B7-H4 antibody and method of use

Abstract

This invention provides a composition for adjusting B7-H4 and a method for using the same. [Solution] For example, immunomodulatory substances are provided that reduce B7-H4 expression, ligand binding, crosslinking, negative signaling, or a combination thereof. Such substances can be used to increase the immune response in subjects that require an increased immune response. Immunomodulatory substances that increase B7-H4 expression, ligand binding, crosslinking, negative signaling, or a combination thereof are also provided. Such substances can be used to reduce the immune response in subjects that require a reduced immune response.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits and priority of U.S. Provisional Patent Application No. 62 / 620,545, filed on 23 January 2018, which is incorporated in its entirety by reference.

[0002] Sequence listing reference The sequence listing submitted on January 23, 2019, as a text file named "064467.005PCT_seqlist_ST25" with a size of 357 MB, created on January 23, 2019, is incorporated herein by reference in accordance with 37 C. FR §1, 52(e)(5).

[0003] Field of Invention The present invention generally relates to the field of immunomodulation, and more specifically to compositions and methods for modulating B7-H4 signaling to increase or decrease the immune response. [Background technology]

[0004] Background of the Invention The B7 family plays a crucial role in both the positive and negative regulation of the immune response by involving various receptors on lymphocytes (Rahbar, R. et al., Cancer Immunol Res, 3(2):184-195 (2015) (Non-Patent Literature 1)). B7-H4 (also known as VTCN1, B7x, or B7S1) is a member of the B7 family and inhibits T cell function. B7-H4 has also been proposed to be upregulated on various tumors and to promote tumor growth. High B7-H4 expression is found in many tumor tissues, providing a correlation between expression levels on tumor cells and adverse clinical and pathological features, including tumor invasiveness.

[0005] The biological activity of B7-H4 is inflammatory CD4 +This is related to a decrease in T cell response and the 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 can favorably alter the tumor microenvironment and enable antigen-specific elimination of tumor cells (Podojil, JR. and Miller, SD, Immunol Rev, 276(l):40-5l (2017) (Non-Patent Literature 2)).

[0006] The cDNA encoding the human B7-H4 protein was identified and cloned from placental cDNA (Sica, G. Letal. (2003) “B7-H4, A Molecule Of The B7 Family, Negatively Regulates T Cell Immunity,” Immunity 18:849-861 (Non-Patent Literature 3); 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 (Non-Patent Literature 4)). B7-H4 is discussed in U.S. Patent No. 7,931,896 (Patent Literature 1), No. 7,875,702 (Patent Literature 2), No. 7,847,081 (Patent Literature 3), and No. 7,622,565 (Patent Literature 4).

[0007] Anti-B7-H4 antibodies are disclosed in U.S. Patent No. 9,574,000 (Patent Document 5), No. 7,888,477 (Patent Document 6), No. 7,737,255 (Patent Document 7), No. 7,619,068 (Patent Document 8), and No. 6,962,980 (Patent Document 9).

[0008] The human B7-H4 protein has 282 amino acid residues, which are classified as containing an amino-terminal extracellular domain, a large hydrophobic transmembrane domain, and a very short intracellular domain (consisting of only two amino acid residues). Like other B7 family members, B7-H4 has a pair of Ig-like regions in its extracellular domain. The B7-H4 protein has the complete structure of a type I transmembrane protein. The protein has minimal (approximately 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 (Non-Patent Literature 5)).

[0009] B7-H4 is also expressed on tumor-associated macrophages (TAMs). TAMs protect tumors from immune recognition by inhibiting the antitumor immune response through the release of fluid-mediated substances and by interfering with cell-mediated immune responses through the cell surface expression of inhibitory molecules such as B7-H4. TAMs originate from resident macrophages or monocytes recruited by the tumor microenvironment and localized to the tumor site. Tumor invasion by TAMs is associated with low patient survival rates, and targeting TAMs represents a promising strategy against cancer. Several methods have already been developed, including depletion by clodronate liposomes, inhibition of tumor recruitment by targeting CFSR-1 and CCL2, and "re-education" by activation via anti-CD40 mAbs, HRG plasma proteins, or mannose receptors (Dangai, D. et al., Cancer Res, 73(15):4820-4829 (2013) (Non-patent Literature 6)).

[0010] Since B7-H4 is expressed on tumor cells and TAMs in various cancer types, targeting B7-H4 with therapeutic agents can have a very significant synergistic effect in favorably altering the tumor microenvironment and eliminating cancer cells. Therefore, novel immunomodulatory agents for B7-H4 are needed.

[0011] Therefore, an object of the present invention is to provide compositions that enhance or promote the immune response by inhibiting B7-H4 mediated inhibitory signaling, thereby inhibiting B7-H4 suppressive signaling. Such compositions are useful in the treatment of cancer and infectious diseases.

[0012] Another object of the present invention is to provide a composition that increases B7-H4-mediated inhibitory signaling, thereby promoting a suppressive immunological response. [Prior art documents] [Patent Documents]

[0013] [Patent Document 1] U.S. Patent No. 7,931,896 [Patent Document 2] U.S. Patent No. 7,875,702 [Patent Document 3] U.S. Patent No. 7,847,081 [Patent Document 4] U.S. Patent No. 7,622,565 [Patent Document 5] U.S. Patent No. 9,574,000 [Patent Document 6] U.S. Patent No. 7,888,477 [Patent Document 7] U.S. Patent No. 7,737,255 [Patent Document 8] U.S. Patent No. 7,619,068 [Patent Document 9] U.S. Patent No. 6,962,980 [Non-patent literature]

[0014]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Summary of the Invention

[0015] Compositions for modulating B7-H4 signaling and methods for using them are provided. Such compositions are useful for treating inflammatory diseases and disorders as well as autoimmune diseases. In one embodiment, an immunomodulatory substance is provided that reduces B7-H4 expression, ligand binding, crosslinking, signaling, or a combination thereof. In one embodiment, the immunomodulatory substance specifically binds to B7-H4. The immunomodulatory substance may be an antibody or its antigen-binding fragment, a fusion protein, an aptamer, or an active agent that specifically binds to B7-H4 and modulates signaling via the B7-H4 signaling pathway. Modulation includes increasing signaling via the B7-H4 signaling pathway or inhibiting signaling via the B7-H4 signaling pathway.

[0016] One embodiment provides a method for treating an infection in a subject requiring treatment of the infection by administering an effective amount of an activator that inhibits or blocks the B7-H4 suppressive immune response in an amount effective to increase neutrophil proliferation and / or innate immunity. The disclosed activators can be used to increase the immune response in a subject requiring an increased immune response by inhibiting the B7-H4 suppressive immune response. The immune response may be, for example, a primary immune response to an antigen, or an increase in effector cell function such as increased antigen-specific proliferation of T cells, enhanced cytokine production by T cells, stimulation of differentiation, neutrophil proliferation, or a combination thereof. Exemplary activators include (i) soluble B7-H4 polypeptides or fusion proteins, (ii) anti-B7-H4 antibodies that block function, (iii) antibodies that can be used to deplete B7-H4 positive cells, and (iv) combinations thereof. In some embodiments, the immunomodulator is a B7-H4 antagonist.

[0017] In certain embodiments, the active ingredient is a B7-H4 fusion protein, for example, a fusion protein comprising the extracellular domain of B7-H4 or a functional variant thereof bound to an immunoglobulin domain. An exemplary fusion protein includes the amino acid sequence or fragment thereof of SEQ ID NO: 1.

[0018] In other embodiments, the active substance is the B7-H4 protein or a functional fragment or variant thereof. For example, the B7-H4 protein or a functional fragment or variant thereof may have at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 1. In one embodiment, the B7-H4 protein or polypeptide modulates signaling via the B7-H4 signaling pathway by specifically binding to a ligand of B7-H4.

[0019] In other embodiments, the active ingredient is a soluble B7-H4 protein or a functional fragment or variant thereof. For example, the soluble B7-H4 protein may consist of the extracellular domain of B7-H4 or a functional fragment or variant thereof.

[0020] Methods for increasing the immune response in a subject typically involve administering an effective amount of an immunomodulator that reduces B7-H4 expression, ligand binding, crosslinking, signaling, or a combination thereof to the subject in need. The subject may, for example, have cancer or an infectious disease.

[0021] In some embodiments, the target, cancer, or disease is characterized by increased expression of B7-H4, increased expression of B7-H4 ligand, or a combination thereof. In certain embodiments, cancers include ovarian cancer, breast cancer, lung cancer, thyroid cancer, gastrointestinal cancer, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), endometrial cancer, brain cancer, head and neck cancer, pancreatic cancer, and bladder cancer. The active agent may be administered simultaneously with or in combination with the vaccine or its components.

[0022] Immunomodulatory substances are also provided that increase B7-H4 expression, ligand binding, crosslinking, immunosuppressive signaling, or combinations thereof. Such substances can be used to reduce the immune response in subjects requiring induction or enhancement of an inhibitory immune B7-H4 response. Exemplary substances include those that activate anti-B7-H4 antibodies or soluble B7-H4 polypeptides.

[0023] Methods for reducing the immune response in a subject typically involve administering an effective amount of an immunomodulator to the subject in need to increase B7-H4 expression, ligand binding, crosslinking, negative signaling, or a combination thereof. In some embodiments, the subject has an inflammatory disorder or autoimmune disorder. In certain embodiments, the subject has rheumatoid arthritis.

[0024] In some embodiments, the subject, disease, or condition is characterized by reduced expression of B7-H4.

[0025] Any of the disclosed methods may include administering an immunomodulatory substance to a subject, either alone or in combination with one or more additional therapeutic agents.

[0026] One embodiment provides a method for treating leukemia in a subject requiring treatment of leukemia by administering an effective amount of a pharmaceutical composition to the subject, comprising a B7-H4 monoclonal antibody, a soluble B7-H4 polypeptide, a B7-H4 fusion protein, or a combination thereof, to inhibit or reduce B7-H4 signaling in leukemia cells, thereby inhibiting the survival of leukemia cells or promoting an anti-tumor immune response against leukemia cells. The leukemia may be acute myeloid leukemia.

[0027] One embodiment provides a method for evaluating or predicting the effectiveness of a treatment using an anti-B7-H4 binding moiety by assaying cells of a subject that require treatment to determine whether the cells express B7-H4, a B7-H4 binding partner, or both. Exemplary cells to be assayed include, but are not limited to, cancer cells obtained from a subject. Exemplary cancer cells include, but are not limited to, acute myeloid leukemia (AML) cells, endometrial cancer, brain cancer, head and neck cancer, and pancreatic cancer.

[0028] One embodiment provides an anti-B7-H4 monoclonal antibody or its antigen-binding fragment having a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 3.

[0029] One embodiment provides a nucleic acid encoding 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 with SEQ ID NO: 7.

[0030] One embodiment provides an anti-B7-H4 monoclonal antibody or its antigen-binding fragment having a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 8.

[0031] One embodiment provides a nucleic acid encoding heavy chain sequence number 8. Another embodiment provides a nucleic acid having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with sequence number 12.

[0032] One embodiment provides an anti-B7-H4 antibody or its antigen-binding fragment having a light chain containing the CDRs of SEQ ID NOs: 4, 5, and 6.

[0033] Another embodiment provides an anti-B7-H4 antibody or its antigen-binding fragment having a heavy chain comprising the CDRs of SEQ ID NOs. 9, 10, and 11.

[0034] In one embodiment, the anti-B7-H4 antibody or its antigen-binding fragment has a light chain containing the CDRs of SEQ ID NOs. 4, 5, and 6, and a heavy chain containing the CDRs of SEQ ID NOs. 9, 10, and 11.

[0035] One embodiment provides an anti-B7-H4 antibody or its antigen-binding fragment, having a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 3, and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 8.

[0036] Another embodiment provides an anti-B7-H4 monoclonal antibody or its antigen-binding fragment having a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 13.

[0037] In one embodiment, the antibody light chain includes a CDR having the amino acid sequences of SEQ ID NOs: 14, 15, and 16.

[0038] One embodiment provides an antibody heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 29.

[0039] Another embodiment provides an antibody heavy chain comprising a CDR having the amino acid sequences of SEQ ID NOs: 9, 11, and 30.

[0040] One embodiment provides an anti-B7-H4 monoclonal antibody or its antigen-binding fragment, comprising 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.

[0041] One embodiment provides an anti-B7-H4 antibody or its antigen-binding fragment, comprising a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 13, and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 29.

[0042] One embodiment provides an anti-B7-H4 antibody light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 46.

[0043] Another embodiment provides an anti-B7-H4 antibody light chain comprising a CDR having the amino acid sequences of SEQ ID NOs. 14, 16, and 47.

[0044] One embodiment provides an antibody heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 49.

[0045] Another embodiment provides an antibody heavy chain comprising a CDR having the amino acid sequences of SEQ ID NOs: 9, 11, and 47.

[0046] One embodiment provides an antibody or its antigen-binding fragment, comprising 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.

[0047] One embodiment provides an anti-B7-H4 antibody or its antigen-binding fragment, comprising a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 46, and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 49.

[0048] One embodiment provides an antibody light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 52.

[0049] In another embodiment, the antibody light chain comprises a CDR having the amino acid sequences of SEQ ID NOs. 53, 54, and 55.

[0050] One embodiment provides an antibody heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 65.

[0051] Another embodiment provides an antibody heavy chain comprising a CDR having the amino acid sequences of SEQ ID NOs. 66, 67, and 68.

[0052] One embodiment provides an anti-B7-H4 antibody or its antigen-binding fragment, comprising 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.

[0053] One embodiment provides an antibody or its antigen-binding fragment, comprising a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 52, and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 65.

[0054] One embodiment provides an antibody light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 85.

[0055] Another embodiment provides an antibody light chain comprising a CDR having the amino acid sequences of SEQ ID NOs: 4, 86, and 87.

[0056] One embodiment provides an antibody heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 89.

[0057] One embodiment provides an antibody heavy chain comprising a CDR having the amino acid sequences of SEQ ID NOs: 9, 90, and 91.

[0058] Another embodiment provides an anti-B7-H4 antibody or its antigen-binding fragment, comprising 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.

[0059] One embodiment provides an anti-B7-H4 antibody or its antigen-binding fragment, comprising a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 85, and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 89.

[0060] One embodiment provides an antibody light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 93.

[0061] Another embodiment provides an antibody light chain comprising a CDR having the amino acid sequences of SEQ ID NOs. 94, 95, and 96.

[0062] One embodiment provides an antibody heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 98.

[0063] Another embodiment provides an antibody heavy chain comprising a CDR having the amino acid sequences of SEQ ID NOs. 9, 99, and 100.

[0064] One embodiment provides an anti-B7-H4 antibody or its antigen-binding fragment 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.

[0065] One embodiment provides an anti-B7-H4 antibody or its antigen-binding fragment, having a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 93, and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 98.

[0066] One embodiment provides an antibody light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 102.

[0067] Another embodiment provides an antibody light chain comprising a CDR having the amino acid sequences of SEQ ID NOs. 103, 104, and 105.

[0068] One embodiment provides an antibody heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 107.

[0069] Another embodiment provides an antibody heavy chain comprising a CDR having the amino acid sequences of SEQ ID NOs. 9, 108, and 109.

[0070] In one embodiment, the anti-B7-H4 antibody or its antigen-binding fragment comprises a light chain CDR according to SEQ ID NOs. 103, 104, and 105, and a heavy chain CDR according to SEQ ID NOs. 9, 108, and 109.

[0071] One embodiment provides an anti-B7-H4 antibody or its antigen-binding fragment, comprising a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 102, and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 107.

[0072] One embodiment provides an antibody light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 111.

[0073] Another embodiment provides an antibody light chain comprising a CDR having the amino acid sequences of SEQ ID NOs. 95, 112, and 113.

[0074] One embodiment provides an antibody heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 115.

[0075] One embodiment provides an antibody heavy chain comprising a CDR having the amino acid sequences of SEQ ID NOs. 116, 117, and 118.

[0076] In one embodiment, the anti-B7-H4 antibody or its antigen-binding fragment comprises a light chain CDR according to SEQ ID NOs. 95, 112, and 113, and a heavy chain CDR according to SEQ ID NOs. 116, 117, and 118.

[0077] One embodiment provides an anti-B7-H4 antibody or its antigen-binding fragment, comprising a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 111, and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 115.

[0078] One embodiment provides an antibody light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 120.

[0079] Another embodiment provides an antibody light chain comprising a CDR having the amino acid sequences of SEQ ID NOs. 54, 55, and 121.

[0080] One embodiment provides an antibody heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 123.

[0081] Another embodiment provides an antibody heavy chain comprising a CDR having the amino acid sequences of SEQ ID NOs. 66, 124, and 125.

[0082] One embodiment provides an anti-B7-H4 antibody or its antigen-binding fragment, comprising 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.

[0083] One embodiment provides an anti-B7-H4 antibody or its antigen-binding fragment, comprising a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 120, and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 123.

[0084] One embodiment provides an anti-B7-H4 antibody heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 128.

[0085] Another embodiment provides an anti-B7-H4 antibody heavy chain comprising a CDR having the amino acid sequences of SEQ ID NOs. 9, 129, and 130.

[0086] In one embodiment, the anti-B7-H4 antibody or its antigen-binding fragment comprises a light chain CDR according to SEQ ID NOs: 14, 15, and 16, and a heavy chain CDR according to SEQ ID NOs: 9, 129, and 130.

[0087] One embodiment provides an anti-B7-H4 antibody or its antigen-binding fragment, comprising a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 13, and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 128.

[0088] One embodiment provides an anti-B7-H4 antibody or its antigen-binding fragment having a light chain having one of the amino acid sequences of SEQ ID NOs: 3, 13, 46, 52, 85, 93, 102, 111, or 120.

[0089] Another embodiment provides an anti-B7-H4 antibody or its antigen-binding fragment having a heavy chain having one of the amino acid sequences of SEQ ID NOs: 8, 29, 49, 65, 89, 98, 107, 115, 123, or 128.

[0090] One embodiment provides an anti-B7-H4 antibody or its antigen-binding fragment, having a light chain having one of the amino acid sequences of SEQ ID NOs: 3, 13, 46, 52, 85, 93, 102, 111, or 120, and a heavy chain having one of the amino acid sequences of SEQ ID NOs: 8, 29, 49, 65, 89, 98, 107, 115, 123, or 128.

[0091] Also provided are an anti-B7-H4 antibody or its antigen-binding fragment having three light chain CDRs having amino acid sequences 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.

[0092] One embodiment provides an anti-B7-H4 antibody or its antigen-binding fragment having three heavy chain CDRs having amino acid sequences 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.

[0093] One embodiment provides an anti-B7-H4 antibody or its antigen-binding fragment, comprising three light chain CDRs having amino acid sequences 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 chain CDRs having amino acid sequences 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.

[0094] Another embodiment provides an anti-B7-H4 antibody or its antigen-binding fragment, having a light chain variable domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with 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 with any one of SEQ ID NOs: 34, 35, 36, or 37.

[0095] One embodiment provides an anti-B7-H4 antibody or its antigen-binding fragment, having a light chain having one of the amino acid sequences of SEQ ID NOs: 24, 25, 26, 27, or 28, and a heavy chain having one of the amino acid sequences of SEQ ID NOs: 38, 39, 40, or 41.

[0096] Another embodiment provides an anti-B7-H4 antibody or its antigen-binding fragment, having a light chain having one of the amino acid sequences of SEQ ID NOs: 24, 25, 26, 27, or 28, and a heavy chain having one of the amino acid sequences of SEQ ID NOs: 42, 43, 44, or 45.

[0097] Also provided are an anti-B7-H4 antibody or its antigen-binding fragment having a light chain variable domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with any one of sequence numbers 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 with any one of sequence numbers 70, 71, 72, 73, or 74.

[0098] One embodiment provides an anti-B7-H4 antibody or its antigen-binding fragment, having a light chain having one of the amino acid sequences of SEQ ID NOs. 62, 63, or 64, and a heavy chain having one of the amino acid sequences of SEQ ID NOs. 75, 76, 77, 78, or 79.

[0099] One embodiment provides an anti-B7-H4 antibody or its antigen-binding fragment, having a light chain having one of the amino acid sequences of SEQ ID NOs. 62, 63, or 64, and a heavy chain having one of the amino acid sequences of SEQ ID NOs. 80, 81, 82, 83, or 84.

[0100] Another embodiment provides an anti-B7-H4 antibody or its antigen-binding fragment having six complementarity-determining regions (CDRs), wherein the CDRs are three light chain CDRs of a polypeptide selected from the group consisting of SEQ ID NOs: 4, 5, 6, 14, 15, 16, 49, 55, 56, 57, 88, 89, 96, 97, 97, 105, 106, 107, 114, 115, or 123, or at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence-matched with SEQ ID NOs: 4, 5, 6, 14, 15, 16, 49, 55, 56, 57, 88, 89, 96, 97, 97, 105, 106, 107, 114, 115, or 123. The antibody or its antigen-binding fragment contains a variant having unisexuality, and three heavy chain CDRs of a polypeptide selected from the group consisting of SEQ ID NOs: 9, 10, 11, 31, 52, 68, 69, 70, 92, 93, 101, 102, 118, 119, 120, 126, 127, 131, or 132, or a variant having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity with SEQ ID NOs: 9, 10, 11, 31, 52, 68, 69, 70, 92, 93, 101, 102, 118, 119, 120, 126, 127, 131, or 132, and the antibody or its antigen-binding fragment binds to B7-H4.

[0101] Another embodiment provides an anti-B7-H4 antibody or its antigen-binding fragment having two light chains and two heavy chains, wherein the two light chains are polypeptides selected from the group consisting of SEQ ID NOs: 24, 25, 26, 27, 28, 62, 63, or 64, or having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity with SEQ ID NOs: 24, 25, 26, 27, 28, 62, 63, or 64. The antibody or its antigen-binding fragment binds to B7-H4.

[0102] One embodiment provides an anti-B7-H4 antibody or its antigen-binding fragment having two light chains and two heavy chains, wherein the two light chains are polypeptides selected from the group consisting of SEQ ID NOs: 24, 25, 26, 27, 28, 62, 63, or 64, or have at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity with SEQ ID NOs: 24, 25, 26, 27, 28, 62, 63, or 64. The antibody or its antigen-binding fragment binds to B7-H4.

[0103] One embodiment provides an anti-B7-H4 antibody or its antigen-binding fragment having two light chains and two heavy chains, wherein the two light chains comprise a polypeptide selected from the group consisting of SEQ ID NOs: 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 with SEQ ID NOs: 24, 25, 26, 27, or 28, and the two heavy chains comprise a polypeptide selected from the group consisting of SEQ ID NOs: 38, 39, 40, or 41, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity with SEQ ID NOs: 38, 39, 40, or 41, and the antibody or its antigen-binding fragment binds to B7-H4.

[0104] Another embodiment provides an anti-B7-H4 antibody or its antigen-binding fragment having two light chains and two heavy chains, wherein the two light chains comprise a polypeptide selected from the group consisting of SEQ ID NOs: 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 with SEQ ID NOs: 24, 25, 26, 27, or 28, and the two heavy chains comprise a polypeptide selected from the group consisting of SEQ ID NOs: 42, 43, 44, or 45, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity with SEQ ID NOs: 42, 43, 44, or 45, and the antibody or its antigen-binding fragment binds to B7-H4.

[0105] One embodiment provides an anti-B7-H4 antibody or its antigen-binding fragment having two light chains and two heavy chains, wherein the two light chains comprise a polypeptide selected from the group consisting of SEQ ID NOs. 62, 63, or 64, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity with SEQ ID NOs. 62, 63, or 64; the two heavy chains comprise a polypeptide selected from the group consisting of SEQ ID NOs. 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 with SEQ ID NOs. 75, 76, 77, 78, or 79; and the antibody or its antigen-binding fragment binds to B7-H4.

[0106] Another embodiment provides an anti-B7-H4 antibody or its antigen-binding fragment having two light chains and two heavy chains, wherein the two light chains comprise a polypeptide selected from the group consisting of SEQ ID NOs. 62, 63, or 64, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity with SEQ ID NOs. 62, 63, or 64, and the two heavy chains comprise a polypeptide selected from the group consisting of SEQ ID NOs. 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 with SEQ ID NOs. 80, 81, 82, 83, or 84, and the antibody or its antigen-binding fragment binds to B7-H4. [Invention 1001] To increase the immune response in subjects that require an increased immune response, (i) Soluble B7-H4 polypeptide or fusion protein, (ii) Anti-B7-H4 antibodies that block function, (iii) Antibodies that deplete B7-H4 positive cells, and (iv) combinations of these An effective amount of immunomodulatory substance selected from the group consisting of reducing B7-H4 expression, ligand binding, crosslinking, negative signaling, or a combination thereof. A pharmaceutical composition containing the following: [Invention 1002] The pharmaceutical composition of the present invention 1001, wherein the immunomodulatory substance is a B7-H4 fusion protein. [Invention 1003] The pharmaceutical composition of the present invention 1002, wherein the B7-H4 fusion protein comprises the extracellular domain of B7-H4 for a functional variant of the B7-H4 bound to the immunoglobulin domain. [Invention 1004] The pharmaceutical composition of the present invention 1003, wherein the B7-H4 fusion protein comprises the amino acid sequence of SEQ ID NO: 1 or a fragment thereof. [Invention 1005] The pharmaceutical composition of the present invention 1001, wherein the immunomodulatory substance is a B7-H4 fusion protein. [Invention 1006] The pharmaceutical composition of the present invention 1005, wherein the B7-H4 fusion protein comprises the extracellular domain of B7-H4 bound to an immunoglobulin domain or a functional variant thereof. [Invention 1007] The pharmaceutical composition of the present invention 1006, wherein the B7-H4 fusion protein comprises the amino acid sequence of SEQ ID NO: 1 or a fragment thereof. [Invention 1008] The pharmaceutical composition of the present invention 1001, wherein the immunomodulatory substance is a soluble B7-H4 protein or a functional fragment or variant thereof. [Invention 1009] The pharmaceutical composition of the present invention 1008, wherein the B7-H4 protein comprises the extracellular domain of B7-H4 or a functional fragment or variant thereof. [Invention 1010] A pharmaceutical composition according to any one of the invention 1001 to 1009, wherein the immune response is a primary immune response to an antigen, or an increase in effector cell function such as increased antigen-specific proliferation of T cells, enhanced cytokine production by T cells, stimulation of differentiation, or a combination thereof. [Invention 1011] A method for increasing an immune response in a subject requiring an increased immune response, comprising administering any of the pharmaceutical compositions of the present invention 1001 to 1010 to the subject. [Invention 1012] The method of the present invention 1011, wherein the subject has cancer. [Invention 1013] The method of the present invention 1012, wherein the cancer is characterized by increased expression of B7-H4. [Invention 1014] The method of the present invention 1012 or 1013, wherein the cancer is ovarian cancer, lung cancer, or gastrointestinal cancer. [Invention 1015] The method of the present invention 1011, wherein the subject has an infectious disease. [Invention 1016] A method according to any of the present invention 1011 to 1015, wherein the pharmaceutical composition is administered to the subject simultaneously with the vaccine or a component thereof. [Invention 1017] Any method according to item 1011 to 1016 of the present invention, further comprising administering a second therapeutic agent to the subject. [Invention 1018] To reduce the immune response in subjects that require a reduction in the immune response, An effective amount of immunomodulatory substance selected from a group consisting of anti-B7-H4 antibodies that activate function, which increases B7-H4 expression, ligand binding, crosslinking, negative signaling, or a combination thereof. A pharmaceutical composition containing the following: [Invention 1019] The pharmaceutical composition of the present invention 1018, wherein the immune response is either a primary immune response to an antigen, or an increase in effector cell function such as increased antigen-specific proliferation of T cells, enhanced cytokine production by T cells, stimulation of differentiation, or a combination thereof. [Invention 1020] A method for reducing an immune response in a subject requiring reduction of the immune response, comprising administering the pharmaceutical composition of the present invention 1018 to the subject. [Invention 1021] The method of the present invention 1020, wherein the subject has inflammation. [Invention 1022] The method of the present invention 1020, wherein the subject has an autoimmune disorder. [Invention 1023] The method of the present invention 1022, wherein the autoimmune disorder is rheumatoid arthritis. [Invention 1024] A method according to any one of the present invention 1020 to 1023, wherein the subject, disease, or condition is characterized by a reduction in the expression of B7-H4. [Invention 1025] Any method according to items 1020 to 1023 of the present invention, further comprising administering a second therapeutic agent to the subject. [Invention 1026] Any of the compositions or methods of the present invention, wherein B7-H4 is expressed on tumor cells. [Invention 1027] A molecule containing an antigen-binding fragment of an anti-human B7-H4 antibody, which binds immunospecifically to human B7-H4. [Invention 1028] (I) Immune-specifically binds to human B7-H4 arranged on the surface of cells. (II) Immune-specifically binds to human B7-H4 arranged on the surface of living cells at endogenous concentrations. (III) Immune-specifically binds to human B7-H4 arranged on the surface of living cells and modulates the binding between B7-H4 and its cell receptor. (IV) It immune-specifically binds to human B7-H4 arranged on the surface of living cells and inhibits immunosuppression by tumor-associated macrophages. (V) Immune-specifically binds to human B7-H4 arranged on the surface of living cells and modulates the activity of tumor-associated macrophages. (VI) Immunospecifically binds to human B7-H4 arranged on the surface of living tumor cells and inhibits tumor-mediated suppression, or (VII) It immune-specifically binds to human B7-H4 arranged on the surface of living tumor cells, causing tumor-specific cell lysis. The molecule of Invention 1027. [Invention 1029] A molecule of the present invention 1027 that can be internally transferred to the aforementioned cells and mediate the death of those cells. [Invention 1030] The molecule of the present invention 1028, wherein the living cell is a tumor cell, a pathogen-infected cell, or a macrophage. [Invention 1031] The molecules of the present invention 1027, comprising toxins, drugs, receptors, enzymes, and receptor ligands that are detectably labeled or conjugated. [Invention 1032] The molecule of the present invention 1027, wherein the molecule is an anti-B7H4 antibody, and the antibody is (I) a monoclonal antibody, a chimeric antibody, or a humanized antibody, or (II) a bispecific antibody, a tripspecific antibody, or a multispecific antibody. [Invention 1033] A molecule of the present invention 1027, which is an IgG1 or IgG4 antibody. [Invention 1034] Having ADCC activity, It can induce tumor or TAM killing activity and / or inhibit TAM or tumor-mediated suppression. The molecule of Invention 1027. [Invention 1035] The molecule of the present invention 1027 is a bispecific, triplicate, or polyspecific antibody capable of binding to B7-H4 and different molecules on the same cell. [Invention 1036] A pharmaceutical composition for the treatment of cancer or infectious disease, comprising a therapeutically effective or preventively effective amount of the molecule of Invention 1027 and a physiologically acceptable carrier or excipient, wherein the molecule upmodulates the immune response by antagonizing B7-H4-mediated inhibition. [Invention 1037] A method for enhancing or inducing an immune response in a subject for which an immune response needs to be enhanced or induced, comprising administering an effective amount of the pharmaceutical composition of the present invention 1036. [Invention 1038] A monoclonal antibody or its antigen-binding fragment having a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 3. [Invention 1039] The nucleic acid that codes for the light chain of Sequence ID No. 3. [Invention 1040] Nucleic acids having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with sequence number 7. [Invention 1041] An anti-B7H4 monoclonal antibody or its antigen-binding fragment having a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 8. [Invention 1042] The nucleic acid encoding the heavy chain of sequence number 8. [Invention 1043] Nucleic acids having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with sequence number 12. [Invention 1044] An anti-B7H4 antibody or its antigen-binding fragment having a light chain containing the CDRs of SEQ ID NOs: 4, 5, and 6. [Invention 1045] An anti-B7H4 antibody or its antigen-binding fragment having a heavy chain containing the CDRs of SEQ ID NOs. 9, 10, and 11. [Invention 1046] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain containing the CDRs of SEQ ID NOs. 4, 5, and 6, and a heavy chain containing the CDRs of SEQ ID NOs. 9, 10, and 11. [Invention 1047] An anti-B7H4 antibody or its antigen-binding fragment having a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 3, and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 8. [Invention 1048] An anti-B7H4 monoclonal antibody or its antigen-binding fragment having a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 13. [Invention 1049] An anti-B7H4 antibody light chain containing a CDR having the amino acid sequences of SEQ ID NOs. 14, 15, and 16. [Invention 1050] An anti-B7H4 antibody heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 29. [Invention 1051] An anti-B7H4 antibody heavy chain containing a CDR having the amino acid sequences of SEQ ID NOs. 9, 11, and 30. [Invention 1052] An anti-B7H4 monoclonal antibody or its antigen-binding fragment, comprising the light chain CDRs of SEQ ID NOs. 14, 15, and 16, and the heavy chain CDRs of SEQ ID NOs. 9, 11, and 30. [Invention 1053] An anti-B7H4 antibody or its antigen-binding fragment comprising a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 13, and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 29. [Invention 1054] An anti-B7H4 antibody light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with sequence number 46. [Invention 1055] An anti-B7H4 antibody light chain containing a CDR having the amino acid sequences of SEQ ID NOs. 14, 16, and 47. [Invention 1056] An anti-B7H4 antibody heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with sequence number 49. [Invention 1057] An anti-B7H4 antibody heavy chain containing a CDR having the amino acid sequences of SEQ ID NOs. 9, 11, and 50. [Invention 1058] An anti-B7H4 antibody or its antigen-binding fragment, comprising the light chain CDRs of SEQ ID NOs. 14, 47, and 16, and the heavy chain CDRs of SEQ ID NOs. 9, 11, and 50. [Invention 1059] An anti-B7H4 antibody or its antigen-binding fragment comprising a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 46, and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 49. [Invention 1060] An anti-B7H4 antibody light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with sequence number 52. [Invention 1061] An anti-B7H4 antibody light chain containing a CDR having the amino acid sequences of SEQ ID NOs. 53, 54, and 55. [Invention 1062] An anti-B7H4 antibody heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 65. [Invention 1063] An anti-B7H4 antibody heavy chain containing a CDR having the amino acid sequences of SEQ ID NOs. 66, 67, and 68. [Invention 1064] An anti-B7H4 antibody or its antigen-binding fragment, comprising the light chain CDRs of SEQ ID NOs. 53, 54, and 55, and the heavy chain CDRs of SEQ ID NOs. 66, 67, and 68. [Invention 1065] An anti-B7H4 antibody or its antigen-binding fragment comprising a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 52, and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 65. [Invention 1066] An antibody light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with sequence number 85. [Invention 1067] An antibody light chain containing a CDR having the amino acid sequences of SEQ ID NOs. 4, 86, and 87. [Invention 1068] An antibody heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with sequence number 89. [Invention 1069] An antibody heavy chain containing a CDR having the amino acid sequences of SEQ ID NOs. 9, 90, and 91. [Invention 1070] An anti-B7H4 antibody or its antigen-binding fragment, comprising the light chain CDRs of SEQ ID NOs. 4, 86, and 87, and the heavy chain CDRs of SEQ ID NOs. 9, 90, and 91. [Invention 1071] An anti-B7H4 antibody or its antigen-binding fragment comprising a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 85, and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 89. [Invention 1072] An antibody light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with sequence number 93. [Invention 1073] Antibody light chains containing CDRs having amino acid sequences of SEQ ID NOs. 94, 95, and 96. [Invention 1074] An antibody heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with sequence number 98. [Invention 1075] Antibody heavy chain containing CDRs having amino acid sequences 9, 99, and 100. [Invention 1076] An anti-B7H4 antibody or its antigen-binding fragment, comprising the light chain CDRs of SEQ ID NOs. 94, 95, and 96, and the heavy chain CDRs of SEQ ID NOs. 9, 99, and 100. [Invention 1077] An anti-B7H4 antibody or its antigen-binding fragment comprising a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 93, and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 98. [Invention 1078] An antibody light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with sequence number 102. [Invention 1079] Antibody light chains containing CDRs having the amino acid sequences of SEQ ID NOs. 103, 104, and 105. [Invention 1080] An antibody heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with sequence number 107. [Invention 1081] Antibody heavy chain containing CDRs having amino acid sequences 9, 108, and 109. [Invention 1082] An anti-B7H4 antibody or its antigen-binding fragment, comprising the light chain CDRs of SEQ ID NOs. 103, 104, and 105, and the heavy chain CDRs of SEQ ID NOs. 9, 108, and 109. [Invention 1083] An anti-B7H4 antibody or its antigen-binding fragment comprising a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 102, and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 107. [Invention 1084] An antibody light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with sequence number 111. [Invention 1085] An antibody light chain containing a CDR having the amino acid sequences of SEQ ID NOs. 95, 112, and 113. [Invention 1086] An antibody heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with sequence number 115. [Invention 1087] An antibody heavy chain containing a CDR having the amino acid sequences of SEQ ID NOs. 116, 117, and 118. [Invention 1088] An anti-B7H4 antibody or its antigen-binding fragment, comprising the light chain CDRs of SEQ ID NOs. 95, 112, and 113, and the heavy chain CDRs of SEQ ID NOs. 116, 117, and 118. [Invention 1089] An anti-B7H4 antibody or its antigen-binding fragment comprising a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 111, and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 115. [Invention 1090] An anti-B7H4 antibody light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with sequence number 120. [Invention 1091] An anti-B7H4 antibody light chain containing a CDR having the amino acid sequences of SEQ ID NOs. 54, 55, and 121. [Invention 1092] An anti-B7H4 antibody heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with sequence number 123. [Invention 1093] An anti-B7H4 antibody heavy chain containing CDRs having amino acid sequences 66, 124, and 125. [Invention 1094] An anti-B7H4 antibody or its antigen-binding fragment, comprising the light chain CDRs of SEQ ID NOs. 54, 55, and 121, and the heavy chain CDRs of SEQ ID NOs. 66, 124, and 125. [Invention 1095] An anti-B7H4 antibody or its antigen-binding fragment comprising a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 120, and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 123. [Invention 1096] An anti-B7H4 antibody heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with sequence number 128. [Invention 1097] An anti-B7H4 antibody heavy chain containing CDRs having amino acid sequences 9, 129, and 130. [Invention 1098] An anti-B7H4 antibody or its antigen-binding fragment, comprising the light chain CDRs of SEQ ID NOs. 14, 15, and 16, and the heavy chain CDRs of SEQ ID NOs. 9, 129, and 130. [Invention 1099] An anti-B7H4 antibody or its antigen-binding fragment comprising a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 13, and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 128. [Invention 1100] An anti-B7H4 antibody or its antigen-binding fragment comprising a light chain having one of the amino acid sequences of SEQ ID NOs: 3, 13, 46, 52, 85, 93, 102, 111, or 120. [Invention 1101] An anti-B7H4 antibody or its antigen-binding fragment comprising a heavy chain having one of the amino acid sequences of SEQ ID NOs: 8, 29, 49, 65, 89, 98, 107, 115, 123, or 128. [Invention 1102] An anti-B7H4 antibody or its antigen-binding fragment comprising a light chain having one of the amino acid sequences of SEQ ID NOs: 3, 13, 46, 52, 85, 93, 102, 111, or 120, and a heavy chain having one of the amino acid sequences of SEQ ID NOs: 8, 29, 49, 65, 89, 98, 107, 115, 123, or 128. [Invention 1103] An anti-B7H4 antibody or its antigen-binding fragment, comprising three light chain CDRs having amino acid sequences 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. [Invention 1104] An anti-B7H4 antibody or its antigen-binding fragment, comprising three heavy chain CDRs having amino acid sequences 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. [Invention 1105] An anti-B7H4 antibody or its antigen-binding fragment comprising three light chain CDRs having amino acid sequences 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 chain CDRs having amino acid sequences 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. [Invention 1106] An anti-B7H4 antibody or its antigen-binding fragment comprising six complementarity-determining regions (CDRs), The aforementioned CDR Three light chain CDRs of a polypeptide selected from the group consisting of SEQ ID NOs: 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 containing at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity with SEQ ID NOs: 4, 5, 6, 14, 15, 16, 49, 55, 56, 57, 88, 89, 96, 97, 97, 105, 106, 107, 114, 115, or 123, and three heavy chain CDRs of a polypeptide selected from the group consisting of SEQ ID NOs. 9, 10, 11, 31, 52, 68, 69, 70, 92, 93, 101, 102, 118, 119, 120, 126, 127, 131, or 132, or variants thereof containing at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity with SEQ ID NOs. 9, 10, 11, 31, 52, 68, 69, 70, 92, 93, 101, 102, 118, 119, 120, 126, 127, 131, or 132. Includes, The antibody or its antigen-binding fragment binds to B7-H4. The anti-B7H4 antibody or its antigen-binding fragment. [Invention 1107] An anti-B7H4 antibody or its antigen-binding fragment comprising a light chain variable domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with 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 with any one of SEQ ID NOs: 34, 35, 36, or 37. [Invention 1108] An anti-B7H4 antibody or its antigen-binding fragment comprising a light chain having one of the amino acid sequences of SEQ ID NOs. 24, 25, 26, 27, or 28, and a heavy chain having one of the amino acid sequences of SEQ ID NOs. 38, 39, 40, or 41. [Invention 1109] An anti-B7H4 antibody or its antigen-binding fragment comprising a light chain having one of the amino acid sequences of SEQ ID NOs. 24, 25, 26, 27, or 28, and a heavy chain having one of the amino acid sequences of SEQ ID NOs. 42, 43, 44, or 45. [Invention 1110] An anti-B7H4 antibody or its antigen-binding fragment comprising a light chain variable domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with any one of sequence numbers 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 with any one of sequence numbers 70, 71, 72, 73, or 74. [Invention 1111] An anti-B7H4 antibody or its antigen-binding fragment comprising a light chain having one of the amino acid sequences of SEQ ID NOs. 62, 63, or 64, and a heavy chain having one of the amino acid sequences of SEQ ID NOs. 75, 76, 77, 78, or 79. [Invention 1112] An anti-B7H4 antibody or its antigen-binding fragment comprising a light chain having one of the amino acid sequences of SEQ ID NOs. 62, 63, or 64, and a heavy chain having one of the amino acid sequences of SEQ ID NOs. 80, 81, 82, 83, or 84. [Invention 1113] An anti-B7H4 antibody or its antigen-binding fragment comprising two light chains and two heavy chains, The two light chains include a polypeptide selected from the group consisting of SEQ ID NOs: 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 with SEQ ID NOs: 24, 25, 26, 27, or 28. The two heavy chains include a polypeptide selected from the group consisting of SEQ ID NOs: 38, 39, 40, or 41, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity with SEQ ID NOs: 38, 39, 40, or 41. The antibody or its antigen-binding fragment binds to B7-H4. The anti-B7H4 antibody or its antigen-binding fragment. [Invention 1114] An anti-B7H4 antibody or its antigen-binding fragment comprising two light chains and two heavy chains, The two light chains include a polypeptide selected from the group consisting of SEQ ID NOs: 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 with SEQ ID NOs: 24, 25, 26, 27, or 28. The two heavy chains include a polypeptide selected from the group consisting of SEQ ID NOs: 42, 43, 44, or 45, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity with SEQ ID NOs: 42, 43, 44, or 45. The antibody or its antigen-binding fragment binds to B7-H4. The anti-B7H4 antibody or its antigen-binding fragment. [Invention 1115] An anti-B7H4 antibody or its antigen-binding fragment comprising two light chains and two heavy chains, The two light chains include a polypeptide selected from the group consisting of SEQ ID NOs. 62, 63, or 64, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity with SEQ ID NOs. 62, 63, or 64. The two heavy chains include a polypeptide selected from the group consisting of SEQ ID NOs. 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 with SEQ ID NOs. 75, 76, 77, 78, or 79. The antibody or its antigen-binding fragment binds to B7-H4. The anti-B7H4 antibody or its antigen-binding fragment. [Invention 1116] An anti-B7H4 antibody or its antigen-binding fragment comprising two light chains and two heavy chains, The two light chains include a polypeptide selected from the group consisting of SEQ ID NOs. 62, 63, or 64, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity with SEQ ID NOs. 62, 63, or 64. The two heavy chains include a polypeptide selected from the group consisting of SEQ ID NOs. 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 with SEQ ID NOs. 80, 81, 82, 83, or 84. The antibody or its antigen-binding fragment binds to B7-H4. The anti-B7H4 antibody or its antigen-binding fragment. [Invention 1117] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 24 and a heavy chain having the amino acid sequence of SEQ ID NO: 38. [Invention 1118] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 24 and a heavy chain having the amino acid sequence of SEQ ID NO: 39. [Invention 1119] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 24 and a heavy chain having the amino acid sequence of SEQ ID NO: 40. [Invention 1120] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 24 and a heavy chain having the amino acid sequence of SEQ ID NO: 41. [Invention 1121] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 24 and a heavy chain having the amino acid sequence of SEQ ID NO: 42. [Invention 1122] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 24 and a heavy chain having the amino acid sequence of SEQ ID NO: 43. [Invention 1123] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 24 and a heavy chain having the amino acid sequence of SEQ ID NO: 44. [Invention 1124] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 24 and a heavy chain having the amino acid sequence of SEQ ID NO: 45. [Invention 1125] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 25 and a heavy chain having the amino acid sequence of SEQ ID NO: 38. [Invention 1126] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 25 and a heavy chain having the amino acid sequence of SEQ ID NO: 39. [Invention 1127] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 25 and a heavy chain having the amino acid sequence of SEQ ID NO: 40. [Invention 1128] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 25 and a heavy chain having the amino acid sequence of SEQ ID NO: 41. [Invention 1129] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 25 and a heavy chain having the amino acid sequence of SEQ ID NO: 42. [Invention 1130] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 25 and a heavy chain having the amino acid sequence of SEQ ID NO: 43. [Invention 1131] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 25 and a heavy chain having the amino acid sequence of SEQ ID NO: 44. [Invention 1132] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 25 and a heavy chain having the amino acid sequence of SEQ ID NO: 45. [Invention 1133] An anti-B7H4 antibody or its antigen-binding fragment comprising a light chain having the amino acid sequence of SEQ ID NO: 26 and a heavy chain having the amino acid sequence of SEQ ID NO: 38. [Invention 1134] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 26 and a heavy chain having the amino acid sequence of SEQ ID NO: 39. [Invention 1135] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 26 and a heavy chain having the amino acid sequence of SEQ ID NO: 40. [Invention 1136] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 26 and a heavy chain having the amino acid sequence of SEQ ID NO: 41. [Invention 1137] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 26 and a heavy chain having the amino acid sequence of SEQ ID NO: 42. [Invention 1138] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 26 and a heavy chain having the amino acid sequence of SEQ ID NO: 43. [Invention 1139] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 26 and a heavy chain having the amino acid sequence of SEQ ID NO: 44. [Invention 1140] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 26 and a heavy chain having the amino acid sequence of SEQ ID NO: 45. [Invention 1141] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 27 and a heavy chain having the amino acid sequence of SEQ ID NO: 38. [Invention 1142] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 27 and a heavy chain having the amino acid sequence of SEQ ID NO: 39. [Invention 1143] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 27 and a heavy chain having the amino acid sequence of SEQ ID NO: 40. [Invention 1144] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 27 and a heavy chain having the amino acid sequence of SEQ ID NO: 41. [Invention 1145] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 27 and a heavy chain having the amino acid sequence of SEQ ID NO: 42. [Invention 1146] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 27 and a heavy chain having the amino acid sequence of SEQ ID NO: 43. [Invention 1147] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 27 and a heavy chain having the amino acid sequence of SEQ ID NO: 44. [Invention 1148] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 27 and a heavy chain having the amino acid sequence of SEQ ID NO: 45. [Invention 1149] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 28 and a heavy chain having the amino acid sequence of SEQ ID NO: 38. [Invention 1150] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 28 and a heavy chain having the amino acid sequence of SEQ ID NO: 39. [Invention 1151] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 28 and a heavy chain having the amino acid sequence of SEQ ID NO: 40. [Invention 1152] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 28 and a heavy chain having the amino acid sequence of SEQ ID NO: 41. [Invention 1153] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 28 and a heavy chain having the amino acid sequence of SEQ ID NO: 42. [Invention 1154] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 28 and a heavy chain having the amino acid sequence of SEQ ID NO: 43. [Invention 1155] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 28 and a heavy chain having the amino acid sequence of SEQ ID NO: 44. [Invention 1156] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 28 and a heavy chain having the amino acid sequence of SEQ ID NO: 45. [Invention 1157] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 62 and a heavy chain having the amino acid sequence of SEQ ID NO: 75. [Invention 1158] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 62 and a heavy chain having the amino acid sequence of SEQ ID NO: 76. [Invention 1159] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 62 and a heavy chain having the amino acid sequence of SEQ ID NO: 77. [Invention 1160] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 62 and a heavy chain having the amino acid sequence of SEQ ID NO: 78. [Invention 1161] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 62 and a heavy chain having the amino acid sequence of SEQ ID NO: 79. [Invention 1162] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 62 and a heavy chain having the amino acid sequence of SEQ ID NO: 80. [Invention 1163] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 62 and a heavy chain having the amino acid sequence of SEQ ID NO: 81. [Invention 1164] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 62 and a heavy chain having the amino acid sequence of SEQ ID NO: 82. [Invention 1165] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 62 and a heavy chain having the amino acid sequence of SEQ ID NO: 83. [Invention 1166] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 62 and a heavy chain having the amino acid sequence of SEQ ID NO: 84. [Invention 1167] An anti-B7H4 antibody or its antigen-binding fragment comprising a light chain having the amino acid sequence of SEQ ID NO: 63 and a heavy chain having the amino acid sequence of SEQ ID NO: 75. [Invention 1168] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 63 and a heavy chain having the amino acid sequence of SEQ ID NO: 76. [Invention 1169] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 63 and a heavy chain having the amino acid sequence of SEQ ID NO: 77. [Invention 1170] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 63 and a heavy chain having the amino acid sequence of SEQ ID NO: 78. [Invention 1171] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 63 and a heavy chain having the amino acid sequence of SEQ ID NO: 79. [Invention 1172] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 63 and a heavy chain having the amino acid sequence of SEQ ID NO: 80. [Invention 1173] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 63 and a heavy chain having the amino acid sequence of SEQ ID NO: 81. [Invention 1174] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 63 and a heavy chain having the amino acid sequence of SEQ ID NO: 82. [Invention 1175] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 63 and a heavy chain having the amino acid sequence of SEQ ID NO: 83. [Invention 1176] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 63 and a heavy chain having the amino acid sequence of SEQ ID NO: 84. [Invention 1177] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 64 and a heavy chain having the amino acid sequence of SEQ ID NO: 75. [Invention 1178] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 64 and a heavy chain having the amino acid sequence of SEQ ID NO: 76. [Invention 1179] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 64 and a heavy chain having the amino acid sequence of SEQ ID NO: 77. [Invention 1180] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 64 and a heavy chain having the amino acid sequence of SEQ ID NO: 78. [Invention 1181] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 64 and a heavy chain having the amino acid sequence of SEQ ID NO: 79. [Invention 1182] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 64 and a heavy chain having the amino acid sequence of SEQ ID NO: 80. [Invention 1183] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 64 and a heavy chain having the amino acid sequence of SEQ ID NO: 81. [Invention 1184] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 64 and a heavy chain having the amino acid sequence of SEQ ID NO: 82. [Invention 1185] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 64 and a heavy chain having the amino acid sequence of SEQ ID NO: 83. [Invention 1186] An anti-B7H4 antibody or its antigen-binding fragment, comprising a light chain having the amino acid sequence of SEQ ID NO: 64 and a heavy chain having the amino acid sequence of SEQ ID NO: 84. [Modes for carrying out the invention]

[0107] Detailed description of the invention I. Definition As used herein, a molecule is said to be able to “immunospecifically bind” to a second molecule if the binding exhibits the specificity and affinity of the antibody to its alloantigen. An antibody is said to be able to immunospecifically bind to a target region or conformation ("epitope") of an antigen if such binding involves an antigen-recognition site on 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 an antigen-recognition site, as determined by, for example, an immunoassay, a BIACORE® assay, or other assay known in the art, but does not bind to completely unrelated antigens. However, in some embodiments, the antibody (and its antigen-binding fragment) does not cross-react with other antigens. Antibodies may also bind to other molecules in ways that are not immunospecific to FcR receptors, for example, by binding domains in other regions / domains of the molecule that do not involve an antigen-recognition site, such as an Fc region.

[0108] As used herein, a molecule is said to “physiospecifically” bind to a second molecule if such binding exhibits the receptor’s specificity and affinity for its homobinding ligand. A molecule can physiologically bind to two or more other molecules.

[0109] As used herein, the term “antibody” is intended to refer to an immunoglobulin molecule having a “variable region” antigen recognition site and containing an antigen-binding fragment of the antibody. The term “variable region” is intended to distinguish such a domain of immunoglobulin from domains widely shared by antibodies (e.g., the antibody Fc domain). The variable region includes a “hypervariable region” in which residues are involved in antigen binding. The hypervariable region consists of amino acid residues from the "complementarity-determining region" or "CDR" (i.e., typically approximately residues 24-34 (Ll), 50-56 (L2), and 89-97 (L3) in the light chain variable domain, and approximately residues 27-35 (Hl), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or residues from the "hypervariable loop" (i.e., residues 26-32 (Ll), 50-52 (L2), and 91-96 (L3) in the light chain variable domain, and residues 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) is included. "Framework region" or "FR" residues are variable domain residues other than the hypervariable region residues as defined herein.The term antibody includes monoclonal antibodies, polyspecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, and camelid antibodies (see, for example, Muyldermans el 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. WO94 / 04678 and WO94 / 25591, and U.S. Patent No. 6,005,079), single-stranded Fvs (scFv) (see, for example, Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New See York, pp. 269-315 (1994), single-chain antibodies, disulfide-linked Fv (sdFv), intra-bodies, diabodies, triabodies, tetrabodies, Bis-scFv, minibodies, Fab2, Fab3, and anti-idiotype (anti-Id) antibodies (e.g., anti-Id and anti-anti-Id antibodies against antibodies). In particular, such antibodies include immunoglobulin molecules of any kind (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass.

[0110] As used herein, the term “antigen-binding fragment” of an antibody refers to one or more portions of an antibody that include the antibody’s complementarity-determining region (“CDR”) and, optionally, framework residues containing the antibody’s “variable region” antigen-recognition site, and that exhibit the ability to bind immunospecifically to an antigen. Such fragments include Fab’, F(ab’)2, Fv, single-stranded (ScFv), and their variants, naturally occurring variants, as well as fusion proteins containing the antibody’s “variable region” antigen-recognition site and heterologous proteins (e.g., toxins, antigen-recognition sites of different antigens, enzymes, receptors, or receptor ligands).

[0111] As used herein, the term “fragment” refers to a peptide or polypeptide comprising an amino acid sequence of at least 5 consecutive amino acid residues, at least 10 consecutive amino acid residues, at least 15 consecutive amino acid residues, at least 20 consecutive amino acid residues, at least 25 consecutive amino acid residues, at least 40 consecutive amino acid residues, at least 50 consecutive amino acid residues, at least 60 consecutive amino acid residues, at least 70 consecutive amino acid residues, at least 80 consecutive amino acid residues, at least 90 consecutive amino acid residues, at least 100 consecutive amino acid residues, at least 125 consecutive amino acid residues, at least 150 consecutive amino acid residues, at least 175 consecutive amino acid residues, at least 200 consecutive amino acid residues, or at least 250 consecutive amino acid residues.

[0112] As used herein, the term “modulate” relates to the ability to alter an effect, outcome, or activity (e.g., signaling). Such modulation may be agonistic or antagonistic. Antagonistic modulation may be partial (i.e., attenuate but not eliminate) or may completely eliminate such activity (e.g., neutralize). Modulation may include the internalization of a receptor after antibody binding or a reduction in receptor expression on target cells. Agonistic modulation may enhance or otherwise increase or enhance activity (e.g., signaling). In further embodiments, such modulation may alter the nature of the induced signaling by altering the nature of the interaction between a ligand and its homologous receptor. For example, a molecule may alter its overall activity by altering its ability to bind to other ligands or receptors by binding to a ligand or receptor. In some embodiments, such modulation provides a change of at least 10% of measurable immune system activity, a change of at least 50% of such activity, or a change of at least 2x, 5x, 10x, or at least 100x of such activity.

[0113] The term "substantially" is intended to indicate that the observed effect is physiologically or therapeutically relevant when used in the context of binding or demonstrated effects. Therefore, for example, if the degree of blockade is physiologically or therapeutically relevant (e.g., if such a degree 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), then the molecule can substantially block the activity of a ligand or receptor. Similarly, a molecule is said to have substantially the same immunospecificity and / or characteristics as another molecule if such immunospecificity 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.

[0114] As used herein, the term "co-stimulus" includes both positive co-stimulus signals (e.g., signals that result in enhanced activity) and negative co-stimulus signals (e.g., signals that result in inhibited activity).

[0115] The term “derivative” refers to an antibody or its antigen-binding fragment that binds immunospecifically to the same target as the parent antibody or reference antibody, but whose amino acid sequence differs from that of the parent antibody or reference antibody or its antigen-binding fragment by including one, two, three, four, five or more amino acid substitutions, additions, deletions, or modifications. In some embodiments, such derivatives will have substantially the same immunospecificity and / or characteristics as the parent antibody or reference antibody or its antigen-binding fragment, or have the same immunospecificity and characteristics. The amino acid substitutions or additions of such derivatives may include naturally occurring (i.e., DNA-encoded) or naturally occurring amino acid residues. The term “derivative” includes, for example, chimeric variants or humanized variants, as well as variants having altered CH1, hinge, CH2, CH3, or CH4 regions, to form antibodies having variant Fc regions that exhibit enhanced or impaired effector or binding characteristics, for example.

[0116] As used herein, "chimeric antibody" refers to a molecule in which different parts of the antibody are derived from different immunoglobulin molecules, such as antibodies having a variable region derived from a non-human antibody and a constant region of human immunoglobulin.

[0117] As used herein, the term “humanized antibody” refers to an immunoglobulin containing a human framework region and one or more CDRs derived from non-human (usually mouse or rat) immunoglobulins. The non-human immunoglobulin providing the CDRs is referred to as the “donor,” and the human immunoglobulin providing the framework is referred to as the “acceptor.” A constant region is not required, but if present, it should be substantially identical to the human immunoglobulin constant region, i.e., at least about 85–99%, or at least about 95% identical. Thus, except possibly in the case of CDRs, all portions of a humanized immunoglobulin are substantially identical to the corresponding portions of the natural human immunoglobulin sequence. A humanized antibody is an antibody containing humanized light chain immunoglobulin and humanized heavy chain immunoglobulin. For example, a humanized antibody would not include a typical chimeric antibody, for example, because the entire variable region of a chimeric antibody is non-human.

[0118] The term "endogenous concentration" refers to the level at which a molecule is naturally expressed by cells (which may be normal cells, cancer cells, or infected cells) in the absence of an expression vector or recombinant promoter.

[0119] As used herein, the terms “treat,” “treating,” “treatment,” and “therapeutic use” refer to the elimination, reduction, or improvement of one or more symptoms of a disease or disorder. As used herein, “therapeutic dose” refers to the amount of therapeutic agent sufficient to mediate a clinically relevant elimination, reduction, or improvement of such symptoms. An effect is clinically relevant if the magnitude of the effect is sufficient to affect the health or prognosis of the recipient. A therapeutic dose may also refer to the amount of therapeutic agent sufficient to delay or minimize the onset of a disease, for example, to delay or minimize the spread of cancer. A therapeutic dose may also refer to the amount of therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease.

[0120] As used herein, the term “preventive agent” refers to an active substance that may be used to prevent a disorder or disease before the detection of any symptom of such disorder or disease. “Preventive effective” amount is the amount of preventive agent sufficient to mediate such protection. Preventive effective amount may also refer to the amount of preventive agent that provides a preventive benefit in the prevention of disease.

[0121] As used herein, the term “cancer” refers to a neoplasm or tumor resulting from the abnormal and uncontrolled proliferation of cells. As used herein, cancer clearly includes leukemia and lymphoma. The term “cancer” refers to a disease in which cells have the potential to metastasize distally and exhibit phenotypic traits distinct from those of non-cancerous cells, such as the formation of colonies in three-dimensional substrates like soft agar, or the formation of tubular networks or web-like matrices in three-dimensional basement membranes or extracellular matrix preparations. Non-cancerous cells do not form colonies in soft agar and form distinct spherical structures in three-dimensional basement membranes or extracellular matrix preparations.

[0122] As used herein, “immune cells” refers to any cell of hematopoietic origin, including, but not limited to, T cells, B cells, monocytes, dendritic cells, and macrophages.

[0123] As used herein, “inflammatory molecules” refers to molecules that induce an inflammatory response, including, but not limited to, cytokines and metalloproteinases, such as, but not limited to, IL-1β, TNF-α, TGF-β, IFN-γ, IL-18, IL-17, IL-6, IL-23, IL-22, IL-21, and MMPs.

[0124] As used herein, "valence" refers to the number of available binding sites per molecule.

[0125] As used herein, the terms “immunologic,” “immunological,” or “immune” response refer to the occurrence of a beneficial humoral (antibody-mediated) and / or cellular (antigen-specific T cell-mediated or secreted product-mediated) response directed to a peptide in a recipient patient. Such a response may be an active response induced by the administration of an immunogen or a passive response induced by the administration of an antibody or primed T cell. Cellular immune responses present polypeptide epitopes associated with class I or class II MHC molecules and antigen-specific CD4 + T helper cells and / or CD8 + It is induced by the activation of cytotoxic T cells. The response may also involve the activation or recruitment of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia, eosinophils, neutrophils, or other components of innate immunity. The presence of a cell-mediated immune response can be detected in proliferation assays (CD4 + This can be determined by a T cell (T cell) or CTL (cytotoxic T lymphocyte) assay. 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 immunized syngenes and measuring their protective or therapeutic effects in a second target.

[0126] "Immunogenic substances" or "immunogens," when administered to mammals in combination with an adjuvant (optional), can induce an immune response against themselves.

[0127] As used herein, the terms “individual,” “host,” “subject,” and “patient” are used interchangeably and are not limited herein, but refer to mammals, including humans, rodents such as mice and rats, and other laboratory animals.

[0128] As used herein, the term “polypeptide” refers to a chain of amino acids of any length, regardless of modifications (e.g., phosphorylation or glycosylation). The term polypeptide includes proteins and their fragments. Polypeptides can be “exogenous,” meaning they are foreign to the host cell that utilizes them, such as human polypeptides produced by bacterial cells. Polypeptides are disclosed herein as amino acid residue sequences. These sequences are written from left to right, from the amino terminus to the carboxyl terminus. According to standard nomenclature, amino acid residue sequences are represented by either three-letter or one-letter codes, as shown below: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, 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 (Val, V).

[0129] As used herein, the term “variant” refers to a polypeptide or polynucleotide that is different from a reference polypeptide or polynucleotide but retains essential properties. A typical variant of a polypeptide differs in its amino acid sequence from another reference polypeptide. Generally, the differences are limited such that the sequences of the reference polypeptide and the variant are closely similar as a whole and identical in many regions. The variant and the reference polypeptide may differ in their amino acid sequence by one or more modifications (e.g., substitution, addition, and / or deletion). The substituted or inserted amino acid residues may or may not be encoded by the genetic code. Variants of a polypeptide may be naturally occurring, such as allele variants, or they may be variants whose natural occurrence is not known.

[0130] Modifications and alterations can be made to the structure of the polypeptides of this disclosure, and molecules with similar characteristics to the polypeptides (e.g., conserved amino acid substitutions) can still be obtained. For example, certain amino acids can be substituted for other amino acids in the sequence without significant loss of activity. Since the interaction ability and properties of a polypeptide determine its biological functional activity, specific amino acid sequence substitutions can be made within the polypeptide sequence, and polypeptides with similar properties can still be obtained.

[0131] When making such modifications, the hydrophilicity index of amino acids can be taken into consideration. The importance of the hydrophilic amino acid index in conferring interactional biological function to polypeptides is generally understood in the art. It is known that certain amino acids can be substituted with other amino acids having similar hydrophilicity indices or scores, resulting in polypeptides with similar biological activity. Each amino acid is assigned a hydrophilicity index based on its hydrophobic and charge properties. These 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), glutamic acid (-3.5), glutamine (-3.5), asparagine (-3.5), lysine (-3.9), and arginine (-4.5).

[0132] The relative hydrophilic characteristics of amino acids are thought to determine the secondary structure of the resulting polypeptide, which then defines the interaction between the polypeptide and other molecules such as enzymes, substrates, receptors, antibodies, antigens, and cofactors. It is known in the art that amino acids can be substituted with other amino acids having similar hydropathic indices, and still functionally equivalent polypeptides can be obtained. In such substitutions, substitutions of amino acids with a hydropathic index of ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0133] Similar amino acid substitutions can also be made based on hydrophilicity, particularly when the polypeptide or peptide having equivalent biological function is intended for use in immunological embodiments. The following hydrophilicity values ​​have been assigned to the amino acid residues: arginine (+3.0), lysine (+3.0), aspartic acid (+3.0±1), glutamic acid (+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), and tryptophan (-3.4). It is understood that amino acids can be substituted with other amino acids having similar hydrophilic values, and that still biologically equivalent, and especially immunologically equivalent, polypeptides can be obtained. In such modifications, substitutions of amino acids with hydrophilic values ​​of ±2 are preferred, those of ±1 are particularly preferred, and those of ±0.5 are even more particularly preferred.

[0134] As described above, amino acid substitutions are generally based on the relative similarity of amino acid side-chain substituents, such as their hydrophobicity, hydrophilicity, charge, and size. Exemplary substitutions considering various aforementioned characteristics are well known to those skilled in the art and include (Ala:Gly,Ser), (Arg:Lys), (Asn:Gln,His), (Asp:Glu,Cys,Ser), (Gln:Asn), (Glu:Asp), (Gly:Ala), (His:Asn,Gln), (Ile:Leu,Val), (Leu:Ile,Val), (Lys:Arg), (Met:Leu,Tyr), (Ser:Thr), (Thr:Ser), (Trp:Tyr), (Tyr:Trp,Phe), and (Val:Ile,Leu) (original residue: exemplary substitution). Accordingly, embodiments of the present disclosure are intended to provide functional or bioequivalents of the polypeptides described above. In particular, the polypeptide embodiments may include variants having approximately 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with the polypeptide of interest.

[0135] The term “sequence identity percentage (%)” is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical to a nucleotide or amino acid in a reference nucleic acid sequence after the sequences have been aligned and gaps introduced as necessary to achieve the maximum sequence identity percentage. Alignment for the purpose of determining sequence identity percentage can be achieved in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithm necessary to achieve the maximum alignment over the entire length of the sequences being compared, can be determined by known methods.

[0136] For the purposes of this specification, the sequence identity % of a given nucleotide or amino acid sequence C to, with, or in relation to a given nucleic acid sequence D (or can be expressed as a given sequence C having or containing a specific sequence identity % to, with, or in relation to a given sequence D) is as follows: Fraction W / Z ×100 It is calculated as follows: In the formula, W is the number of nucleotides or amino acids scored as a perfect match by the sequence alignment program in the alignment of C and D of that program, and Z is the total number of nucleotides or amino acids in D. Please note that if the length of sequence C is not equal to the length of sequence D, the sequence identity % from C to D is not equal to the sequence identity % from D to C.

[0137] As used herein, the term “pharmaceutically acceptable carrier” includes any of the standard pharmaceutically acceptable carriers, such as phosphate-buffered saline, water and emulsions such as oil / water or water / oil emulsions, and various types of wetting agents.

[0138] II. Composition The high levels of B7-H4 expression found in many tumor tissues, such as human ovarian cancer, indicate the important role of B7-H4 in mediating immunosuppression. Furthermore, 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):87l-88l). The strength of B7-H4 expression in TAMs is significantly correlated with the number of Treg cells in the tumor. Furthermore, B7-H4 expressed on TAMs is associated with poor patient outcomes (Kryczek, I. et al. (2006) “B7-H4 Expression Identifies A Novel Suppressive Macrophage Population In Human Ovarian Carcinoma,” J.Exp.Med.203(4): 87 l-881). In addition, B7-H4 can be expressed on myeloid-derived suppressor cells (MDSCs), where it can exert immunosuppressive effects in viral infections (Garg, A et al. (2017) “Human Immunodeficiency Virus Type-l Myeloid Derived Suppressor Cells Inhibit Cytomegalovirus Inflammation through Interleukin-27 and B7-H4” Sci.Rep.Mar 24;7:44485). In uterine cancer studies, B7-H4 expression in the tumor microenvironment was associated with increased MDSC invasion (Vanderstraeten, A. et al. (2014) “Mapping the immunosuppressive environment in uterine tumors: effects for immunotherapy,” Cancer ImmunolImmunother. Jun;63(6):545-57).Therefore, B7-H4 may be expressed on tumor cells, TAMs, and / or MDSCs, where it may exert immunosuppressive signaling in cancer.

[0139] Neutrophils are a major component of the host's natural defense against infection and also contribute to autoimmune pathogenesis and chronic inflammation. During infection, neutrophils rapidly migrate to the site of inflammation, become activated, and initiate a cascade of defense mechanisms, including phagocytosis, toxicization, and degradation of microorganisms by antimicrobial and proteolytic proteins, along with the generation of reactive oxygen species. Neutrophils are also involved in the tissue degradation, remodeling, wound healing, and regulation of other inflammatory and adaptive immune components. Due to their short lifespan, neutrophils must be continuously replenished during infection and inflammation by proliferation from myeloid progenitor cells in the bone marrow. In vitro B7-H4 inhibits the proliferation of myeloid-derived neutrophil precursors, suggesting an inhibitory function of B7-H4 in neutrophil proliferation (Zhu, G. et al., Blood, 113:1759-1767 (2009)). Therefore, compositions that modulate B7-H4 signaling are provided.

[0140] A.B7-H4 sequence One embodiment provides a B7-H4 polypeptide. The polypeptide may contain the full-length B7-H4 amino acid sequence, a fragment or variant thereof, or a fusion protein thereof.

[0141] One embodiment provides a human B7-H4 protein or polypeptide thereof. The sequence of human B7-H4 is known in the art. For example, the consensus sequence of B7-H4 is: TIFF2026098127000001.tif17132 (SEQ ID NO: 1, Q7Z7D3 (VTCNl_HUMAN)) contains a signal sequence with amino acids 1-27 (underlined according to U.S. Patent Publication No. 2016 / 0039905, which is incorporated in its entirety by reference). Sica et al. show that amino acids 1-20 contain the signal sequence. UniProtKB accession number Q7Z7D3 indicates that the extracellular domain contains amino acids 25-259.

[0142] Another embodiment provides a murine B7-H4 protein and polypeptide. The sequence of murine B7-H4 is known in the art. For example, the consensus sequence of murine B7-H4 is TIFF2026098127000002.tif19128 (SEQ ID NO: 2, Q7TSP5 (VTCN1_MOUSE)), which is incorporated herein by reference in its entirety.

[0143] 1. Anti-B7-H4 antibody sequence a. B1A1 sequence One embodiment provides a murine monoclonal antibody produced by the hybridoma clone B1A1, which comprises two light chains and two heavy chains and specifically binds to B7-H4.

[0144] i. Light chain One embodiment provides a murine 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 the following amino acid sequence: TIFF2026098127000003.tif12164 and specifically binds to B7-H4.

[0145] The CDRs of SEQ ID NO: 3 are in bold and underlined, TIFF2026098127000004.tif19128.

[0146] Another embodiment provides a nucleic acid encoding the light chain (SEQ ID NO: 3).

[0147] An exemplary nucleic acid encoding the light chain (SEQ ID NO: 3) is TIFF2026098127000005.tif41165.

[0148] ii. Heavy chain One embodiment provides the following amino acid sequence: The present invention provides a mouse monoclonal antibody or its antigen-binding fragment having a heavy chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with TIFF2026098127000006.tif19164, and specifically binding to B7-H4.

[0149] The CDR of sequence number 8 is in bold and underlined. The filename is TIFF2026098127000007.tif19128.

[0150] Another embodiment provides a nucleic acid encoding a heavy chain (SEQ ID NO: 8).

[0151] An example nucleic acid encoding the heavy chain (SEQ ID NO: 8) is: The filename is TIFF2026098127000008.tif48165.

[0152] One embodiment provides an antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain with CDRs of SEQ ID NOs. 4, 5, and 6, and specifically binding to B7-H4.

[0153] One embodiment provides an antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a heavy chain with CDRs of SEQ ID NOs. 9, 10, and 11, and specifically binding to B7-H4.

[0154] One embodiment provides an antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, which has a light chain having CDRs of SEQ ID NOs. 4, 5, and 6, and a heavy chain having CDRs of SEQ ID NOs. 9, 10, and 11, and which specifically binds to B7-H4.

[0155] 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 with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 8 and specifically binding to B7-H4.

[0156] b. B1H1 sequence In one embodiment, the mouse monoclonal antibody is produced by hybridoma clone B1H1 and comprises two light chains and two heavy chains.

[0157] i. Light chain One embodiment provides a mouse 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 the following amino acid sequence: TIFF2026098127000009.tif12165.

[0158] The CDRs of SEQ ID NO: 13 are in bold and underlined and are TIFF2026098127000010.tif19128.

[0159] Another embodiment provides a nucleic acid encoding the light chain (SEQ ID NO: 13).

[0160] An exemplary nucleic acid encoding the light chain (SEQ ID NO: 13) is TIFF2026098127000011.tif41165.

[0161] One embodiment provides the following amino acid sequence: The present invention provides a monoclonal antibody or its antigen-binding fragment having a light chain constant domain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with TIFF2026098127000012.tif12164, and specifically binding to B7-H4.

[0162] ii. Humanized light chains One embodiment has the following amino acid sequence: The present invention provides an anti-B7-H4 monoclonal antibody or its antigen-binding fragment having a humanized light chain variable domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with one of TIFF2026098127000013.tif107165.

[0163] One embodiment has the following amino acid sequence: The present invention provides an anti-B7-H4 monoclonal antibody or its antigen-binding fragment having a humanized light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with one of TIFF2026098127000014.tif180167.

[0164] iii. Heavy chain One embodiment has the following amino acid sequence: The present invention provides a mouse monoclonal antibody or its antigen-binding fragment having a heavy chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with TIFF2026098127000015.tif19165, and specifically binding to B7-H4.

[0165] The CDR for sequence number 29 is in bold and underlined. The filename is TIFF2026098127000016.tif19128.

[0166] Another embodiment provides a nucleic acid that codes for a heavy chain (SEQ ID NO: 29).

[0167] An example nucleic acid encoding the heavy chain (SEQ ID NO: 29) is: The filename is TIFF2026098127000017.tif48166.

[0168] One embodiment has the following amino acid sequence: The present invention provides a mouse monoclonal antibody or its antigen-binding fragment having a heavy chain constant domain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with TIFF2026098127000018.tif41165, and specifically binding to B7-H4.

[0169] Underlined and bolded amino acids represent amino acids that differ from the mutated sequence.

[0170] Another embodiment has the following amino acid sequence: The present invention provides a mutant heavy chain constant domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with TIFF2026098127000019.tif41165.

[0171] Underlined and bolded amino acids represent amino acids that differ from the wild-type sequence.

[0172] iv. Humanized heavy chain One embodiment has the following amino acid sequence: The present invention provides an anti-B7-H4 monoclonal antibody or its antigen-binding fragment having a humanized heavy chain variable domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with one of TIFF2026098127000020.tif114165.

[0173] One embodiment has the following amino acid sequence: The present invention provides an anti-B7-H4 monoclonal antibody or its antigen-binding fragment having a humanized heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with one of TIFF2026098127000021.tif34165 or TIFF2026098127000022.tif223165.

[0174] In another embodiment, the monoclonal antibody or its antigen-binding fragment has the following amino acid sequence: It has a humanized heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with one of TIFF2026098127000023.tif201165 or TIFF2026098127000024.tif55165.

[0175] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain having the CDRs of SEQ ID NOs. 14, 15, and 16.

[0176] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a heavy chain containing the CDRs of SEQ ID NOs. 9, 11, and 30.

[0177] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or its antigen-binding fragment, having a light chain containing the CDRs of SEQ ID NOs. 14, 15, and 16, and a heavy chain containing the CDRs of SEQ ID NOs. 9, 11, and 30.

[0178] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or its antigen-binding fragment, having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 13, and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 29.

[0179] 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 with 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 with any one of SEQ ID NOs: 34, 35, 36, or 37.

[0180] One embodiment provides an anti-B7H4 antibody or its antigen-binding fragment, having a light chain having one of the amino acid sequences of SEQ ID NOs: 24, 25, 26, 27, or 28, and a heavy chain having one of the amino acid sequences of SEQ ID NOs: 38, 39, 40, or 41.

[0181] Another embodiment provides an anti-B7H4 antibody or its antigen-binding fragment, having a light chain having one of the amino acid sequences of SEQ ID NOs: 24, 25, 26, 27, or 28, and a heavy chain having one of the amino acid sequences of SEQ ID NOs: 42, 43, 44, or 45.

[0182] One embodiment provides an anti-B7H4 antibody or its antigen-binding fragment having two light chains and two heavy chains, wherein the two light chains comprise a polypeptide selected from the group consisting of SEQ ID NOs: 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 with SEQ ID NOs: 24, 25, 26, 27, or 28, and the two heavy chains comprise a polypeptide selected from the group consisting of SEQ ID NOs: 38, 39, 40, or 41, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity with SEQ ID NOs: 38, 39, 40, or 41, and the antibody or its antigen-binding fragment binds to B7-H4.

[0183] Another embodiment provides an antibody or an antigen-binding fragment having two light chains and two heavy chains, wherein the two light chains comprise a polypeptide selected from the group consisting of SEQ ID NOs: 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 with SEQ ID NOs: 24, 25, 26, 27, or 28, and the two heavy chains comprise a polypeptide selected from the group consisting of SEQ ID NOs: 42, 43, 44, or 45, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity with SEQ ID NOs: 42, 43, 44, or 45, and the antibody or antigen-binding fragment is bound to B7-H4.

[0184] c.B1H3 array In one embodiment, the mouse monoclonal antibody is produced by the hybridoma clone B1H3, contains two light chains and two heavy chains, and specifically binds to B7-H4.

[0185] i. Light chain One embodiment has the following amino acid sequence: The present invention provides an anti-B7-H4 mouse monoclonal antibody or its antigen-binding fragment having a light chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with TIFF2026098127000025.tif12164.

[0186] The CDR with sequence number 46 is in bold and underlined. The filename is TIFF2026098127000026.tif20128.

[0187] Another embodiment provides a nucleic acid encoding a light chain (SEQ ID NO: 46).

[0188] An exemplary nucleic acid encoding the light chain (SEQ ID NO: 46) is: The filename is TIFF2026098127000027.tif41165.

[0189] ii. Heavy chain One embodiment has the following amino acid sequence: The present invention provides an anti-B7-H4 mouse monoclonal antibody or its antigen-binding fragment having a heavy chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with TIFF2026098127000028.tif19165.

[0190] The CDR with sequence number 49 is in bold and underlined. The filename is TIFF2026098127000029.tif19128.

[0191] Another embodiment provides a nucleic acid encoding a heavy chain (SEQ ID NO: 49).

[0192] An example nucleic acid encoding the heavy chain (SEQ ID NO: 49) is: The filename is TIFF2026098127000030.tif48166.

[0193] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain containing the CDRs of SEQ ID NOs. 14, 16, and 47.

[0194] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a heavy chain containing the CDRs of SEQ ID NOs. 9, 11, and 50.

[0195] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or its antigen-binding fragment, having a light chain containing CDRs of SEQ ID NOs. 14, 16, and 47, and a heavy chain containing CDRs of SEQ ID NOs. 9, 11, and 50.

[0196] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 46, and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 49.

[0197] d.B1H10 sequence In one embodiment, the anti-B7H4 mouse monoclonal antibody is produced by the hybridoma clone B1H10 and contains two light chains and two heavy chains.

[0198] i. Light chain One embodiment has the following amino acid sequence: The present invention provides an anti-B7H4 mouse monoclonal antibody or its antigen-binding fragment having a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with TIFF2026098127000031.tif12164.

[0199] The CDR with sequence number 52 is in bold and underlined. The filename is TIFF2026098127000032.tif19128.

[0200] Another embodiment provides a nucleic acid that codes for a light chain (SEQ ID NO: 52).

[0201] An example nucleic acid encoding the light chain (SEQ ID NO: 52) is: The filename is TIFF2026098127000033.tif41166.

[0202] One embodiment has the following amino acid sequence: The present invention provides an anti-B7H4 monoclonal antibody or its antigen-binding fragment having a light chain constant domain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with TIFF2026098127000034.tif26165.

[0203] ii. Humanized B1H10 light chain In another embodiment, the anti-B7H4 monoclonal antibody or its antigen-binding fragment has the following amino acid sequence: It has a humanized light chain variable domain that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with one of TIFF2026098127000035.tif85165.

[0204] One embodiment has the following amino acid sequence: The present invention provides an anti-B7H4 monoclonal antibody or its antigen-binding fragment having a humanized light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with one of TIFF2026098127000036.tif107165.

[0205] iii. Heavy chain One embodiment has the following amino acid sequence: The present invention provides an anti-B7H4 mouse monoclonal antibody or its antigen-binding fragment having a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with TIFF2026098127000037.tif19166.

[0206] The CDR with sequence number 65 is in bold and underlined. The filename is TIFF2026098127000038.tif19128.

[0207] Another embodiment provides a nucleic acid encoding a heavy chain (SEQ ID NO: 65).

[0208] An example nucleic acid encoding the heavy chain (SEQ ID NO: 65) is: The filename is TIFF2026098127000039.tif48165.

[0209] iv. Humanized B1H10 heavy chain One embodiment has the following amino acid sequence: The present invention provides an anti-B7H4 monoclonal antibody or its antigen-binding fragment having a humanized heavy chain variable domain variant having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with one of TIFF2026098127000040.tif143165.

[0210] One embodiment has the following amino acid sequence: The present invention provides an anti-B7H4 monoclonal antibody or its antigen-binding fragment having a humanized heavy chain variant having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with one of TIFF2026098127000041.tif173165 or TIFF2026098127000042.tif150165.

[0211] One embodiment has the following amino acid sequence: The present invention provides an anti-B7H4 monoclonal antibody or its antigen-binding fragment having a humanized mutant heavy chain variant having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with one of TIFF2026098127000043.tif41165, TIFF2026098127000044.tif223165, or TIFF2026098127000045.tif55165.

[0212] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or its antigen-binding fragment, having a light chain containing the CDR of SEQ ID NO: 53, 54, or 55.

[0213] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or its antigen-binding fragment, having a heavy chain containing the CDR of SEQ ID NO: 66, 67, or 68.

[0214] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or its antigen-binding fragment, having a light chain containing the CDR of SEQ ID NO: 53, 54, or 55, and a heavy chain containing the CDR of SEQ ID NO: 66, 67, or 68.

[0215] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 52, and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 65.

[0216] Another embodiment provides an anti-B7H4 antibody or its antigen-binding fragment having a light chain variable domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with 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 with any one of SEQ ID NOs. 70, 71, 72, 73, or 74.

[0217] One embodiment provides an anti-B7H4 antibody or its antigen-binding fragment, having a light chain having one of the amino acid sequences of SEQ ID NOs. 62, 63, or 64, and a heavy chain having one of the amino acid sequences of SEQ ID NOs. 75, 76, 77, 78, or 79.

[0218] Another embodiment provides an anti-B7H4 antibody or its antigen-binding fragment, having a light chain having one of the amino acid sequences of SEQ ID NOs. 62, 63, or 64, and a heavy chain having one of the amino acid sequences of SEQ ID NOs. 80, 81, 82, 83, or 84.

[0219] One embodiment provides an anti-B7H4 antibody or its antigen-binding fragment having two light chains and two heavy chains, wherein the two light chains comprise a polypeptide selected from the group consisting of SEQ ID NOs. 62, 63, or 64, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity with SEQ ID NOs. 62, 63, or 64, and the two heavy chains comprise a polypeptide selected from the group consisting of SEQ ID NOs. 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 with SEQ ID NOs.

[0220] Another embodiment provides an anti-B7H4 antibody or its antigen-binding fragment having two light chains and two heavy chains, wherein the two light chains comprise a polypeptide selected from the group consisting of SEQ ID NOs. 62, 63, or 64, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity with SEQ ID NOs. 62, 63, or 64, and the two heavy chains comprise a polypeptide selected from the group consisting of SEQ ID NOs. 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 with SEQ ID NOs. 80, 81, 82, 83, or 84.

[0221] e.B2E6 array In one embodiment, the anti-B7H4 mouse monoclonal antibody is produced by the hybridoma clone B2E6 and contains two light chains and two heavy chains.

[0222] i. Light chain One embodiment has the following amino acid sequence: The present invention provides an anti-B7H4 mouse monoclonal antibody or its antigen-binding fragment having a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with TIFF2026098127000046.tif12164.

[0223] The CDR with sequence number 85 is in bold and underlined. The filename is TIFF2026098127000047.tif19128.

[0224] Another embodiment provides a nucleic acid that codes for a light chain (SEQ ID NO: 85).

[0225] An example nucleic acid encoding the light chain (SEQ ID NO: 85) is: The filename is TIFF2026098127000048.tif41164.

[0226] ii. Heavy chain One embodiment has the following amino acid sequence: The present invention provides an anti-B7H4 mouse monoclonal antibody or its antigen-binding fragment having a heavy chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with TIFF2026098127000049.tif19163.

[0227] The CDR with sequence number 89 is in bold and underlined. The filename is TIFF2026098127000050.tif19128.

[0228] Another embodiment provides a nucleic acid that codes for a heavy chain (SEQ ID NO: 89).

[0229] An example nucleic acid encoding the heavy chain (SEQ ID NO: 89) is: The filename is TIFF2026098127000051.tif48165.

[0230] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain having the CDRs of SEQ ID NOs. 4, 86, and 87.

[0231] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a heavy chain having the CDRs of SEQ ID NOs. 9, 90, and 91.

[0232] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or its antigen-binding fragment, having a light chain containing the CDRs of SEQ ID NOs. 4, 86, and 87, and a heavy chain containing the CDRs of SEQ ID NOs. 9, 90, and 91.

[0233] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 85, and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 89.

[0234] f.B4B3 array In one embodiment, the anti-B7H4 mouse monoclonal antibody is produced by the hybridoma clone B4B3 and contains two light chains and two heavy chains.

[0235] i. Light chain One embodiment has the following amino acid sequence: The present invention provides an anti-B7H4 mouse monoclonal antibody or its antigen-binding fragment having a light chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with TIFF2026098127000052.tif19163.

[0236] The CDR with sequence number 93 is in bold and underlined. The filename is TIFF2026098127000053.tif19128.

[0237] Another embodiment provides a nucleic acid encoding a light chain (SEQ ID NO: 93).

[0238] An example nucleic acid encoding the light chain (sequence number 93) is: The filename is TIFF2026098127000054.tif48166.

[0239] ii. Heavy chain One embodiment has the following amino acid sequence: The present invention provides an anti-B7H4 mouse monoclonal antibody or its antigen-binding fragment having a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with TIFF2026098127000055.tif19163.

[0240] The CDR with sequence number 98 is in bold and underlined. The filename is TIFF2026098127000056.tif19128.

[0241] Another embodiment provides a nucleic acid that codes for a heavy chain (SEQ ID NO: 98).

[0242] An example nucleic acid encoding the heavy chain (SEQ ID NO: 98) is: The filename is TIFF2026098127000057.tif48165.

[0243] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain containing the CDRs of SEQ ID NOs. 94, 95, and 96.

[0244] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a heavy chain containing the CDRs of SEQ ID NOs. 9, 99, and 100.

[0245] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or its antigen-binding fragment, having a light chain containing CDRs of SEQ ID NOs. 94, 95, and 96, and a heavy chain containing CDRs of SEQ ID NOs. 9, 99, and 100.

[0246] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 93, and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 98.

[0247] g.B4E11 array In one embodiment, the anti-B7H4 mouse monoclonal antibody is produced by the hybridoma clone B4E11 and contains two light chains and two heavy chains.

[0248] i. Light chain One embodiment has the following amino acid sequence: The present invention provides an anti-B7H4 mouse monoclonal antibody or its antigen-binding fragment having a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with TIFF2026098127000058.tif12164.

[0249] The CDR with sequence number 102 is in bold and underlined. The filename is TIFF2026098127000059.tif19128.

[0250] Another embodiment provides a nucleic acid encoding a light chain (SEQ ID NO: 102).

[0251] An example nucleic acid encoding the light chain (sequence number 102) is: The filename is TIFF2026098127000060.tif41165.

[0252] ii. Heavy chain One embodiment has the following amino acid sequence: The present invention provides an anti-B7H4 mouse monoclonal antibody or its antigen-binding fragment having a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with TIFF2026098127000061.tif19165.

[0253] The CDR with sequence number 107 is in bold and underlined. The filename is TIFF2026098127000062.tif19128.

[0254] Another embodiment provides a nucleic acid encoding a heavy chain (SEQ ID NO: 107).

[0255] An example nucleic acid encoding the heavy chain (sequence number 107) is: The filename is TIFF2026098127000063.tif48165.

[0256] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain containing the CDRs of SEQ ID NOs. 103, 104, and 105.

[0257] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a heavy chain containing the CDRs of SEQ ID NOs. 9, 108, and 109.

[0258] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or its antigen-binding fragment, having a light chain containing the CDRs of SEQ ID NOs. 103, 104, and 105, and a heavy chain containing the CDRs of SEQ ID NOs. 9, 108, and 109.

[0259] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 102, and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 107.

[0260] h.B6C8 array In one embodiment, the anti-B7H4 mouse monoclonal antibody is produced by the hybridoma clone B6C8 and contains two light chains and two heavy chains.

[0261] i. Light chain One embodiment has the following amino acid sequence: The present invention provides an anti-B7H4 mouse monoclonal antibody or its antigen-binding fragment having a light chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with TIFF2026098127000064.tif19165.

[0262] The CDR with sequence number 111 is in bold and underlined. The filename is TIFF2026098127000065.tif19128.

[0263] Another embodiment provides a nucleic acid encoding a light chain (SEQ ID NO: 111).

[0264] An example nucleic acid encoding the light chain (sequence number 111) is: The filename is TIFF2026098127000066.tif48165.

[0265] ii. Heavy chain One embodiment has the following amino acid sequence: The present invention provides an anti-B7H4 mouse monoclonal antibody or its antigen-binding fragment having a heavy chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with TIFF2026098127000067.tif19165.

[0266] The CDR with sequence number 115 is in bold and underlined. The filename is TIFF2026098127000068.tif19128.

[0267] Another embodiment provides a nucleic acid encoding a heavy chain (SEQ ID NO: 115).

[0268] An example nucleic acid encoding the heavy chain (SEQ ID NO: 115) is: The filename is TIFF2026098127000069.tif48164.

[0269] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain containing the CDRs of SEQ ID NOs. 97, 116, and 117.

[0270] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a heavy chain containing the CDRs of SEQ ID NOs. 118, 119, and 120.

[0271] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or its antigen-binding fragment, having a light chain containing the CDRs of SEQ ID NOs. 97, 114, and 115, and a heavy chain containing the CDRs of SEQ ID NOs. 118, 119, and 120.

[0272] One embodiment provides an antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 113, and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 117.

[0273] i.B9H1 array In one embodiment, the anti-B7H4 mouse monoclonal antibody is produced by the hybridoma clone B9H1 and contains two light chains and two heavy chains.

[0274] i. Light chain One embodiment has the following amino acid sequence: The present invention provides an anti-B7H4 mouse monoclonal antibody or its antigen-binding fragment having a light chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with TIFF2026098127000070.tif12165.

[0275] The CDR with sequence number 120 is in bold and underlined. The filename is TIFF2026098127000071.tif19128.

[0276] Another embodiment provides a nucleic acid encoding a light chain (SEQ ID NO: 120).

[0277] An example nucleic acid encoding the light chain (sequence number 120) is: The filename is TIFF2026098127000072.tif41165.

[0278] ii. Heavy chain One embodiment has the following amino acid sequence: The present invention provides an anti-B7H4 mouse monoclonal antibody or its antigen-binding fragment having a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with TIFF2026098127000073.tif19164.

[0279] The CDR with sequence number 123 is in bold and underlined. The filename is TIFF2026098127000074.tif19128.

[0280] Another embodiment provides a nucleic acid encoding a heavy chain (SEQ ID NO: 125).

[0281] An example nucleic acid encoding the heavy chain (SEQ ID NO: 125) is: The filename is TIFF2026098127000075.tif48164.

[0282] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain containing the CDRs of SEQ ID NOs. 54, 55, and 121.

[0283] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a heavy chain containing the CDRs of SEQ ID NOs. 66, 124, and 125.

[0284] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or its antigen-binding fragment, having a light chain containing the CDRs of SEQ ID NOs. 54, 55, and 121, and a heavy chain containing the CDRs of SEQ ID NOs. 66, 124, and 125.

[0285] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 120, and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 123.

[0286] j.B10D7 array In one embodiment, the anti-B7H4 mouse monoclonal antibody is produced by the hybridoma clone B10D7 and contains two light chains and two heavy chains.

[0287] i. Light chain One embodiment has the following amino acid sequence: The present invention provides an anti-B7H4 monoclonal antibody or its antigen-binding fragment having a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with TIFF2026098127000076.tif12165.

[0288] The CDR of sequence number 13 is in bold and underlined. The filename is TIFF2026098127000077.tif19128.

[0289] Another embodiment provides a nucleic acid encoding a light chain (SEQ ID NO: 13).

[0290] An exemplary nucleic acid encoding the light chain (SEQ ID NO: 13) is: The filename is TIFF2026098127000078.tif41165.

[0291] ii. Heavy chain One embodiment has the following amino acid sequence: The present invention provides an anti-B7H4 mouse monoclonal antibody or its antigen-binding fragment having a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with TIFF2026098127000079.tif19165.

[0292] The CDR with sequence number 128 is in bold and underlined. The filename is TIFF2026098127000080.tif20128.

[0293] Another embodiment provides a nucleic acid that encodes a heavy chain (SEQ ID NO: 128). An example nucleic acid encoding the heavy chain (SEQ ID NO: 130) is: The filename is TIFF2026098127000081.tif48164.

[0294] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain containing the CDRs of SEQ ID NOs. 14, 15, and 16.

[0295] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a heavy chain containing the CDRs of SEQ ID NOs. 9, 129, and 130.

[0296] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or its antigen-binding fragment, having a light chain containing the CDRs of SEQ ID NOs. 14, 15, and 16, and a heavy chain containing the CDRs of SEQ ID NOs. 9, 129, and 130.

[0297] One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 13, and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 128.

[0298] 2. Antibody composition The disclosed B7-H4 binding molecule may be an antibody or its antigen-binding fragment. The disclosed antibody and its antigen-binding fragment include an entire immunoglobulin of any class (i.e., an intact antibody), a fragment thereof, and a synthetic protein containing at least the antigen-binding variable domain of the antibody. In some embodiments, the disclosed molecule includes both the antibody light chain and at least the variable domain of the antibody heavy chain. In other embodiments, such a molecule may further include one or more of the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain (in particular, the CH1 and hinge region, or the CH1, hinge, and CH2 region, or the CH1, hinge, CH2, and CH3 region). The antibody can be selected from any class of immunoglobulin, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4. In some embodiments, the constant domain is a complementary fixed constant domain from which the antibody is desired to exhibit cytotoxic activity, and the class is typically IgG1. In other embodiments, if such cytotoxic activity is undesirable, the constant domain may be of the IgG2 or IgG4 class. The antibody may contain sequences derived from two or more classes or isotypes, and selecting specific constant domains to optimize desired effector function is within the scope of the art.

[0299] Variable domains have different sequences among antibodies and are used for the binding and specificity of each particular antibody to its specific antigen. However, variability is not usually evenly distributed throughout the variable domains of an antibody. Typically, variability is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in both the light chain and heavy chain variable domains. The more highly conserved portion of the variable domain is called the framework (FR). The native heavy chain and light chain variable domains each contain four FR regions, primarily in a β-sheet configuration and linked by three CDRs, which connect the β-sheet structure and, in some cases, form loops that form part of it. The CDRs in each chain are held together in close proximity by the FR regions and, together with CDRs from other chains, contribute to the formation of the antibody's antigen-binding site.

[0300] Antibody fragments possessing biological activity are also disclosed. The fragments include insertions, deletions, substitutions, or other selected modifications of specific regions or specific amino acid residues, whether or not they are bound to other sequences, provided that the activity of the fragment is not significantly altered or impaired compared to an unmodified antibody or antibody fragment.

[0301] The technology can also be adapted to produce single-chain antibodies specific to B7-H4. Methods for producing single-chain antibodies are well known to those skilled in the art. Single-chain antibodies can be produced by fusing the variable domains of the heavy and light chains together using a short peptide linker, thereby reconstituted an antigen-binding site on a single molecule. Variable fragments of single-chain antibodies (scFv) have been developed in which the C-terminus of one variable domain is linked to the N-terminus of another variable domain via 15-25 amino acid peptides or linkers, which does not significantly interfere with antigen binding or binding specificity. The linker is selected so as to allow the heavy and light chains to bind together in the appropriate conformational orientation.

[0302] Divalent single-stranded variable fragments (di-scFv) can be manipulated by joining two scFvs. This can be done by producing a single peptide chain with two VH regions and two VL regions, thereby producing a tandem scFv. scFvs can also be designed with a linker peptide (approximately 5 amino acids) that is too short for the two variable regions to fold together, forcing the scFv to dimerize. This type is known as a diabody. Diabodies have been shown to have dissociation constants up to 40 times lower than their corresponding scFvs, meaning they have a much higher affinity for their target. Even shorter linkers (1 or 2 amino acids) result in the formation of trimers (tribodies or tribodies). Tetrabodies have also been produced. Tetrabodies exhibit even higher affinity for their target than diabodies.

[0303] One embodiment provides a monoclonal antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies within the population are identical except for 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 chain and / or light chain is identical or homologous to a corresponding sequence in an antibody originating from a particular species or belonging to a particular antibody class or subclass, while the rest of the chain is identical or homologous to a corresponding sequence in an antibody originating from another species or belonging to another antibody class or subclass, and fragments of such antibodies insofar as they exhibit the desired antagonistic activity.

[0304] In one embodiment, an antibody that specifically binds to human B7-H4 and an antigen-binding fragment thereof are provided.

[0305] One embodiment provides antibodies produced by hybridomas from the group consisting of B1A1, B1H1, B1H3, B1H10, B2E6, B4B2, B4E11, B6C8, B9H1, and B10D7.

[0306] a.Antibodies Immunomodulatory substances can be antibodies. Suitable antibodies are known in the art or can be prepared by those skilled in the art. The nucleic acid and polypeptide sequences of B7-H4 are known in the art, and exemplary protein sequences are provided above. These sequences can be used by those skilled in the art to prepare antibodies or antigen-binding fragments specific to B7-H4, as will be discussed in more detail below. Thus, antibodies or antigen-binding fragments can be agonists or antagonists of B7-H4 signaling.

[0307] The activity (i.e., agonist or antagonist) of an antibody or antigen-binding fragment specific to B7-H4 can be measured using functional assays known in the art, including those discussed below. Typically, the assay involves determining whether the antibody or antigen-binding fragment increases (i.e., agonists) or decreases (i.e., antagonists) signaling via B7-H4. Since B7-H4 signaling results in a suppressive immune response, agonizing B7-H4 results in a suppressed or reduced immune response. Antagonizing B7-H4 signaling inhibits the immunosuppressive response and results in an overall increase in the immune response.

[0308] In some embodiments, the disclosed antibody and its antigen-binding fragment bind immunospecifically to B7-H4. In some embodiments, the antibody binds to the extracellular domain of B7-H4.

[0309] for example, (I) It can immunospecifically bind to B7-H4 arranged on the surface of cells (especially living cells), (II) It can immunospecifically bind to B7-H4 arranged on the surface of cells (especially living cells) at endogenous concentrations. (III) It can immunospecifically bind to B7-H4 arranged on the surface of living cells and regulate the binding between B7-H4 and its ligand. (IV) It can immune-specifically bind to B7-H4 arranged on the surface of living cells, and reduce or inhibit immunosuppression mediated by B7-H4. (V) It can immune-specifically bind to B7-H4 arranged on the surface of living cells and induce or enhance immunosuppression by B7-H4. (VI) It can immune-specifically bind to B7-H4 arranged on the surface of living tumor cells, (VII) A combination of I-IV and VI. (VIII) combinations of I-III and V-IV, and (IX) It can immune-specifically bind to B7-H4 arranged on the surface of myeloid cells or lymphoid-derived cancer cells (AML or ALL), and enhances apoptosis and differentiation, leading to reduced self-renewal of cancer stem cells. A molecule is provided.

[0310] In some embodiments, the molecule can induce antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cell-mediated cytotoxicity (CDC), or other mechanisms of cell apoptosis in B7-H4 expressing cells.

[0311] To prepare antibodies or antigen-binding fragments that specifically bind to B7-H4, purified proteins, polypeptides, fragments, fusions, or epitopes for B7-H4, or polypeptides expressed from their nucleic acid sequences, can be used. The antibodies or antigen-binding fragments can be prepared using any suitable method known in the art, for example, the method discussed in more detail below.

[0312] i. Human antibodies and humanized antibodies Many non-human antibodies (e.g., those derived from mice, rats, or rabbits) are naturally antigenic in humans and therefore can trigger undesirable immune responses when administered to humans. Thus, the use of human antibodies or humanized antibodies in this method acts to reduce the chances of antibodies administered to humans inducing undesirable immune responses.

[0313] During immunization, transgenic animals (e.g., mice) capable of producing a complete repertoire of human antibodies in the absence of endogenous immunoglobulin production can be used. For example, homozygous deletion of the antibody heavy chain binding region (J(H)) gene in chimeric and germline mutant mice has been shown to result in complete inhibition of endogenous antibody production. Transplantation of a human germline immunoglobulin gene array into such germline mutant mice results in the production of human antibodies upon antigen challenge.

[0314] Optionally, antibodies are produced in other species and “humanized” for administration in humans. Humanized forms of non-human (e.g., mouse) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of the antibody) containing minimal sequences derived from non-human immunoglobulins. Humanized antibodies include human immunoglobulin (recipient antibody) in which residues from the complementarity-determining region (CDR) of the recipient antibody are replaced with residues from the CDR of a non-human species (donor antibody), such as mouse, rat, or rabbit, possessing the desired specificity, affinity, and capability. In some cases, Fv framework residues of human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also contain residues not found in the recipient antibody and not in the imported CDR or framework sequence. Generally, humanized antibodies contain substantially all, typically two, variable domains, of at least one variable domain where all or substantially all of the CDR region corresponds to that of a non-human immunoglobulin, or where all or substantially all of the FR region corresponds to the human immunoglobulin consensus sequence. Humanized antibodies also optimally contain at least a portion of the immunoglobulin constant region (Fc), and typically contain at least a portion of the human immunoglobulin.

[0315] 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 the humanized antibody from a non-human source. These non-human amino acid residues are often referred to as “import” residues, typically derived from “import” variable domains. Antibody humanization techniques generally involve the use of recombinant DNA techniques to manipulate the DNA sequences encoding one or more polypeptide chains of the antibody molecule. Humanization can be carried out essentially by substituting rodent CDRs or CDR sequences with corresponding sequences in a human antibody. Thus, the humanized form of a non-human antibody (or fragment thereof) is a chimeric antibody or fragment in which a human variable domain, far below intact, is substituted with corresponding sequences derived from a non-human species. In practice, a humanized antibody is typically a human antibody in which several CDR residues and possibly several FR residues are substituted with residues derived from similar sites in the rodent antibody.

[0316] To reduce antigenicity, the selection of human variable domains in both the light and heavy chains used when producing humanized antibodies is crucial. According to the "best-fit" method, the variable domain sequences of rodent antibodies are screened against an entire library of known human variable domain sequences. The human sequence most closely resembling the rodent sequence is then accepted as the human framework (FR) for the humanized antibody. Another method uses a specific framework derived from the consensus sequences of all human antibodies in a particular subgroup of the light or heavy chain. The same framework may be used for several different humanized antibodies.

[0317] It is even more important to humanize antibodies to retain high affinity for antigens and other desirable biological properties. To achieve this objective, humanized antibodies can be prepared by analytical processes of parental sequences and various conceptual humanized products using three-dimensional models of parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and well known to those skilled in the art. Computer programs are available that illustrate and display the possible three-dimensional structures of selected candidate immunoglobulin sequences. By examining these displays, it is possible to analyze the possible roles of residues in the function of the candidate immunoglobulin sequence, i.e., the residues that affect the candidate immunoglobulin's ability to bind to its antigen. In this way, FR residues can be selected and combined from consensus and imported sequences so that desired antibody characteristics, such as increased affinity for target antigens (multiple antigens), are achieved. Generally, CDR residues are directly and most substantially involved in influencing antigen binding.

[0318] Antibodies can be bound to a substrate, labeled with a detectable moiety, or both. The detectable moieties intended by this composition include fluorescent markers, enzyme markers, and radioactive markers.

[0319] ii. Single chain antibody Methods for producing single-chain antibodies are well known to those skilled in the art. Single-chain antibodies are produced by fusing the variable domains of the heavy and light chains together using a short peptide linker, thereby reconstituted an antigen-binding site on a single molecule. Variable fragments of single-chain antibodies (scFv) have been developed in which the C-terminus of one variable domain is linked to the N-terminus of another variable domain via 15-25 amino acid peptides or linkers, which do not significantly interfere with antigen binding or binding specificity. The linker is selected to allow the heavy and light chains to bind together in the appropriate conformational orientation. These Fv lack the constant region (Fc) present in the heavy and light chains of native antibodies.

[0320] iii. Monovalent antibodies In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce antibody fragments, particularly Fab fragments, can be achieved using routine techniques known in the art. For example, digestion can be performed using papain. Papain digestion of an antibody typically produces two identical antigen-binding fragments, each referred to as a single antigen-binding site, called a Fab fragment, and a residual Fc fragment. Pepsin treatment produces a fragment referred to as an F(ab')2 fragment, which has two antigen-binding sites and can still crosslink antigens.

[0321] The Fab fragment produced by antibody digestion includes the constant domain of the light chain and the first constant domain of the heavy chain. The Fab' fragment differs from the Fab fragment in that several residues are added at the carboxyl terminus of the heavy chain domain, which contains one or more cysteines derived from the antibody hinge region. The F(ab')2 fragment is a bivalent fragment containing two Fab' fragments linked by a disulfide crosslink in the hinge region. Fab'-SH is the herein designation for Fab', in which the cysteine ​​residue(s) of the constant domain have a free thiol group. Antibody fragments were originally produced as pairs of Fab' fragments having a hinge cysteine ​​between them. Other chemical bonds of antibody fragments are also known.

[0322] iv. Hybrid antibodies Antibodies can be hybrid antibodies. In a hybrid antibody, one heavy-and-light chain pair is homologous to a pair found in an antibody produced against one epitope, and the other heavy-and-light chain pair is homologous to a pair found in an antibody produced against another epitope. This results in a multifunctional titer, i.e., the ability to bind to at least two different epitopes simultaneously. Such hybrids can be formed by the fusion of hybridomas producing each component antibody, or by recombinant technology. Such hybrids may, of course, be formed using chimeric chains.

[0323] v. Conjugate or fusion of antibody fragments The targeting function of an antibody can be used therapeutically by conjugating the antibody or a fragment thereof with a therapeutic agent. Such conjugation of an antibody or fragment (e.g., at least a portion of the constant region (Fc) of an immunoglobulin) with a therapeutic agent can be achieved by creating an immunoconjugate comprising the antibody or antibody fragment and the therapeutic agent, or by creating a fusion protein.

[0324] Such conjugation of an antibody or fragment with a therapeutic agent can be achieved by creating an immunoconjugate containing the antibody or antibody fragment and the therapeutic agent, by creating a fusion protein, or by conjugating the antibody or fragment to a nucleic acid such as siRNA.

[0325] In some embodiments, antibodies are modified to alter their half-life. In some embodiments, it is desirable to increase the half-life of the antibody so that it remains in circulation or at the treatment site for a longer period. For example, it may be desirable to maintain the antibody titer in circulation or at the treated site for a longer period. Antibodies can be manipulated using Fc variants that extend the half-life, for example, using Xtend® antibody half-life extension technology (Xencor, Monrovia, CA). In other embodiments, the half-life of an anti-DNA antibody is shortened to reduce potential side effects. The disclosed conjugates can be used to modify a given biological response. The drug moiety should not be interpreted as being limited to classical chemotherapeutic agents. For example, the drug moiety may be a protein or polypeptide having the desired biological activity. Such proteins may include toxins such as abrin, lysine A, Pseudomonas exotoxin, or diphtheria toxin.

[0326] vi. Exemplary B7H4 antibody Exemplary B7H4 antibodies or antigen-binding fragments are disclosed herein. The antibodies may include one or more heavy chains and one or more light chains of mouse anti-human B7H4 antibodies B1H1 or B1H10. In some embodiments, the B7H45 antibody includes part or all of the light chain CDR, the entire light chain variable region, part or all of the heavy chain CDR, the entire heavy chain variable region, or any combination thereof of mouse anti-human B7H4 antibodies B1H1 or B1H10. Exemplary combinations are disclosed below.

[0327] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 24 and a heavy chain having the amino acid sequence of SEQ ID NO: 38.

[0328] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 24 and a heavy chain having the amino acid sequence of SEQ ID NO: 39.

[0329] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 24 and a heavy chain having the amino acid sequence of SEQ ID NO: 40.

[0330] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 24 and a heavy chain having the amino acid sequence of SEQ ID NO: 41.

[0331] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 24 and a heavy chain having the amino acid sequence of SEQ ID NO: 42.

[0332] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 24 and a heavy chain having the amino acid sequence of SEQ ID NO: 43.

[0333] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 24 and a heavy chain having the amino acid sequence of SEQ ID NO: 44.

[0334] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 24 and a heavy chain having the amino acid sequence of SEQ ID NO: 45.

[0335] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 25 and a heavy chain having the amino acid sequence of SEQ ID NO: 38.

[0336] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 25 and a heavy chain having the amino acid sequence of SEQ ID NO: 39.

[0337] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 25 and a heavy chain having the amino acid sequence of SEQ ID NO: 40.

[0338] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 25 and a heavy chain having the amino acid sequence of SEQ ID NO: 41.

[0339] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 25 and a heavy chain having the amino acid sequence of SEQ ID NO: 42.

[0340] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 25 and a heavy chain having the amino acid sequence of SEQ ID NO: 43.

[0341] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 25 and a heavy chain having the amino acid sequence of SEQ ID NO: 44.

[0342] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 25 and a heavy chain having the amino acid sequence of SEQ ID NO: 45.

[0343] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 26 and a heavy chain having the amino acid sequence of SEQ ID NO: 38.

[0344] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 26 and a heavy chain having the amino acid sequence of SEQ ID NO: 39.

[0345] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 26 and a heavy chain having the amino acid sequence of SEQ ID NO: 40.

[0346] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 26 and a heavy chain having the amino acid sequence of SEQ ID NO: 41.

[0347] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 26 and a heavy chain having the amino acid sequence of SEQ ID NO: 42.

[0348] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 26 and a heavy chain having the amino acid sequence of SEQ ID NO: 43.

[0349] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 26 and a heavy chain having the amino acid sequence of SEQ ID NO: 44.

[0350] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 26 and a heavy chain having the amino acid sequence of SEQ ID NO: 45.

[0351] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 27 and a heavy chain having the amino acid sequence of SEQ ID NO: 38.

[0352] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 27 and a heavy chain having the amino acid sequence of SEQ ID NO: 39.

[0353] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 27 and a heavy chain having the amino acid sequence of SEQ ID NO: 40.

[0354] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 27 and a heavy chain having the amino acid sequence of SEQ ID NO: 41.

[0355] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 27 and a heavy chain having the amino acid sequence of SEQ ID NO: 42.

[0356] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 27 and a heavy chain having the amino acid sequence of SEQ ID NO: 43.

[0357] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 27 and a heavy chain having the amino acid sequence of SEQ ID NO: 44.

[0358] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 27 and a heavy chain having the amino acid sequence of SEQ ID NO: 45.

[0359] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 28 and a heavy chain having the amino acid sequence of SEQ ID NO: 38.

[0360] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 28 and a heavy chain having the amino acid sequence of SEQ ID NO: 39.

[0361] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 28 and a heavy chain having the amino acid sequence of SEQ ID NO: 40.

[0362] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 28 and a heavy chain having the amino acid sequence of SEQ ID NO: 41.

[0363] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 28 and a heavy chain having the amino acid sequence of SEQ ID NO: 42.

[0364] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 28 and a heavy chain having the amino acid sequence of SEQ ID NO: 43.

[0365] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 28 and a heavy chain having the amino acid sequence of SEQ ID NO: 44.

[0366] An anti-B7H4 antibody or its antigen-binding fragment, having a light chain having the amino acid sequence of SEQ ID NO: 28 and a heavy chain having the amino acid sequence of SEQ ID NO: 45.

[0367] 2. Proteins and polypeptides a. Protein and polypeptide compositions Immunomodulatory substances may be proteins, polypeptides, or fusion proteins. For example, an immunomodulatory substance may be an isolated or recombinant B7-H4 protein or polypeptide, or a functional fragment, variant, or fusion protein thereof.

[0368] A protein or polypeptide, or a functional fragment, variant, or fusion protein thereof, can be an agonist or an antagonist. For example, in some embodiments, the 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 may be a soluble fragment, such as 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 function as an agonist and increase signal transduction via B7-H4.

[0369] The activity (i.e., agonist or antagonist) of a B7-H4 protein or polypeptide, or any fragment, variant, or fusion protein thereof, can be determined using functional assays known in the art, including those discussed below. Typically, an assay involves determining whether a protein, polypeptide, or any fragment, variant, or fusion protein thereof increases (i.e., agonists) or decreases (i.e., antagonists) signaling via the B7-H4 receptor. In some embodiments, an assay involves determining whether a protein, polypeptide, or any fragment, variant, or fusion protein thereof increases (i.e., agonists) or decreases (i.e., antagonists) the immune response associated with B7-H4. Typically, an assay involves determining whether a protein, polypeptide, or any fragment, variant, or fusion protein thereof increases (i.e., agonists) or decreases (i.e., antagonists) signaling via B7-H4. In some embodiments, the assay includes determining whether a protein, polypeptide, or fragment, variant, or fusion protein thereof reduces (i.e., acts as an agonist) or increases (i.e., acts as an antagonist) the immune response negatively regulated by B7-H4. In some embodiments, the assay includes determining whether a protein, polypeptide, or fragment, variant, or fusion protein thereof increases apoptosis and differentiation of acute myeloid leukemia cells and acute lymphoblastic leukemia cells (i.e., acts as an antagonist) and reduces the self-renewal capacity of AML and ALL stem cells.

[0370] The nucleic acid and polypeptide sequences of B7-H4 are known in the art, and exemplary protein and peptide sequences are provided above. These sequences can be used by those skilled in the art to prepare any B7-H4 protein or polypeptide, or any fragment, variant, or fusion protein thereof, as will be discussed in more detail below. Generally, B7-H4 proteins, polypeptides, fragments, variants, and fusions are expressed from nucleic acids containing a sequence encoding a signal sequence. The signal sequence is generally cleaved from an immature polypeptide to produce a mature polypeptide lacking the signal sequence. The signal sequence can be replaced by the signal sequence of another polypeptide using standard molecular biological techniques to affect the expression level, secretion, solubility, or other properties of the polypeptide B7-H4 protein having or lacking the disclosed signal sequence. In some cases, it should be understood that a mature protein known or described in the art, i.e., a protein sequence without a signal sequence, is a putative mature protein. During normal cell expression, the signal sequence can be removed by cellular peptidases to obtain a mature protein. The sequences of mature proteins can be determined or confirmed using methods known in the art.

[0371] i. Fragment As used herein, a B7-H4 fragment refers to any subset of polypeptides that is at least one amino acid shorter than the full-length protein. Useful fragments include those that retain the ability to bind to their native ligand or ligand(s). Any full-length B7-H4 fragment polypeptide typically has a binding ability to its native ligand of 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 more than 100 percent compared to the full-length protein.

[0372] The B7-H4 fragments include cell-free fragments. Cell-free polypeptides can be fragments of full-length transmembrane polypeptides, which can be removed, secreted, or otherwise extracted from producing cells. Cell-free fragments of polypeptides may contain 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 one embodiment, the polypeptide fragment contains the entire extracellular domain of the full-length protein. In other embodiments, the cell-free fragment of a polypeptide contains a fragment of the extracellular domain that retains the biological activity of the full-length protein. The extracellular domain may contain 1, 2, 3, 4, or 5 consecutive amino acids derived from the transmembrane domain and / or 1, 2, 3, 4, or 5 consecutive amino acids derived from a signal sequence. Alternatively, the extracellular domain may 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 sole functional domain of the fragment (e.g., the ligand-binding domain).

[0373] ii. Variants Variants and fragments of B7-H4 are also provided. In some embodiments, the variants are at least 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, or 99 percent identical to any one of Sequence ID No. 1. Useful variants include those that increase biological activity as shown by any of the assays described herein, or those that increase the half-life or stability of the protein. B7-H4 proteins and polypeptides, as well as their fragments, variants, and fusion proteins, can be manipulated to increase biological activity. For example, in some embodiments, B7-H4 polypeptides, proteins, or their fragments, variants, or fusions are modified by substitution, deletion, or insertion of at least one amino acid to increase their function.

[0374] Finally, variant polypeptides can be manipulated to have an increased half-life compared to the wild type. These variants are typically modified to be resistant to enzymatic degradation. Exemplary modifications include modified amino acid residues and modified peptide bonds that are resistant to enzymatic degradation. Various modifications to achieve this are known in the art. Variants can be modified to modulate the effect of receptor affinity on the half-life of proteins, polypeptides, their fragments, or fusions in serum and endosomal pH.

[0375] iii. Fusion protein The fusion polypeptide has a first fusion partner which includes all or part of polypeptide B7-H4, either directly fused to the second polypeptide or fused via a linker peptide sequence fused to the second polypeptide. The fusion protein optionally includes a domain which functions to dimerize or polymerize two or more fusion proteins. The peptide / polypeptide linker domain may be a separate domain or may be contained within one of the other domains of the fusion protein (the first polypeptide or the second polypeptide). Similarly, the domain which functions to dimerize or polymerize the fusion protein may be a separate domain or may be contained within one of the other domains of the fusion protein (the first polypeptide, the second polypeptide, or the peptide / polypeptide linker domain). In one embodiment, the dimerization / polymerization domain and the peptide / polypeptide linker domain are the same.

[0376] The fusion proteins disclosed herein are of formula I: N-R1-R2-R3-C In the formula, "N" represents the N-terminus of the fusion protein and "C" represents the C-terminus of the fusion protein. In some embodiments, "R1" is a polypeptide or protein of B7-H4 or a 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 a fragment or variant thereof, and R1 may be a second polypeptide. In some embodiments, the B7-H4 polypeptide is an extracellular domain or fragment thereof, such as an Ig-like C2 domain, or a region framed by cysteine ​​forming the disulfide bond described above.

[0377] Dimerization or multimerization can occur between or between two or more fusion proteins via dimerizing or multimerizing domains. Alternatively, dimerization or multimerization of fusion proteins can occur by chemical crosslinking. The resulting dimers or multimers may be homodimers / homopolymers or heterodimers / heteropolymers.

[0378] In some embodiments, the fusion protein comprises an extracellular domain or fragment or variant of B7-H4 fused to the Ig Fc region. Recombinant Ig fusion proteins can be prepared by fusioning the coding region of the extracellular domain or fragment or variant of the extracellular domain to the Fc region of human IgG1, IgG2, IgG3, or IgG4, or mouse IgG2a, or other preferred Ig domains previously described (Chapoval, et al., Methods Mol. Med., 45:247-255 (2000)).

[0379] iv. Polypeptide modification Polypeptides and fusion proteins can be modified by chemical moieties that may be present in polypeptides in a normal cellular environment, for example, by phosphorylation, methylation, amidation, sulfation, acylation, glycosylation, sumoylation, and ubiquitination. Fusion proteins can also be modified with labels that can provide a detectable signal, either directly or indirectly, including, but not limited to, radioisotopes and fluorescent compounds.

[0380] Polypeptides and fusion proteins can also be modified by chemical moieties not normally attached to polypeptides in the cellular environment. For example, the disclosed fusion proteins can also be modified by covalent bonding of polymer chains, including, but not limited to, polyethylene glycol polymer (PEG) chains (i.e., pegylation). The conjugation of macromolecules to PEG has recently emerged as an effective strategy to alter the pharmacokinetic (PK) profiles of various drugs and thereby improve their therapeutic potential. PEG conjugations increase the retention of drugs in circulation by protecting them from enzymatic digestion, slowing renal filtration, and reducing the production of neutralizing antibodies. Furthermore, PEG conjugates can be used to enable the multimerization of fusion proteins.

[0381] Modifications can be introduced into a molecule by reacting a target amino acid residue of a polypeptide with an organic derivatizing agent that can react with a selected side chain or terminal residue. Another modification is protein cyclization.

[0382] Examples of chemical derivatives of polypeptides include amino-terminal residues derivatized with lysinyl and succinic acid or other carboxylic acid anhydrides. Derivatization with cyclic carboxylic acid anhydrides has the effect of reversing the charge of the lysinyl residue. Other suitable reagents for derivatizing amino-containing residues include transaminase-catalyzed reactions with imide esters, such as methyl picoline imide, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione, and glyoxylates. The carboxyl side group, aspartyl, or glutamyl can be selectively modified by reacting with a carbodiimide (RN=C=N-R') such as 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore, aspartyl and glutamyl residues can be converted to asparaginyl and glutamyl residues by reaction with ammonia. The fusion protein may also contain one or more D-amino acids substituted with one or more L-amino acids.

[0383] v. Modification of bonding properties The binding properties of proteins, polypeptides, their fragments, variants, and fusions are related to the administered dose and dose regimen. In one embodiment, the disclosed proteins, polypeptides, their fragments, variants, and fusions have binding properties to the B7-H4 ligand that show a higher percentage or higher percentage occupancy of the binding site (e.g., on the ligand) compared to other receptor molecules that bind to them. In other embodiments, the disclosed proteins, polypeptides, their fragments, variants, and fusions have reduced binding affinity to the B7-H4 ligand compared to the wild-type protein.

[0384] In some embodiments, proteins, polypeptides, their fragments, variants, and fusions may have a relatively high affinity for the B7-H4 ligand and therefore a relatively slow off-rate. In other embodiments, protein polypeptides, fragments, variants, and fusions may be administered intermittently over days, weeks, or months to suppress the immune response, thereby allowing for recovery before the next administration and potentially altering the immune response without completely turning it on or off, thus avoiding long-term side effects.

[0385] 3. Isolated nucleic acid molecules Isolated nucleic acid sequences encoding proteins, polypeptides, their fragments, variants, and fusions are disclosed herein. As used herein, “isolated nucleic acid” refers to nucleic acids isolated from other nucleic acid molecules present in the mammalian genome, including nucleic acids that are typically adjacent to one or both sides of a nucleic acid in the mammalian genome. The term “isolated” as used herein with respect to nucleic acids also includes combinations with any non-naturally occurring nucleic acid sequence, since non-naturally occurring sequences are not found in nature and do not have immediately contiguous sequences in naturally occurring genomes.

[0386] An isolated nucleic acid can be, for example, a DNA molecule, but with one of the nucleic acid sequences that would normally be immediately adjacent to that DNA molecule in a naturally occurring genome removed or absent. Therefore, isolated nucleic acids include, but are not limited to, DNA molecules that exist as separate molecules independent of other sequences (e.g., chemically synthesized nucleic acids, or cDNA or genomic DNA fragments produced by PCR or restriction endonuclease treatment), and recombinant DNA that is incorporated into vectors, autonomously replicating plasmids, viruses (e.g., retroviruses, lentiviruses, adenoviruses, or herpesviruses), or into the genomic DNA of prokaryotes or eukaryotes. Furthermore, isolated nucleic acids can include engineered nucleic acids, such as recombinant DNA molecules that are part of a hybrid nucleic acid or fusion nucleic acid. For example, nucleic acids present among hundreds to millions of other nucleic acids in a cDNA library or genomic library or in a gel slice containing genomic DNA restriction digestion are not considered isolated nucleic acids.

[0387] Nucleic acids encoding proteins, polypeptides, their fragments, variants, and fusions can be optimized for expression in a preferred expression host. Codons can be substituted with alternative codons encoding the same amino acids to account for differences in codon usage between the mammalian source of the nucleic acid sequence and the expression host. Thus, nucleic acids can be synthesized using codons preferred for the expression host.

[0388] Nucleic acids can be sense-oriented or antisense-oriented, or complementary to a reference sequence encoding a B7-H4 polypeptide or protein. Nucleic acids can be DNA, RNA, or nucleic acid analogs. Nucleic acid analogs can be modified in the base moiety, sugar moiety, or phosphate backbone. Such modifications can, for example, improve the stability, hybridization, or solubility of the nucleic acid. Modifications in the base moiety can include deoxyuridine in the case of deoxythymidine, and 5-methyl-2'-deoxycytidine or 5-bromo-2'-deoxycytidine in the case of deoxycytidine. Modifications in the sugar moiety can include modification of the 2'-hydroxyl of the ribose sugar to form a 2'-O-methyl or 2'-O-allyl sugar. The deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, where each base moiety is bonded to a 6-membered morpholino ring or peptide nucleic acid, and the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone, retaining four bases. 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. Furthermore, the deoxyphosphate skeleton can be replaced with, for example, a phosphorothioate or phosphorodithioate skeleton, a phosphoramidite, or an alkylphosphotryster skeleton.

[0389] Nucleic acids encoding polypeptides can be administered to targets that require them. Nucleic acid delivery involves introducing "foreign" nucleic acids into cells and ultimately into living animals. Compositions and methods for delivering nucleic acids to targets are known in the art (see Understanding Gene Therapy, Lemoine, NR, ed., BIOS Scientific Publishers, Oxford, 2008).

[0390] 4. Vectors and host cells Vectors encoding proteins, polypeptides, their fragments, variants, and fusions are also provided. Nucleic acids, such as those described above, can be inserted into vectors for expression in cells. As used herein, “vector” is a replicon such as a plasmid, phage, virus, or cosmid into which another DNA segment may be inserted to result in replication of the inserted segment. A vector can also be an expression vector. An “expression vector” is a vector containing one or more expression regulatory sequences, where “expression regulatory sequences” are DNA sequences that control and regulate the transcription and / or translation of another DNA sequence.

[0391] Nucleic acids in a vector can be operably bound to one or more regulatory sequences. As used herein, “operably bound” means that the regulatory sequence is incorporated into the gene construct so as to effectively control the expression of the coding sequence of interest. Examples of regulatory sequences include promoters, enhancers, and transcription termination regions. A promoter is a regulatory sequence consisting of a region of a DNA molecule, typically within 100 nucleotides upstream of the point where transcription begins (generally near the start site of RNA polymerase II). To place a coding sequence under the control of a promoter, the translation start site of the polypeptide’s translation reading frame must be located 1 to about 50 nucleotides downstream of the promoter. Enhancers provide expression specificity with respect to time, place, and level. Unlike promoters, enhancers can function when located at various distances from the transcription site. Enhancers can also be located downstream of the transcription start site. When RNA polymerase can transcribe the coding sequence into mRNA, and then translate it into the protein encoded by the coding sequence, the coding sequence is “operably bound” and “under the control” of a regulatory sequence in the cell.

[0392] Suitable expression vectors include, but are not limited to, plasmids and viral vectors derived from bacteriophages, baculoviruses, tobacco mosaic viruses, herpesviruses, cytomegaloviruses, retroviruses, vaccinia viruses, adenoviruses, and adeno-associated viruses. Many vectors and expression systems are commercially available from companies such as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA), and Invitrogen Life Technologies (Carlsbad, CA).

[0393] Expression vectors may include tag sequences. Tag sequences are typically expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide, including either a carboxyl terminus or an amino terminus. Examples of useful tags, but not limited to, include 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 encoding one or more domains of the constant region of the Ig heavy chain having amino acid sequences corresponding to the hinge, CH2, and CH3 regions of the human immunoglobulin Cγ1 chain.

[0394] Vectors containing the nucleic acids to be expressed can be transferred into host cells. The term “host cells” is intended to include prokaryotic and eukaryotic cells into which recombinant expression vectors can be introduced. As used herein, “transformed” and “transfected” encompass the introduction of nucleic acid molecules (e.g., vectors) into cells by one of many techniques. Many of these techniques are well established within the art, but are not limited to any particular technique. Prokaryotic cells can be transformed with nucleic acids, for example, by electroporation or calcium chloride-mediated transformation. Nucleic acids can be transfected into mammalian cells by techniques including, for example, calcium phosphate coprecipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, or microinjection. Host cells (e.g., prokaryotic cells or eukaryotic cells such as CHO cells) can be used, for example, to produce proteins, polypeptides, their fragments, variants, and fusions as described herein.

[0395] The vectors described can be used to express proteins, polypeptides, their fragments, variants, and fusions in cells. Exemplary vectors include, but are not limited to, adenovirus vectors. One approach involves nucleic acid transfer into primary cells in culture, followed by autotransplantation of ex vivo transformed cells into a host, either systemically or into a specific organ or tissue. Ex vivo methods may 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 the expression of the encoded polypeptide. These methods are known in the field of molecular biology. The transduction step can be achieved by any standard means used in ex vivo gene therapy, including, for example, calcium phosphate, lipofection, electroporation, viral infection, and biolistic gene transfer. Alternatively, liposomes or polymer microparticles can be used. Successfully transduced cells can then be selected for, for example, the expression of a coding sequence or a drug resistance gene. The cells can then be lethally irradiated (if desired) and injected or transplanted into a subject. In one embodiment, an expression vector containing a nucleic acid encoding a fusion protein is transfected into cells that are to be administered to a target that requires it.

[0396] In vivo nucleic acid therapy can be achieved by directly transferring functionally active DNA into mammalian somatic cells, tissues, or organs in vivo. For example, nucleic acids encoding polypeptides disclosed herein can be directly administered to lymphoid tissue. Alternatively, lymphoid tissue-specific targeting can be achieved using lymphoid tissue-specific transcriptional regulators (TREs), such as B lymphocyte-specific, T lymphocyte-specific, or dendritic cell-specific TREs. Lymphoid tissue-specific TREs are known in the art.

[0397] Nucleic acids can also be administered in vivo by viruses. Nucleic acid molecules encoding fusion proteins can be packaged into retroviral vectors using packaging cell lines that produce replication-defective retroviruses, as is well known in the art. Other viral vectors, including recombinant adenoviruses and vaccinia viruses, which may not replicate, can also be used. In addition to naked DNA or RNA, or viral vectors, engineered bacteria can be used as vectors.

[0398] Nucleic acids can also be delivered by other carriers, including liposomes, polymer microparticles and nanoparticles, as well as polycations such as asialoclycoproteins / polylyridine.

[0399] In addition to in vivo viral and carrier-mediated gene transfer, physical means known in the art, including plasmid DNA administration and particle bombardment-mediated gene transcription, can be used for direct DNA transfer.

[0400] 5.Small molecules Immunomodulatory substances can be small molecules. Small molecule agonists and antagonists B7-H4 are known in the art or can be identified using routine screening methods.

[0401] In some embodiments, the screening assay may include random screening of a large library of test compounds. Alternatively, the assay may be used to focus on a specific class of compounds suspected of modulating B7-H4 levels. The assay may include determining B7-H4 signaling activity or inhibitory responses mediated by B7-H4. Other assays may include determining nucleic acid transcription or translation, mRNA levels, mRNA stability, mRNA degradation, transcription rate, and translation rate.

[0402] C. Pharmaceutical Compositions A pharmaceutical composition containing an immunomodulatory substance is provided. The pharmaceutical composition containing the immunomodulatory substance can be administered parenterally (intramuscular, intraperitoneal, intravenous (IV), or subcutaneous injection), transdermally (passively or using either ion electrophoresis or electroporation), via a mucosal route (nasal, vaginal, rectal, or sublingual), or using a bioerodible insert, and can be formulated in a dosage form suitable for each route of administration.

[0403] In some in vivo approaches, the compositions disclosed herein are administered to a subject in a therapeutically effective dose. As used herein, the terms “effective dose” or “therapeutically effective dose” mean a dose sufficient to treat, inhibit, or alleviate one or more symptoms of the disorder being treated, or otherwise sufficient to provide the desired pharmacological and / or physiological effect. The exact dose will vary depending on various factors, including subject-dependent variables (e.g., age, immune system health), the disease, and the treatment being performed.

[0404] As further research is conducted on the disclosed immunomodulatory substances, information will emerge regarding appropriate dosage levels for treating various conditions in various patients, and those skilled in the art will be able to determine the appropriate dosage considering the recipient's therapeutic background, age, and overall health. The selected dosage depends on the desired therapeutic effect, route of administration, and desired duration of treatment. For the disclosed immunomodulatory substances, the dosage level for mammals is generally 0.001 to 20 mg / kg body weight per day. Generally, dosages may be lower when administered by intravenous injection or infusion.

[0405] In certain embodiments, the immunomodulatory substance is administered locally, for example, by direct injection at the site of treatment. Typically, injection results in a greater increase in the local concentration of the immunomodulatory substance composition than can be achieved by systemic administration. The immunomodulatory substance composition can be combined with the matrix described above to help increase the local concentration of the polypeptide composition by reducing the passive diffusion of polypeptides from the site of treatment.

[0406] 1. Parenteral administration formulations In some embodiments, compositions disclosed herein, including those comprising peptides and polypeptides, are administered by parenteral injection into aqueous solutions. Formulations may also be in the form of suspensions or emulsions. Generally, pharmaceutical compositions are provided comprising an effective amount of peptide or polypeptide and optionally comprising pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants, and / or carriers. Such compositions optionally comprise one or more of the following: diluents, sterile water, buffered salines of varying buffer content, pH, and ionic strength (e.g., Tris-HCl, acetates, phosphates), as well as additives, e.g., detergents and solubilizers (e.g., TWEEN20 (Polysorbate-20), TWEEN80 (Polysorbate-80)), antioxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g., thimerosal, benzyl alcohol), as well as swelling agents (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles include propylene glycol, polyethylene glycol, vegetable oils such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. The formulations can be lyophilized and redissolved / resuspended immediately before use. The formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, by incorporating a sterilizing agent into the composition, by irradiation of the composition, or by heating the composition.

[0407] 2. Oral administration preparations In embodiments, the composition is formulated for oral delivery. Oral solid dosage forms are generally described in Chapter 89 of Remington's Pharmaceutical Sciences, 18th Ed. 1990 (Mack Publishing Co., Easton, Pa. 18042). Solid dosage forms include tablets, capsules, pills, lozenges, or lozenges, cachets, pellets, powders, or granules, or the incorporation of the material into particulate preparations of high molecular weight compounds such as polylactic acid, polyglycolic acid, or into liposomes. Such compositions may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the disclosed composition. See, for example, pages 1435–1712 of Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042), incorporated herein by reference. The composition may be prepared in liquid form or in dry powder form (e.g., lyophilized). Liposomes or protein encapsulation can be used to formulate compositions. Liposome encapsulation may be used, and liposomes can be derivatized with various polymers (e.g., U.S. Patent No. 5,013,556). See also Marshall, K. In: Modem Pharmaceutics Edited by GSBanker and CTRhodes Chapter 10, 1979. Generally, formulations include peptides (or chemically modified forms thereof) and inert components that protect the peptide in the gastric environment, as well as the release of bioactive substances in the intestines.

[0408] The active substance can be chemically modified to facilitate the oral delivery of the derivative. Generally, the intended chemical modification involves adding at least one moiety to the component molecule itself, which enables uptake from the stomach or intestines into the bloodstream or direct uptake into the intestinal mucosa. Increased overall stability of the component or component(s) and increased circulation time in the body are also desired. PEGylation is an exemplary chemical modification for pharmaceutical use. Other parts that may be used include propylene glycol, ethylene glycol and propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyproline, poly-1,3-dioxolane, and poly-1,3,6-thioxocan [see, for example, Abuchowski and Davis (1981) “Soluble Polymer-Enzyme Adducts,” in Enzymes as Drugs. Hocenberg and Roberts, eds. (Wiley-Interscience: New York, NY) pp.367-383; and Newmark, et al. (1982) J. Appl. Biochem. 4:185-189].

[0409] Another embodiment provides a liquid dosage form for oral administration, comprising pharmaceutically acceptable emulsions, solutions, suspensions, and syrups, which may include auxiliary agents such as inert diluents, wetting agents, emulsifiers, and suspending agents, as well as other components including sweeteners, flavorings, and flavorings.

[0410] Controlled-release oral formulations may be preferable. The active ingredient can be incorporated into an inert matrix, such as a viscous substance (gum), which allows for release by either diffusion or leaching mechanisms. Slowly denaturing matrices can also be incorporated into formulations. Another form of controlled release is based on the Oros therapeutic system (Alza Corp.), in which the drug is encapsulated in a semipermeable membrane that allows water to enter through a single small opening and push the drug out by osmotic effect.

[0411] In the case of oral formulations, the release site may be the stomach, small intestine (duodenum, jejunum, or ileum), or large intestine. In some embodiments, release is carried out beyond the gastric environment to avoid adverse effects on the gastric environment, for example, by protecting the active substance (or derivative) in the intestines or releasing the active substance (or derivative) in the intestines. To ensure sufficient tolerance in the stomach, a coating that is impermeable to at least pH 5.0 is essential. Examples of more common inert components used as enteric coatings include cellulose trimellitate acetate (CAT), hydroxypropyl methylcellulose phthalate (HPMCP), HPMCP50, HPMCP55, polyvinyl acetate phthalate (PVAP), Eudragit L30D™, Aquateric™, cellulose acetate phthalate (CAP), Eudragit L™, Eudragit S™, and Shellac™. These coatings can be used as a membrane for the mixture.

[0412] 3. Locally administered preparations The disclosed immunomodulatory substances can be applied topically. Topical administration is not effective for most peptide formulations, but it may be effective, especially when applied to the lungs, nose, oral (sublingual, oral cavity), vagina, or rectal mucosa.

[0413] When the composition is delivered as either an aerosol or spray-dried particles having an aerodynamic diameter of less than approximately 5 microns, it can be delivered to the lungs upon inhalation and traverse the epithelial lining into the bloodstream.

[0414] A wide variety of mechanical devices designed to deliver therapeutic products to the lungs may be used, including, but not limited to, nebulizers, metered-dose inhalers, and powder inhalers (all of which are well known to those skilled in the art). Some specific examples of commercially available devices include 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, NC), and the Spinhaler powder inhaler (Fisons Corp., Bedford, Mass.). Nektar, Alkermes, and Mannkind all have inhalable insulin powder preparations in clinical trials that have been approved or in which their technology can be applied to the formulations described herein.

[0415] Formulations for administration to mucous membranes are typically spray-dried drug particles that can be incorporated into tablets, gels, capsules, suspensions, or emulsions. Standard pharmaceutically acceptable excipients are available from any formulation manufacturer.

[0416] Transdermal formulations can also be prepared. These are typically ointments, lotions, sprays, or patches, all of which can be prepared using standard techniques. Transdermal formulations may require the inclusion of penetration enhancers.

[0417] 4. Controlled Delivery Polymer Matrix The immunomodulatory substances disclosed herein may also be administered in controlled-release formulations. Controlled-release polymer devices can be fabricated for long-term systemic release after implantation or injection (microparticles) of the polymer device (rod, cylinder, film, disk). The matrix may be in the form of microparticles such as microspheres, where the active ingredient is dispersed within a solid polymer matrix or microcapsule, and the core is a different material from the polymer shell, with the peptide dispersed or suspended within the core, which may be liquid or solid. Unless otherwise defined herein, microparticles, microspheres, and microcapsules are used interchangeably. Alternatively, the polymer may be molded as thin slabs or films ranging from nanometers to 4 centimeters, powders produced by grinding or other standard techniques, or even gels such as hydrogels.

[0418] Either a non-biodegradable or biodegradable matrix can be used for the delivery of a fusion polypeptide or nucleic acid encoding a fusion polypeptide, but in some embodiments, a biodegradable matrix is ​​preferred. These may be natural or synthetic polymers, but in some embodiments, synthetic polymers are preferred for better characterization of the degradation and release profile. The polymer is selected based on the period over which release is desired. In some cases, linear release may be most useful, but in others, pulsed release or "bulk release" may yield more effective results. The polymer may be in the form of a hydrogel (typically absorbing up to about 90% by weight of water) and may optionally be crosslinked with polyvalent ions or polymers.

[0419] The matrix can be formed by evaporation of the solvent, spray drying, solvent extraction, and other methods known to those skilled in the art. Biodegradable microparticles can be prepared using any of the methods developed for producing microparticles for drug delivery, such as those described in 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).

[0420] The devices can typically be formulated for local release to treat implantation or injection areas, or for systemic delivery, delivering doses much smaller than those for systemic treatment. They can be implanted into muscle or fat, injected subcutaneously, or swallowed.

[0421] III. Manufacturing method A. Method for producing antibodies Antibodies can be produced in cell cultures, phages, or in a variety of animals, including, but not limited to, cattle, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, and primates. Therefore, in one embodiment, the antibody is a mammalian antibody. Phage techniques can be used to isolate initial antibodies or to generate variants with altered specificity or affinity characteristics. Such techniques are commonplace and well-known in the art. In one embodiment, the antibody is produced by recombinant means known in the art. For example, recombinant antibodies can be produced by transfecting host cells with a vector containing a DNA sequence encoding the antibody. One or more vectors can be used to transfect host cells with DNA sequences expressing at least one VL region and one VH region. Exemplary descriptions of recombinant methods for 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), and Current Protocols in Immunology (John Wiley & Sons, latest edition).

[0422] The disclosed antibodies can be modified by recombinant means to increase their greater potency in mediating a desired function. Therefore, the modification of antibodies by substitution using recombinant means falls within the scope of the present invention. Typically, the substitutions are conservative substitutions. For example, at least one amino acid in the constant region of an antibody can be replaced with a different residue. See, for example, U.S. Patent No. 5,624,821, U.S. Patent No. 6,194,551, Application No. WO9958572, and Angal, et al., Mol. Immunol. 30:105-08 (1993). Amino acid modifications include amino acid deletions, additions, and substitutions. In some cases, such changes are made to reduce undesirable activity, such as complement-dependent cytotoxicity. Frequently, antibodies are labeled by covalently or noncovalently binding a substance that provides a detectable signal. A wide variety of labeling and binding techniques are known and have been widely reported in both scientific and patent literature. These antibodies can be screened for binding to B7-H4 proteins, polypeptides, or fusion proteins. See, for example, Antibody Engineering: A Practical Approach (Oxford University Press, 1996).

[0423] For example, suitable antibodies with desired biological activity can be identified using in vitro assays, including, but not limited to, those involving proliferation, migration, adhesion, soft agar growth, angiogenesis, intercellular communication, apoptosis, transport, and signal transduction, as well as in vivo assays such as inhibition of tumor growth. The antibodies provided herein may also be useful for diagnostic applications. As capture antibodies or non-neutralizing antibodies, they can be screened for their ability to bind to specific antigens without inhibiting receptor binding or biological activity of the antigen. As neutralizing antibodies, antibodies may be useful in competitive binding assays.

[0424] Antibodies that can be used in the disclosed compositions and methods include whole immunoglobulins of any class (i.e., intact antibodies), fragments thereof, and synthetic proteins containing at least the antigen-binding variable domain of an antibody. The variable domains have different sequences among antibodies and are used for the binding and specificity of each particular antibody to its particular antigen. However, variability is not usually evenly distributed throughout the variable domain of an antibody. Typically, variability is aggregated into three segments called complementarity-determining regions (CDRs) or hypervariable regions in both the light chain and heavy chain variable domains. The more highly conserved portion of the variable domain is called the framework (FR). The native heavy chain and light chain variable domains each contain four FR regions, primarily in a β-sheet configuration and linked by three CDRs, which connect the β-sheet structures and, in some cases, form loops that form part of them. The CDRs in each chain are held together in close proximity by the FR regions and, together with CDRs from other chains, contribute to the formation of the antibody's antigen-binding site.

[0425] Antibody fragments possessing biological activity are also disclosed. The fragments include insertions, deletions, substitutions, or other selected modifications of specific regions or specific amino acid residues, whether or not they are bound to other sequences, provided that the activity of the fragment is not significantly altered or impaired compared to an unmodified antibody or antibody fragment.

[0426] The technique can also be adapted to produce single-chain antibodies specific to antigen peptides. Methods for producing single-chain antibodies are well known to those skilled in the art. Single-chain antibodies can be produced by fusing the variable domains of the heavy and light chains together using a short peptide linker, thereby reconstituted the antigen-binding site on a single molecule. Variable fragments of single-chain antibodies (scFv) have been developed in which the C-terminus of one variable domain is linked to the N-terminus of another variable domain via 15-25 amino acid peptides or a linker, which does not significantly interfere with antigen binding or binding specificity. The linker is selected so as to allow the heavy and light chains to bind together in the appropriate conformational orientation.

[0427] Divalent single-stranded variable fragments (di-scFv) can be manipulated by joining two scFvs. This can be done by producing a single peptide chain with two VH regions and two VL regions, thereby producing a tandem scFv. scFvs can also be designed with a linker peptide (approximately 5 amino acids) that is too short for the two variable regions to fold together, forcing the scFv to dimerize. This type is known as a diabody. Diabodies have been shown to have dissociation constants up to 40 times lower than their corresponding scFvs, meaning they have a much higher affinity for their target. Even shorter linkers (1 or 2 amino acids) result in the formation of trimers (tribodies or tribodies). Tetrabodies have also been produced. Tetrabodies exhibit even higher affinity for their target than diabodies.

[0428] Monoclonal antibodies are obtained from a substantially homogeneous antibody population; that is, individual antibodies within the population are identical except for naturally occurring mutations that may exist in a small subset of the antibody molecule. Monoclonal antibodies include "chimeric" antibodies in which parts of the heavy and / or light chain are identical or homologous to corresponding sequences in antibodies originating from a particular species or belonging to a particular antibody class or subclass, while the rest of the chain is identical or homologous to corresponding sequences in antibodies originating from another species or belonging to another antibody class or subclass, and fragments of such antibodies insofar as they exhibit the desired antagonistic activity.

[0429] Monoclonal antibodies can be produced using any procedure for producing monoclonal antibodies. In the hybridoma method, mice or other suitable host animals are typically immunized with an immunizer to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizer. Alternatively, lymphocytes can be immunized in vitro.

[0430] Antibodies can also be produced by recombinant DNA methods. The DNA encoding the disclosed antibodies can be readily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of mouse antibodies). Libraries of antibodies or active antibody fragments can also be generated and screened using phage display technology.

[0431] Methods for producing antibodies using protein chemistry are also known in the art. One method for producing antibodies-containing proteins is to combine two or more peptides or polypeptides by protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert-butyloxycarbonyl) chemistry and currently available laboratory equipment (Applied Biosystems, Inc., Foster City, CA). Those skilled in the art will readily understand, for example, that peptides or polypeptides corresponding to antibodies can be synthesized by standard chemical reactions. For example, a peptide or polypeptide may be synthesized and not cleaved from its synthetic resin, while other fragments of an antibody may be cleaved from the resin after synthesis, thereby exposing terminal groups that are functionally blocked on the other fragment. By peptide condensation reactions, these two fragments can be covalently linked by peptide bonds at their carboxyl and amino termini, respectively, to form an antibody or fragment thereof. Alternatively, peptides or polypeptides can be synthesized independently in vivo as described above. Once isolated, these independent peptides or polypeptides can be combined by similar peptide condensation reactions to form antibodies or their antigen-binding fragments.

[0432] For example, cloning or enzymatic linking of synthetic peptide segments allows relatively short peptide fragments to be linked to produce larger peptide fragments, polypeptides, or entire protein domains. Alternatively, the innate chemical bonds of synthetic peptides can be utilized to synthetically construct larger peptides or polypeptides from shorter peptide fragments. This method involves a two-step chemical reaction. The first step is a chemoselective reaction between an unprotected synthetic peptide-α-thioester and another unprotected peptide segment containing an amino-terminal Cys residue, yielding a thioester-linked intermediate as the initial covalent product. Without changing the reaction conditions, this intermediate undergoes a spontaneous and rapid intramolecular reaction to form a native peptide bond at the binding site.

[0433] B. Methods for producing proteins The disclosed proteins, polypeptides, their fragments, variants, and fusions can be produced using prior art known in the art. Isolated fusion proteins can be obtained, for example, by chemosynthesis or by recombinant production in host cells. To produce proteins, polypeptides, their fragments, variants, or fusions by recombinant production, nucleic acids containing nucleotide sequences encoding proteins, polypeptides, their fragments, variants, or fusions can be used to transform, transduce, or transfect bacterial or eukaryotic host cells (e.g., insect, yeast, or mammalian cells). Generally, nucleic acid constructs include regulatory sequences operably bound to nucleotide sequences encoding proteins, polypeptides, their fragments, variants, or fusions. Regulatory sequences (also referred herein as expression control sequences) typically do not encode gene products, but instead affect the expression of the nucleic acid sequences to which they are operably bound.

[0434] Useful prokaryotic and eukaryotic systems for the expression and production of polypeptides are well known in the art and include, for example, Escherichia coli strains such as BL-21 and cultured mammalian cells such as CHO cells.

[0435] In eukaryotic host cells, several virus-based expression systems can be used to express fusion proteins. These virus-based expression systems are well-known in the art and include, but are not limited to, baculoviruses, SV40, retroviruses, or vaccinia-derived viral vectors.

[0436] Mammalian cell lines that stably express proteins, polypeptides, their fragments, variants, or fusions can be produced using expression vectors with appropriate regulatory elements and selectable markers. For example, eukaryotic expression vectors pCR3.1 (Invitrogen Life Technologies) and p91023(B) (see Wong et al. (1985) Science 228:810-815) are suitable for the expression of proteins, polypeptides, their fragments, variants, or fusions 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 from Lonza Group Ltd.

[0437] After introducing an expression vector by electroporation, lipofection, calcium phosphate or calcium chloride coprecipitation, DEAE dextran, or other suitable transfection methods, a stable cell line can be selected (e.g., by metabolic selection or by antibiotic resistance to G418, kanamycin, or hygromycin). The transfected cells can be cultured to express the polypeptide of interest, which can be recovered, for example, from the cell culture supernatant or lysed cells. Alternatively, proteins, polypeptides, their fragments, variants, or fusions can be produced by (a) conjugating an amplified sequence to a mammalian expression vector such as pcDNA3 (Invitrogen Life Technologies), and (b) transcribing and translating it in vitro using wheat germ extract or rabbit reticulocyte lysate.

[0438] Proteins, polypeptides, their fragments, variants, or fusions can be isolated using 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, their fragments, variants, or fusions can be manipulated to include additional domains containing amino acid sequences that allow the polypeptide to be captured in an affinity matrix. For example, Fc fusion polypeptides in cell culture supernatant or cytoplasmic extracts can be isolated using a protein A column. Furthermore, tags such as c-myc, hemagglutinin, polyhistidine, or Flag® (Kodak) can be used to aid in polypeptide purification. Such tags can be inserted anywhere within a polypeptide, including either a carboxyl terminus or an amino terminus. Other potentially useful fusions include enzymes that aid in polypeptide detection, such as alkaline phosphatases. Immunoaffinity chromatography can also be used to purify polypeptides. Fusion proteins can be further manipulated to include secretory signals (if no secretory signals already exist) that cause the protein, polypeptide, their fragments, variants, or fusions to be secreted by the cells producing them. The secreted proteins, polypeptides, their fragments, variants, or fusions can then be conveniently isolated from the cell culture medium.

[0439] C. Method for producing isolated nucleic acid molecules Isolated nucleic acid molecules can be produced by standard techniques, including, but not limited to, general molecular cloning and chemical nucleic acid synthesis techniques. For example, polymerase chain reaction (PCR) techniques can be used to obtain isolated nucleic acids encoding variant polypeptides. PCR is a technique for enzymatically amplifying target nucleic acids. Typically, sequence information from the end or beyond of the target region can be used to design oligonucleotide primers whose sequence is identical to that of the opposite strand of the template being amplified. PCR can be used to amplify DNA and RNA-derived specific sequences, including sequences from whole-genomic DNA or whole-cellular RNA. Primers are typically 14–40 nucleotides long, but can range from 10 to several hundred nucleotides long. General PCR techniques are described, for example, in *PCR Primer: A Laboratory Manual* ed. by Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995. When RNA is used as the template source, a complementary DNA (cDNA) strand can be synthesized using reverse transcriptase. Isolated nucleic acids can also be obtained using ligase chain reaction, strand substitution amplification, auto-persistent sequence replication, or nucleic acid sequence-based amplification. For example, see 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.

[0440] Isolated nucleic acids can be synthesized chemically (e.g., using phosphoramidite techniques for automated DNA synthesis in the 3'-5' direction) as a single nucleic acid molecule or as a series of oligonucleotides. For example, one or more pairs of long oligonucleotides (e.g., >100 nucleotides) containing the desired sequence can be synthesized, each pair containing a complementary short segment (e.g., about 15 nucleotides) such that a double helix is ​​formed when the oligonucleotide pair is annealed. Oligonucleotides can be extended using DNA polymerase, thereby yielding a single double-stranded nucleic acid molecule per pair of oligonucleotides, which can then be conjugated to a vector. Isolated nucleic acids can also be obtained by mutagenesis. Protein-coding nucleic acids can be mutated using standard techniques, including oligonucleotide-specific mutagenesis and / or site-specific mutagenesis by PCR. See Short Protocols in Molecular Biology. Chapter 8, Green Publishing Associates and John Wiley & Sons, edited by Ausubel et al, 1992.

[0441] IV. Assays and Antibody Screening Assays for antibody screening include: 1. Analysis of the binding affinity of B7-H4-Fc to its ligand compared with B7-H4. 2. Functional assays to identify B7-H4-Fc inhibit signaling by B7-H4 expressing cells. Reporter cells can be used in these assays, or primary B7-H4+ cells are an alternative.

[0442] B7-H4-deficient ("knockout") mice or wild-type mice can be used to generate high-affinity mAbs against B7-H4 using proprietary immunization techniques. Autoimmune susceptible mice NZB / WF1 can be used to generate mAbs to overcome "resistance."

[0443] 1. Phase I screening: mAb binding to cell lines transfected to express cell surface B7-H4. Furthermore, the mAbs must have the ability to bind to endogenously expressed B7-H4 on the surface of a primary human cell subset. These mAbs must be highly specific to B7-H4. To detect anti-B7-H4 antibodies, purified B7-H4 protein can be used to screen for antibodies using ELISA.

[0444] 2. Phase II screening: B7-H4 specific mAbs must block B7-H4 binding to the ligand and / or target cells.

[0445] 3. Phase III Screening: Functional assays to confirm that B7-H4 mAbs or combinations of mAbs modulate B7-H4 signaling. These assays utilize cell lines expressing endogenous B7-H4 or primary cells such as human monocytes, macrophages, and dendritic cell subsets to evaluate function in the presence of B7-H4 mAbs. Furthermore, reporter cell lines may be used to determine whether signaling pathways such as NFκB (NFκB reporter) or NFAT (NFAT reporter) are altered after culture with B7-H4 mAbs.

[0446] 4. Phase IV Screening: A functional assay to determine whether B7-H4 mAbs can induce antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cell-mediated cytotoxicity (CDC), or other mechanisms of cell apoptosis in B7-H4-expressing cell lines. In particular, B7-H4 mAbs are tested for their ability to deplete leukemia cell lines known to express B7-H4 on their cell surface using one of these methods. B7-H4 mAbs can also be engineered to deplete B7-H4-expressing cells and tested using known methods as described later in this document.

[0447] 5. Phase V Screening: Functional assays to determine whether B7-H4 mAbs can inhibit cellular function by delivering or inducing negative signals (agonists) to B7-H4-expressing cells via B7-H4. Cell lines endogenously expressing B7-H4 or transfectants of cell lines are evaluated for changes in phenotype and survival after being cultured with B7-H4 mAbs. Other assays use reporter cell lines to determine whether B7-H4 mAbs modulate positive signaling pathways such as NF-κB (NF-κB reporter) or other known cellular signaling reporters. Induction of apoptosis in cell lines is also evaluated.

[0448] Phase II and Phase III assays can be used to predict the concentration of B7-H4 mAb required to block physiological levels of ligand in vivo.

[0449] V. How to use B7-H4 antagonists or agonists can be used to modulate the immune response in subjects requiring treatment to regulate the immune response.

[0450] The example method will be discussed in more detail below.

[0451] A. Immune response stimulation 1. Treatment Strategy Methods are provided for inducing or enhancing an immune response in a subject. Typically, the method involves administering to the subject an effective amount of an immunomodulator, or cells primed ex vivo with an immunomodulator. The immune response may be, for example, a primary immune response to an antigen, or an increase in effector cell function, such as increased antigen-specific proliferation of T cells, enhanced cytokine production by T cells, stimulation of differentiation, or a combination thereof. In some embodiments, the active agent can increase the development of naive T cells to Th1, Th17, Th22, or other cells that secrete or cause other cells to secrete inflammatory molecules, including, but not limited to, IL-1β, TNF-α, TGF-β, IFN-γ, IL-17, IL-6, IL-23, IL-22, IL-21, and MMPs. In some embodiments, the active ingredient can reduce or inhibit Treg activity, reduce the production of cytokines such as IL-10 from Tregs, decrease Treg differentiation, reduce the number of Tregs, decrease the proportion of Tregs in the immune cell population, or decrease Treg viability. Immunomodulatory substances can be administered in effective doses to subjects in need to overcome T cell depletion and / or T cell anergy. Overcoming T cell depletion or T cell anergy can be determined by measuring T cell function using known techniques.

[0452] The method can be used in vivo or ex vivo as an immune response stimulating therapeutic application. Thus, in some embodiments, the active agent, or nucleic acid encoding the active agent, is administered directly to the subject. In some embodiments, the active agent, or nucleic acid encoding the active agent, is brought into contact with cells (e.g., immune cells) ex vivo, and the treated cells are administered to the subject (e.g., adoptive transfer). Generally, the disclosed immunomodulatory substances can be used to treat subjects who have or are prone to any disease or disorder in which the subject's immune system gives an immune response. The active agents enable a more robust immune response. The disclosed compositions are useful for stimulating or enhancing T cell-involved immune responses.

[0453] Immunomodulatory substances used to enhance the immune response typically reduce B7-H4 expression, ligand binding, crosslinking, negative signaling, or a combination thereof. For example, the activator may be a B7-H4 antagonist, such as an antagonist (blocker) anti-B7-H4 antibody or its antigen-binding fragment. This activator may also be a B7-H4 polypeptide, such as a soluble polypeptide or its fusion protein, that can act as a decoy receptor for one or more B7-H4 ligands or receptors.

[0454] For example, B7-H4 blockade using an anti-B7-H4 antibody that blocks function can be an alternative or complementary activator 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 its fusion protein. Combined treatments (e.g., B7-H4-Fc and B7-H4 blockade) can be complementary.

[0455] In some embodiments, immune response stimulating therapy (e.g., in the treatment of cancer or infection) includes depletion of B7-H4+ cells.

[0456] The development and identification of B7-H4 depletion mAbs can be carried out according to known construction and screening methods, including those discussed herein. For example, see Reff, et al, Blood. Vol 83, No 2, 1994: pp435-445, which describes the preparation of an anti-CD20 chimeric antibody, which binds to human Clq and mediates complement-dependent cell lysis (CDCC) in the presence of human complement, and antibody-dependent cell-mediated cytotoxicity (ADCC) with human effector cells. Rituximab destroys B cells and is therefore used to treat diseases characterized by hyperactivity, dysfunction, or an excess number of B cells. Other B cell depletion antibodies include ocrelizumab and ofatumumab. In another example, CD3 Ab is CD4 + Foxp3 +While retaining Treg cells, activated effector T cells can be preferentially targeted and depleted. Antibodies transiently deplete T cells but show little to no complement-dependent cytotoxicity and antibody-dependent cytotoxicity. Redirected cytolysis has been shown, resulting from the ability to crosslink CD3 molecules expressed by two different cells (one cytotoxic CD8+ T cell and the other other target T cell), but T cell depletion has been shown to be mostly due to AICD (reviewed in You, Front Immunol. 2015;6:242).

[0457] For subjects requiring enhanced immune response, substances that inhibit or block the B7-H4 suppressive immune response can be administered in amounts effective in increasing antigen uptake by antigen-presenting cells (APCs).

[0458] 2. Subjects to Treatment a. Cancer treatment The disclosed compositions and methods can be used to treat cancer. Generally, the active agents are used to stimulate or enhance the immune response against cancer in a subject by administering to the subject an amount of immunomodulatory substance that reduces B7-H4 expression, ligand binding, crosslinking, negative signaling, or a combination thereof. Cancer cells acquire a characteristic set of functional capabilities during their development, albeit through a variety of mechanisms. Such capabilities include evasion of apoptosis, self-sufficiency of proliferation signals, insensitivity to anti-proliferative signals, tissue invasion / metastasis, unlimited replication capacity, and persistent angiogenesis. The term “cancer cells” means to encompass both pre-malignant and malignant cancer cells. In some embodiments, cancer refers to a benign tumor that maintains localization. In other embodiments, cancer refers to a malignant tumor that invades and destroys adjacent body structures and spreads to distal sites. In yet another embodiment, cancer is associated with specific cancer antigens (e.g., pan-cancer antigen (KS1 / 4), ovarian cancer antigen (CA125), prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), CD19, CD20, HER2 / neu, etc.).

[0459] The methods and compositions disclosed herein include, but are not limited to, cancers including bladder cancer, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid cancer, and skin cancer; cancers including squamous cell carcinoma; lymphoid hematopoietic malignancies including leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, and Burkitt lymphoma; myeloid hematopoietic malignancies including acute and chronic myeloid leukemia and promyelocytic leukemia; and fibromas. It is useful in the treatment or prevention of various cancers or other abnormal proliferative diseases, including mesenchymal tumors, including rhabdomyosarcoma; other tumors, including melanoma, seminomas, teratocarcinomas, neurotumors, and gliomas; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannoma; mesenchymal tumors, including fibromas, rhabdomyosarcomas, and osteosarcomas; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminomas, follicular carcinoma of the thyroid, and teratocarcinomas.

[0460] Cancers caused by abnormalities in apoptosis can also be treated by the disclosed methods and compositions. Such cancers may include, but are not limited to, follicular lymphoma, cancers with p53 mutations, hormone-dependent mammary gland tumors, prostate and ovarian cancers, precancerous lesions such as familial adenomatous polyposis, and myelodysplastic syndromes. In certain embodiments, malignant tumors or dysproliferative changes (such as metaplasia and dysplasia), or hyperproliferative disorders are treated or prevented by the methods and compositions in the ovaries, bladder, breast, colon, lungs, skin, pancreas, or uterus. In other specific embodiments, sarcomas, melanomas, or leukemias are treated or prevented by the methods and compositions.

[0461] The disclosed compositions and methods are particularly useful for treating cancers associated with cells expressing abnormally high levels of B7-H4.

[0462] Certain cancers and related disorders that can be treated or prevented by the methods and compositions disclosed herein include, but are not limited to, acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, such as myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, and myelodysplastic syndromes, chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia, and chronic leukemia; polycythemia vera; Hodgkin's disease or non-Hodgkin's lymphoma (e.g., diffuse anaplastic lymphoma kinase (ALK)-negative large B-cell lymphoma (DLBCL)); and Lymphomas including but not limited to diffuse anaplastic lymphoma kinase (ALK)-positive large B-cell lymphoma (DLBCL), anaplastic lymphoma kinase (ALK)-positive ALK+ anaplastic large cell lymphoma (ALCL), and acute myeloid lymphoma (AML); multiple myelomas including but not limited to smoldering multiple myeloma, non-secretory myeloma, osteosclerosing myeloma, plasma cell leukemia, solitary plasmacytoma, and extramedullary plasmacytoma; Waldenström macroglobulinemia; monoclonal immunoglobulinemia of unknown significance; benign monoclonal immunoglobulinemia; heavy chain disease; osteosarcoma (bone sarcomas), osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, osteofibrosarcoma, chordoma, periosteosarcoma, soft tissue sarcoma, angiosarcoma (including but not limited to hemangiosarcoma, fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, schwannoma, rhabdomyosarcoma, synovial sarcoma, etc., osteosarcoma and connective tissue sarcoma; glioma, astrocytoma, brainstem glioma, ependymoma, oligodendroglioma, non-glioma, auditory Brain tumors, including but not limited to neuromas, craniopharyngiomas, medulloblastomas, meningiomas, pineal cell tumors, pineal blastomas, and primary brain lymphomas; breast cancers, including but not limited to adenocarcinomas, lobular (small cell) carcinomas, ductal carcinomas, medullary breast carcinomas, mucinous breast carcinomas, tubular breast carcinomas, papillary breast carcinomas, Paget's disease, and inflammatory breast carcinomas; adrenal carcinomas, including but not limited to pheochromocytomas and adrenocortical carcinomas; thyroid cancers, including but not limited to papillary or follicular thyroid carcinomas, medullary thyroid carcinomas, and anaplastic thyroid carcinomas;Pancreatic cancer, including but not limited to insulinoma, gastrinoma, glucagonoma, VIP-producing tumor (vipoma), somatostatin-secreting tumor, and carcinoid or islet cell tumor; pituitary adenoma, including but not limited to Cushing's disease, prolactin-secreting tumor, acromegaly, and diabetes insipidus; ocular cancer, including but not limited to ocular melanoma, such as ciliary melanoma, choroidal malignant melanoma, and uveal melanoma, as well as retinoblastoma; vaginal cancer, including but not limited to squamous cell carcinoma, adenocarcinoma, and melanoma; squamous cell carcinoma Vulvar cancer, including but not limited to melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; uterine sarcoma, including but not limited to squamous cell carcinoma and adenocarcinoma; uterine cancer, including but not limited to endometrial cancer and uterine sarcoma; ovarian cancer, including but not limited to ovarian epithelial carcinoma, borderline malignant tumor, germ cell tumor, and stromal tumor; esophageal cancer, including but not limited to squamous cell carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucosal epidermal carcinoma, adenosquamous cell carcinoma, sarcoma, melanoma, plasmacytoma, verrucous, and oat cell (small cell) carcinoma; adenocarcinoma, fungal (po) Gastric cancer, including but not limited to leaping, ulcerative, superficial spreading, diffuse spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancer; rectal cancer; liver cancer, including hepatocellular carcinoma and hepatoblastoma; gallbladder cancer, including but not limited to adenocarcinoma; cholangiocarcinoma, including but not limited to papillary, nodular, and diffuse; lung cancer, including but not limited to non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large cell carcinoma, and small cell lung cancer; germ cell tumors, seminoma, undifferentiated carcinoma, classical (typical), spermatocyte, non-seminoma Testicular cancer, including but not limited to cerebrospinal carcinoma, fetal carcinoma, teratoma, choriocarcinoma (yolk sac tumor); prostate cancer, including but not limited to adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; renal cancer; oral cancer, including but not limited to squamous cell carcinoma; basal carcinoma; salivary gland cancer, including but not limited to adenocarcinoma, mucosal epidermal carcinoma, and adenoid cystic carcinoma; pharyngeal cancer, including but not limited to squamous cell carcinoma and verrucous carcinoma; cutaneous cancer, including but not limited to basal cell carcinoma, squamous cell carcinoma, and melanoma, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, and acral lentigo melanoma;This includes kidney cancer, including but not limited to renal cell carcinoma, adenocarcinoma, adrenal tumor, fibrosarcoma, and transitional cell carcinoma (renal pelvis and / or ureter); Wilms' tumor; and bladder cancer, including but not limited to transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, and carcinosarcoma. Furthermore, cancers include myxosarcoma, osteosarcoma, endosarcoma, lymphangiosarcoma, mesothelioma, synoviomas, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchial carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, and papillary carcinoma (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., JBLippincott Co., Philadelphia and Murphy et al., 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books USA, Inc., United States of America).

[0463] b. Treatment of infectious diseases The disclosed compositions and methods can be used to treat infectious diseases and infectious illnesses. Generally, the active agents are used to stimulate or enhance the immune response to infectivity factors in a subject by administering an amount of immunomodulatory substance to the subject that reduces B7-H4 expression, ligand binding, crosslinking, negative signaling, or a combination thereof. The methods can alleviate one or more symptoms of an infection.

[0464] Neutrophils are a major component of the host's natural defense against infection and also contribute to autoimmune pathogenicity and chronic inflammation. During infection, neutrophils rapidly migrate to the site of inflammation, are activated, and initiate a cascade of defense mechanisms, including phagocytosis, death, and degradation of microorganisms by antimicrobial and proteolytic proteins, accompanied by the generation of reactive oxygen species. Neutrophils are also involved in tissue degradation, remodeling, wound healing, and the regulation of other inflammatory and adaptive immune components. Due to their short lifespan, neutrophils must be continuously replenished during infection and inflammation by proliferation from myeloid progenitor cells in the bone marrow. In vitro B7-H4 inhibits the proliferation of myeloid-derived neutrophil precursors, suggesting an inhibitory function of B7-H4 in neutrophil proliferation (Zhu, G. et al., Blood, 113:1759-1767 (2009)).

[0465] One embodiment provides a method for treating infection in subjects requiring treatment of infection by administering an effective amount of a substance that inhibits or blocks the B7-H4 suppressive immune response in an amount effective in promoting neutrophil proliferation and / or increasing innate immunity.

[0466] Infection or disease can be caused by bacteria, viruses, protozoa, helminths, or other microbial pathogens that invade cells and are attacked, i.e., by cytotoxic T lymphocytes.

[0467] Infections or diseases can be acute or chronic. Acute infections are typically short-lived infections. During acute microbial infection, immune cells initiate the expression of immunomodulatory receptors. Therefore, in some embodiments, the method involves increasing the immune-stimulating response to acute infections.

[0468] Infections can be caused by, for example, but are not limited to, Candida albicans, Listeria monocytogenes, Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria meningitidis, Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, or Mycobacterium.

[0469] In some embodiments, the disclosed compositions are used to treat chronic infections, such as those resulting in T-cell depletion or T-cell anergy, which in turn cause the infection to persist in the host for an extended period.

[0470] Exemplary infections treated include chronic infections caused by hepatitis viruses, human immunodeficiency viruses (HIV), human T-lymphotropic viruses (HTLV), herpesviruses, Epstein-Barr virus, or human papillomavirus.

[0471] Since viral infections are primarily eliminated by T cells, increased T cell activity would be therapeutically useful in situations where more rapid or complete elimination of infectious viral agents is beneficial to animal or human subjects. Therefore, the disclosed compositions, though not limited to, can be administered for the treatment of topical or systemic infections, including, but are not limited to, immunodeficiency (e.g., HIV), papillomas (e.g., HPV), herpes (e.g., HSV), encephalitis, influenza (e.g., human influenza A virus), and the common cold (e.g., human rhinovirus), and other viral infections caused by, for example, HTLV, hepatitis viruses, respiratory syncytial viruses, vaccinia viruses, and rabies viruses. The molecules can be administered topically to treat herpes lesions or viral skin diseases such as herpes zoster or genital warts. The molecules can also be administered systemically to treat systemic viral diseases, including, but are not limited to, AIDS, influenza, the common cold, or encephalitis.

[0472] The typical infectious diseases that can be treated include, but are not limited to, Actinomyces, Anabaena (Bacillus), Bacteroides, Bdellovibrio, Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium, and Chlorobium. Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus, Hemophilus influenzae type BB:HIB), Hyphomicrobium, Legionella, Leptospirosis, Listeria, Meningococcus A, B and C, Methanobacterium, Micrococcus, Myobacterium, Mycoplasma, Myxococcus, Neisseria, Nitrobacter, Oscillatoria, Prochloron, Proteus, Pseudomonas nas), Phodospirillum, Rickettsia, Salmonella, Shigella, Spirillum, Spirochaeta, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus, Thermoplasma, Thiobacillus, Treponema, Vibrio, Yersinia, Cryptococcus neoformans *Candida neoformans*, *Histoplasma capsulatum*, *Candida albicans*, *Candida tropicalis*, *Nocardia asteroides*, *Rickettsia ricketsii*, *Rickettsia typhi*, *Mycoplasma pneumoniae*, *Chlamydial psittaci*, *Chlamydial trachomatis*This includes infections caused by Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis, and Schistosoma mansoni.

[0473] Other microorganisms that can be treated using the compositions and methods of this disclosure include bacteria such as Klebsiella, Serratia, and Pasteurella; pathogens associated with cholera, tetanus, botulism, anthrax, plague, and Lyme disease; or fungi or parasitic pathogens such as Candida, e.g., Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus, Aspergillus (fumigatus, niger, etc.), and Mucorals. es) (Mucor, Absidia, Rhizophus), Sporothrix (Schenkii), Blastomyces (dermatitidis), Paracoccidioides (brasiliensis), Coccidioides (immitis), and Histoplasma (capsulatuma), Entomoeba historica, Balantidium *), Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brusey, Trypanosoma cruzi, Toxoplasma gondii This includes bacteria such as those belonging to the genera Sporotrichus, Blastomyces, Paracoccidioides, Coccidioides, Histoplasma, Entoamoeba historica, Balantidium, Naegleria, Acanthamoeba, Giardia, Cryptosporidium, Pneumocystis, Plasmodium vivax, Babesia, or Trypanosoma.

[0474] B. Inhibition of the immune response 1. Treatment Strategy Methods are provided for reducing or inhibiting the immune response in a subject. Typically, these methods involve administering an effective amount of an immunomodulator, or cells primed ex vivo with an immunomodulator, to the subject. The immune response may be, for example, a primary immune response to an antigen, or an increase in effector cell function, such as increased antigen-specific proliferation of T cells, enhanced cytokine production by T cells, stimulation of differentiation, or a combination thereof. Thus, in some embodiments, the activator reduces T cell proliferation, T cell cytokine production, T cell differentiation, or a combination thereof. In some embodiments, the activator can reduce the differentiation of naive T cells into Th1, Th17, Th22, or other cells that secrete or cause other cells to secrete inflammatory molecules, including, but not limited to, IL-1β, TNF-α, TGF-β, IFN-γ, IL-17, IL-6, IL-23, IL-22, IL-21, and MMPs. In some embodiments, the active ingredient can increase or promote Treg activity, increase the production of cytokines such as IL-10 from Tregs, increase Treg differentiation, increase the number of Tregs, increase the proportion of Tregs in the immune cell population, or increase Treg viability.

[0475] This method can be used in vivo or ex vivo for immune response inhibitory therapeutic applications. Therefore, in some embodiments, the active agent, or nucleic acid encoding the active agent, is administered directly to the subject. In some embodiments, the active agent, or nucleic acid encoding the active agent, is brought into contact with cells (e.g., immune cells) ex vivo, and the treated cells are administered to the subject (e.g., adoptive transfer). Generally, the disclosed immunomodulatory substances can be used to treat subjects who have, or are prone to, any disease or disorder in which the subject's immune system gives an overactive or inadequate immune response. The active agents can enable a less robust immune response. The disclosed compositions are useful for reducing or inhibiting immune responses involving T cells.

[0476] Immunomodulatory substances used to reduce the immune response typically increase B7-H4 expression, ligand binding, cross-linking, negative signaling, or a combination of these. For example, the agonist may be a B7-H4 agonist, such as an anti-B7-H4 antibody or its antigen-binding fragment.

[0477] a. Inflammatory response The disclosed compositions and methods can be used to treat inflammation. Generally, active agents are used to reduce or inhibit the immune response in a subject by administering an amount of immunomodulatory substances to the subject that increase B7-H4 expression, ligand binding, crosslinking, negative signaling, or a combination thereof. The methods can alleviate or reduce inflammatory symptoms. Inflammation can be acute, chronic, or persistent.

[0478] In some embodiments, immunomodulatory substances weaken the immune system. For example, the substance can be used to control an over-inflammatory response that damages healthy tissue. Therefore, in some embodiments, the substance is administered to a subject with an excessive inflammatory response. In such cases, controlling the excessive immune response may be beneficial to the subject.

[0479] b. Inflammatory and autoimmune diseases / disorders Substances that increase B7-H4 expression, ligand binding, crosslinking, negative signaling, or combinations thereof can also be used to treat inflammatory or autoimmune diseases and disorders. Representative inflammatory or autoimmune diseases / disorders include, but are not limited to, rheumatoid arthritis, systemic lupus erythematosus, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behçet's disease, bullous pemphigoid, cardiomyopathy, celiac disease-dermatitis, chronic fatigue syndrome-immunodeficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, scarring pemphigoid, cold agglutinin disease, CRESTO syndrome, Crohn's disease, Dego's disease, dermatomyositis, juvenile dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, and G This includes Leavs' disease, Guillain-Barré disease, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, insulin-dependent diabetes mellitus (Type 1), juvenile arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjögren's syndrome, Stiffman syndrome, Takayasu's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis.

[0480] In some embodiments, inflammation or autoimmune disease is caused by a pathogen or is a result of an infection.

[0481] VI. Combination Therapy The disclosed immunomodulatory substances can be administered alone or in combination with one or more additional therapeutic agents to subjects requiring them. In some embodiments, the immunomodulatory substance and the additional therapeutic agents are administered separately but simultaneously. The immunomodulatory substance and the additional therapeutic agents can also be administered as part of the same composition. In other embodiments, the immunomodulatory substance and the second therapeutic agent are administered separately and at different times but as part of the same treatment regimen.

[0482] The subject may be administered the first therapeutic agent 1, 2, 3, 4, 5, 6 hours or more before the administration of the second therapeutic agent, or 1, 2, 3, 4, 5, 6, 7 days or more before the administration of the second therapeutic agent. In some embodiments, the subject may be administered one or more doses of the first therapeutic agent every 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, or 48 days before the first administration of the second therapeutic agent. The immunomodulatory substance may be the first therapeutic agent or the second therapeutic agent.

[0483] Immunomodulatory substances and additional therapeutic agents may be administered as part of a treatment regimen. For example, if the first therapeutic agent can be administered every four days, the second therapeutic agent may be administered on day 1, day 2, day 3, or day 4, or in combination thereafter. The first or second therapeutic agent may be administered repeatedly throughout the entire treatment regimen.

[0484] Exemplary molecules include, but are not limited to, cytokines, chemotherapeutic agents, radionuclides, other immunotherapeutic agents, enzymes, antibiotics, antiviral drugs (in particular protease inhibitors alone or in combination with nucleosides for the treatment of HIV or hepatitis B or C), antiparasitic agents (helmins, protozoa), growth factors, growth inhibitors, hormones, hormone antagonists, antibodies and their bioactive fragments (including humanized, single-stranded, and chimeric antibodies), antigens and vaccine preparations (including adjuvants), peptide drugs, anti-inflammatory drugs, ligands that bind to Toll-like receptors to activate the innate immune system (including, but not limited to, CpG oligonucleotides), molecules that mobilize and optimize the adaptive immune system, other molecules that activate or upregulate the action of cytotoxic T lymphocytes, natural killer cells and helper T cells, and other molecules that inactivate or downregulate suppressor or regulatory T cells.

[0485] Additional therapeutic agents are selected based on the condition, disorder, or disease being treated. For example, immunomodulatory substances may be administered concurrently with one or more additional agents that function to enhance or promote the immune response, or to reduce or inhibit the immune response.

[0486] A. Increased immune response 1. Antibacterial agents For example, B7-H4 immunomodulatory substances can be used in a preventive or prophylactic role in the treatment and prevention of the diseases discussed above, and can also be used in the context of severe trauma such as major burns, open fractures, accidental amputations, or other wounds. Therefore, B7-H4 immunomodulatory substances can be administered to subjects in combination with antimicrobial agents, such as antibiotics, antifungals, antivirals, antiparasitic agents, or essential oils.

[0487] In some embodiments, to prevent further bacterial, fungal, or viral complications, subjects are administered B7-H4 immunomodulatory agents and / or antibacterial agents upon admission. Antibiotics can target pathogens, while B7-H4 immunomodulatory agents can stimulate the immune system to provide an enhanced response to treat or prevent further infections or diseases.

[0488] 2. Chemotherapy agents B7-H4 immunomodulatory substances can be combined with one or more chemotherapeutic agents and apoptosis promoters. Representative chemotherapeutic agents include, but are not limited to, amsacrin, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, chrysanthanspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide, irinotecan, leucovorin, and ri These include posomal doxorubicin, liposomal daunorubicin, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, larcitrexed, satoraplatin, streptozocin, tegafur-uracil, temozolomide, teniposide, thiotepa, thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, or combinations thereof. Representative apoptosis promoters include, but are not limited to, fludarabinetaurosporine, cycloheximide, actinomycin D, lactosylceramide, l5d-PGJ(2), and combinations thereof.

[0489] 3. Other immunomodulatory substances a. PD-1 Antagonist In some embodiments, the B7-H4 immunomodulator is administered co-administered with a PD-1 antagonist. Programmed Death-1 (PD-1) is a member of the CD28 receptor family that delivers a negative immune response when induced on T cells. Contact between PD-1 and one of its ligands (B7-H1 or B7-DC) induces an inhibitory response that enhances T cell proliferation and / or reduces the duration of the T cell response. Suitable PD-1 antagonists are described in U.S. Patents 8,114,845, 8,609,089, and 8,709,416, which are incorporated herein by reference in whole, and include compounds or agents that either interfere with or inhibit ligand binding to the PD-1 receptor, or directly bind to the PD-1 receptor without inducing inhibitory signaling via the PD-1 receptor, thereby blocking the PD-1 receptor.

[0490] In some embodiments, PD-1 receptor antagonists bind directly to the PD-1 receptor without inducing inhibitory signaling, and also bind to ligands of the PD-1 receptor to reduce or inhibit the ligands from inducing PD-1 receptor-mediated signaling. By reducing the number and / or amount of ligands that bind to the PD-1 receptor and induce transduction of the inhibitory signal, fewer cells can be attenuated by the negative signal delivered by PD-1 signal transduction, resulting in a more robust immune response.

[0491] PD-1 signaling is thought to be driven by the binding of PD-1 ligands (e.g., B7-H1 or B7-DC) to peptide antigens presented by the major histocompatibility complex (MHC) (see, e.g., Freeman, Proc. Natl. Acad. Sci. USA, 105:10275-10276 (2008)). Therefore, proteins, antibodies, or small molecules that prevent the co-binding of PD-1 and the TCR on the T cell membrane are also useful PD-1 antagonists.

[0492] In some embodiments, a PD-1 receptor antagonist is a small molecule antagonist or antibody that reduces or interferes with PD-1 receptor signaling by binding to a ligand for PD-1 or to PD-1 itself, and in particular, does not induce inhibitory signaling via the PD-1 receptor if co-binding of PD-1 and the TCR does not follow such binding. Other PD-1 antagonists intended by the method of the present invention include antibodies that bind to PD-1 or a ligand for PD-1, and other antibodies.

[0493] Suitable anti-PD-1 antibodies include, but are not limited to, those described in the following publications: PCT / IL03 / 00425(Hardy et al.,WO / 2003 / 099196) PCT / JP2006 / 309606(Korman et al.,WO / 2006 / 121168) PCT / US2008 / 008925(Li et al.,WO / 2009 / 014708) PCT / JP03 / 08420(Honjo et al.,WO / 2004 / 004771) PCT / JP04 / 00549(Honjo et al.,WO / 2004 / 072286) PCT / IB2003 / 006304(Collins et al.,WO / 2004 / 056875) PCT / US2007 / 088851(Ahmed et al.,WO / 2008 / 083174) PCT / US2006 / 026046(Korman et al.,WO / 2007 / 005874) PCT / US2008 / 084923(Terrett et al.,WO / 2009 / 073533) Berger et al., Clin. Cancer Res., 14:30443051 (2008).

[0494] A specific example of an anti-PD-1 antibody is the human anti-PD-1 antibody described in Kosak, US20070166281 (July 19, 2007, paragraph 42), which is administered at a dose of 3 mg / kg in some embodiments.

[0495] Examples of anti-B7-Hl antibodies include, but are not limited to, those described in the following publications: PCT / US06 / 022423 (WO / 2006 / 133396, released December 14, 2006) PCT / US07 / 088851 (WO / 2008 / 083174, released July 10, 2008) US2006 / 0110383 (Released May 25, 2006).

[0496] A specific example of an anti-B7-Hl antibody is the human anti-B7-Hl antibody, which is the antibody described in (WO / 2007 / 005874, published January 11, 2007).

[0497] Additional anti-PD-1 and anti-B7-H1 antibodies are disclosed in 2014 / 0044738, which are specifically incorporated herein by reference in their entirety.

[0498] For information on anti-B7-DC antibodies, see 7,411,051, 7,052,694, 7,390,888, and U.S. Publication No. 2006 / 0099203.

[0499] Other exemplary PD-1 receptor antagonists include, but are not limited to, B7-DC polypeptides, their homologs and variants, and any of the aforementioned active fragments, as well as fusion proteins incorporating any of these. In some embodiments, the fusion protein comprises a soluble portion of B7-DC bound to the Fc portion of an antibody such as human IgG, and does not incorporate all or part of the transmembrane portion of human B7-DC.

[0500] PD-1 antagonists can also be fragments of mammalian B7-H1 derived from mice or primates, such as humans, which bind to and block PD-1 but do not confer inhibitory signaling via PD-1. The fragments can also be part of fusion proteins, such as Ig fusion proteins.

[0501] Other useful polypeptide PD-1 antagonists include those that bind to ligands of the PD-1 receptor. These include the PD-1 receptor protein or soluble fragments thereof, which can bind to PD-1 ligands such as B7-H1 or B7-DC, preventing binding to the endogenous PD-1 receptor and thereby preventing inhibitory signaling. B7-H1 has also been shown to bind to protein B7.1 (Butte et al., Immunity, Vol.27, pp.111-122, (2007)). Such fragments also include soluble ECD moieties of the PD-1 protein, including mutations such as the A99L mutation that increases binding to the native ligand (Molnar et al., PNAS, 105:10483-10488 (2008)). B7-1 or soluble fragments thereof are also useful, as they can bind to the B7-H1 ligand and prevent binding to the endogenous PD-1 receptor and thereby prevent inhibitory signaling.

[0502] Both DNA and RNA PD-1 and B7-H1 antisense nucleic acids, as well as siRNA molecules, can be PD-1 antagonists. Such antisense molecules prevent the expression of PD-1 on T cells, as well as the production of T cell ligands such as B7-H1, PD-L1, and / or PD-L2. For example, siRNA complexed with a carrier such as polyethyleneimine (e.g., approximately 21-nucleotide-long siRNAs specific to genes encoding PD-1 or PD-1 ligands; either oligonucleotide is readily available commercially) (see Cubillos-Ruiz et al., J.Clin.Invest. 119(8):2231-2244 (2009)) is readily taken up by cells expressing PD-1 and its ligands, reducing the expression of these receptors and ligands, thereby achieving a decrease in inhibitory signaling in T cells and thus activating T cells.

[0503] b.CTLA4 Antagonist Other molecules useful for mediating the effects of T cells in the immune response are also intended as additional therapeutic agents. In some embodiments, the molecule is a CTLA4 antagonist, such as an antagonistic anti-CTLA4 antibody. Examples of anti-CTLA4 antibodies intended for use in the methods of the present invention include the antibody described in PCT / US2006 / 043690 (Fischkoff et al., WO / 2007 / 056539).

[0504] Dosages of anti-PD-1, anti-B7-H1, and anti-CTLA4 antibodies are known in the art and may be, for example, in the range of 0.1 to 100 mg / kg, or in a shorter range of 1 to 50 mg / kg or 10 to 20 mg / kg. Appropriate doses for human subjects may be 5 to 15 mg / kg, or 10 mg / kg of antibody in a specific embodiment (e.g., human anti-PD-1 antibody).

[0505] Specific examples of anti-CTLA4 antibodies useful in the method of the present invention include ipilimumab, a human anti-CTLA4 antibody administered at a dose of approximately 10 mg / kg, and tremelimumab, a human anti-CTLA4 antibody administered at a dose of approximately 15 mg / kg. See also Sammartino, et al., Clinical Kidney Journal, 3(2):135-137 (2010), published online in December 2009.

[0506] In other embodiments, the antagonist is a small molecule. A series of small organic compounds have been shown to bind to the B7-1 ligand and prevent binding to CTLA4 (see Erbe et al., J. Biol. Chem., 277:7363-7368 (2002)). Such small organic compounds can be administered alone or in combination with an anti-CTLA4 antibody to reduce inhibitory signaling on T cells.

[0507] 4. Enhancers In some embodiments, additional therapeutic agents include enhancers. Enhancers may act to increase the potency of immune response-boosting regulators by two or more possible mechanisms, but the exact mechanism of action is not essential to the broad implementation of the invention.

[0508] In some embodiments, the enhancer is cyclophosphamide. Cyclophosphamide (CTX, Cytoxan®, or Neosar®) is an oxaphosphorine drug, and its analogues include ifosphamide (IFO, Ifex), perosphamide, trophosphamide (trofosfamide, Ixoten), and their pharmaceutically acceptable salts, solvates, prodrugs, and metabolites (all incorporated in U.S. Patent Application 200702077). Ifosphamide (MITOXANA®) is a structural analogue of cyclophosphamide, and its mechanism of action is considered to be identical or substantially similar to that of cyclophosphamide. Perphosphamide (4-hydroperoxycyclophosphamide) and trophosphamide are also alkylating agents structurally related to cyclophosphamide. For example, perosphamide alkylates DNA, thereby inhibiting DNA replication and RNA and protein synthesis. Novel oxazaphosphorine derivatives are being designed and evaluated in an attempt to improve selectivity and responsiveness with reduced host toxicity (Liang J, Huang M, Duan W, Yu XQ, Zhou S. Design of new oxazaphosphorine anticancer drugs. Curr Pharm Des. 2007;13(9):963-78. Review). These include maphosphamide (NSC345842), gluphosphamide (D19575, β-D-glucosylisophosphoramide mustard), S-(-)-bromosphamide (CBM-11), NSC612567 (aldophosphamide perhydrothiazine), and NSC613060 (aldophosphamide thiazolidinedion). Maphosphamide is an oxafoline analog, a chemically stable 4-thioethanesulfonate of 4-hydroxy-CPA. Gluphosphamide is an IFO derivative in which isophosphoramide mustard, an alkylated metabolite of IFO, is glycosidically linked to a β-D-glucose molecule.Additional cyclophosphamide analogs are described in U.S. Patent No. 5,190,929, entitled "Cyclophosphamide analogs useful as anti-tumor agents," which is incorporated herein by reference in its entirety.

[0509] While CTX itself is non-toxic, some of its metabolites are cytotoxic alkylating agents that induce DNA crosslinking and, at higher doses, strand breaks. Many cells are resistant to CTX because they express high levels of the detoxification enzyme aldehyde dehydrogenase (ALDH). CTX targets proliferating lymphocytes because lymphocytes (though not hematopoietic stem cells) express only low levels of ALDH, and cycling cells are the most sensitive to DNA alkylating agents.

[0510] Low doses of CTX (<200 mg / kg) can have immunostimulatory effects, including stimulation of antitumor immune responses in human and mouse models of cancer (Brode & Cooke Crit Rev. Immunol. 28:109-126 (2008)). These low doses are subthermal and do not have direct antitumor activity. In contrast, high doses of CTX inhibit antitumor responses. Several mechanisms of action may explain the role of CTX in enhancing antitumor immune responses: (a) depletion of CD4+CD25+FoxP3+Treg (and particularly suppressive, specifically proliferating Treg), (b) depletion of B lymphocytes, (c) induction of nitric oxide (NO) resulting in inhibition of tumor cell proliferation, and (d) recruitment and proliferation of CD1 1b+Gr-1+MDSCs. These primary effects have many secondary effects; for example, after Treg depletion, macrophages produce more IFN-γ and less IL-10. CTX has also been shown to induce type I IFN expression and promote homeostatic proliferation of lymphocytes.

[0511] Treg depletion is most frequently cited as a mechanism by which CTX enhances the anti-tumor immune response. This conclusion is based in part on the results of adoptive transfer experiments. In the AB1-HA tumor model, CTX treatment on day 9 resulted in a 75% cure rate. The transfer of purified Tregs on day 12 almost completely inhibited the CTX response (van der Most et al. Cancer Immunol. Immunother. 58:1219-1228 (2009)). Similar results were observed in the HHD2 tumor model: adoptive transfer of CD4+CD25+ Tregs after CTX pretreatment abolished the therapeutic response to the vaccine (Taieb, J. J. Immunol. 176:2722-2729 (2006)).

[0512] Multiple human clinical trials have demonstrated that low-dose CTX is a safe, well-tolerated, and effective agent for promoting the anti-tumor immune response (Bas, & Mastrangelo Cancer Immunol. Immunother. 47:1-12 (1998)).

[0513] The optimal dose of CTX for enhancing the anti-tumor immune response is a dose that reduces the total T cell count by decreasing Treg levels below the normal range but is below the therapeutic dose (see Machiels et al. Cancer Res. 61:3689-3697 (2001)).

[0514] In human clinical trials where CTX is used as an immune enhancer, a dose of 300 mg / m 2 is commonly used. In an average male (6 feet, 170 pounds (78 kg), body surface area 1.98 m 2 )), 300 mg / m 2This corresponds to 8 mg / kg or 624 mg of total protein. In mouse models of cancer, efficacy has been found in 30 g mice at doses ranging from 15 to 150 mg / kg for 0.45 to 4.5 mg of total protein (Machiels et al. Cancer Res. 61:3689-3697 (2001), Hengst et al. Cancer Res. 41:2163-2167 (1981), Hengst Cancer Res. 40:2135-2141 (1980)).

[0515] In larger mammals, such as primates, such as human patients, such mg / m 2 While doses may be used, unit doses administered over a limited time interval may be used. Such unit doses may be administered on a daily basis over a limited period such as up to 3 days, or up to 5 days, or up to 7 days, or up to 10 days, or up to 15 days, or up to 20 days, or up to 25 days, all as specifically intended by the present invention. The same regimen may be applied to other enhancers described herein.

[0516] In other embodiments, the enhancer is an agent that reduces the activity and / or number of regulatory T lymphocytes (T-Regs), such as sunitinib (SUTENT®), anti-TGFβ, or imatinib (GLEEVAC®). The described therapeutic regimens may also include the administration of an adjuvant.

[0517] Useful enhancers include mitotic inhibitors such as paclitaxol, aromatase inhibitors (e.g., letrozole), and angiogenesis inhibitors (VEGF inhibitors, e.g., Avastin, VEGF-Trap) (see, for example, Li et al., Vascular endothelial growth factor blockade reduces intratumoral regulatory T cells and enhances the efficacy of a GM-CSF-secreting cancer immunotherapy. Clin Cancer Res. 2006 Nov 15;12(22):6808-16.), as well as anthracyclines, oxaliplatin, doxorubicin, TLR4 antagonists, and IL-18 antagonists.

[0518] B. Reduction of the immune response 1. Immunosuppressants In some embodiments, an immune response or inflammatory / autoimmune disease / disorder is treated by targeting a B7-H4 immunomodulator and a second active agent which is an immunosuppressant. Immunosuppressants include, but are not limited to, antibodies against other lymphocyte surface markers (e.g., CD40, α-4 integrin) or cytokines), fusion proteins (e.g., CTLA-4-Ig (Orencia®), TNFR-Ig (Enbrel®)), TNF-α blockers (e.g., Enbrel, Remicade, Cimzia, and Humira), cyclophosphamide (CTX) (i.e., Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune®), methotrexate (MTX) (i.e., Rheumatrex®, Trexall®), belimumab (i.e., Benlysta®), or other immunosuppressants (e.g., cyclosporine A, FK506-like compounds, rapamycin compounds, steroids), antiproliferative agents, cytotoxic agents, or other compounds that aid in immunosuppression.

[0519] The therapeutic agent may be a CTLA-4 fusion protein, such as CTLA-4-Ig (abatacept). The CTLA-4-Ig fusion protein competes with the costimulatory receptor CD28 on T cells for binding to CD80 / CD86 (B7-1 / B7-2) on antigen-presenting cells, and thus functions to inhibit T cell activation. In another embodiment, the therapeutic agent is a CTLA-4-Ig fusion protein known as beratacept. Beratacept contains two amino acid substitutions (L104E and A29Y) that significantly increase its affinity for CD86 in vivo. In another embodiment, the therapeutic agent is Maxy-4.

[0520] In another embodiment, the therapeutic agent is cyclophosphamide (CTX). Cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune®), also known as cyclophosphan, is a nitrogen mustard alkylating agent derived from an oxazofolin group. It is used to treat various types of cancer and several autoimmune disorders. Cyclophosphamide (CTX) is the primary agent used for diffuse proliferative glomerulonephritis in patients with lupus nephritis.

[0521] Therapeutic agents can be administered in effective doses to reduce blood or serum levels of anti-double-stranded DNA (anti-dsDNA) autoantibodies and / or to reduce proteinuria in patients who require it.

[0522] In another embodiment, the therapeutic agent increases the amount of adenosine in the serum. See, for example, WO08 / 147482. For example, the second therapeutic agent may be CD73-Ig, recombinant CD73, or another agent that increases the expression of CD73 (e.g., a cytokine, monoclonal antibody, or small molecule). See, for example, WO04 / 084933. In another embodiment, the therapeutic agent is interferon-β.

[0523] The therapeutic agent may be a small molecule that inhibits or reduces the differentiation, proliferation, activity, and / or cytokine production and / or secretion by other cells that secrete or cause other cells to secrete inflammatory molecules, including but not limited to Th1, Th17, Th22, IL-1β, TNF-α, TGF-β, IFN-γ, IL-18, IL-17, IL-6, IL-23, IL-22, IL-21, and MMPs. In another embodiment, the therapeutic agent is a small molecule that interacts with Tregs, enhances Treg activity, promotes or enhances IL-10 secretion by Tregs, increases the number of Tregs, increases the inhibitory capacity of Tregs, or a combination thereof.

[0524] In some embodiments, the composition increases Treg activity or production. Exemplary Treg enhancers include, but are not limited to, the glucocorticoid fluticasone, salmetolal, antibodies against IL-12, IFN-γ, and IL-4, vitamin D3, and dexamethasone, as well as combinations thereof.

[0525] In some embodiments, the therapeutic agent is an antibody that blocks the function of pro-inflammatory molecules, such as IL-6, IL-23, IL-22, or IL-21.

[0526] As used herein, the term “rapamycin compound” includes the neutral tricyclic compound rapamycin, rapamycin derivatives, rapamycin analogs, and other macrolide compounds that are thought to have the same mechanism of action as rapamycin (e.g., inhibition of cytokine function). The term “rapamycin compound” also includes compounds that are structurally similar to rapamycin, such as compounds having a similar macrocyclic structure that have been modified to enhance its therapeutic effect. Exemplary rapamycin compounds are known in the art (see, for example, W095122972, WO95116691, WO95104738, U.S. Patent Nos. 6,015,809, 5,989,591, 5,567,709, 5,559,112, 5,530,006, 5,484,790, 5,385,908, 5,202,332, 5,162,333, 5,780,462, and 5,120,727).

[0527] The term "FK506-like compound" includes FK506 and its derivatives and analogues, such as compounds structurally similar to FK506, for example, compounds having a similar macrocyclic structure that have been modified to enhance their therapeutic effect. Examples of FK506-like compounds include, for example, those described in WO00101385. In some embodiments, the term "rapamycin compound" as used herein does not include FK506-like compounds.

[0528] 2. Anti-inflammatory drugs Other suitable therapeutic agents include, but are not limited to, anti-inflammatory agents. The anti-inflammatory agent may be a non-steroidal agent, a steroidal agent, or a combination thereof. One embodiment provides an oral composition containing about 1% (w / w) to about 5% (w / w), typically about 2.5% (w / w), of an anti-inflammatory agent. Representative examples of nonsteroidal anti-inflammatory drugs (NSAIDs) include, but are not limited to, oxicam, e.g., piroxicam, isoxicam, tenoxicam, sudoxicam; salicylates, e.g., aspirin, disalside, benolilate, trilysate, sapphin, sorprin, diflunisal, and fendosal; acetic acid derivatives, e.g., diclofenac, fenclofenac, indomethacin, sulindac, tolmetin, isoxepac, flofenac, thiopinac, zidomethacin, acematacin, fentiazac, zomepirac, clindanac, oxepinac, felbinac, and ketorolac; fename Examples include mefenamic acid, meclofenamic acid, flufenamic acid, diflumic acid, and tolfenamic acid; propionic acid derivatives, such as ibuprofen, naproxen, benoxaprofen, flurbiprofen, ketoprofen, fenoprofen, fenbufen, indoprofen, pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen, thioxaprofen, suprofen, aluminoprofen, and thiaprofen; and pyrazoles, such as phenylbutazone, oxyfenbutazone, feprazone, azapropazone, and trimethazone. Mixtures of these nonsteroidal anti-inflammatory drugs may also be used.

[0529] Representative examples of steroid anti-inflammatory drugs include, but are not limited to, corticosteroids such as hydrocortisone, hydroxyl-triamcinolone, alpha-methyldexamethasone, dexamethasone phosphate, beclomethasone dipropionate, clobetasol vivalent, desonide, desoxymethasone, desoxycorticosterone acetate, dexamethasone, dichlorolysone, diflorasone diacetate, diflucortolone vivalent, fluadrenolone, fluchlorolone acetonide, fludrocortisone, flumethasone vivalent, fluocinolone acetonide, fluocinonide, flucortin butyl ester, fluocortolone, fluprednilidene acetate, fludrenolone, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, and methylprednilidene. This includes nisolone, triamcinolone acetonide, cortisone, cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate, fluradrenolon, fludrocortisone, diflurosone diacetate, fluradrenolon acetonide, medrizone, amsinafel, amsinafid, betamethasone and its esters in balance, chloroprednisone, chlorprednisone acetate, crocorterone, cresinolone, dichlorizone, diflurepdonate, fluchloronide, flunisolide, fluoromethalone, fluperolon, fluprednisolone, hydrocortisone valerate, hydrocortisone cyclopentylpropionate, hydrocoltamate, meprednisone, paramethasone, prednisolone, prednisone, beclomethasone dipropionate, triamcinolone, and mixtures thereof.

[0530] VII. Kit The disclosed B7-H4 immunomodulatory substances can be packaged in sealed containers such as ampoules or sachets indicating the quantity. The active ingredient can be supplied as a dry, sterile, lyophilized powder or a water-free concentrate in a sealed container, which can be reconstituted, for example, with water or saline to an appropriate concentration for administration to a subject. For example, the active ingredient can be supplied as a dry, sterile, lyophilized powder in a sealed container in unit doses of at least 5 mg, or at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, or at least 75 mg. The lyophilized agent can be stored in the original container at 2-8°C and is typically administered within 12 hours, or 6 hours, or 5 hours, or 3 hours, or 1 hour after reconstitution.

[0531] In alternative embodiments, the active substance may be supplied in liquid form in a sealed container indicating the volume and concentration. In some embodiments, the liquid form of the active substance may be supplied in a sealed container containing at least 1 mg / ml, or at least 2.5 mg / ml, at least 5 mg / ml, at least 8 mg / ml, at least 10 mg / ml, at least 15 mg / ml, at least 25 mg / ml, at least 50 mg / ml, at least 100 mg / ml, at least 150 mg / ml, or at least 200 mg / ml of the active substance.

[0532] Pharmaceutical packaging and kits are also provided, which include one or more containers filled with an active substance. Furthermore, one or more other prophylactic or therapeutic agents useful for treating a disease may also be included in the pharmaceutical packaging or kit. Pharmaceutical packaging or kits may also include one or more containers filled with one or more of the components of the disclosed pharmaceutical composition. Optionally, a warning in the form prescribed by the government agency that controls the manufacture, use, or sale of a pharmaceutical or biological product may be attached to such container(s), and such warning reflects the approval by the agency for manufacture, use, or sale for human administration.

[0533] Kits designed for the methods described above are also provided. Embodiments typically include one or more B7-H4 immunomodulatory substances. In certain embodiments, the kit also includes one or more other prophylactic or therapeutic agents useful for treating cancer in one or more containers. In other embodiments, the kit also includes one or more anti-inflammatory agents useful for treating inflammatory and autoimmune diseases in one or more containers.

[0534] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in the field to which the disclosed invention pertains. Publications cited herein and documents citing them are incorporated specifically by reference.

[0535] Those skilled in the art will recognize many equivalents to the specific embodiments of the invention described herein, or can confirm them through mere routine experimentation. Such equivalents are intended to be covered by the following claims.

[0536] Sequence information SEQUENCE LISTING <110> NextCure, Inc. <120> B7-H4 Antibodies and Methods of Use Thereof <150> US 62 / 620,545 <151> 2018-01-23 <160> 131 <170> PatentIn version 3.5 <210> 1 <211> 282 <212> PRT <213> Homo sapiens <400> 1 Met Ala Ser Leu Gly Gln Ile Leu Phe Trp Ser Ile Ile Ser Ile Ile 1 5 10 15 Ile Ile Leu Ala Gly Ala Ile Ala Leu Ile Ile Gly Phe Gly Ile Ser 20 25 30 Gly Arg His Ser Ile Thr Val Thr Thr Val Ala Ser Ala Gly Asn Ile 35 40 45 Gly Glu Asp Gly Ile Leu Ser Cys Thr Phe Glu Pro Asp Ile Lys Leu 50 55 60 Ser Asp Ile Val Ile Gln Trp Leu Lys Glu Gly Val Leu Gly Leu Val 65 70 75 80 His Glu Phe Lys Glu Gly Lys Asp Glu Leu Ser Glu Gln Asp Glu Met 85 90 95 Phe Arg Gly Arg Thr Ala Val Phe Ala Asp Gln Val Ile Val Gly Asn 100 105 110 Ala Ser Leu Arg Leu Lys Asn Val Gln Leu Thr Asp Ala Gly Thr Tyr 115 120 125 Lys Cys Tyr Ile Ile Thr Ser Lys Gly Lys Gly Asn Ala Asn Leu Glu 130 135 140 Tyr Lys Thr Gly Ala Phe Ser Met Pro Glu Val Asn Val Asp Tyr Asn 145 150 155 160 Ala Ser Ser Glu Thr Leu Arg Cys Glu Ala Pro Arg Trp Phe Pro Gln 165 170 175 Pro Thr Val Val Trp Ala Ser Gln Val Asp Gln Gly Ala Asn Phe Ser 180 185 190 Glu Val Ser Asn Thr Ser Phe Glu Leu Asn Ser Glu Asn Val Thr Met 195 200 205 Lys Val Val Ser Val Leu Tyr Asn Val Thr Ile Asn Asn Thr Tyr Ser 210 215 220 Cys Met Ile Glu Asn Asp Ile Ala Lys Ala Thr Gly Asp Ile Lys Val 225 230 235 240 Thr Glu Ser Glu Ile Lys Arg Arg Ser His Leu Gln Leu Leu Asn Ser 245 250 255 Lys Ala Ser Leu Cys Val Ser Ser Phe Phe Ala Ile Ser Trp Ala Leu 260 265 270 Leu Pro Leu Ser Pro Tyr Leu Met Leu Lys 275 280 <210> 2 <211> 283 <212> PRT <213> Mus musculus <400> 2 Met Ala Ser Leu Gly Gln Ile Ile Phe Trp Ser Ile Ile Asn Ile Ile 1 5 10 15 Ile Ile Leu Ala Gly Ala Ile Ala Leu Ile Ile Gly Phe Gly Ile Ser 20 25 30 Gly Lys His Phe Ile Thr Val Thr Thr Phe Thr Ser Ala Gly Asn Ile 35 40 45 Gly Glu Asp Gly Thr Leu Ser Cys Thr Phe Glu Pro Asp Ile Lys Leu 50 55 60 Asn Gly Ile Val Ile Gln Trp Leu Lys Glu Gly Ile Lys Gly Leu Val 65 70 75 80 His Glu Phe Lys Glu Gly Lys Asp Asp Leu Ser Gln Gln His Glu Met 85 90 95 Phe Arg Gly Arg Thr Ala Val Phe Ala Asp Gln Val Val Val Gly Asn 100 105 110 Ala Ser Leu Arg Leu Lys Asn Val Gln Leu Thr Asp Ala Gly Thr Tyr 115 120 125 Thr Cys Tyr Ile Arg Thr Ser Lys Gly Lys Gly Asn Ala Asn Leu Glu 130 135 140 Tyr Lys Thr Gly Ala Phe Ser Met Pro Glu Ile Asn Val Asp Tyr Asn 145 150 155 160 Ala Ser Ser Glu Ser Leu Arg Cys Glu Ala Pro Arg Trp Phe Pro Gln 165 170 175 Pro Thr Val Ala Trp Ala Ser Gln Val Asp Gln Gly Ala Asn Phe Ser 180 185 190 Glu Val Ser Asn Thr Ser Phe Glu Leu Asn Ser Glu Asn Val Thr Met 195 200 205 Lys Val Val Ser Val Leu Tyr Asn Val Thr Ile Asn Asn Thr Tyr Ser 210 215 220 Cys Met Ile Glu Asn Asp Ile Ala Lys Ala Thr Gly Asp Ile Lys Val 225 230 235 240 Thr Asp Ser Glu Val Lys Arg Arg Ser Gln Leu Gln Leu Leu Asn Ser 245 250 255 Gly Pro Ser Pro Cys Val Phe Ser Ser Ala Phe Val Ala Gly Trp Ala 260 265 270 Leu Leu Ser Leu Ser Cys Cys Leu Met Leu Arg 275 280 <210> 3 <211> 107 <212> PRT <213> Mus musculus <400> 3 Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Arg Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Thr Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Phe Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Val Thr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 4 <211> 11 <212> PRT <213> Mus musculus <400> 4 Lys Ala Ser Gln Asp Val Arg Thr Ala Val Ala 1 5 10 <210> 5 <211> 7 <212> PRT <213> Mus musculus <400> 5 Ser Thr Ser Tyr Arg Tyr Thr 1 5 <210> 6 <211> 9 <212> PRT <213> Mus musculus <400> 6 Gln Gln Tyr Tyr Val Thr Pro Leu Thr 1 5 <210> 7 <211> 321 <212> DNA <213> Mus musculus <400> 7 gacattgtga tgacccagtc tcacaaattc atgtccacat cagtaggaga cagggtcagt 60 atcacctgca aggccagtca ggatgtgaga actgctgtag cctggtatca acagaaacca 120 ggacaatctc ctaaactact gatttactcg acatcctacc ggtacactgg agtccctgat 180 cgcttcactg gcagtggatc tgggacggaa ttcactttca ccatcagcag tgtgcaggct 240 gaagacctgg cagtttatta ctgtcagcaa tattatgtta ctccgctcac gttcggtgct 300 gggaccaagc tggagctgaa a 321 <210> 8 <211> 117 <212> PRT <213> Mus musculus <400> 8 Glu Val Gln Leu Gln Gln Ser Gly Thr Val Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Tyr Pro Gly Asn Ser Asp Thr Lys Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Lys Leu Thr Ala Val Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Asn Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Ser Thr Val Arg Asn Val Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser 115 <210> 9 <211> 5 <212> PRT <213> Mus musculus <400> 9 Ser Tyr Trp Met His 1 5 <210> 10 <211> 18 <212> PRT <213> Mus musculus <400> 10 Ala Ile Tyr Pro Gly Asn Ser Asp Thr Lys Tyr Asn Gln Lys Phe Lys 1 5 10 15 Aspen Light <210> 11 <211> 8 <212> PRT <213> Mus musculus <400> 11 Thr Val Arg Asn Val Met Asp Tyr 1 5 <210> 12 <211> 351 <212> DNA <213> Mus musculus <400> 12 gaggttcagc tccagcagtc tgggactgtg ctggcaaggc ctggggcttc agtgaagatg 60 tcctgcaagg cttctggcta cacctttacc agctactgga tgcactggat aaaacagagg 120 cctggacagg gtctggaatg gattggcgct attatcctg gaaatagtga tactaaatac 180 aaccagaagt tcaaggacaa ggccaaactg actgcagtca catctgccag cactgcctac 240 atggagctca gcagcctgac aaatgaggac tctgcggtct attactgtac atctacggta 300 cggaatgtta tggactactg gggtcaagga acctcagtca ccgtctcctc a 351 <210> 13 <211> 107 <212> PRT <213> Mus musculus <400> 13 Glu Ile Gln Met Thr Gln Sir Pro Sister Sister Sister Sir Ala Sir Leu Gly 1 5 10 15 Asp Arg And Thr And Thr Cys Gln To Serve Thr Gln Asp And Val Lys 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Ser Phe Leu Ile 35 40 45 Tyr Tyr Thr Ala Gln Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Ser 65 70 75 80 Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Phe Tyr Glu Phe Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 14 <211> 11 <212> PRT <213> Mus musculus <400> 14 Gln Ala Thr Gln Asp Ile Val Lys Ser Leu Asn 1 5 10 <210> 15 <211> 7 <212> PRT <213> Mus musculus <400> 15 Tyr Thr Ala Gln Leu Ala Glu 1 5 <210> 16 <211> 9 <212> PRT <213> Mus musculus <400> 16 Leu Gln Phe Tyr Glu Phe Pro Pro Thr 1 5 <210> 17 <211> 321 <212> DNA <213> Mus musculus <400> 17 gaaatccaga tgacccagtc tccatcctct atgtctgcat ctctgggaga cagaataacc 60 atcacttgcc aggcaactca agacattgtt aagagtttaa actggtatca acaaaaacca 120 gggaaacccc cttcattcct gatctattat acagctcaac tggcagaagg ggtcccatca 180 aggttcagtg gcagtgggtc tgggtcagac tattctctga caatcagcaa cctggagtct 240 gaagattttg cagactatta ctgtctacag ttttatgagt ttcctccgac gttcggtgga 300 ggcaccaagc tggaaatcaa a 321 <210> 18 <211> 107 <212> PRT <213> Mus musculus <400> 18 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 19 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 19 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Thr Gln Asp Ile Val Lys Ser 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Lys Phe Leu Ile 35 40 45 Tyr Tyr Thr Ala Gln Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Phe Tyr Glu Phe Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 20 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 20 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Thr Gln Asp Ile Val Lys Ser 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Lys Phe Leu Ile 35 40 45 Tyr Tyr Thr Ala Gln Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Phe Tyr Glu Phe Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 21 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 21 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Thr Gln Asp Ile Val Lys Ser 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Ile 35 40 45 Tyr Tyr Thr Ala Gln Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Phe Tyr Glu Phe Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 22 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 22 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Thr Gln Asp Ile Val Lys Ser 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Lys Phe Leu Ile 35 40 45 Tyr Tyr Thr Ala Gln Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Phe Tyr Glu Phe Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 23 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 23 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Thr Gln Asp Ile Val Lys Ser 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Lys Phe Ala Ile 35 40 45 Tyr Tyr Thr Ala Gln Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Phe Tyr Glu Phe Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 24 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 24 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Thr Gln Asp Ile Val Lys Ser 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Lys Phe Leu Ile 35 40 45 Tyr Tyr Thr Ala Gln Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Phe Tyr Glu Phe Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 25 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 25 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Thr Gln Asp Ile Val Lys Ser 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Lys Phe Leu Ile 35 40 45 Tyr Tyr Thr Ala Gln Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Phe Tyr Glu Phe Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 26 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 26 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Thr Gln Asp Ile Val Lys Ser 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Ile 35 40 45 Tyr Tyr Thr Ala Gln Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Phe Tyr Glu Phe Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 27 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 27 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Thr Gln Asp Ile Val Lys Ser 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Lys Phe Leu Ile 35 40 45 Tyr Tyr Thr Ala Gln Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Phe Tyr Glu Phe Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 28 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 28 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Thr Gln Asp Ile Val Lys Ser 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Lys Phe Leu Ile 35 40 45 Tyr Tyr Thr Ala Gln Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Phe Tyr Glu Phe Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Se...

Claims

1. (a) A light chain having the amino acid sequence of SEQ ID NO: 63, (b) comprising a heavy chain having the amino acid sequence of SEQ ID NO: 81, Anti-B7H4 antibody.

2. A pharmaceutical composition comprising the anti-B7H4 antibody described in claim 1.

3. One or more nucleic acids encoding the anti-B7H4 antibody described in claim 1.

4. A vector comprising one or more nucleic acids as described in claim 3.

5. A host cell comprising one or more nucleic acids as described in claim 3 or the vector as described in claim 4.

6. A pharmaceutical composition comprising the anti-B7H4 antibody according to claim 1, for use in a method for increasing an immune response in a subject requiring an increased immune response, or in a method for reducing an immune response in a subject requiring a decreased immune response.

7. The pharmaceutical composition according to claim 6 for increasing the immune response in a subject having cancer.

8. The pharmaceutical composition according to claim 7, wherein the cancer is ovarian cancer, lung cancer, or gastrointestinal cancer.

9. The pharmaceutical composition according to claim 6 for increasing the immune response in a subject having an infectious disease.

10. A pharmaceutical composition according to claim 6, administered simultaneously with a vaccine or a component thereof, for increasing the immune response.

11. (a) The subject has inflammation, (b) The subject has an autoimmune disease, (c) The subject has an autoimmune disorder, A pharmaceutical composition according to claim 6 for reducing the immune response.

12. A pharmaceutical composition according to any one of claims 6 to 10, to be used in combination with a second therapeutic agent.

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