, second polypeptide or peptide / polypeptide linker domain) of the fusion protein. In one embodiment, the dimerization / multimerization domain and the peptide / polypeptide linker domain are the same.

[0404] Fusion proteins disclosed herein are of formula I:N-R1-R2-R3-C wherein “N” represents the N-terminus of the fusion protein, “C” represents the C- terminus of the fusion protein. In some embodiments, “Ri” is a polypeptide or protein of B7-H4 or fragment or variant thereof, “R2” is an optional peptide / polypeptide linker domain, and “R3” is a second polypeptide. Alternatively, R3 may be a polypeptide or protein of B7-H4, or fragment or variant thereof and Ri may be a second polypeptide. In some embodiments, the B7-H4 polypeptide is the extracellular domain or a fragment thereof such as the Ig-like C2-domain, or the region framed by the cysteines that form a disulfide bond as discussed above.

[0405] Dimerization or multimerization can occur between or among two or more fusion proteins through dimerization or multimerization domains. Alternatively, dimerization or multimerization of fusion proteins can occur by chemical crosslinking. The dimers or multimers that are formed can be homodimeric / homomultimeric or heterodimeric / heteromultimeric.

[0406] In some embodiments, the fusion protein includes the extracellular domain of B7-H4, or a fragment or variant thereof, fused to an Ig Fc region. Recombinant Ig fusion proteins can be prepared by fusing the coding region of the extracellular domain of an extracellular domain or a fragment or variant thereof to the Fc region of human IgGl, IgG2, IgG3 or IgG4 or mouse IgG2a, or other suitable Ig domain, as described previously (Chapoval, et al., Methods Mol. Med., 45:247-255 (2000)). iv. Polypeptide Modifications

[0407] The polypeptides and fusion proteins may be modified by chemical moieties that may be present in polypeptides in a normal cellular environment, for example, phosphorylation, methylation, amidation, sulfation, acylation, glycosylation, sumoylation and ubiquitylation. Fusion proteins may also be modified with a label capable of providing a detectable signal, either directly or indirectly, including, but not limited to, radioisotopes and fluorescent compounds.

[0408] The polypeptides and fusion proteins may also be modified by chemical moieties that are not normally added to polypeptides in a cellular environment. Forexample, the disclosed fusion proteins may also be modified by covalent attachment of polymer chains, including, but not limited to, polyethylene glycol polymer (PEG) chains (i.e., pegylation). Conjugation of macromolecules to PEG has emerged recently as an effective strategy to alter the pharmacokinetic (PK) profiles of a variety of drugs, and thereby to improve their therapeutic potential. PEG conjugation increases retention of drugs in the circulation by protecting against enzymatic digestion, slowing filtration by the kidneys and reducing the generation of neutralizing antibodies. In addition, PEG conjugates can be used to allow multimerization of the fusion proteins.

[0409] Modifications may be introduced into the molecule by reacting targeted amino acid residues of the polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Another modification is cyclization of the protein.

[0410] Examples of chemical derivatives of polypeptides include lysinyl and amino terminal residues derivatized with succinic or other carboxylic acid anhydrides. Derivatization with a cyclic carboxylic anhydride has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate. Carboxyl side groups, aspartyl or glutamyl, may be selectively modified by reaction with carbodiimides (R — N=C=N— R') such as l-cyclohexyl-3-(2-morpholinyl-(4-ethyl)carbodiimide or l-ethyl-3-(4-azonia-4,4- dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residues can be converted to asparaginyl and glutaminyl residues by reaction with ammonia. Fusion proteins may also include one or more D-amino acids that are substituted for one or more L-amino acids. v. Modified Binding Properties

[0411] Binding properties of the proteins, polypeptides, fragments, variants and fusions thereof are relevant to the dose and dose regimen to be administered. In one embodiment the disclosed the proteins, polypeptides, fragments, variants and fusions thereof have binding properties to a B7-H4 ligand that demonstrate a higher term, or higher percentage, of occupancy of a binding site (e.g., on the ligand) relative to other receptor molecules that bind thereto. In other embodiments, the disclosed proteins, polypeptides, fragments, variants and fusions thereof have reduced binding affinity to a B7-H4 ligand relative to wild type protein.

[0412] In some embodiments the proteins, polypeptides, fragments, variants and fusions thereof have a relatively high affinity for B7-H4 ligand and may therefore have a relatively slow off rate. In other embodiments, the proteins polypeptides, fragments, variants and fusions thereof are administered intermittently over a period of days, weeks or months to dampen immune responses which are allowed to recover prior to the next administration, which may serve to alter the immune response without completely turning the immune response on or off and may avoid long term side effects.4. Isolated Nucleic Acid Molecules

[0413] Isolated nucleic acid sequences encoding the proteins, polypeptides, fragments, variants and fusions thereof are disclosed herein. As used herein, “isolated nucleic acid” refers to a nucleic acid that is separated from other nucleic acid molecules that are present in a mammalian genome, including nucleic acids that normally flank one or both sides of the nucleic acid in a mammalian genome. The term “isolated” as used herein with respect to nucleic acids also includes the combination with any non-naturally occurring nucleic acid sequence, since such non-naturally-occurring sequences are not found in nature and do not have immediately contiguous sequences in a naturally- occurring genome.

[0414] An isolated nucleic acid can be, for example, a DNA molecule, provided one of the nucleic acid sequences normally found immediately flanking that DNA molecule in a naturally occurring genome is removed or absent. Thus, an isolated nucleic acid includes, without limitation, a DNA molecule that exists as a separate molecule independent of other sequences (e.g., a chemically synthesized nucleic acid, or a cDNA or genomic DNA fragment produced by PCR or restriction endonuclease treatment), as well as recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, lentivirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include an engineered nucleic acid such as a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid. A nucleic acid existing among hundreds to millions of other nucleic acids within, for example, a cDNA library or a genomic library, or a gel slice containing a genomic DNA restriction digest, is not to be considered an isolated nucleic acid.

[0415] Nucleic acids encoding the proteins, polypeptides, fragments, variants and fusions thereof may be optimized for expression in the expression host of choice. Codons may be substituted with alternative codons encoding the same amino acid to account fordifferences in codon usage between the mammal from which the nucleic acid sequence is derived and the expression host. In this manner, the nucleic acids may be synthesized using expression host-preferred codons.

[0416] Nucleic acids can be in sense or antisense orientation or can be complementary to a reference sequence encoding a polypeptide or protein of B7-H4. Nucleic acids can be DNA, RNA, or nucleic acid analogs. Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone. Such modification can improve, for example, stability, hybridization, or solubility of nucleic acid. Modifications at the base moiety can include deoxyuridine for deoxythymidine, and 5- methyl-2’-deoxycytidine or 5 -bromo-2’ -deoxy cytidine for deoxycytidine. Modifications of the sugar moiety can include modification of the 2’ hydroxyl of the ribose sugar to form 2’-O-methyl or 2’-O-allyl sugars. The deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six membered, morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained. See, for example, Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev. 7: 187- 195; and Hyrup et al. (1996) Bioorgan. Med. Chem. 4:5-23. In addition, the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone.

[0417] Nucleic acids encoding polypeptides can be administered to subjects in need thereof. Nucleic delivery involves introduction of “foreign” nucleic acids into a cell and ultimately, into a live animal. Compositions and methods for delivering nucleic acids to a subject are known in the art (see Understanding Gene Therapy, Lemoine, N.R., ed., BIOS Scientific Publishers, Oxford, 2008).7. Cytotoxic Agents a. Auristatins

[0418] Anti-B7-H4 antibodies may be conjugated to at least one auristatin. Auristatins represent a group of dolastatin analogs that have generally been shown to possess anticancer activity by interfering with microtubule dynamics and GTP hydrolysis, thereby inhibiting cellular division. For example, Auristatin E (U.S. Pat. No. 5,635,483) is a synthetic analogue of the marine natural product dolastatin 10, a compound that inhibits tubulin polymerization by binding to the same site on tubulin as the anticancer drug vincristine (G. R. Pettit, Prog. Chem. Org. Nat. Prod, 70: 1-79 (1997)). Dolastatin 10, auristatin PE, and auristatin E are linear peptides having four amino acids, three ofwhich are unique to the dolastatin class of compounds. Exemplary embodiments of the auristatin subclass of mitotic inhibitors include, but are not limited to, monomethyl auristatin D (MMAD or auristatin D derivative), monomethyl auristatin E (MMAE or auristatin E derivative), monomethyl auristatin F (MMAF or auristatin F derivative), auristatin F phenylenediamine (AFP), auristatin EB (AEB), auristatin EFP (AEFP), and 5-benzoylvaleric acid-AE ester (AEVB). The synthesis and structure of auristatin derivatives are described in U.S. Patent Application Publication Nos. 2003- 0083263, 2005-0238649 and 2005-0009751; International Patent Publication No. WO 04 / 010957, International Patent Publication No. WO 02 / 088172, and U.S. Pat. Nos. 6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,097; 5,521,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444; and 4,486,414, each of which is incorporated by reference herein.

[0419] In one embodiment, anti-B7-H4 antibodies are conjugated to at least one MMAE (mono-methyl auristatin E). Monomethyl auristatin E (MMAE, vedotin) inhibits cell division by blocking the polymerization of tubulin. Because of its super toxicity, it also cannot be used as a drug itself. In recent cancer therapy developments, it is linked to a monoclonal antibody (mAb) that recognizes a specific marker expression in cancer cells and directs MMAE to the cancer cells. In one embodiment, the linker linking MMAE to the anti-B7-H4 antibody is stable in extracellular fluid (i.e., the medium or environment that is external to cells), but is cleaved by cathepsin once the ADC has bound to the specific cancer cell antigen and entered the cancer cell, thus releasing the toxic MMAE and activating the potent anti-mitotic mechanism.

[0420] In one embodiment, the antibody is coupled to a single drug and, therefore, has a DAR of 1. In certain embodiments, the ADC will have a DAR of 2 to 8, or, alternatively, 2 to 4. b. Maytansinoids

[0421] The anti-B7-H4 antibodies may be conjugated to at least one maytansinoid to form an ADC. Maytansinoids are potent antitumor agents that were originally isolated from members of the higher plant families Celastraceae, Rhamnaceae and Euphorbiaceae, as well as some species of mosses (Kupchan et al, J. Am. Chem. Soc. 94: 1354-1356

[1972] ; Wani et al, J. Chem. Soc. Chem. Commun. 390:

[1973] ; Powell et al, J. Nat. Prod. 46:660-666

[1983] ; Sakai et al, J. Nat. Prod. 51 :845-850

[1988] ; and Suwanborirux et al, Experientia 46: 117-120

[1990] ). Evidence suggests that maytansinoids inhibit mitosis byinhibiting polymerization of the microtubule protein tubulin, thereby preventing formation of microtubules (see, e.g., U.S. Pat. No. 6,441,163 and Remillard et al., Science, 189, 1002-1005 (1975)). Maytansinoids have been shown to inhibit tumor cell growth in vitro using cell culture models, and in vivo using laboratory animal systems. Moreover, the cytotoxicity of maytansinoids is 1.000-fold greater than conventional chemotherapeutic agents, such as, for example, methotrexate, daunorubicin, and vincristine (see. e.g., U.S. Pat. No. 5,208,020).

[0422] Maytansinoids to include maytansine, maytansinol, C-3 esters of maytansinol, and other maytansinol analogues and derivatives (see, e.g., U.S. Pat. Nos. 5,208,020 and 6,441,163, each of which is incorporated by reference herein). C-3 esters of maytansinol can be naturally occurring or synthetically derived. Moreover, both naturally occurring and synthetic C-3 maytansinol esters can be classified as a C-3 ester with simple carboxylic acids, or a C-3 ester with derivatives of N-methyl-L-alanine, the latter being more cytotoxic than the former. Synthetic maytansinoid analogues are described in, for example, Kupchan et al., J. Med. Chem., 21, 31-37 (1978).

[0423] Suitable maytansinoids for use in antibody-drug conjugates can be isolated from natural sources, synthetically produced, or semi-synthetically produced. Moreover, the maytansinoid can be modified in any suitable manner, so long as sufficient cytotoxicity is preserved in the ultimate conjugate molecule. In this regard, maytansinoids lack suitable functional groups to which antibodies can be linked. A linking moiety desirably is utilized to link the maytansinoid to the antibody to form the conjugate.

[0424] The structure of an exemplary maytansinoid, 4-methyl-4-mercapto-l- oxopentyl)-maytansine, (DM4), is provided below.

[0425] Representative examples of maytansinoids include, but are not limited, to DM1 (N2'-deacetyl-N2'-(3 -mercapto- l-oxopropyl)-maytansine; also referred to as mertansine, drug maytansinoid 1; ImmunoGen, Inc.; see also Chari et al. (1992) Cancer Res 52 2T), DM2, DM3 (N2-deacetyl-N2-(4-mercapto-l-oxopentyl)-maytansine), DM4 (4-methyl-4-mercapto-l-oxopentyl)-maytansine) and maytansinol (a synthetic maytansinoid analog). Other examples of maytansinoids are described in U.S. Pat. No. 8,142,784, incorporated by reference herein.

[0426] Ansamitocins are a group of maytansinoid antibiotics that have been isolated from various bacterial sources. These compounds have potent antitumor activities. Representative examples include, but are not limited to ansamitocin Pl, ansamitocin P2, ansamitocin P3, and ansamitocin P4. In one embodiment, an anti-B7-H4 antibody isconjugated to at least one DM1. In one embodiment, an anti-B7-H4 antibody is conjugated to at least one DM2. In one embodiment, an anti-B7-H4 antibody is conjugated to at least one DM3. In one embodiment, an anti-B7-H4 antibody is conjugated to at least one DM4. c. DNA Alkylating Agents

[0427] The term “DNA alkylating agent”, as used herein, includes a family of DNA alkylating agents including indolino-benzodiazepines (IGNs). IGNs represent a chemical class of cytotoxic molecules with high in vitro potency (IC.sub.50 values in the low pmol / L range) toward cancer cells. Examples of IGN DNA alkylating agents that can be used as a cytotoxic payload in an ADC are described in Miller et al. (2016) Molecular Cancer Therapeutics, 15(8)). The IGN compounds described in Miller et al. bind to the minor groove of DNA followed by covalent reaction of guanine residues with the two imine functionalities in the molecule resulting in cross-linking of DNA. The structure of an exemplary IGN is provided below. d. Other Drugs for Conjugation

[0428] Examples of drugs that may be used in ADCs, i.e., drugs that may be conjugated to the anti-B7-H4 antibodies, are provided below, and include mitotic inhibitors, antitumor antibiotics, immunomodulating agents, gene therapy vectors, alkylating agents, anti angiogenic agents, antimetabolites, boron-containing agents, chemoprotective agents, hormone agents, glucocorticoids, photoactive therapeutic agents, oligonucleotides, radioactive isotopes, radiosensitizers, topoisomerase inhibitors, tyrosine kinase inhibitors, and combinations thereof. e. Mitotic Inhibitors

[0429] In one aspect, anti-B7-H4 antibodies may be conjugated to one or more mitotic inhibitor(s) to form an ADC for the treatment of cancer. The term “mitotic inhibitor”, as used herein, refers to a cytotoxic and / or therapeutic agent that blocks mitosis or cell division, a biological process particularly important to cancer cells. A mitotic inhibitor disrupts microtubules such that cell division is prevented, often by affecting microtubule polymerization or microtubule depolymerization. Thus, in one embodiment, an anti-B7-H4 antibody is conjugated to one or more mitotic inhibitor(s) that disrupts microtubule formation by inhibiting tubulin polymerization. In one embodiment, the mitotic inhibitor used in the ADCs is IXEMPRA® (ixabepilone). Examples of mitotic inhibitors that may be used in the anti-B7-H4 ADCs include dolastatins, e.g., dolastatin 10 and dolastatin 15, and plant alkaloids, e.g., a taxane andvinca alkaloid, e.g., indesine sulfate, vincristine, vinblastine and vinorelbine. Included in the genus of mitotic inhibitors are auristatins and maytansinoids, described above.

[0430] Anti-B7-H4 antibodies described herein may be conjugated to at least one taxane. The term “taxane” as used herein refers to the class of antineoplastic agents having a mechanism of microtubule action and having a structure that includes the taxane ring structure and a stereospecific side chain that is required for cytostatic activity. Also included within the term “taxane” are a variety of known derivatives, including both hydrophilic derivatives, and hydrophobic derivatives. Taxane derivatives include, but not limited to, galactose and mannose derivatives described in International Patent Application No. WO 99 / 18113; piperazino and other derivatives described in WO 99 / 14209; taxane derivatives described in WO 99 / 09021, WO 98 / 22451, and U.S. Pat. No. 5,869,680; 6-thio derivatives described in WO 98 / 28288; sulfenamide derivatives described in U.S. Pat. No. 5,821,263; and TAXOL™ derivative described in U.S. Pat. No. 5,415,869, each of which is incorporated by reference herein. Taxane compounds have also previously been described in U.S. Pat. Nos. 5,641,803, 5,665,671, 5,380,751, 5,728,687, 5,415,869, 5,407,683, 5,399,363, 5,424,073, 5,157,049, 5,773,464, 5,821,263, 5,840,929, 4,814,470, 5,438,072, 5,403,858, 4,960,790, 5,433,364, 4,942,184, 5,362,831, 5,705,503, and 5,278,324, all of which are expressly incorporated by reference. Further examples of taxanes include, but are not limited to, docetaxel (TAXOTERE®; Sanofi Aventis), paclitaxel (ABRAXANE® or TAXOL™; Abraxis Oncology), and nanoparticle paclitaxel (ABI-007 / ABRAXANE®; Abraxis Bioscience). f. Antitumor Antibiotics

[0431] Anti-B7-H4 antibodies may be conjugated to one or more antitumor antibiotic(s) for the treatment of cancer. As used herein, the term “antitumor antibiotic” means an antineoplastic drug that blocks cell growth by interfering with DNA and is made from a microorganism. Often, antitumor antibiotics either break up DNA strands or slow down or stop DNA synthesis. Examples of antitumor antibiotics that may be included in the anti-B7-H4 ADCs include, but are not limited to, actinomycines (e.g., pyrrolo[2,l-c][l,4]benzodiazepines), anthracyclines, calicheamicins, and duocarmycins. In addition to the foregoing, additional antitumor antibiotics that may be used in the anti- B7-H4 ADCs include bleomycin (BLENOXANE™, Bristol-Myers Squibb), mitomycin, and plicamycin (also known as mithramycin).g. Immunomodulating Agents

[0432] In one aspect, anti-B7-H4 antibodies may be conjugated to at least one immunomodulating agent. As used herein, the term “immunomodulating agent” refers to an agent that can stimulate or modify an immune response. In one embodiment, an immunomodulating agent is an immunostimuator which enhances a subject's immune response. In another embodiment, an immunomodulating agent is an immunosuppressant which prevents or decreases a subject's immune response. An immunomodulating agent may modulate myeloid cells (monocytes, macrophages, dendritic cells, megakaryocytes and granulocytes) or lymphoid cells (T cells, B cells and natural killer (NK) cells) and any further differentiated cell thereof. Representative examples include, but are not limited to, bacillus calmette-guerin (BCG) and levamisole (ERGAMISOL™). Other examples of immunomodulating agents that may be used in the ADCs include, but are not limited to, cancer vaccines, and cytokines.

[0433] As used herein, the term “cancer vaccine” refers to a composition (e.g., a tumor antigen and a cytokine) that elicits a tumor-specific immune response. The response is elicited from the subject's own immune system by administering the cancer vaccine, or, in the case of the instant disclosure, administering an ADC comprising an anti-B7-H4 antibody and a cancer vaccine. In preferred embodiments, the immune response results in the eradication of tumor cells in the body (e.g., primary or metastatic tumor cells). The use of cancer vaccines generally involves the administration of a particular antigen or group of antigens that are, for example, present on the surface a particular cancer cell, or present on the surface of a particular infectious agent shown to facilitate cancer formation. In some embodiments, the use of cancer vaccines is for prophylactic purposes, while in other embodiments, the use is for therapeutic purposes. Non-limiting examples of cancer vaccines that may be used in the anti-B7-H4 ADCs include, recombinant bivalent human papillomavirus (HPV) vaccine types 16 and 18 vaccine (CERVARIX®, GlaxoSmithKline), recombinant quadrivalent human papillomavirus (HPV) types 6, 11, 16, and 18 vaccine (GARDASIL®, Merck & Company), and sipuleucel-T (PROVENGE®, Dendreon). Thus, in one embodiment, the anti-B7-H4 antibody is conjugated to at least one cancer vaccine that is either an immunostimulator or is an immunosuppressant.

[0434] The anti-B7-H4 antibodies may be conjugated to at least one cytokine. The term “cytokine” generally refers to proteins released by one cell population which act on another cell as intercellular mediators. Cytokines directly stimulate immune effector cellsand stromal cells at the tumor site and enhance tumor cell recognition by cytotoxic effector cells (Lee and Margolin (2011) Cancers 3 :3856). Numerous animal tumor model studies have demonstrated that cytokines have broad anti-tumor activity and this has been translated into a number of cytokine-based approaches for cancer therapy (Lee and Margoli, supra). Recent years have seen a number of cytokines, including GM-CSF, IL-7, IL-12, IL-15, IL-18 and IL-21, enter clinical trials for patients with advanced cancer (Lee and Margoli, supra).

[0435] Examples of cytokines that may be used in the ADCs include, but are not limited to, parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor: integrin; thrombopoietin (TPO); nerve growth factors such as NGF; platelet-growth factor; transforming growth factors (TGFs); insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon a, P, and y, colony stimulating factors (CSFs); granulocyte-macrophage-C-SF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-la, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12; tumor necrosis factor; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines. Thus, in one embodiment, the disclosure provides an ADC comprising an anti-B7-H4 antibody described herein and a cytokine.

[0436] The anti-B7-H4 antibodies may be conjugated to at least one colony stimulating factor (CSF). Colony stimulating factors (CSFs) are growth factors that assist the bone marrow in making red blood cells. Because some cancer treatments (e.g., chemotherapy) can affect white blood cells (which help fight infection), colonystimulating factors may be introduced to help support white blood cell levels and strengthen the immune system. Colony-stimulating factors may also be used following a bone marrow transplant to help the new marrow start producing white blood cells. Representative examples of CSFs that may be used in the anti-B7-H4 ADCs include, but are not limited to erythropoietin (Epoetin), filgrastim (NEUPOGEN® (also known as granulocyte colony-stimulating factor (G-CSF); Amgen, Inc.), sargramostim (LEUKINE® (granulocyte-macrophage colony-stimulating factor and GM-CSF);Genzyme Corporation), prom egapoi etin, and Oprelvekin (recombinant IL-11; Pfizer, Inc.). Thus, in one embodiment, an ADC may comprise an anti-B7-H4 antibody described herein and a CSF. h. Alkylating Agents

[0437] The anti-B7-H4 antibodies may be conjugated to one or more alkylating agent(s). Alkylating agents are a class of antineoplastic compounds that attaches an alkyl group to DNA. Examples of alkylating agents that may be used in the ADCs include, but are not limited to, alkyl sulfonates, ethylenimimes, methylamine derivatives, epoxides, nitrogen mustards, nitrosoureas, triazines and hydrazines. i. Antiangiogenic Agents

[0438] In one aspect, the anti-B7-H4 antibodies described herein are conjugated to at least one antiangiogenic agent. Antiangiogenic agents inhibit the growth of new blood vessels. Antiangiogenic agents exert their effects in a variety of ways. In some embodiments, these agents interfere with the ability of a growth factor to reach its target. For example, vascular endothelial growth factor (VEGF) is one of the primary proteins involved in initiating angiogenesis by binding to particular receptors on a cell surface. Thus, certain antiangiogenic agents, which prevent the interaction of VEGF with its cognate receptor, prevent VEGF from initiating angiogenesis. In other embodiments, these agents interfere with intracellular signaling cascades. For example, once a particular receptor on a cell surface has been triggered, a cascade of other chemical signals is initiated to promote the growth of blood vessels. Thus, certain enzymes, for example, some tyrosine kinases, which are known to facilitate intracellular signaling cascades that contribute to, for example, cell proliferation, are targets for cancer treatment. In other embodiments, these agents interfere with intercellular signaling cascades. Yet, in other embodiments, these agents disable specific targets that activate and promote cell growth or by directly interfering with the growth of blood vessel cells. Angiogenesis inhibitory properties have been discovered in more than 300 substances with numerous direct and indirect inhibitory effects.

[0439] Representative examples of antiangiogenic agents that may be used in the ADCs include, but are not limited to, angiostatin, ABX EGF, C 1-1033, PKI-166, EGF vaccine, EKB-569, GW2016, ICR-62, EMD 55900. CP358, PD153035, AG1478, IMC- C225 (ERBITUX®, ZD1839 (IRESSA®), OSI-774. Erlotinib (TARCEVA®), angiostatin, arrestin, endostatin, BAY 12-9566 and w / fluorouracil or doxorubicin, canstatin, carboxyamidotriozole and with paclitaxel, EMD 121974, S-24, vitaxin,dimethylxanthenone acetic acid, IM862, Interleukin- 12, Interleukin-2, NM-3, HuMV833, PTK787, RhuMab, angiozyme (ribozyme), IMC-1C11, Neovastat, marimstat, prinomastat, BMS-275291, COL-3, MM1270, SU101, SU6668, SU11248, SU5416, with paclitaxel, with gemcitabine and cisplatin, and with irinotecan and cisplatin and with radiation, tecogalan, temozolomide and PEG interferon a2b, tetrathiomolybdate, TNP- 470, thalidomide, CC-5013 and with TAXOTERE®, tumstatin, 2-methoxyestradiol, VEGF trap, mTOR inhibitors (deforolimus, everolimus (AFINITOR®, Novartis Pharmaceutical Corporation), and temsirolimus (TORISEL®, Pfizer. Inc.)), tyrosine kinase inhibitors (e.g., erlotinib (TARCEVA®, Genentech, Inc.), imatinib (GLEEVEC®, Novartis Pharmaceutical Corporation), gefitinib (IRESSA®, AstraZeneca Pharmaceuticals), dasatinib (SPRYCEL®, Brystol-Myers Squibb), sunitinib (SUTENT®, Pfizer, Inc.), nilotinib (TASIGNA®, Novartis Pharmaceutical Corporation), lapatinib (TYKERB®, GlaxoSmithKline Pharmaceuticals), sorafenib (NEXAVAR®, Bayer and Onyx), phosphoinositide 3 -kinases (PI3K). j. Antimetabolites

[0440] The anti-B7-H4 antibodies may be conjugated to at least one antimetabolite. Antimetabolites are types of chemotherapy treatments that are very similar to normal substances within the cell. When the cells incorporate an antimetabolite into the cellular metabolism, the result is negative for the cell, e.g., the cell is unable to divide. Antimetabolites are classified according to the substances with which they interfere. Examples of antimetabolies that may be used in the ADCs include, but are not limited to, a folic acid antagonist (e.g., methotrexate), a pyrimidine antagonist (e.g., 5 -Fluorouracil, Foxuridine, Cytarabine, Capecitabine, and Gemcitabine), a purine antagonist (e.g., 6- Mercaptopurine and 6-Thioguanine) and an adenosine deaminase inhibitor (e.g., Cladribine, Fludarabine, Nelarabine and Pentostatin), as described in more detail below. k. Boron-Containing Agents

[0441] The anti-B7-H4 antibody may be conjugated to at least one boron containing agent. Boron-containing agents comprise a class of cancer therapeutic compounds which interfere with cell proliferation. Representative examples of boron containing agents include, but are not limited, to borophycin and bortezomib (VELCADE®, Millenium Pharmaceuticals). l. Chemoprotective Agents

[0442] The anti-B7-H4 antibodies may be conjugated to at least one chemoprotective agent. Chemoprotective drugs are a class of compounds, which helpprotect the body against specific toxic effects of chemotherapy. Chemoprotective agents may be administered with various chemotherapies in order to protect healthy cells from the toxic effects of chemotherapy drugs, while simultaneously allowing the cancer cells to be treated with the administered chemotherapeutic. Representative chemoprotective agents include, but are not limited to amifostine (ETHYOL®, Medimmune, Inc.), which is used to reduce renal toxicity associated with cumulative doses of cisplatin, dexrazoxane (TOTECT®, Apricus Pharma; ZINECARD®), for the treatment of extravasation caused by the administration of anthracycline (TOTECT®), and for the treatment of cardiac- related complications caused by the administration of the antitumor antibiotic doxorubicin (ZINECARD®), and mesna (MESNEX®, Bristol-Myers Squibb), which is used to prevent hemorrhagic cystitis during chemotherapy treatment with ifocfamide. m. Photoactive Therapeutic Agents

[0443] The anti-B7-H4 antibodies may be conjugated to at least one photoactive therapeutic agent. Photoactive therapeutic agents include compounds that can be deployed to kill treated cells upon exposure to electromagnetic radiation of a particular wavelength. Therapeutically relevant compounds absorb electromagnetic radiation at wavelengths which penetrate tissue. In preferred embodiments, the compound is administered in a non-toxic form that is capable of producing a photochemical effect that is toxic to cells or tissue upon sufficient activation. In other preferred embodiments, these compounds are retained by cancerous tissue and are readily cleared from normal tissues. Non-limiting examples include various chromagens and dyes. n. Radionuclide Agents (Radioactive Isotopes)

[0444] The anti-B7-H4 antibodies may be conjugated to at least one radionuclide agent. Radionuclide agents comprise agents that are characterized by an unstable nucleus that is capable of undergoing radioactive decay. The basis for successful radionuclide treatment depends on sufficient concentration and prolonged retention of the radionuclide by the cancer cell. Other factors to consider include the radionuclide half-life, the energy of the emitted particles, and the maximum range that the emitted particle can travel. In preferred embodiments, the therapeutic agent is a radionuclide selected from the group consisting ofn iIn,177Lu,212Bi,213Bi,211At,62Cu,64Cu,67Cu,90Y,125I,1311,32P,33P,47Sc,mAg,67Ga,142Pr,153Sm,161Th,166Dy,166Ho,186Re,188Re,189Re,212Pb,223Ra,225Ac,59Fe,75Se,77As,89Sr, "Mo,105Rh,109Pd,143Pr,149Pm,169Er,194Ir,198Au,199Au, and211Pb. Also preferred are radionuclides that substantially decay with Auger-emitting particles. For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111 1, Sb-119. 1-125, Ho-161, Os- 189m and Ir-192. Decay energies of useful beta-particle-emitting nuclides are preferably Dy-152. At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-21 1, Ac-225, Fr-221, At-217, Bi-213 and Fm-255. Decay energies of useful alpha-particle-emitting radionuclides are preferably 2,000-10,000 keV, more preferably 3,000-8.000 keV, and most preferably 4,000-7,000 keV. Additional potential radioisotopes of use includenC,199Au,57Co,58Co,51Cr,59Fe,75Se,2O1T1,225Ac,76Br,169Yb, and the like. o. Radiosensitizers

[0445] The anti-B7-H4 antibodies may be conjugated to at least one radiosensitizer. The term “radiosensitizer,” as used herein, is defined as a molecule, preferably a low molecular weight molecule, administered to animals in therapeutically effective amounts to increase the sensitivity of the cells to be radiosensitized to electromagnetic radiation and / or to promote the treatment of diseases that are treatable with electromagnetic radiation. Radiosensitizers are agents that make cancer cells more sensitive to radiation therapy, while typically having much less of an effect on normal cells. Thus, the radiosensitizer can be used in combination with a radiolabeled antibody or ADC. The addition of the radiosensitizer can result in enhanced efficacy when compared to treatment with the radiolabeled antibody or antibody fragment alone. Radiosensitizers are described in D. M. Goldberg (ed.), Cancer Therapy with Radiolabeled Antibodies, CRC Press (1995). Examples of radiosensitizers include gemcitabine, 5-fluorouracil, taxane, and cisplatin.

[0446] Radiosensitizers may be activated by electromagnetic radiation of X-rays. Representative examples of X-ray activated radiosensitizers include, but are not limited to, the following: metronidazole, misonidazole, desmethylmisonidazole, pimonidazole, etanidazole, nimorazole, mitomycin C. RSU 1069, SR 4233, E09, RB 6145, nicotinamide, 5-bromodeoxyuridine (BUdR), 5-iododeoxyuridine (lUdR), bromodeoxycytidine, fluorodeoxyuridine (FUdR), hydroxyurea, cisplatin, and therapeutically effective analogs and derivatives of the same. Alternatively, radiosensitizers may be activated using photodynamic therapy (PDT). Representative examples of photodynamic radiosensitizers include, but are not limited to, hematoporphyrin derivatives, Photofrin®, benzoporphyrin derivatives, NPe6, tin etioporphyrin (SnET2), pheoborbide a, bacteriochlorophyll a, naphthalocyanines,phthalocyanines, zinc phthalocyanine, and therapeutically effective analogs and derivatives of the same. p. Topoisomerase Inhibitors

[0447] The anti-B7-H4 antibodies may be conjugated to at least one topoisomerase inhibitor. Topoisomerase inhibitors are chemotherapy agents designed to interfere with the action of topoisomerase enzymes (topoisomerase I and II), which are enzymes that control the changes in DNA structure by catalyzing then breaking and rejoining of the phosphodiester backbone of DNA strands during the normal cell cycle. Representative examples of DNA topoisomerase I inhibitors include, but are not limited to, camptothecins and its derivatives irinotecan (CPT-11, CAMPTOSAR®, Pfizer, Inc.) and topotecan (HYCAMTIN®, GlaxoSmithKline Pharmaceuticals). Representative examples of DNA topoisomerase II inhibitors include, but are not limited to, amsacrine, daunorubicin, doxotrubicin, epipodophyllotoxins, ellipticines, epirubicin, etoposide, razoxane, and teniposide. q. Tyrosine Kinase Inhibitors

[0448] The anti-B7-H4 antibodies may be conjugated to at least one tyrosine kinase inhibitor. Tyrosine kinases are enzymes within the cell that function to attach phosphate groups to the amino acid tyrosine. By blocking the ability of protein tyrosine kinases to function, tumor growth may be inhibited. Examples of tyrosine kinases that may be used on the ADCs include, but are not limited to, Axitinib, Bosutinib, Cediranib, Dasatinib. Erlotinib, Gefitinib, Imatinib. Lapatinib, Lestaurtinib, Nilotinib. Semaxanib, Sunitinib, and Vandetanib. r. Other Agents

[0449] Examples of other agents that may be used in the ADCs include, but are not limited to, abrin (e.g. abrin A chain), alpha toxin. Aleurites fordii proteins, amatoxin, crotin, curcin, dianthin proteins, diptheria toxin (e.g. diphtheria A chain and nonbinding active fragments of diphtheria toxin), deoxyribonuclease (Dnase), gelonin, mitogellin, modeccin A chain. Momordica charantia inhibitor, neomycin, onconase, phenomycin, Phytolaca americana proteins (PAP I, PAPII, and PAP-S), pokeweed antiviral protein. Pseudonomas endotoxin, Pseudomonas exotoxin (e.g. exotoxin A chain (from Pseudomonas aeruginosa)), restrictocin, ricin A chain, ribonuclease (Rnase), Sapaonaria officinalis inhibitor, saporin, alpha-sarcin. Staphylcoccal enterotoxin-A, tetanus toxin, cisplatin, carboplatin, and oxaliplatin (ELOXATIN®, Sanofi Aventis), proteasome inhibitors (e.g. PS-341 [bortezomib or VELCADE®]), HD AC inhibitors (vorinostat (ZOLINZA®, Merck & Company. Inc.)), belinostat,entinostat, mocetinostat, and panobinostat), COX-2 inhibitors, substituted ureas, heat shock protein inhibitors (e.g. Geldanamycin and its numerous analogs), adrenocortical suppressants, and the tricothecenes. (See, for example, WO 93 / 21232). Other agents also include asparaginase (Espar. Lundbeck Inc.), hydroxyurea, levamisole, mitotane (LYSODREN®, Bristol-Myers Squibb), and tretinoin (RENOVA®, Valeant Pharmaceuticals Inc.).

[0450] In one embodiment, the agent is pyrrolobenzodiazepine (PBD). In one embodiment, the agent is a PARP inhibitor, e.g., olaparib, rucaparib, niraparib, or iniparib. In one embodiment, the PARP inhibitor is olaparib. In one embodiment, the PARP inhibitor is rucaparib. In one embodiment, the PARP inhibitor is niraparib. In one embodiment, the PARP inhibitor is iniparib. In one embodiment, the agent is saporin toxin.

[0451] It should be noted that the aforementioned groups of drug moieties that may be used in the anti-B7-H4 ADCs are not exclusive, in that certain examples of drugs may be found in more than one category, e.g., ansamitocins are both mitotic inhibitors and antitumor antibiotics.

[0452] All stereoisomers of the above drug moieties are contemplated for use herein, i.e. any combination of R and S configurations at the chiral carbons of D.

[0453] The above agents (i.e., naked agents not conjugated to an antibody) may also be used in combination therapies with the anti-B7-H4 antibodies described herein. In one embodiment, anti-B7-H4 antibodies or ADCs are used with any of the foregoing agents in a combination therapy to treat cancer, where the agent is administered prior to, at the same time as, or following administration of the anti-B7-H4 antibody or ADC to the subject.8. Linkers

[0454] An anti-B7-H4 ADC comprises an anti-B7-H4 antibody and at least one drug(s), whereby the antibody and the at least one drug are conjugated by a linker. The term “linker,” as used herein, refers to a chemical moiety that may be bifunctional or multifunctional, and is used to attach an antibody to a drug moiety. A linker may include one conjugating component or may include multiple components.

[0455] For example, the linker may include a spacer, which is a moiety that extends the drug linkage to avoid, for example, shielding the active site of the antibody or improving the solubility of the ADC. Other examples of components of linkers include a stretcher unit and an amino acid unit.

[0456] The linker described herein may be cleavable, non-cleavable and hydrophilic or hydrophobic.

[0457] In certain embodiments, the cleavable linker is cleavable under intracellular or extracellular conditions, by which an active agent is released from an antibody construct-active agent conjugate in the intracellular environment.

[0458] The cleavable linker can be cleaved by a cleaving agent present in an intracellular environment (e.g., lysosomes, endosomes, or caveolea). The cleavable linker may be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not being limited to, a lysosomal or endosomal protease. Generally, the peptidyl linker has a length of at least two amino acids or a length of at least three amino acids. Cleaving agents may include cathepsin B, cathepsin D, and plasmin, all of which are known to hydrolyze dipeptide drug derivatives to release an active drug in target cells (e.g., see Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). The most common are peptidyl linkers cleavable by enzymes present in antigen-expressing cells. For example, peptidyl linkers cleavable by thiol-dependent protease cathepsin-B, which is highly expressed in cancer tissue, may be used (e.g., a Phe-Leu or Gly-Phe-Leu-Gly linker). Other examples of these linkers are described in, for example, U.S. Patent No.6, 214, 345. In addition, the peptidyl linker cleavable by an intracellular protease may be, for example, a Val-Cit linker, a Phe-Lys linker (e.g., see U.S. Patent No. 6,214,345, which describes the synthesis of doxorubicin using a Val-Cit linker), or a Vai-Ala linker. The Val-Cit linker or the Val-Ala linker may contain a pentafluorophenyl group and may contain a succinimide group or a maleimide group. Alternatively, the Val-Cit linker or the Val-Ala linker may contain a pentafluorophenyl group, may contain a 4-aminobenzoic acid (PAB A) group and a maleimide group, and may contain a PABA group and a succinimide group.

[0459] In addition, a cleavable linker may be easily hydrolyzed in a pH-sensitive manner, i.e., at certain pH values. Generally, the pH-sensitive linker may be hydrolyzed under acidic conditions. For example, acid-labile linkers that can be hydrolyzed in lysosomes (e.g., hydrazone, semicarbazone, thiosemicarbazone, cis-aconic amides, orthoesters, acetals, and ketals) may be used (e.g., see: U.S. Patent NOs. 5,122,368, 5,824,805, and 5,622,929; and Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67- 123; and Neville et al., 1989, Biol. Chem.264: 14653-14661). These linkers are relatively stable under neutral pH conditions, such as in blood, but are unstable at pH 5.5, which is the approximate pH of lysosomes, or less than pH 5.0. Examples of hydrolysable linkersinclude thioether linkers (e.g., thioethers attached to a therapeutic agent via an acylhydrazone bond) (e.g., see U.S. Patent No.5, 622, 929).

[0460] Also, the linker is cleavable under reducing conditions (e.g., a disulfide linker). For example, various disulfide linkers including N-succinimidyl-5- acetylthioacetate (SATA), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N- succinimidyl-3-(2-pyridyldithio)butyrate (SPDB), and N-succinimidyl-oxycarbonyl- alpha-methyl-alpha-(2-pyridyl-thio)toluene)- (SMPT), and those that can be formed using SPDB and SMPT (e.g., see: Thorpe et al., 1987, Cancer Res.47: 5924-5931; and U.S. Patent No.4, 880, 935).

[0461] In addition, the linker may be a malonate linker (Johnson et al., 1995, Anticancer Res.15: 1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med- Chem. 3(10): 1299-1304), a 3'-N-amide analogue (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1305-12), a P-glucuronide linker (Jeffery et al., 2006, Bioconjug Chem. 17(3):832- 40), or a P-galactoside linker (Kolodych et al., 2017, Eur J Med Chem. Dec 15;142:376- 382).

[0462] The non-cleavable linker may be a maleimidocaproyl linker. The maleimidocaproyl linker may include N-maleimidomethylcyclohexane-1 -carboxylate. The maleimidocaproyl linker may contain a succinimide group. The maleimidocaproyl linker may contain a pentafluorophenyl group. The linker may be a combination of a maleimide group and one or more polyethylene glycol molecules. The linker may be a combination of a maleimidocaproyl group and one or more polyethylene glycol molecules. The linker may be a maleimide-PEG4 linker. The linker may be a combination of a maleimidocaproyl linker containing a succinimide group and one or more polyethylene glycol molecules. The linker may be a combination of a pentafluorophenyl group and a maleimidocaproyl linker containing one or more polyethylene glycol molecules. The linker may contain a maleimide linked to a polyethylene glycol molecule, wherein the polyethylene glycol allows for more linker flexibility or allows longer linkers to be used. The linker may be a (maleimidocaproyl)-(valine-citrulline)-(para- aminobenzyloxycarbonyl) linker.

[0463] In certain embodiments, the linker may be a cleavable linker.

[0464] In certain embodiments, the linker may be a protease cleavable linker, an acid-cleavable linker, a disulfide linker, a self-immolative linker or a self-stabilizing linker, a malonate linker, a maleimidobenzoyl linker, a 3'-N-amide analogue, a P- glucuronide linker, or a P-galactoside linker.

[0465] In certain embodiments, the protease cleavable linker may include a thiolreactive spacer or a dipeptide, and more specifically, the protease cleavable linker may include a thiol -reactive maleimidocaproyl spacer, a valine-citrulline dipeptide, or a p- amino-benzyloxycarbonyl spacer.

[0466] In certain embodiments, the acid-cleavable linker may be a hydrazine linker or a quaternary ammonium linker.

[0467] Exemplary antibody drug conjugates are disclosed in US 10,583,197, US 9,993,568, US 9,951,072, US 9,919,057, US 9,669,107, US 11,413,353, US 11,173,214, US 11,167,040, US 10,980,890, US 10,583,197, US 10,383,949, US 10,273,235, US 10,183,997, and US 10,118,965, the contents of each of which is fully incorporated by reference herein.

[0468] Multiple methods are commonly used for conjugating drugs to antibodies: i) alkylation of reduced interchain cysteine disulfides through an enzymatically non- cleavable maleimido or simple and cleavable disulfide linker, ii) acylation of lysines by cleavable linear amino acids and iii) prenylation of a linker to a CAAX sequence, wherein C is a cysteine residue, A is an aliphatic amino acid, and X is selected from glutamine, glutamate, serine, cysteine, methionine, alanine, and leucine. An exemplary CAAX sequence is CVIM.

[0469] In one aspect, a linker covalently attaches an antibody to a drug moiety. An ADC is prepared using a linker having reactive functionality for binding to the antibody and the drug. For example, a cysteine thiol, or an amine, e.g., N-terminus or amino acid side chain such as lysine, of the antibody may form a bond with a functional group of the linker.

[0470] In one aspect, a linker has a functionality that is capable of reacting with a free cysteine present on an antibody to form a covalent bond. Nonlimiting exemplary such reactive functionalities include maleimide, haloacetamides, a-haloacetyl, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. See, e.g., the conjugation method at page 766 of Klussman, et al (2004), Bioconjugate Chemistry 15(4):765-773.

[0471] In some embodiments, a linker has a functionality that is capable of reacting with an electrophilic group present on an antibody. Exemplary electrophilic groups include, but are not limited to, aldehyde and ketone carbonyl groups. In some embodiments, a heteroatom of the reactive functionality of the linker can react with anelectrophilic group on an antibody and form a covalent bond to an antibody unit. Nonlimiting exemplary such reactive functionalities include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.

[0472] In one aspect, an anti-B7-H4 antibody is conjugated to an auristatin, e.g., MMAE, via a linker comprising maleimidocaproyl (“me”), valine citrulline (val-cit or “vc”). Maleimidocaproyl acts as a linker to the anti-B7-H4 antibody and is not cleavable. Val-cit is a dipeptide that is an amino acid unit of the linker and allows for cleavage of the linker by a protease, specifically the protease cathepsin B. Thus, the val-cit component of the linker provides a means for releasing the auristatin from the ADC upon exposure to the intracellular environment. In one embodiment, within the linker, p- aminobenzylalcohol (PABA) acts as a spacer and is self immolative, allowing for the release of the MMAE.

[0473] In another aspect, an anti-B7-H4 antibody is conjugated to a cytotoxin, via a charged hindered disulfide N-succinimidyl-4-(2-pyridyldithio)butanoate (sSPDB) linker, sSPDB is a cleavable linker that allows the conjugate to be cleaved inside the target cell in the cytosol due to the reducing intracellular environment.

[0474] In another aspect, an anti-B7-H4 antibody is conjugated to a cytotoxin, via a cleavable peptide linker such as D-Ala-L-dpa.

[0475] In another aspect, an anti-B7-H4 antibody is conjugated to an IGN via a cleavable peptide linker such as D-Ala-L-dpa.

[0476] Suitable linkers include, for example, cleavable and non-cleavable linkers. A linker may be a “cleavable linker,” facilitating release of a drug. Nonlimiting exemplary cleavable linkers include acid-labile linkers (e.g., comprising hydrazone), proteasesensitive (e.g., peptidase-sensitive) linkers, photolabile linkers, or disulfide-containing linkers (Chari et al., Cancer Research 52: 127-131 (1992); U.S. Pat. No. 5,208,020). A cleavable linker is typically susceptible to cleavage under intracellular conditions.Suitable cleavable linkers include, for example, a peptide linker cleavable by an intracellular protease, such as lysosomal protease or an endosomal protease. In exemplary embodiments, the linker can be a dipeptide linker, such as a valine-citrulline (val-cit) or a phenylalanine-lysine (phe-lys) linker.

[0477] Linkers are preferably stable extracellularly in a sufficient manner to be therapeutically effective. Before transport or delivery into a cell, the ADC is preferably stable and remains intact, i.e. the antibody remains conjugated to the drug moiety. Linkersthat are stable outside the target cell may be cleaved at some efficacious rate once inside the cell. Thus, an effective linker will: (i) maintain the specific binding properties of the antibody; (ii) allow delivery, e.g., intracellular delivery, of the drug moiety; and (iii) maintain the therapeutic effect, e.g., cytotoxic effect, of a drug moiety.

[0478] In one embodiment, the linker is cleavable under intracellular conditions, such that cleavage of the linker sufficiently releases the drug from the antibody in the intracellular environment to be therapeutically effective. In some embodiments, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at certain pH values. Typically, the pH-sensitive linker is hydrolyzable under acidic conditions. For example, an acid-labile linker that is hydrolyzable in the lysosome (e.g., a hydrazone, semi carb azone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or the like) can be used. (See, e.g., U.S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol.Chem. 264: 14653-14661.) Such linkers are relatively stable under neutral pH conditions, such as those in the blood, but are unstable at below pH 5.5 or 5.0, the approximate pH of the lysosome. In certain embodiments, the hydrolyzable linker is a thioether linker (such as, e.g., a thioether attached to the therapeutic agent via an acylhydrazone bond (see, e.g., U.S. Pat. No. 5,622,929).

[0479] In other embodiments, the linker is cleavable under reducing conditions (e.g., a disulfide linker). A variety of disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-5-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2- pyridyldithio)butyrate) and SMPT (N-succinimidyloxycarbonyl-alpha-methyl-alpha-(2- pyridyl-dithio)toluene), SPDB and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed., Oxford U. Press. 1987. See also U.S. Pat. No. 4,880,935).

[0480] In some embodiments, the linker is cleavable by a cleaving agent, e.g., an enzyme, which is present in the intracellular environment (e.g., within a lysosome or endosome or caveolca). The linker can be, e.g., a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease. In some embodiments, the peptidyl linker is at least two amino acids long or at least three amino acids long. Cleaving agents can include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives resulting inthe release of active drug inside target cells (see. e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). Most typical are peptidyl linkers that are cleavable by enzymes that are present in B7-H4-expressing cells. Examples of such linkers are described, e.g., in U.S. Pat. No. 6,214,345, incorporated herein by reference in its entirety and for all purposes. In a specific embodiment, the peptidyl linker cleavable by an intracellular protease is a Val-Cit linker or a Phe-Lys linker (see, e.g., U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin with the val-cit linker). One advantage of using intracellular proteolytic release of the therapeutic agent is that the agent is typically attenuated when conjugated and the serum stabilities of the conjugates are typically high.

[0481] In other embodiments, the linker is a malonate linker (Johnson et al., 1995, Anticancer Res. 15: 1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med- Chem. 3(10): 1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med- Chem. 3(10): 1305-12).

[0482] In yet other embodiments, the linker unit is not cleavable, and the drug is released, for example, by antibody degradation. See U.S. Publication No. 20050238649 incorporated by reference herein in its entirety. An ADC comprising a non-cleavable linker may be designed such that the ADC remains substantially outside the cell and interacts with certain receptors on a target cell surface such that the binding of the ADC initiates (or prevents) a particular cellular signaling pathway.

[0483] In some embodiments, the linker is substantially hydrophilic linker (e.g., PEG4Mal and sulfo-SPDB). A hydrophilic linker may be used to reduce the extent to which the drug may be pumped out of resistant cancer cells through MDR (multiple drug resistance) or functionally similar transporters.

[0484] In other embodiments, upon cleavage, the linker functions to directly or indirectly inhibit cell growth and / or cell proliferation. For example, in some embodiments, the linker, upon cleavage, can function as an intercalating agent, thereby inhibiting macromolecular biosynthesis (e.g. DNA replication, RNA transcription, and / or protein synthesis).

[0485] In other embodiments, the linker is designed to facilitate bystander killing (the killing of neighboring cells) through diffusion of the linker-drug and / or the drug alone to neighboring cells. In other embodiments, the linker promotes cellular internalization.

[0486] The presence of a sterically hindered disulfide can increase the stability of a particular disulfide bond, enhancing the potency of the ADC. Thus, in one embodiment, the linker includes a sterically hindered disulfide linkage. A sterically hindered disulfide refers to a disulfide bond present within a particular molecular environment, wherein the environment is characterized by a particular spatial arrangement or orientation of atoms, typically within the same molecule or compound, which prevents or at least partially inhibits the reduction of the disulfide bond. Thus, the presence of bulky (or sterically hindering) chemical moieties and / or bulky amino acid side chains proximal to the disulfide bond prevents or at least partially inhibits the disulfide bond from potential interactions that would result in the reduction of the disulfide bond.

[0487] Notably, the aforementioned linker types are not mutually exclusive. For example, in one embodiment, the linker used in the anti-B7-H4 ADCs described herein is a non-cleavable linker that promotes cellular internalization.

[0488] In some embodiments, the ADC has the following formula (formula I):Ab-(L-D)n(I)

[0489] or a pharmaceutically acceptable salt or solvate thereof; wherein Ab is the antibody, e.g., anti-B7-H4 antibody, and (L-D) is a Linker-Drug moiety. The Linker-Drug moiety is made of L- which is a Linker, and -D, which is a drug moiety having, for example, cytostatic, cytotoxic, or otherwise therapeutic activity against a target cell, e.g., a cell expressing B7-H4; and n is an integer from 1 to 20.

[0490] In some embodiments, n ranges from 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or is 1.

[0491] In some embodiments, the -D moieties are the same. In yet another embodiment, the -D moieties are different.

[0492] In some embodiments, a linker component comprises an “amino acid unit.” In some such embodiments, the amino acid unit allows for cleavage of the linker by a protease, thereby facilitating release of the drug from the immunoconjugate upon exposure to intracellular proteases, such as lysosomal enzymes (Doronina et al. (2003) Nat. Biotechnol. 21 :778-784). Exemplary amino acid units include, but are not limited to, dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptides include, but are not limited to, valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala- phe); phenylalanine-lysine (fk or phe-lys); phenylalanine-homolysine (phe-homolys); andN-methyl-valine-citrulline (Me-val-cit). Exemplary tripeptides include, but are not limited to, glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). An amino acid unit may comprise amino acid residues that occur naturally and / or minor amino acids and / or non-naturally occurring amino acid analogs, such as citrulline Amino acid units can be designed and optimized for enzymatic cleavage by a particular enzyme, for example, a tumor-associated protease, cathepsin B, C and D, or a plasmin protease.

[0493] In one embodiment, the amino acid unit is valine-citrulline (vc or val-cit). In another aspect, the amino acid unit is phenylalanine-lysine (i.e., fk). In yet another aspect of the amino acid unit, the amino acid unit is N-methylvaline-citrulline. In yet another aspect, the amino acid unit is 5 -aminovaleric acid, homo phenylalanine lysine, tetraisoquinolinecarboxylate lysine, cyclohexylalanine lysine, isonepecotic acid lysine, beta-alanine lysine, glycine serine valine glutamine and isonepecotic acid.

[0494] Another approach for the generation of ADCs involves the use of heterobifunctional cross-linkers which link the anti-B7-H4 antibody to the drug moiety. Examples of cross-linkers that may be used include N-succinimidyl 4-(5-nitro-2- pyridyldithio)-pentanoate or the highly water-soluble analog N-sulfosuccinimidyl 4-(5- nitro-2-pyridyldithio)-pentanoate. N-succinimidyl-4-(2 -pyridyldithio) butyrate (SPDB), N-succinimidyl-4-(5-nitro-2-pyridyldithio) butyrate (SNPB), and N-sulfosuccinimidyl-4- (5-nitro-2-pyridyldithio) butyrate (SSNPB), N-succinimidyl-4-methyl-4-(5-nitro-2- pyridyldithio)pentanoate (SMNP), N-succinimidyl -4-(5-N,N-dimethylcarboxamido-2- pyridyldithio) butyrate (SCPB) or N-sulfosuccinimidyl4-(5-N,N-dimethylcarboxamido-2- pyridyldithio) butyrate (SSCPB)). The antibodies may be modified with the cross-linkers N-succinimidyl 4-(5-nitro-2-pyridyldithio)-pentanoate, N-sulfosuccinimidyl 4-(5-nitro-2- pyridyldithio)-pentanoate, SPDB, SNPB, SSNPB, SMNP, SCPB, or SSCPB can then react with a small excess of a particular drug that contains a thiol moiety to give excellent yields of an ADC (see also U.S. Pat. No. 6,913,748, incorporated by reference herein).

[0495] In one embodiment, charged linkers (also referred to as pro-charged linkers) are used to conjugate anti-B7-H4 antibodies to drugs to form ADCs. Charged linkers include linkers that become charged after cell processing. The presence of a charged group(s) in the linker of a particular ADC or on the drug after cellular processing provides several advantages, such as (i) greater water solubility of the ADC, (ii) ability to operate at a higher concentration in aqueous solutions, (iii) ability to link a greater number of drug molecules per antibody, potentially resulting in higher potency, (iv) potential for the charged conjugate species to be retained inside the target cell, resultingin higher potency, and (v) improved sensitivity of multidrug resistant cells, which would be unable to export the charged drug species from the cell. Examples of some suitable charged or pro-charged cross-linkers and their synthesis are shown in FIGS. 1 to 10 of U.S. Pat. No. 8,236,319, and are incorporated by reference herein. Preferably, the charged or pro-charged cross-linkers are those containing sulfonate, phosphate, carboxyl or quaternary amine substituents that significantly increase the solubility of the ADCs, especially for ADCs with 2 to 20 conjugated drugs. Conjugates prepared from linkers containing a pro-charged moiety would produce one or more charged moieties after the conjugate is metabolized in a cell.

[0496] Additional examples of linkers that can be used with the compositions and methods include valine-citrulline; maleimidocaproyl; amino benzoic acids; p- aminobenzylcarbamoyl (PAB); lysosomal enzyme-cleavable linkers; maleimidocaproyl- polyethylene glycol (MC(PEG)6-OH); N-methyl-valine citrulline; N-succinimidyl 4-(N- mal eimidom ethyl )cy cl ohexane-1 -carboxylate (SMCC); N-Succinimidyl 4-(2- pyridyldithiojbutanoate (SPDB); and N-Succinimidyl 4-(2-pyridylthio)pentanoate (SPP) (See also US 2011 / 0076232). Another linker for use includes an avidin-biotin linkage to provide an avidin-biotin-containing ADC (See also U.S. Pat. No. 4,676,980, PCT publication Nos. WO 1992 / 022332 A2, WO 1994 / 016729A1, WO 1995 / 015770A 1, WO 1997 / 031655 A2, WO 1998 / 035704A1, WO 1999 / 019500A1, WO2001 / 09785 A2, WO200 1 / 090198 Al, W02003 / 093793A2, W02004 / 050016A2, W02005 / 081898A2, W02006 / 083562A2, W02006 / 089668A1, W02007 / 150020A1, WO2008 / 135237A1, WO2010 / 111198A1, WO2011 / 057216A1, WO2011 / 058321 Al, WO2012 / 027494 Al, and EP77671B1), wherein some such linkers are resistant to biotinidase cleavage. Additional linkers that may be used include a cohesin / dockerin pair to provide a cohesion-dockerin- containing ADC (See PCT publication Nos. W02008 / 097866A2, W02008 / 097870A2, W02008 / 103947A2, and W02008 / 103953A2).

[0497] Additional linkers may contain non-peptide polymers (examples include, but are not limited to, polyethylene glycol, polypropylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, PLA (poly(lactic acid)), PLGA (poly(lactic acid-glycolic acid)), and combinations thereof, wherein a preferred polymer is polyethylene glycol) (See also PCT publication No.WO201 1 / 000370). Additional linkers are also described in WO 2004-010957, U.S. Publication No. 20060074008, U.S. Publication No. 20050238649, and U.S. Publication No. 20060024317, each of which is incorporated by reference herein in its entirety).

[0498] For an ADC comprising a maytansinoid, many positions on maytansinoids can serve as the position to chemically link the linking moiety. In one embodiment, maytansinoids comprise a linking moiety that contains a reactive chemical group are C-3 esters of maytansinol and its analogs where the linking moiety contains a disulfide bond, and the chemical reactive group comprises a N-succinimidyl or N-sulfosuccinimidyl ester. For example, the C-3 position having a hydroxyl group, the C-14 position modified with hydroxymethyl, the C-15 position modified with hydroxy and the C-20 position having a hydroxy group are all useful. The linking moiety most preferably is linked to the C-3 position of maytansinol.

[0499] The conjugation of the drug to the antibody via a linker can be accomplished by any technique known in the art. A number of different reactions are available for covalent attachment of drugs and linkers to antibodies. This may be accomplished by reaction of the amino acid residues of the antibody, including the amine groups of lysine, the free carboxylic acid groups of glutamic and aspartic acid, the sulfhydryl groups of cysteine and the various moieties of the aromatic amino acids. One of the most commonly used non-specific methods of covalent attachment is the carbodiimide reaction to link a carboxy (or amino) group of a compound to amino (or carboxy) groups of the antibody. Additionally, bifunctional agents such as dialdehydes or imidoesters have been used to link the amino group of a compound to amino groups of an antibody. Also available for attachment of drugs to antibodies is the Schiff base reaction. This method involves the periodate oxidation of a drug that contains glycol or hydroxy groups, thus forming an aldehyde which is then reacted with the binding agent. Attachment occurs via formation of a Schiff base with amino groups of the antibody. Isothiocyanates can also be used as coupling agents for covalently attaching drugs to antibodies. Other techniques are known to the skilled artisan and within the scope of the disclosure.

[0500] In certain embodiments, an intermediate, which is the precursor of the linker, is reacted with the drug under appropriate conditions. In certain embodiments, reactive groups are used on the drug or the intermediate. The product of the reaction between the drug and the intermediate, or the derivatized drug, is subsequently reacted with the anti- B7-H4 antibody under appropriate conditions. The synthesis and structure of exemplary linkers, stretcher units, amino acid units, self-immolative spacer units are described in U.S. Patent Application Publication Nos. 20030083263, 20050238649 and 20050009751, each if which is incorporated herein by reference.

[0501] Stability of the ADC may be measured by standard analytical techniques such as mass spectroscopy, HPLC, and the separation / analysis technique LC / MS.

[0502] In one aspect, the antibody drug conjugate has a structure represented by Formula la or pharmaceutically acceptable salt thereof:Formula la whereinAb is an anti-B7-H4 antibody comprising a light chain having an amino acid sequence according to any one of SEQ ID NOs: 62, 63, or 64, and a heavy chain having an amino acid sequence according to any one of SEQ ID NOs: 75, 76, 77, 78, or 79, each instance of W is -C(O)-, -C(O)NR'-, -C(O)O-, -SO2NR'-, -P(O)R"NR'-, -SONR'-, - PO2NR’-, or -NR’C(O)-; each instance of R1and R" is independently hydrogen, C1-8 alkyl, C3-8 cycloalkyl, C1-8 alkoxy, C1-8 alkylthio, mono- or di-Ci-8 alkylamino, heteroaryl or aryl; each instance of Z is independently C1-8 alkyl, halogen, cyano, or nitro; each instance of nl and n2 is independently an integer from 1 to 10, preferably 1 to 4; each instance of n3 is an integer from 0 to 3, preferably 0; each instance of B is independently a therapeutically active substance (e.g., a drug or diagnostic agent), preferably a drug; andY is an alkylene or heteroalkylene, preferably a C1-50 alkylene or C1-50 heteroalkylene, comprising one or more of the following:(i) one or more unsaturated bonds;(ii) a heteroarylene (e.g., a heteroarylene in the alkylene or heteroalkylene chain);(iii) at least one C1-20 alkyl sunstituent; and(iv) at least one isoprenyl group having a structure represented by Formula II:Formula II wherein n4 is an integer from 1 to 20, preferably 1 to 4.

[0503] In certain embodiments, Ab is an anti-B7-H4 antibody comprising a light chain having an amino acid sequence according to any one of SEQ ID NO: 63, and a heavy chain having an amino acid sequence according to any one of SEQ ID NO: 78.

[0504] In certain embodiments, W is -C(O)NR'-, further wherein the C is directly bonded to the phenyl ring of Formula la, and NR' is bonded to Y.

[0505] In certain embodiments, Y comprises a peptide and the peptide comprises at least one hydrophilic amino acid, preferably an amino acid having a side chain having a moiety that bears a charge at neutral pH in aqueous solution (e.g., an amine, guanidine, or carboxyl moiety), most preferably each amino acid of the peptide is independently selected from alanine, aspartate, asparagine, glutamate, glutamine, glycine, lysine, ornithine, proline, serine, and threonine.

[0506] In certain embodiments, Y is covalently bonded to the antibody by a thioether bond, and the thioether bond comprises a sulfur atom of a cysteine of the antibody.

[0507] In certain embodiments, Y comprises an oxime and: the oxygen atom of the oxime is on the side of Y that is linked to W and the carbon atom of the oxime is on the side of Y that is linked to Ab; or the carbon atom of the oxime is on the side of Y that is linked to W and the oxygen atom of the oxime is on the side of Y that is linked to Ab.

[0508] In certain embodiments, Y comprises a connection unit represented by Formula III or Formula IV:-(CH2)r(V(CH2)P)q-Formula III,-(CH2CH2X)W-Formula IV;V is a single bond, -O-, -S-, -NR1-, -C(O)NR2-, -NR3C(O)-, -NR4SO2-, or -SO2NR5-, preferably -O-;X is -O-, Ci-8 alkylene, or -NR1-, preferably -O-;R1to R5are each independently hydrogen, C1-6 alkyl, C1-6 alkyl C6-20 aryl, or C1-6 alkyl C3-20 heteroaryl; r is an integer from 1 to 10, preferably 2; p is an integer from 0 to 12, preferably 2; q is an integer from 1 to 20, preferably 2, 5, or 11; and w is an integer from 1 to 20, preferably 6 to 20.

[0509] In certain embodiments, Y comprises -(CH2CH2O)w-, -O(CH2CH2O)W-, or - (CH2CH2O)WCH2-, wherein w is an integer from 1 to 20, preferably 2 to 10.

[0510] In certain embodiments, Y comprises a moiety represented by Formula V,VI, VII, VIII or IX:Formula V,Formula VI,Formula VII,Formula VIIIFormula IXLi is a single bond or C1-30 alkylene; and R11is hydrogen or C1-10 alkyl.

[0511] In certain embodiments, Y is linear. As used herein, the term “linear”, when used in the context of variable Y, refers to a unit that couples the antibody to a single therapeutically active agent (e.g., a drug or diagnostic agent) via an unbranched covalent moiety (e.g., a via C1-50 alkylene). The term “linear”, when used in the context of variable Y, does not preclude substitution (e.g., alkyl, aryl, or heteroaryls) on Y, provided that said substituents are not therapeutically active agents or coupled to further active agents.

[0512] In other embodiments, Y is branched. As used herein, the term “branched”, when used in the context of variable Y, refers to a unit that couples the antibody to multiple therapeutically active agents (e.g., drugs and / or diagnostic agents) via a covalent moiety. For example, Y may comprise an alkylene that splits at a branching point into multiple alkylene chains, each of which covalently links the antibody to one or more therapeutically active agents (e.g., drugs).

[0513] In certain embodiments, Y comprises: i) a branching unit covalently coupled to Ab by a primary linker; ii) a first branch, which couples a first therapeutically active substance, via a first cleavage group, to the branching unit; and iiia) a second branch, which couples a second therapeutically active substance, via a second cleavage group, to the branching unit; or iiib) a second branch which couples an alkyl or heteroalkyl (e.g., a polyethylene glycol monomer or a polyethylene glycol oligomer) to the branching unit.

[0514] In certain embodiments, at least one branching unit has a structure representedR30is hydrogen or C1-30 alkyl;R40is hydrogen or L5-COOR50;R50is hydrogen or C1-30 alkyl; andL2, L3, L4, and L5are each independently a bond or alkylene.

[0515] In certain embodiments, the branching unit is a nitrogen atom. In other embodiments, the branching unit is an amide and the primary linker comprises the carbonyl of the amide. In yet other embodiments, the branching unit is an amide and the secondary linker comprises the carbonyl of the amide. In certain preferred embodiments, the branching unit is lysine.

[0516] In certain embodiments wherein Y is covalently bonded to the antibody by a thioether bond, the antibody comprises an amino acid motif recognizable by an isoprenoid transferase at the C-terminus of the antibody, and the thioether bond comprises a sulfur atom of a cysteine of the amino acid motif.

[0517] In certain embodiments, the isoprenoid transferase is famesyl protein transferase (FTase) or geranylgeranyl transferase (GGTase).

[0518] In certain embodiments, the amino acid motif has a CY1Y1X sequence, further wherein:C is cysteine; each Yi independently is an aliphatic amino acid;X is selected from glutamine, glutamate, serine, cysteine, methionine, alanine, and leucine; and the thioether bond comprises a sulfur atom of a cysteine of the amino acid motif.

[0519] In certain embodiments, each Yi is independently selected from alanine, isoleucine, leucine, methionine, and valine.

[0520] In certain embodiments, the amino acid motif comprises a CVIM or CVLL.

[0521] In certain embodiments, the conjugate comprises at least one of 1 to 20 amino acids between the antibody and the amino acid motif, and at least one of the amino acids is glycine.

[0522] In certain embodiments, the amino acid motif has the sequence GGGGGGGCVIM. In certain embodiments, the antibody conjugate comprises a structure represented bywherein MMAE is monomethyl auri statin E; represents a connection point to Y.

[0523] In one embodiment, glucuronide-based linker connects drug units to an antibody in which its glucuronide unit comprises a glycosidase recognition site that is cleavable by an enzyme having P-glucuronidase activity thereby releasing free drug. Glucuronide-based linkers improve the solubility of ADCs and exhibit sufficient serum stability to provide targeted delivery of a conjugated drug to a targeted cell.9. Vectors and Host Cells

[0524] Vectors encoding the proteins, polypeptides, fragments, variants and fusions thereof are also provided. Nucleic acids, such as those described above, can be inserted into vectors for expression in cells. As used herein, a “vector” is a replicon, such as a plasmid, phage, virus or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. Vectors can be expression vectors. An “expression vector” is a vector that includes one or more expression control sequences, and an “expression control sequence” is a DNA sequence that controls and regulates the transcription and / or translation of another DNA sequence.

[0525] Nucleic acids in vectors can be operably linked to one or more expression control sequences. As used herein, “operably linked” means incorporated into a genetic construct so that expression control sequences effectively control expression of a coding sequence of interest. Examples of expression control sequences include promoters, enhancers, and transcription terminating regions. A promoter is an expression control sequence composed of a region of a DNA molecule, typically within 100 nucleotides upstream of the point at which transcription starts (generally near the initiation site for RNA polymerase II). To bring a coding sequence under the control of a promoter, it isnecessary to position the translation initiation site of the translational reading frame of the polypeptide between one and about fifty nucleotides downstream of the promoter.Enhancers provide expression specificity in terms of time, location, and level. Unlike promoters, enhancers can function when located at various distances from the transcription site. An enhancer also can be located downstream from the transcription initiation site. A coding sequence is “operably linked” and “under the control” of expression control sequences in a cell when RNA polymerase is able to transcribe the coding sequence into mRNA, which then can be translated into the protein encoded by the coding sequence.

[0526] Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, tobacco mosaic virus, herpes viruses, cytomegalo virus, retroviruses, vaccinia viruses, adenoviruses, and adeno- associated viruses. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA), and Invitrogen Life Technologies (Carlsbad, CA).

[0527] An expression vector can include a tag sequence. Tag sequences, are typically expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino terminus. Examples of useful tags include, but are not limited to, green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, Flag™ tag (Kodak, New Haven, CT), maltose E binding protein and protein A. In one embodiment, a nucleic acid molecule encoding one of the disclosed polypeptides is present in a vector containing nucleic acids that encode one or more domains of an Ig heavy chain constant region, for example, having an amino acid sequence corresponding to the hinge, CH2 and CH3 regions of a human immunoglobulin Cyl chain.

[0528] Vectors containing nucleic acids to be expressed can be transferred into host cells. The term “host cell” is intended to include prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced. As used herein, “transformed” and “transfected” encompass the introduction of a nucleic acid molecule (e.g., a vector) into a cell by one of a number of techniques. Although not limited to a particular technique, a number of these techniques are well established within the art. Prokaryotic cells can be transformed with nucleic acids by, for example, electroporation or calcium chloride mediated transformation. Nucleic acids can be transfected into mammalian cells by techniques including, for example, calcium phosphate co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, or microinjection. Host cells (e.g., a prokaryotic cell or a eukaryotic cell such as a CHO cell) can be used to, for example, produce the proteins, polypeptides, fragments, variants and fusions thereof described herein.

[0529] The vectors described can be used to express the proteins, polypeptides, fragments, variants and fusions thereof in cells. An exemplary vector includes, but is not limited to, an adenoviral vector. One approach includes nucleic acid transfer into primary cells in culture followed by autologous transplantation of the ex vivo transformed cells into the host, either systemically or into a particular organ or tissue. Ex vivo methods can include, for example, the steps of harvesting cells from a subject, culturing the cells, transducing them with an expression vector, and maintaining the cells under conditions suitable for expression of the encoded polypeptides. These methods are known in the art of molecular biology. The transduction step can be accomplished by any standard means used for ex vivo gene therapy, including, for example, calcium phosphate, lipofection, electroporation, viral infection, and biolistic gene transfer. Alternatively, liposomes or polymeric microparticles can be used. Cells that have been successfully transduced then can be selected, for example, for expression of the coding sequence or of a drug resistance gene. The cells then can be lethally irradiated (if desired) and injected or implanted into the subject. In one embodiment, expression vectors containing nucleic acids encoding fusion proteins are transfected into cells that are administered to a subject in need thereof.

[0530] In vivo nucleic acid therapy can be accomplished by direct transfer of a functionally active DNA into mammalian somatic tissue or organ in vivo. For example, nucleic acids encoding polypeptides disclosed herein can be administered directly to lymphoid tissues. Alternatively, lymphoid tissue specific targeting can be achieved using lymphoid tissue-specific transcriptional regulatory elements (TREs) such as a B lymphocyte-, T lymphocyte-, or dendritic cell-specific TRE. Lymphoid tissue specific TREs are known in the art.

[0531] Nucleic acids may also be administered in vivo by viral means. Nucleic acid molecules encoding fusion proteins may be packaged into retrovirus vectors using packaging cell lines that produce replication-defective retroviruses, as is well-known in the art. Other virus vectors may also be used, including recombinant adenoviruses and vaccinia virus, which can be rendered non-replicating. In addition to naked DNA or RNA, or viral vectors, engineered bacteria may be used as vectors.

[0532] Nucleic acids may also be delivered by other carriers, including liposomes, polymeric micro- and nanoparticles and polycations such as asialoglycoprotein / polylysine.

[0533] In addition to virus- and carrier-mediated gene transfer in vivo, physical means well-known in the art can be used for direct transfer of DNA, including administration of plasmid DNA and particle-bombardment mediated gene transfer.10. Small Molecules

[0534] The immunomodulatory agent can be a small molecule. Small molecules agonists and antagonists B7-H4 are known in the art or can be identified using routine screening methods.

[0535] In some embodiments, screening assays can include random screening of large libraries of test compounds. Alternatively, the assays may be used to focus on particular classes of compounds suspected of modulating the level of B7-H4. Assays can include determinations of B7-H4 signaling activity, or inhibitory response mediated by B7-H4. Other assays can include determinations of nucleic acid transcription or translation, mRNA levels, mRNA stability, mRNA degradation, transcription rates, and translation rates.B. Pharmaceutical Compositions

[0536] Pharmaceutical compositions including the disclosed immunomodulatory agents are provided. Pharmaceutical compositions containing the immunomodulatory agent can be for administration by parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), transdermal (either passively or using iontophoresis or electroporation), or transmucosal (nasal, vaginal, rectal, or sublingual) routes of administration or using bioerodible inserts and can be formulated in dosage forms appropriate for each route of administration.

[0537] In some in vivo approaches, the compositions disclosed herein are administered to a subject in a therapeutically effective amount. As used herein the term “effective amount” or “therapeutically effective amount” means a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of the disorder being treated or to otherwise provide a desired pharmacologic and / or physiologic effect. The precise dosage will vary according to a variety of factors such as subject-dependent variables (e.g., age, immune system health, etc.), the disease, and the treatment being effected.

[0538] For the disclosed immunomodulatory agents, as further studies are conducted, information will emerge regarding appropriate dosage levels for treatment ofI l lvarious conditions in various patients, and the ordinary skilled worker, considering the therapeutic context, age, and general health of the recipient, will be able to ascertain proper dosing. The selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment desired. For the disclosed immunomodulatory agents, generally dosage levels of 0.001 to 20 mg / kg of body weight daily are administered to mammals. Generally, for intravenous injection or infusion, dosage may be lower.

[0539] In certain embodiments, the immunomodulatory agent is administered locally, for example by injection directly into a site to be treated. Typically, the injection causes an increased localized concentration of the immunomodulatory agent composition which is greater than that which can be achieved by systemic administration. The immunomodulatory agent compositions can be combined with a matrix as described above to assist in creating an increased localized concentration of the polypeptide compositions by reducing the passive diffusion of the polypeptides out of the site to be treated.1. Formulations for Parenteral Administration

[0540] In some embodiments, compositions disclosed herein, including those containing peptides and polypeptides, are administered in an aqueous solution, by parenteral injection. The formulation may also be in the form of a suspension or emulsion. In general, pharmaceutical compositions are provided including effective amounts of a peptide or polypeptide, and optionally include pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers. Such compositions optionally include one or more for the following: diluents, sterile water, buffered saline of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; and additives such as detergents and solubilizing agents (e.g., TWEEN 20 (polysorbate-20), TWEEN 80 (polysorbate-80)), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and com oil, gelatin, and injectable organic esters such as ethyl oleate. The formulations may be lyophilized and redissolved / resuspended immediately before use. The formulation may be sterilized by, for example, filtration through a bacteria retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions.2. Formulations for Oral Administration

[0541] In embodiments the compositions are formulated for oral delivery. Oral solid dosage forms are described generally in Remington's Pharmaceutical Sciences, 18th Ed. 1990 (Mack Publishing Co. Easton Pa. 18042) at Chapter 89. Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets, pellets, powders, or granules or incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the disclosed. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712 which are herein incorporated by reference. The compositions may be prepared in liquid form, or may be in dried powder (e.g., lyophilized) form. Liposomal or proteinoid encapsulation may be used to formulate the compositions. Liposomal encapsulation may be used, and the liposomes may be derivatized with various polymers (e.g., U.S. Patent No. 5,013,556). See also Marshall, K. In: Modern Pharmaceutics Edited by G. S. Banker and C. T. Rhodes Chapter 10, 1979. In general, the formulation will include the peptide (or chemically modified forms thereof) and inert ingredients which protect peptide in the stomach environment, and release of the biologically active material in the intestine.

[0542] The agents can be chemically modified so that oral delivery of the derivative is efficacious. Generally, the chemical modification contemplated is the attachment of at least one moiety to the component molecule itself, where the moiety permits uptake into the blood stream from the stomach or intestine, or uptake directly into the intestinal mucosa. Also desired is an increase in overall stability of the component or components and increase in circulation time in the body. PEGylation is an exemplary chemical modification for pharmaceutical usage. Other moieties that may be used include: propylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, polyproline, poly-1, 3- dioxolane and poly-1, 3, 6-tioxocane [see, e.g., Abuchowski and Davis (1981) "Soluble Polymer-Enzyme Adducts," in Enzymes as Drugs. Hocenberg and Roberts, eds. (Wiley - Interscience: New York, N.Y.) pp. 367-383; and Newmark, et al. (1982) J. Appl. Biochem. 4: 185-189],

[0543] Another embodiment provides liquid dosage forms for oral administration, including pharmaceutically acceptable emulsions, solutions, suspensions, and syrups, which may contain other components including inert diluents; adjuvants such as wettingagents, emulsifying and suspending agents; and sweetening, flavoring, and perfuming agents.

[0544] Controlled release oral formulations may be desirable. The agent can be incorporated into an inert matrix which permits release by either diffusion or leaching mechanisms, e.g., gums. Slowly degenerating matrices may also be incorporated into the formulation. Another form of a controlled release is based on the Oros therapeutic system (Alza Corp.), i.e., the drug is enclosed in a semipermeable membrane which allows water to enter and push drug out through a single small opening due to osmotic effects.

[0545] For oral formulations, the location of release may be the stomach, the small intestine (the duodenum, the jejunum, or the ileum), or the large intestine. In some embodiments, the release will avoid the deleterious effects of the stomach environment, either by protection of the agent (or derivative) or by release of the agent (or derivative) beyond the stomach environment, such as in the intestine. To ensure full gastric resistance a coating impermeable to at least pH 5.0 is essential. Examples of the more common inert ingredients that are used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D™, Aquateric™, cellulose acetate phthalate (CAP), Eudragit L™, Eudragit S™, and Shellac™. These coatings may be used as mixed films.3. Formulations for Topical Administration

[0546] The disclosed immunomodulatory agents can be applied topically. Topical administration does not work well for most peptide formulations, although it can be effective especially if applied to the lungs, nasal, oral (sublingual, buccal), vaginal, or rectal mucosa.

[0547] Compositions can be delivered to the lungs while inhaling and traverse across the lung epithelial lining to the blood stream when delivered either as an aerosol or spray dried particles having an aerodynamic diameter of less than about 5 microns.

[0548] A wide range of mechanical devices designed for pulmonary delivery of therapeutic products can be used, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. Some specific examples of commercially available devices are the Ultravent nebulizer (Mallinckrodt Inc., St. Louis, Mo.); the Acorn II nebulizer (Marquest Medical Products, Englewood, Colo.); the Ventolin metered dose inhaler (Glaxo Inc., Research Triangle Park, N.C.); and the Spinhaler powder inhaler (Fisons Corp., Bedford, Mass.). Nektar,Alkermes and Mannkind all have inhalable insulin powder preparations approved or in clinical trials where the technology could be applied to the formulations described herein.

[0549] Formulations for administration to the mucosa will typically be spray dried drug particles, which may be incorporated into a tablet, gel, capsule, suspension or emulsion. Standard pharmaceutical excipients are available from any formulator.

[0550] Transdermal formulations may also be prepared. These will typically be ointments, lotions, sprays, or patches, all of which can be prepared using standard technology. Transdermal formulations may require the inclusion of penetration enhancers.4. Controlled Delivery Polymeric Matrices

[0551] The immunomodulatory agents disclosed herein can also be administered in controlled release formulations. Controlled release polymeric devices can be made for long term release systemically following implantation of a polymeric device (rod, cylinder, film, disk) or injection (microparticles). The matrix can be in the form of microparticles such as microspheres, where the agent is dispersed within a solid polymeric matrix or microcapsules, where the core is of a different material than the polymeric shell, and the peptide is dispersed or suspended in the core, which may be liquid or solid in nature. Unless specifically defined herein, microparticles, microspheres, and microcapsules are used interchangeably. Alternatively, the polymer may be cast as a thin slab or film, ranging from nanometers to four centimeters, a powder produced by grinding or other standard techniques, or even a gel such as a hydrogel.

[0552] Either non-biodegradable or biodegradable matrices can be used for delivery of fusion polypeptides or nucleic acids encoding the fusion polypeptides, although in some embodiments biodegradable matrices are preferred. These may be natural or synthetic polymers, although synthetic polymers are preferred in some embodiments due to the better characterization of degradation and release profiles. The polymer is selected based on the period over which release is desired. In some cases linear release may be most useful, although in others a pulse release or “bulk release” may provide more effective results. The polymer may be in the form of a hydrogel (typically absorbing up to about 90% by weight of water) and can optionally be crosslinked with multivalent ions or polymers.

[0553] The matrices can be formed by solvent evaporation, spray drying, solvent extraction and other methods known to those skilled in the art. Bioerodible microspheres can be prepared using any of the methods developed for making microspheres for drugdelivery, for example, as described by Mathiowitz and Langer, J. Controlled Release, 5: 13-22 (1987); Mathiowitz, et al., Reactive Polymers, 6:275-283 (1987); and Mathiowitz, et al., J. Appl. Polymer Sci., 35:755-774 (1988).

[0554] The devices can be formulated for local release to treat the area of implantation or injection - which will typically deliver a dosage that is much less than the dosage for treatment of an entire body - or systemic delivery. These can be implanted or injected subcutaneously, into the muscle, fat, or swallowed.III. Methods of ManufactureA. Methods of Making Antibodies

[0555] The antibodies can be generated in cell culture, in phage, or in various animals, including but not limited to cows, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, apes. Therefore, in one embodiment, an antibody is a mammalian antibody. Phage techniques can be used to isolate an initial antibody or to generate variants with altered specificity or avidity characteristics. Such techniques are routine and well known in the art. In one embodiment, the antibody is produced by recombinant means known in the art. For example, a recombinant antibody can be produced by transfecting a host cell with a vector comprising a DNA sequence encoding the antibody. One or more vectors can be used to transfect the DNA sequence expressing at least one VL and one VH region in the host cell. Exemplary descriptions of recombinant means of antibody generation and production include Delves, Antibody Production: Essential Techniques (Wiley, 1997); Shephard, et al., Monoclonal Antibodies (Oxford University Press, 2000); Goding, Monoclonal Antibodies: Principles and Practice (Academic Press, 1993); Current Protocols in Immunology (John Wiley & Sons, most recent edition).

[0556] The disclosed antibodies can be modified by recombinant means to increase greater efficacy of the antibody in mediating the desired function. Thus, it is within the scope of the invention that antibodies can be modified by substitutions using recombinant means. Typically, the substitutions will be conservative substitutions. For example, at least one amino acid in the constant region of the antibody can be replaced with a different residue. See, e.g., U.S. Pat. No. 5,624,821, U.S. Pat. No. 6,194,551, Application No. WO 9958572; and Angal, et al., Mol. Immunol. 30: 105-08 (1993). The modification in amino acids includes deletions, additions, and substitutions of amino acids. In some cases, such changes are made to reduce undesired activities, e.g., complement-dependent cytotoxicity. Frequently, the antibodies are labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in both scientific and patent literature. These antibodies can be screened for binding to proteins, polypeptides, or fusion proteins of B7-H4. See, e.g., Antibody Engineering: A Practical Approach (Oxford University Press, 1996).

[0557] For example, suitable antibodies with the desired biologic activities can be identified using in vitro assays including but not limited to: proliferation, migration, adhesion, soft agar growth, angiogenesis, cell-cell communication, apoptosis, transport, signal transduction, and in vivo assays such as the inhibition of tumor growth. The antibodies provided herein can also be useful in diagnostic applications. As capture or non-neutralizing antibodies, they can be screened for the ability to bind to the specific antigen without inhibiting the receptor-binding or biological activity of the antigen. As neutralizing antibodies, the antibodies can be useful in competitive binding assays.

[0558] Antibodies that can be used in the disclosed compositions and methods include whole immunoglobulin (i.e., an intact antibody) of any class, fragments thereof, and synthetic proteins containing at least the antigen binding variable domain of an antibody. The variable domains differ in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not usually evenly distributed through the variable domains of antibodies. It is typically concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of the variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies.

[0559] Also disclosed are fragments of antibodies which have bioactivity. The fragments, whether attached to other sequences or not, include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment.

[0560] Techniques can also be adapted for the production of single-chain antibodies specific to an antigenic peptide. Methods for the production of single-chain antibodies are well known to those of skill in the art. A single chain antibody can be created by fusing together the variable domains of the heavy and light chains using a short peptide linker, thereby reconstituting an antigen binding site on a single molecule. Single-chain antibody variable fragments (scFvs) in which the C-terminus of one variable domain is tethered to the N-terminus of the other variable domain via a 15 to 25 amino acid peptide or linker have been developed without significantly disrupting antigen binding or specificity of the binding. The linker is chosen to permit the heavy chain and light chain to bind together in their proper conformational orientation.

[0561] Divalent single-chain variable fragments (di-scFvs) can be engineered by linking two scFvs. This can be done by producing a single peptide chain with two VH and two VL regions, yielding tandem scFvs. ScFvs can also be designed with linker peptides that are too short for the two variable regions to fold together (about five amino acids), forcing scFvs to dimerize. This type is known as diabodies. Diabodies have been shown to have dissociation constants up to 40-fold lower than corresponding scFvs, meaning that they have a much higher affinity to their target. Still shorter linkers (one or two amino acids) lead to the formation of trimers (triabodies or tribodies). Tetrabodies have also been produced. They exhibit an even higher affinity to their targets than diabodies.

[0562] A monoclonal antibody is obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules. Monoclonal antibodies include “chimeric” antibodies in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity.

[0563] Monoclonal antibodies can be made using any procedure which produces monoclonal antibodies. In a hybridoma method, a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.

[0564] Antibodies may also be made by recombinant DNA methods. DNA encoding the disclosed antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques.

[0565] Methods of making antibodies using protein chemistry are also known in the art. One method of producing proteins comprising the antibodies is to link two or more peptides or polypeptides together by protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert - butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc., Foster City, CA). One skilled in the art can readily appreciate that a peptide or polypeptide corresponding to the antibody, for example, can be synthesized by standard chemical reactions. For example, a peptide or polypeptide can be synthesized and not cleaved from its synthesis resin whereas the other fragment of an antibody can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group which is functionally blocked on the other fragment. By peptide condensation reactions, these two fragments can be covalently joined via a peptide bond at their carboxyl and amino termini, respectively, to form an antibody, or fragment thereof. Alternatively, the peptide or polypeptide is independently synthesized in vivo as described above. Once isolated, these independent peptides or polypeptides may be linked to form an antibody or antigen binding fragment thereof via similar peptide condensation reactions.

[0566] For example, enzymatic ligation of cloned or synthetic peptide segments allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides or whole protein domains. Alternatively, native chemical ligation of synthetic peptides can be utilized to synthetically construct large peptides or polypeptides from shorter peptide fragments. This method includes a two-step chemical reaction. The first step is the chemoselective reaction of an unprotected synthetic peptide-alpha- thioester with another unprotected peptide segment containing an amino-terminal Cys residue to give a thioester-linked intermediate as the initial covalent product. Without a change in the reaction conditions, this intermediate undergoes spontaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site.B. Methods for Producing Proteins

[0567] The disclosed proteins, polypeptides, fragments, variants and fusions thereof can be manufactured using conventional techniques that are known in the art. Isolated fusion proteins can be obtained by, for example, chemical synthesis or by recombinant production in a host cell. To recombinantly produce a protein, polypeptide, fragment, variant or fusion thereof, a nucleic acid containing a nucleotide sequence encoding the protein, polypeptide, fragment, variant or fusion thereof can be used to transform, transduce, or transfect a bacterial or eukaryotic host cell (e.g., an insect, yeast, or mammalian cell). In general, nucleic acid constructs include a regulatory sequence operably linked to a nucleotide sequence encoding the protein, polypeptide, fragment, variant or fusion thereof. Regulatory sequences (also referred to herein as expression control sequences) typically do not encode a gene product, but instead affect the expression of the nucleic acid sequences to which they are operably linked.

[0568] Useful prokaryotic and eukaryotic systems for expressing and producing polypeptides are well known in the art include, for example, Escherichia coli strains such as BL-21, and cultured mammalian cells such as CHO cells.

[0569] In eukaryotic host cells, a number of viral -based expression systems can be utilized to express fusion proteins. Viral based expression systems are well known in the art and include, but are not limited to, baculoviral, SV40, retroviral, or vaccinia based viral vectors.

[0570] Mammalian cell lines that stably express proteins, polypeptides, fragments, variants or fusions thereof, can be produced using expression vectors with appropriate control elements and a selectable marker. For example, the eukaryotic expression vectors pCR3.1 (Invitrogen Life Technologies) and p91023(B) (see Wong et al. (1985) Science 228:810-815) are suitable for expression of proteins, polypeptides, fragments, variants or fusions thereof, in, for example, Chinese hamster ovary (CHO) cells, COS-1 cells, human embryonic kidney 293 cells, NIH3T3 cells, BHK21 cells, MDCK cells, and human vascular endothelial cells (HUVEC). Additional suitable expression systems include the GS Gene Expression System™ available through Lonza Group Ltd.

[0571] Following introduction of an expression vector by electroporation, lipofection, calcium phosphate, or calcium chloride co-precipitation, DEAE dextran, or other suitable transfection method, stable cell lines can be selected (e.g., by metabolic selection, or antibiotic resistance to G418, kanamycin, or hygromycin). The transfected cells can be cultured such that the polypeptide of interest is expressed, and the polypeptide can be recovered from, for example, the cell culture supernatant or fromlysed cells. Alternatively, a protein, polypeptide, fragment, variant or fusion thereof, can be produced by (a) ligating amplified sequences into a mammalian expression vector such as pcDNA3 (Invitrogen Life Technologies), and (b) transcribing and translating in vitro using wheat germ extract or rabbit reticulocyte lysate.

[0572] Proteins, polypeptides, fragments, variants or fusions thereof, can be isolated using, for example, chromatographic methods such as affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, DEAE ion exchange, gel filtration, and hydroxylapatite chromatography. In some embodiments, Proteins, polypeptides, fragments, variants or fusions thereof can be engineered to contain an additional domain containing amino acid sequence that allows the polypeptides to be captured onto an affinity matrix. For example, an Fc-fusion polypeptide in a cell culture supernatant or a cytoplasmic extract can be isolated using a protein A column. In addition, a tag such as c-myc, hemagglutinin, polyhistidine, or Flag™ (Kodak) can be used to aid polypeptide purification. Such tags can be inserted anywhere within the polypeptide, including at either the carboxyl or amino terminus. Other fusions that can be useful include enzymes that aid in the detection of the polypeptide, such as alkaline phosphatase. Immunoaffinity chromatography also can be used to purify polypeptides. Fusion proteins can additionally be engineered to contain a secretory signal (if there is not a secretory signal already present) that causes the Proteins, polypeptides, fragments, variants or fusions thereof to be secreted by the cells in which it is produced. The secreted Proteins, polypeptides, fragments, variants or fusions thereof can then conveniently be isolated from the cell media.C. Methods for Producing Isolated Nucleic Acid Molecules

[0573] Isolated nucleic acid molecules can be produced by standard techniques, including, without limitation, common molecular cloning and chemical nucleic acid synthesis techniques. For example, polymerase chain reaction (PCR) techniques can be used to obtain an isolated nucleic acid encoding a variant polypeptide. PCR is a technique in which target nucleic acids are enzymatically amplified. Typically, sequence information from the ends of the region of interest or beyond can be employed to design oligonucleotide primers that are identical in sequence to opposite strands of the template to be amplified. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Primers typically are 14 to 40 nucleotides in length but can range from 10 nucleotides to hundreds of nucleotides in length. General PCR techniques are described, for example in PCRPrimer: A Laboratory Manual, ed. by Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995. When using RNA as a source of template, reverse transcriptase can be used to synthesize a complementary DNA (cDNA) strand. Ligase chain reaction, strand displacement amplification, self-sustained sequence replication or nucleic acid sequence-based amplification also can be used to obtain isolated nucleic acids. See, for example, Lewis (1992) Genetic Engineering News 12: 1; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87: 1874-1878; and Weiss (1991) Science 254: 1292-1293.

[0574] Isolated nucleic acids can be chemically synthesized, either as a single nucleic acid molecule or as a series of oligonucleotides (e.g., using phosphoramidite technology for automated DNA synthesis in the 3’ to 5’ direction). For example, one or more pairs of long oligonucleotides (e.g., >100 nucleotides) can be synthesized that contain the desired sequence, with each pair containing a short segment of complementarity (e.g., about 15 nucleotides) such that a duplex is formed when the oligonucleotide pair is annealed. DNA polymerase can be used to extend the oligonucleotides, resulting in a single, double-stranded nucleic acid molecule per oligonucleotide pair, which then can be ligated into a vector. Isolated nucleic acids can also obtained by mutagenesis. Protein-encoding nucleic acids can be mutated using standard techniques, including oligonucleotide-directed mutagenesis and / or site-directed mutagenesis through PCR. See, Short Protocols in Molecular Biology. Chapter 8, Green Publishing Associates and John Wiley & Sons, edited by Ausubel et al, 1992.IV. Assays and Antibody Screening

[0575] Assays for antibody screening include:1. Analysis of binding affinity of B7-H4-Fc to ligands in comparison to B7-H4.2. Functional assays to confirm B7-H4-Fc prevents signaling by B7- H4 expressing cells. Reporter cells may be utilized for these assays, or primary B7-H4+ cells are another option.

[0576] B7-H4 deficient (“knockout) mice or wild type mice can be utilized for the generation of high affinity mAbs against B7-H4 using proprietary immunization techniques. Autoimmune prone mice NZB / WF1 can be used to generate mAbs to overcome “tolerance”.1. Phase I screening: mAb binding to cell lines transfected to express cell surface B7-H4. Additionally, mAbs should have the capacity to bind endogenously expressed B7-H4 on the surface of primary human cell subsets.These mAbs should be highly specific for B7-H4. Antibodies can be screened using ELISA with purified B7-H4 protein to detect ant-B7-H4 antibodies.2. Phase II screening: B7-H4 specific mAbs should block the binding of B7-H4 to its ligands and / or target cells.3. Phase III screening: Functional assays to confirm that B7-H4 mAbs or combination of mAbs modulate B7-H4 signaling. These assays will utilize cell lines that express endogenous B7-H4, or primary cells such as human monocytes, macrophages and dendritic cell subsets to assess function in the presence of B7-H4 mAbs. Additionally, reporter cells lines may be used to determine if signaling pathways such as NFkB (NFkB reporter) or NF AT (NF AT reporter) are altered following culture with B7-H4 mAbs.4. Phase IV screening: Functional assays to determine if B7-H4 mAbs are capable of inducing antibody dependent cell cytotoxicity (ADCC), complement dependent cytotoxicity (CDC) or cellular apoptosis through other mechanisms, of B7-H4 expressing cell lines. In particular, B7-H4 mAbs will be tested for the ability to deplete through one of these methods leukemia cell lines, known to express B7-H4 on the cell surface. B7-H4 mAbs may also be engineered to deplete B7-H4 expressing cells and tested as described later in this document through known methods.5. Phase V screening: Functional assays to determine if B7-H4 mAbs are capable of delivering or inducing a negative signal (agonist) via B7-H4 into B7-H4 expressing cells to inhibit cellular function. Cell lines that endogenously express B7-H4, or transfectants of cell lines will be assessed for changes in phenotype and survival following culture with B7-H4 mAbs. In other assays, reporter cell lines will be used to determine in B7-H4 mAbs modulate positive signaling pathways such as NF-kB (NF-kB reporter) or other known cell signaling reporters. Induction of apoptosis in cell lines will be also be evaluated.

[0577] Phase II and III assays can be used to predict the concentrations of B7-H4 mAb(s) required to block physiological levels of ligands in vivo.V. Method of Use

[0578] Antagonists or agonists of B7-H4 can be used to modulate immune responses in subjects in need of such treatment.

[0579] Exemplary methods are discussed in more detail below.A. Immune Response Stimulation1. Therapeutic Strategies

[0580] Methods of inducing or enhancing an immune response in a subject are provided. Typically, the methods include administering a subject an effective amount of immunomodulatory agent, or cells primed ex vivo with the immunomodulatory agent. The immune response can be, for example, a primary immune response to an antigen or an increase effector cell function such as increasing antigen-specific proliferation of T cells, enhancing cytokine production by T cells, stimulating differentiation, or a combination thereof. In some embodiments, the agent can increase the development of naive T cells into Th 1, Th 17, Th22, or other cells that secrete, or cause other cells to secrete, inflammatory molecules, including, but not limited to, IL-ip, TNF-a, TGF-beta, IFN-y, IL- 17, IL-6, IL-23, IL-22, IL-21, and MMPs. In some embodiments, the agent can reduce or inhibit the activity of Tregs, reduce the production of cytokines such as IL- 10 from Tregs, reduce the differentiation of Tregs, reduce the number of Tregs, reduce the ratio of Tregs within an immune cell population, or reduce the survival of Tregs. The immunomodulatory agent can be administered to a subject in need thereof in an effective amount to overcome T cell exhaustion and / or T cell anergy. Overcoming T cell exhaustion or T cell anergy can be determined by measuring T cell function using known techniques.

[0581] The methods can be used in vivo or ex vivo as immune response-stimulating therapeutic applications. Thus in some embodiments, the agent, or nucleic acid encoding the agent, is administered directly to the subject. In some embodiments, the agent or nucleic acid encoding the agent, is contacted with cells (e.g., immune cells) ex vivo, and the treat cells are administered to the subject (e.g. adoptive transfer). In general, the disclosed immunomodulatory agents can be used for treating a subject having or being predisposed to any disease or disorder to which the subject's immune system mounts an immune response. The agents can enable a more robust immune response to be possible. The disclosed compositions are useful to stimulate or enhance immune responses involving T cells.

[0582] The immunomodulatory agents utilized for increasing an immune response are typically those that reduce B7-H4 expression, ligand binding, crosslinking, negative signaling, or a combination thereof. For example, the agent can be an antagonist of B7- H4, such as an antagonist (blocking) anti- B7-H4 antibody or antigen binding fragment thereof. The agent can also be a B7-H4 polypeptide, for example, a soluble polypeptide,or fusion protein thereof that can serve as a decoy receptor for one or more B7-H4 ligands or receptors.

[0583] B7-H4 blockade, for example using function blocking anti- B7-H4 antibodies, can be an alternative agent or complementary agent to soluble B7-H4 polypeptides and fusion proteins. For example, in some embodiments, B7-H4 blockade is combined with a decoy receptor such as soluble B7-H4 or fusion protein thereof. The combined treatment (e.g., B7-H4-Fc and B7-H4 blockade) may be complementary.

[0584] In some embodiments, immune response stimulating therapy (e.g., in the treatment of cancer or infections) includes depletion of B7-H4+ cells.

[0585] Development and identification of B7-H4 depleting mAbs can be carried out according to known construction and screening methods including those discussed herein. See, for example, Reff, et al, Blood. Vol83, No 2, 1994: pp 435-445, which describes preparation of an anti-CD20 chimeric antibody that binds to human Clq, and mediates complement-dependent cell lysis (CDCC) in the presence of human complement, and anti-body-dependent cellular cytotoxicity (ADCC) with human effector cells. Rituximab destroys B cells and is therefore used to treat diseases which are characterized by overactive, dysfunctional, or excessive numbers of B cells. Other B cell -depleting antibodies include ocrelizumab and ofatumumab. In another example, CD3 Abs can preferentially target and deplete activated effector T cells while preserving CD4+Foxp3+Tregs. The antibodies transiently deplete T cells although they display no or little complement-dependent and antibody-dependent cellular cytotoxicity. Redirected cell lysis due to the ability to crosslink CD3 molecules expressed by two different cells (cytotoxic CD8+ T cells on one side and other target T cells on the other side) has been shown, however, T cell depletion mostly results from AICD (reviewed in You, Front Immunol. 2015; 6: 242).

[0586] A subject in need of enhancing their immune response can be administered with an agent that inhibits or blocks B7-H4 suppressive immune response in an amount effective to increase the uptake of antigen by antigen presenting cells (APCs).2. Subjects to be Treated a. Treatment of Cancer

[0587] The disclosed compositions and methods can be used to treat cancer. Generally, the agents are used to stimulate or enhance an immune response to cancer in the subject by administering to the subject an amount of an immunomodulatory agent that reduces B7-H4 expression, ligand binding, crosslinking, negative signaling, or acombination thereof Cancer cells acquire a characteristic set of functional capabilities during their development, albeit through various mechanisms. Such capabilities include evading apoptosis, self-sufficiency in growth signals, insensitivity to anti -growth signals, tissue invasion / metastasis, limitless replicative potential, and sustained angiogenesis. The term “cancer cell” is meant to encompass both pre-malignant and malignant cancer cells. In some embodiments, cancer refers to a benign tumor, which has remained localized. In other embodiments, cancer refers to a malignant tumor, which has invaded and destroyed neighboring body structures and spread to distant sites. In yet other embodiments, the cancer is associated with a specific cancer antigen (e.g., pan-carcinoma antigen (KS 1 / 4), ovarian carcinoma antigen (CA125), prostate specific antigen (PSA), carcinoembryonic antigen (CEA), CD 19, CD20, HER2 / neu, etc.).

[0588] The methods and compositions disclosed herein are useful in the treatment or prevention of a variety of cancers or other abnormal proliferative diseases, including (but not limited to) the following: carcinoma, including that of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, head and neck, thyroid and skin; including squamous cell carcinoma; hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Berketts lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; other tumors, including melanoma, seminoma, tetratocarcinoma, neuroblastoma and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyoscarama, and osteosarcoma; and other tumors, including melanoma, xenoderma pegmentosum, keratoactanthoma, seminoma, thyroid follicular cancer and teratocarcinoma.

[0589] Cancers caused by aberrations in apoptosis can also be treated by the disclosed methods and compositions. Such cancers may include, but are not limited to, follicular lymphomas, carcinomas with p53 mutations, hormone dependent tumors of the breast, prostate and ovary, and precancerous lesions such as familial adenomatous polyposis, and myelodysplastic syndromes. In specific embodiments, malignancy or dysproliferative changes (such as metaplasias and dysplasias), or hyperproliferative disorders, are treated or prevented by the methods and compositions in the ovary, bladder,breast, colon, lung, skin, pancreas, or uterus. In other specific embodiments, sarcoma, melanoma, or leukemia is treated or prevented by the methods and compositions.

[0590] The disclosed compositions and methods are particularly useful for the treatment of cancers that are associated with cells that express abnormally high levels of B7-H4.

[0591] Specific cancers and related disorders that can be treated or prevented by methods and compositions disclosed herein include, but are not limited to, leukemias including, but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia leukemias and myelodysplastic syndrome, chronic leukemias such as but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as, but not limited to, Hodgkin's disease or non-Hodgkin's disease lymphomas (e.g., diffuse anaplastic lymphoma kinase (ALK) negative, large B-cell lymphoma (DLBCL); diffuse anaplastic lymphoma kinase (ALK) positive, large B-cell lymphoma (DLBCL); anaplastic lymphoma kinase (ALK) positive, ALK+ anaplastic large-cell lymphoma (ALCL), acute myeloid lymphoma (AML)); multiple myelomas such as, but not limited to, smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedullary plasmacytoma; Waldenstrom's macroglobulinemia; monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; bone and connective tissue sarcomas such as, but not limited to, bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft- tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial sarcoma; brain tumors including but not limited to, glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic neurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, primary brain lymphoma; breast cancer including, but not limited to, adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget's disease, and inflammatory breast cancer; adrenal cancer, including but not limited to, pheochromocytom and adrenocortical carcinoma; thyroid cancer such as but not limited to papillary or follicular thyroid cancer, medullary thyroid cancer and anaplastic thyroidcancer; pancreatic cancer, including but not limited to, insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor; pituitary cancers including but not limited to, Cushing's disease, prolactin-secreting tumor, acromegaly, and diabetes insipius; eye cancers including, but not limited to, ocular melanoma such as iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancers, including, but not limited to, squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancer, including but not limited to, squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancers including, but not limited to, squamous cell carcinoma, and adenocarcinoma; uterine cancers including, but not limited to, endometrial carcinoma and uterine sarcoma; ovarian cancers including, but not limited to, ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and stromal tumor; esophageal cancers including, but not limited to, squamous cancer, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; stomach cancers including, but not limited to, adenocarcinoma, fungating (polypoid), ulcerating, superficial spreading, diffusely spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancers; rectal cancers; liver cancers including, but not limited to, hepatocellular carcinoma and hepatoblastoma, gallbladder cancers including, but not limited to, adenocarcinoma; cholangiocarcinomas including, but not limited to, papillary, nodular, and diffuse; lung cancers including but not limited to, nonsmall cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large-cell carcinoma and small-cell lung cancer; testicular cancers including, but not limited to, germinal tumor, seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma, embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sac tumor), prostate cancers including, but not limited to, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penal cancers; oral cancers including, but not limited to, squamous cell carcinoma; basal cancers; salivary gland cancers including, but not limited to, adenocarcinoma, mucoepidermoid carcinoma, and adenoidcystic carcinoma; pharynx cancers including, but not limited to, squamous cell cancer, and verrucous; skin cancers including, but not limited to, basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, acral lentiginous melanoma; kidney cancers ...

Claims

We claim:

1. An anti-B7-H4 antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-B7-H4 antibody comprising a light chain having an amino acid sequence according to any one of SEQ ID NOs: 62, 63, 64, or 132, and a heavy chain having an amino acid sequence according to any one of SEQ ID NOs: 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 133, a linker and a cytotoxic payload.

2. The anti-B7-H4 antibody-drug conjugate of claim 1, wherein the cytotoxic payload is an auristatin.

3. The anti-B7-H4 antibody-drug conjugate of claim 2, wherein the auristatin is monomethyl auristatin E.

4. The anti-B7-H4 antibody-drug conjugate of claim 1, wherein a) the light chain has an amino acid sequence according to SEQ ID NO: 62 and the heavy chain is selected from heavy chains having an amino acid sequence according to SEQ ID NO:75, 76, 77, 78, or 79; b) the light chain has an amino acid sequence according to SEQ ID NO: 63 and the heavy chain is selected from heavy chains having an amino acid sequence according to any one of SEQ ID NO:75, 76, 77, 78, or 79; c) the light chain has an amino acid sequence according to SEQ ID NO: 64 and the heavy chain is selected from heavy chains having an amino acid sequence according to any one of SEQ ID NO:75, 76, 76, 77, 78, or 79; d) the light chain has an amino acid sequence according to SEQ ID NO: 132 and the heavy chain is selected from heavy chains having an amino acid sequence according to any one of SEQ ID NO:75, 76, 77, 78, or 79; e) the light chain has an amino acid sequence according to SEQ ID NO: 62 and the heavy chain has an amino acid sequence according to SEQ ID NO: 80, 81, 82, 83, or 84; f) the light chain has an amino acid sequence according to SEQ ID NO: 63 and the heavy chain has an amino acid sequence according to SEQ ID NO: 80, 81, 82, 83, or 84;g) the light chain has an amino acid sequence according to SEQ ID NO: 64 and the heavy chain has an amino acid sequence according to SEQ ID NO: 80, 81, 82, 83, or 84; h) the light chain has an amino acid sequence according to SEQ ID NO: 132 and the heavy chain has an amino acid sequence according to SEQ ID NO: 80, 81, 82, 83, or 84; i) the light chain has an amino acid sequence according to SEQ ID NO: 62 and the heavy chain has an amino acid sequence according to SEQ ID NO: 133; j) the light chain has an amino acid sequence according to SEQ ID NO: 63 and the heavy chain has an amino acid sequence according to SEQ ID NO: 133; k) the light chain has an amino acid sequence according to SEQ ID NO: 64 and the heavy chain has an amino acid sequence according to SEQ ID NO: 133; or l) the light chain has an amino acid sequence according to SEQ ID NO: 132 and the heavy chain has an amino acid sequence according to SEQ ID NO: 133.

5. The anti-B7-H4 antibody-drug conjugate of claim 1, wherein the conjugate has a structure represented by Formula la or pharmaceutically acceptable salt thereof:Formula la whereinAb is an anti-B7-H4 antibody comprising a light chain having an amino acid sequence according to any one of SEQ ID NOs: 62, 63, or 64, and a heavy chain having an amino acid sequence according to any one of SEQ ID NOs: 75, 76, 77, 78, or 79, each instance of W is -C(O)-, -C(O)NR'-, -C(O)O-, -SO2NR'-, -P(O)R"NR'-, - SONR'-, -PO2NR'-, or -NR'C(O)-;each instance of R' and R" is independently hydrogen, Ci-8 alkyl, C3-8 cycloalkyl, C1-8 alkoxy, C1-8 alkylthio, mono- or di-Ci-8 alkylamino, heteroaryl or aryl; each instance of Z is independently C1-8 alkyl, halogen, cyano, or nitro; each instance of nl and n2 is independently an integer from 1 to 10, preferably 1 to 4; each instance of n3 is an integer from 0 to 3, preferably 0; each instance of B is independently a cytotoxic payload (e.g., a drug or diagnostic agent), preferably a drug; and each instance of Y is an alkylene or heteroalkylene, preferably a C1-50 alkylene or Ci-50 heteroalkylene, comprising one or more of the following:(i) one or more unsaturated bonds;(ii) a heteroarylene (e.g., a heteroarylene in the alkylene or heteroalkylene chain);(iii) at least one C1-20 alkyl substituent; and(iv) comprises at least one isoprenyl group having a structure represented byFormula II:Formula II wherein n4 is an integer from 1 to 20, preferably 1 to 4.

6. The anti-B7-H4 antibody-drug conjugate of claim 5, wherein W is -C(0)NR'-, further wherein the C is directly bonded to the phenyl ring of Formula la, and NR' is bonded to Y.

7. The anti-B7-H4 antibody-drug conjugate of claim 5, wherein Y comprises a peptide and the peptide comprises at least one hydrophilic amino acid, preferably an amino acid having a side chain having a moiety that bears a charge at neutral pH in aqueous solution (e.g., an amine, guanidine, or carboxyl moiety), most preferably each amino acid of the peptide is independently selected from alanine, aspartate, asparagine, glutamate, glutamine, glycine, lysine, ornithine, proline, serine, and threonine.

8. The anti-B7-H4 antibody-drug conjugate of claim 5, wherein Y is covalently bonded to the antibody by a thioether bond, and the thioether bond comprises a sulfur atom of a cysteine of the antibody.

9. The anti-B7-H4 antibody-drug conjugate of claim 5, wherein Y comprises an oxime and: the oxygen atom of the oxime is on the side of Y that is linked to W and the carbon atom of the oxime is on the side of Y that is linked to Ab; or the carbon atom of the oxime is on the side of Y that is linked to W and the oxygen atom of the oxime is on the side of Y that is linked to Ab.

10. The anti-B7-H4 antibody-drug conjugate of claim 5, wherein Y comprises a connection unit represented by Formula III or Formula IV:-(CH2)r(V(CH2)P)q-Formula III,-(CH2CH2X)W-Formula IV;V is a single bond, -O-, -S-, -NR1-, -C(O)NR2-, -NR3C(O)-, -NR4SO2-, or - SO2NR5-, preferably -O-;X is -O-, Ci-8 alkylene, or -NR1-, preferably -O-;R1to R5are each independently hydrogen, Ci-6 alkyl, Ci-6 alkyl Ce-2o aryl, or Ci-6 alkyl C3-2o heteroaryl; r is an integer from 1 to 10, preferably 2; p is an integer from 0 to 12, preferably 2; q is an integer from 1 to 20, preferably 2, 5, or 11; and w is an integer from 1 to 20, preferably from 6 to 20.

11. The anti-B7-H4 antibody-drug conjugate of claim 5, wherein Y comprises a moiety represented by Formula V, VI, VII, VIII or IX:Formula V,Formula VI,Formula VII,Formula VIIIFormula IXL1is a single bond or C1-30 alkylene; and R11is hydrogen or C1-10 alkyl.

12. The anti-B7-H4 antibody-drug conjugate of claim 5, wherein Y is linear or branched.

13. The anti-B7-H4 antibody-drug conjugate of claim 12, wherein Y comprises: i) a branching unit covalently coupled to Ab by a primary linker; ii) a first branch, which couples a first therapeutically active substance, via a first cleavage group, to the branching unit; and iiia) a second branch, which couples a second therapeutically active substance, via a second cleavage group, to the branching unit; or iiib) a second branch which couples an alkyl or heteroalkyl (e.g., a polyethylene glycol monomer or a polyethylene glycol oligomer) to the branching unit.

14. The anti-B7-H4 antibody-drug conjugate of claim 13, wherein at least one branching unit has a structure representedR30is hydrogen or C1-30 alkyl;R40is hydrogen or L5-COOR50;R50is hydrogen or C1-30 alkyl; andL2, L3, L4, and L5are each independently a bond or alkylene.

15. The anti-B7-H4 antibody-drug conjugate of claim 5, wherein the antibody comprises an amino acid motif recognizable by an isoprenoid transferase at the C- terminus of the antibody, and the thioether bond comprises a sulfur atom of a cysteine of the amino acid motif.

16. The anti-B7-H4 antibody-drug conjugate of claim 15, wherein the amino acid motif has a CY1Y1X sequence, further wherein:C is cysteine; each Yi independently is an aliphatic amino acid;X is selected from glutamine, glutamate, serine, cysteine, methionine, alanine, and leucine; and the thioether bond comprises a sulfur atom of a cysteine of the amino acid motif.

17. The anti-B7-H4 antibody-drug conjugate of claim 16, wherein each Yi independently is selected from alanine, isoleucine, leucine, methionine, and valine.

18. The anti-B7-H4 antibody-drug conjugate of claim 16, wherein the amino acid motif comprises a CVIM.

19. The anti-B7-H4 antibody-drug conjugate of claim 16, wherein the conjugate comprises at least one of 1 to 20 amino acids between the antibody and the amino acid motif, and at least one of the amino acids is glycine.

20. The anti-B7-H4 antibody-drug conjugate of claim 16, wherein the amino acid motif has the sequence GGGGGGGCVIM.

21. The anti-B7-H4 antibody-drug conjugate of claim 16, wherein the conjugate comprises a structure represented bywherein MMAE is monomethyl auri statin E; and represents a connection point to Y.

22. An anti-B7-H4 antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-B7-H4 antibody comprising a light chain having an amino acid sequence according to SEQ ID NOs: 63 and a heavy chain having an amino acidsequence according to SEQ ID NOs: 78, a linker and a cytotoxic payload, wherein the linker and payload comprises a structure represented bywherein MMAE is monomethyl auri statin E; and represents a connection point to Y.

23. An anti-B7-H4 antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-B7-H4 antibody comprising a light chain having an amino acid sequence according to SEQ ID NOs: 132 and a heavy chain having an amino acid sequence according to SEQ ID NOs: 133, a linker and a cytotoxic payload, wherein the linker and payload comprises a structure represented bywherein MMAE is monomethyl auri statin E; and represents a connection point to Y.

24. A method of treating a patient having a cancer that expresses B7-H4 by administering an antibody of claim 1.