B7-H4 antibodies and methods of use thereof

Abstract

Compositions for modulating B7-H4 and methods of using the same are provided. For example, provided are immunomodulators that reduce B7-H4 expression, ligand binding, crosslinking, negative signaling, or a combination thereof. Such immunomodulators may be used to enhance the immune response in a subject in need thereof. Also provided are immunomodulators that increase B7-H4 expression, ligand binding, crosslinking, negative signaling, or a combination thereof. Such immunomodulators may be used to reduce the immune response in a subject in need thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is a divisional application of the Chinese invention patent application with application number 201980021260.8, application date January 23, 2019, and invention name “B7-H4 antibody and method of use thereof”. The original application is the national phase application PCT / US2019 / 014748. This application claims the benefit and priority of the U.S. provisional patent application with application number 62 / 620,545 filed on January 23, 2018, the entire contents of which are incorporated by reference into this application.

[0003] References to sequence listings

[0004] Pursuant to 37 CFR §1,52(e)(5), the Sequence Listing filed on January 23, 2019, as a 357 MB text file named “064467.005PCT_seqlist_ST25” created on January 23, 2019, is incorporated by reference into this application. Technical Field

[0005] The present invention relates generally to the field of immunomodulation, and more particularly to compositions and methods for modulating B7-H4 signaling to increase or decrease immune responses. Background Art

[0006] The B7 family plays an important role in the positive and negative regulation of immune responses by engaging multiple receptors on lymphocytes (Rahbar, R. et al., Cancer Immunol Res, 3(2):184-195 (2015)). B7-H4 (also known as VTCN1, B7x or B7S1) is a member of the B7 family that inhibits T cell function. It is also upregulated in a variety of tumors and is thought to promote tumor growth. B7-H4 is found to be highly expressed in many tumor tissues, indicating that its expression level in tumor cells is associated with adverse clinical and pathological characteristics, including tumor aggressiveness.

[0007] The biological activity of B7-H4 is associated with reduced inflammatory CD4+ T cell responses and the correlation between tumor-associated macrophages expressing B7-H4 and FoxP3+ regulatory T cells (Treg) in the tumor microenvironment. Since B7-H4 is expressed in tumor cells and tumor-associated macrophages of various cancer types, therapeutic blockade of B7-H4 can favorably alter the tumor microenvironment, thereby achieving antigen-specific clearance of tumor cells (Podojil, JR. and Miller, SD, Immunol Rev, 276(1): 40-51(2017)).

[0008] The cDNA encoding the human B7-H4 protein has been identified and cloned from placental cDNA (Sica, GL et al., (2003) "B7-H4, A Molecule Of The B7 Family, Negatively Regulates T Cell Immunity," Immunity 18:849-861; Zang, X. et al., (2003) B7x: A Widely Expressed B7 Family Member That Inhibits T Cell Activation," Proc. Natl. Acad. Sci. (USA) 100:10388-10392). B7-H4 is discussed in U.S. Pat. Nos. 7,931,896, 7,875,702, 7,847,081, and 7,622,565.

[0009] Anti-B7-H4 antibodies are disclosed in U.S. Pat. Nos. 9,574,000, 7,888,477, 7,737,255, 7,619,068, and 6,962,980.

[0010] The human B7-H4 protein has 282 amino acid residues and is classified as including an amino-terminal extracellular domain, a large hydrophobic transmembrane domain, and an ultrashort intracellular domain (consisting of only 2 amino acid residues). Like other B7 family members, B7-H4 also has a pair of immunoglobulin-like regions in its extracellular domain. The B7-H4 protein has the overall structure of a type I transmembrane protein. The protein has very low homology with other B7 family members (about 25%) (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).

[0011] B7-H4 is also expressed in tumor-associated macrophages (TAMs). TAMs inhibit antitumor immune responses by releasing humoral mediators and hinder cell-mediated immune responses by expressing inhibitory molecules (such as B7-H4) on the cell surface, thereby protecting tumors from immune recognition. TAMs are derived from resident macrophages or monocytes recruited by the tumor microenvironment and polarized at the tumor site. Tumor TAM infiltration is associated with poor patient survival, and targeting TAMs is a promising anticancer strategy. Several methods have been developed, including clodronate disodium liposome depletion; CFSR-1 and CCL2 targeted tumor recruitment inhibition; and "re-education" methods activated by anti-CD40 mAb or HRG plasma protein or mannose receptor (Dangai, D. et al., Cancer Res, 73 (15): 4820-4829 (2013)).

[0012] Since B7-H4 is expressed in tumor cells and TAMs in various cancer types, targeted therapy against B7-H4 could have a tremendous synergistic effect in favorably altering the tumor microenvironment and eliminating cancer cells. Therefore, new B7-H4 immunomodulators are needed.

[0013] Therefore, one of the objects of the present invention is to provide a composition that can inhibit B7-H4-mediated inhibitory signal transduction to enhance or promote immune response by inhibiting B7-H4 inhibitory signal transduction. Such a composition is beneficial for treating cancer and infectious diseases.

[0014] Another object of the present invention is to provide a composition that can enhance B7-H4-mediated inhibitory signal transduction, thereby promoting an inhibitory immune response. Summary of the invention

[0015] The present invention provides compositions for modulating B7-H4 signal transduction and methods of using the same. Such compositions are beneficial for treating inflammatory diseases and disorders and autoimmune diseases. In one embodiment, an immunomodulator that can reduce B7-H4 expression, ligand binding, cross-linking, signal transduction, or a combination thereof is provided. In one embodiment, the immunomodulator specifically binds to B7-H4. The immunomodulator can be an antibody or antigen binding fragment thereof, a fusion protein, an aptamer, or an agent that specifically binds to B7-H4 and modulates signal transduction through the B7-H4 signal transduction pathway. Modulation includes enhancing signal transduction through the B7-H4 signal transduction pathway, or inhibiting signal transduction through the B7-H4 signal transduction pathway.

[0016] One embodiment provides a method for treating an infection in a subject in need thereof, specifically by administering an effective amount of an agent that can inhibit or block a B7-H4 inhibitory immune response, the effective amount can increase neutrophils and / or enhance innate immunity. By inhibiting the B7-H4 inhibitory immune response, the disclosed agent can be used to enhance the immune response of a subject in need thereof. For example, the immune response can be a primary immune response to an antigen or enhance effector cell function, such as increasing antigen-specific proliferation of T cells, enhancing cytokine production by T cells, stimulating differentiation, increasing neutrophils, or a combination thereof. Exemplary agents include (i) soluble B7-H4 polypeptides or fusion proteins, (ii) function-blocking anti-B7-H4 antibodies, (iii) antibodies that can be used to deplete B7-H4 positive cells, and (iv) combinations thereof. In some embodiments, the immunomodulator is an antagonist of B7-H4.

[0017] In certain embodiments, the immunomodulator is a B7-H4 fusion protein, such as a fusion protein comprising an extracellular domain of B7-H4 or a functional variant thereof connected to an immunoglobulin domain. An exemplary fusion protein comprises the amino acid sequence SEQ ID NO: 1 or a fragment thereof.

[0018] In other embodiments, the immunomodulator is a 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 regulates signal transduction via the B7-H4 signal transduction pathway by specifically binding to a ligand of B7-H4.

[0019] In other embodiments, the immunomodulator is a soluble B7-H4 protein or a functional fragment or variant thereof. For example, the soluble B7-H4 protein can be composed of the extracellular domain of B7-H4 or a functional fragment or variant thereof.

[0020] The method of enhancing the immune response of a subject generally comprises administering to a subject in need thereof an effective amount of an immunosuppressant that can reduce B7-H4 expression, ligand binding, cross-linking, signal transduction, or a combination thereof. The subject can suffer from, for example, cancer or an infectious disease.

[0021] In some embodiments, the subject, cancer or disease is characterized by increased expression of B7-H4, increased expression of a B7-H4 ligand, or a combination thereof. In specific embodiments, the cancer is ovarian cancer, breast cancer, lung cancer, thyroid cancer, gastrointestinal cancer, acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), endometrial cancer, brain cancer, head and neck cancer, pancreatic cancer, and bladder cancer. The immunomodulator can be administered simultaneously or in combination with a vaccine or a component thereof.

[0022] Also provided are immunomodulators that increase B7-H4 expression, ligand binding, cross-linking, immunosuppressive signaling, or a combination thereof. Such agents can be used to attenuate an immune response in a subject in need thereof, thereby inducing or enhancing an inhibitory immune B7-H4 response. Exemplary agents include functionally activated anti-B7-H4 antibodies or soluble B7-H4 polypeptides.

[0023] Methods of reducing an immune response in a subject generally include administering to a subject in need thereof an effective amount of an immunosuppressant that increases B7-H4 expression, ligand binding, cross-linking, negative signaling, or a combination thereof. In some embodiments, the subject suffers from an inflammatory or autoimmune disease. In specific embodiments, the subject suffers from rheumatoid arthritis.

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

[0025] Any of the disclosed methods can include administering to the subject an immunosuppressant alone or in combination with one or more other therapeutic agents.

[0026] One embodiment provides a method for treating leukemia in a subject in need thereof, specifically by administering to the subject an effective amount of a pharmaceutical composition 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 signal transduction in leukemia cells, thereby inhibiting leukemia cell survival or promoting an anti-tumor immune response to the leukemia cells. The leukemia may be acute myeloid leukemia.

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

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

[0029] One embodiment provides a nucleic acid encoding a light chain as set forth in SEQ ID NO: 3. Another embodiment provides a nucleic acid having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:7.

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

[0031] One embodiment provides a nucleic acid encoding the heavy chain of SEQ ID NO: 8. Another embodiment provides a nucleic acid having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:12.

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

[0033] Another embodiment provides an anti-B7-H4 antibody or an antigen-binding fragment thereof having a heavy chain comprising CDRs as listed in SEQ ID NOs: 9, 10 and 11.

[0034] In one embodiment, an anti-B7-H4 antibody or antigen-binding fragment thereof has a light chain and a heavy chain, wherein the light chain contains CDRs as listed in SEQ ID NOs: 4, 5 and 6, and the heavy chain contains CDRs as listed in SEQ ID NOs: 9, 10 and 11.

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

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

[0037] In one embodiment, the antibody light chain contains CDRs whose amino acid sequences are listed in 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 to SEQ ID NO:29.

[0039] Another embodiment provides an antibody heavy chain comprising CDRs with amino acid sequences as listed in SEQ ID NOs: 9, 11 and 30.

[0040] One embodiment provides an anti-B7-H4 monoclonal antibody or an antigen-binding fragment thereof, comprising light chain CDRs as listed in SEQ ID NOs: 14, 15 and 16 and heavy chain CDRs as listed in SEQ ID NOs: 9, 11 and 30.

[0041] One embodiment provides an anti-B7-H4 antibody or antigen-binding fragment thereof comprising a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:13 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: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 to SEQ ID NO:46.

[0043] Another embodiment provides an anti-B7-H4 antibody light chain comprising CDRs with amino acid sequences as listed in 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 to SEQ ID NO:49.

[0045] Another embodiment provides an antibody heavy chain comprising CDRs with amino acid sequences as listed in SEQ ID NOs: 9, 11 and 47.

[0046] One embodiment provides an antibody or antigen-binding fragment thereof comprising light chain CDRs as listed in SEQ ID NOs: 14, 16 and 47 and heavy chain CDRs as listed in SEQ ID NOs: 9, 11 and 50.

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

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

[0049] In another embodiment, the antibody light chain contains CDRs whose amino acid sequences are set forth in 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 to SEQ ID NO:65.

[0051] Another embodiment provides an antibody heavy chain comprising CDRs having amino acid sequences as listed in SEQ ID NOs: 66, 67 and 68.

[0052] One embodiment provides an anti-B7-H4 antibody or an antigen-binding fragment thereof, comprising light chain CDRs as listed in SEQ ID NOs: 53, 54 and 55 and heavy chain CDRs as listed in SEQ ID NOs: 66, 67 and 68.

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

[0055] Another embodiment provides an antibody light chain comprising CDRs having amino acid sequences as listed in 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 to SEQ ID NO:89.

[0057] One embodiment provides an antibody heavy chain comprising CDRs having amino acid sequences as listed in SEQ ID NOs: 9, 90 and 91.

[0058] Another embodiment provides an anti-B7-H4 antibody or an antigen-binding fragment thereof, comprising light chain CDRs as listed in SEQ ID NOs: 4, 86 and 87 and heavy chain CDRs as listed in SEQ ID NOs: 9, 90 and 91.

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

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

[0061] Another embodiment provides an antibody light chain comprising CDRs having amino acid sequences as listed in 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 to SEQ ID NO:98.

[0063] Another embodiment provides an antibody heavy chain comprising CDRs with amino acid sequences as listed in SEQ ID NOs: 9, 99 and 100.

[0064] One embodiment provides an anti-B7-H4 antibody or an antigen-binding fragment thereof having light chain CDRs as listed in SEQ ID NOs: 94, 95 and 96 and heavy chain CDRs as listed in SEQ ID NOs: 9, 99 and 100.

[0065] One embodiment provides an anti-B7-H4 antibody or an antigen-binding fragment thereof having a light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:93 and a heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to 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 to SEQ ID NO:102.

[0067] Another embodiment provides an antibody light chain comprising CDRs having amino acid sequences as listed in 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 to SEQ ID NO:107.

[0069] Another embodiment provides an antibody heavy chain comprising CDRs of amino acid sequences such as SEQ ID NOs: 9, 108 and 109.

[0070] In one embodiment, the anti-B7-H4 antibody or antigen-binding fragment thereof contains the light chain CDRs set forth in SEQ ID NOs: 103, 104, and 105 and the heavy chain CDRs set forth in SEQ ID NOs: 9, 108, and 109.

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

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

[0073] Another embodiment provides an antibody light chain comprising CDRs having amino acid sequences as listed in 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 to SEQ ID NO:115.

[0075] One embodiment provides an antibody heavy chain comprising CDRs with amino acid sequences as listed in SEQ ID NOs: 116, 117 and 118.

[0076] In one embodiment, the anti-B7-H4 antibody or antigen-binding fragment thereof contains the light chain CDRs set forth in SEQ ID NOs:95, 112, and 113 and the heavy chain CDRs set forth in SEQ ID NOs:116, 117, and 118.

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

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

[0079] Another embodiment provides an antibody light chain comprising CDRs having amino acid sequences as listed in 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 to SEQ ID NO:123.

[0081] Another embodiment provides an antibody heavy chain comprising CDRs with amino acid sequences as listed in SEQ ID NOs: 66, 124 and 125.

[0082] One embodiment provides an anti-B7-H4 antibody or an antigen-binding fragment thereof, comprising light chain CDRs as listed in SEQ ID NOs: 54, 55 and 121 and heavy chain CDRs as listed in SEQ ID NOs: 66, 124 and 125.

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

[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 to SEQ ID NO:128.

[0085] Another embodiment provides an anti-B7-H4 antibody heavy chain comprising CDRs with amino acid sequences as listed in SEQ ID NOs: 9, 129 and 130.

[0086] In one embodiment, the anti-B7-H4 antibody or antigen-binding fragment thereof contains the light chain CDRs listed in SEQ ID NOs: 14, 15, and 16 and the heavy chain CDRs listed in SEQ ID NOs: 9, 129, 130.

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

[0088] One embodiment provides an anti-B7-H4 antibody or an antigen-binding fragment thereof having a light chain with an amino acid sequence as set forth in any one of SEQ ID NOs: 3, 13, 46, 52, 85, 93, 102, 111 or 120.

[0089] Another embodiment provides an anti-B7-H4 antibody or an antigen-binding fragment thereof having a heavy chain with an amino acid sequence as set forth in any one of SEQ ID NO: 8, 29, 49, 65, 89, 98, 107, 115, 123 or 128.

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

[0091] Also provided is an anti-B7-H4 antibody or antigen-binding fragment thereof having three light chain CDRs, wherein the amino acid sequence is selected from the group consisting of SEQ ID NO: 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 an antigen-binding fragment thereof having three heavy chain CDRs, wherein the amino acid sequence is selected from the group consisting of SEQ ID NO: 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 an antigen-binding fragment thereof having three light chain CDRs and three heavy chain CDRs, wherein the amino acid sequences of the three light chain CDRs are selected from the group consisting of SEQ ID NOs: 4, 5, 6, 14, 15, 16, 47, 53, 54, 55, 86, 87, 94, 95, 96, 103, 104, 105, 112, 113 and 121; and the amino acid sequences of the three heavy chain CDRs are selected from the group consisting of SEQ ID NOs: 9, 10, 11, 30, 50, 66, 67, 68, 90, 91, 99, 100, 108, 109, 116, 117, 118, 124, 125, 129 and 130.

[0094] Another embodiment provides an anti-B7-H4 antibody or antigen-binding fragment thereof having a light chain variable domain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to any one of SEQ ID NOs: 19, 20, 21, 22 or 23 and a heavy chain variable domain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to any one of SEQ ID NOs: 34, 35, 36 or 37.

[0095] One embodiment provides an anti-B7-H4 antibody or an antigen-binding fragment thereof having a light chain with an amino acid sequence as set forth in any one of SEQ ID NOs: 24, 25, 26, 27 or 28 and a heavy chain with an amino acid sequence as set forth in any one of SEQ ID NOs: 38, 39, 40 or 41.

[0096] Another embodiment provides an anti-B7-H4 antibody or an antigen-binding fragment thereof having a light chain with an amino acid sequence as set forth in any one of SEQ ID NOs: 24, 25, 26, 27 or 28 and a heavy chain with an amino acid sequence as set forth in any one of SEQ ID NOs: 42, 43, 44 or 45.

[0097] Also provided is an anti-B7-H4 antibody or antigen-binding fragment thereof having a light chain variable domain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to any one of SEQ ID NOs:58, 59, 60 or 61 and a heavy chain variable domain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to any one of SEQ ID NOs:70, 71, 72, 73 or 74.

[0098] One embodiment provides an anti-B7-H4 antibody or an antigen-binding fragment thereof having a light chain with an amino acid sequence as set forth in any one of SEQ ID NOs: 62, 63, or 64 and a heavy chain with an amino acid sequence as set forth in any one of SEQ ID NOs: 75, 76, 77, 78, or 79.

[0099] One embodiment provides an anti-B7-H4 antibody or an antigen-binding fragment thereof having a light chain with an amino acid sequence as set forth in any one of SEQ ID NOs: 62, 63, or 64 and a heavy chain with an amino acid sequence as set forth in any one of SEQ ID NOs: 80, 81, 82, 83, or 84.

[0100] Another embodiment provides an anti-B7-H4 antibody or antigen-binding fragment thereof having six complementarity determining regions (CDRs), wherein the CDRs include a polypeptide selected from the group consisting of SEQ ID NO: 4, 5, 6, 14, 15, 16, 49, 55, 56, 57, 88, 89, 96, 97, 97, 105, 106, 107, 114, 115 or 123, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 4, 5, 6, 14, 15, 16, 49, 55, 56, 57, 88, 89, 96, 97, 97, 105, 106, 107, 114, 115 or 123, and three light chain CDRs, and a polypeptide selected from the group consisting of SEQ ID NO: NO:9, 10, 11, 31, 52, 68, 69, 70, 92, 93, 101, 102, 118, 119, 120, 126, 127, 131 or 132, or three heavy chain CDRs of a variant thereof that comprises at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO:9, 10, 11, 31, 52, 68, 69, 70, 92, 93, 101, 102, 118, 119, 120, 126, 127, 131 or 132; and wherein the antibody or antigen binding fragment thereof binds B7-H4.

[0101] Another embodiment provides an anti-B7-H4 antibody or an antigen-binding fragment thereof having two light chains and two heavy chains, wherein the two light chains comprise a polypeptide selected from the group consisting of SEQ ID NO: 24, 25, 26, 27, 28, 62, 63 or 64 or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 24, 25, 26, 27, 28, 62, 63 or 64, and the two heavy chains comprise a polypeptide selected from the group consisting of SEQ ID NO: 38, 39, 40, 41, 75, 76, 77, 78 or 79 or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: NO:38, 39, 40, 41, 75, 76, 77, 78 or 79 has a variant with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity, and wherein the antibody or antigen-binding fragment thereof binds B7-H4.

[0102] One embodiment provides an anti-B7-H4 antibody or an antigen-binding fragment thereof having two light chains and two heavy chains, wherein the two light chains comprise a polypeptide selected from the group consisting of SEQ ID NO: 24, 25, 26, 27, 28, 62, 63 or 64, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 24, 25, 26, 27, 28, 62, 63 or 64, and the two heavy chains comprise a polypeptide selected from the group consisting of SEQ ID NO: 42, 43, 44, 45, 80, 81, 82, 83 or 84, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: NO:42, 43, 44, 45, 80, 81, 82, 83 or 84 have a variant with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity, and wherein the antibody or antigen-binding fragment thereof binds B7-H4.

[0103] One embodiment provides an anti-B7-H4 antibody or an antigen-binding fragment thereof having two light chains and two heavy chains, wherein the two light chains comprise a polypeptide selected from the group consisting of SEQ ID NO: 24, 25, 26, 27 or 28, or a variant thereof comprising at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 24, 25, 26, 27 or 28, and the two heavy chains comprise a polypeptide selected from the group consisting of SEQ ID NO: 38, 39, 40 or 41, or a variant thereof comprising at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 38, 39, 40 or 41, and wherein the antibody or antigen-binding fragment thereof binds to B7-H4.

[0104] Another embodiment provides an anti-B7-H4 antibody or an antigen-binding fragment thereof having two light chains and two heavy chains, wherein the two light chains comprise a polypeptide selected from the group consisting of SEQ ID NO: 24, 25, 26, 27 or 28, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 24, 25, 26, 27 or 28, and the two heavy chains comprise a polypeptide selected from the group consisting of SEQ ID NO: 42, 43, 44 or 45, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 42, 43, 44 or 45, and wherein the antibody or antigen-binding fragment thereof binds to B7-H4.

[0105] One embodiment provides an anti-B7-H4 antibody or an antigen-binding fragment thereof having two light chains and two heavy chains, wherein the two light chains comprise a polypeptide selected from the group consisting of SEQ ID NO: 62, 63 or 64 or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity with SEQ ID NO: 62, 63 or 64, and the two heavy chains comprise a polypeptide selected from the group consisting of SEQ ID NO: 75, 76, 77, 78 or 79 or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity with SEQ ID NO: 75, 76, 77, 78 or 79, and the antibody or antigen-binding fragment thereof binds to B7-H4.

[0106] Another embodiment provides an anti-B7-H4 antibody or antigen-binding fragment thereof having two light chains and two heavy chains, wherein the two light chains comprise a polypeptide selected from the group consisting of SEQ ID NO: 62, 63 or 64 or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 62, 63 or 64, and the two heavy chains comprise a polypeptide selected from the group consisting of SEQ ID NO: 80, 81, 82, 83 or 84 or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 80, 81, 82, 83 or 84, and wherein the antibody or antigen-binding fragment thereof binds B7-H4.

[0107] This application also includes the following implementation methods:

[0108] 1. A pharmaceutical composition comprising an effective amount of an immunomodulator that reduces B7-H4 expression, ligand binding, cross-linking, negative signaling, or a combination thereof, wherein the immunomodulator is selected from the group consisting of:

[0109] (i) a soluble B7-H4 polypeptide or fusion protein,

[0110] (ii) function-blocking anti-B7-H4 antibodies,

[0111] (iii) antibodies that deplete B7-H4 positive cells, and

[0112] (iv) any combination thereof;

[0113] To enhance the immune response of a subject in need thereof.

[0114] 2. A pharmaceutical composition according to embodiment 1, wherein the immunomodulator is a B7-H4 fusion protein.

[0115] 3. A pharmaceutical composition according to embodiment 2, wherein the B7-H4 fusion protein comprises the extracellular domain of B7-H4 connected to an immunoglobulin domain for its functional variant.

[0116] 4. A pharmaceutical composition according to embodiment 3, wherein the B7-H4 fusion protein comprises the amino acid sequence SEQ ID NO: 1 or a fragment thereof.

[0117] 5. A pharmaceutical composition according to embodiment 1, wherein the immunomodulator is a B7-H4 fusion protein.

[0118] 6. A pharmaceutical composition according to embodiment 5, wherein the B7-H4 fusion protein comprises the extracellular domain of B7-H4 or a functional variant thereof connected to an immunoglobulin domain.

[0119] 7. A pharmaceutical composition according to embodiment 6, wherein the B7-H4 fusion protein comprises the amino acid sequence SEQ ID NO: 1 or a fragment thereof.

[0120] 8. A pharmaceutical composition according to embodiment 1, wherein the immunomodulator is a soluble B7-H4 protein or a functional fragment or variant thereof.

[0121] 9. A pharmaceutical composition according to embodiment 8, wherein the B7-H4 protein consists of the extracellular domain of B7-H4 or a functional fragment or variant thereof.

[0122] 10. A pharmaceutical composition according to any one of embodiments 1-9, wherein the immune response is a primary immune response to an antigen or enhances effector cell function, such as increasing antigen-specific proliferation of T cells, enhancing cytokine production by T cells, stimulating differentiation, or a combination thereof.

[0123] 11. A method of enhancing an immune response in a subject in need thereof, comprising administering to the subject a pharmaceutical composition as described in any one of embodiments 1-10.

[0124] 12. A method according to embodiment 11, wherein the subject suffers from cancer.

[0125] 13. The method of embodiment 12, wherein the cancer is characterized by increased expression of B7-H4.

[0126] 14. The method of embodiment 12 or 13, wherein the cancer is ovarian cancer, lung cancer, or gastrointestinal cancer.

[0127] 15. The method of embodiment 11, wherein the subject suffers from an infectious disease.

[0128] 16. The method of any one of embodiments 11-15, wherein a vaccine or a component thereof is administered to the subject simultaneously with the administration of the pharmaceutical composition.

[0129] 17. The method of any one of embodiments 11-16, further comprising administering a second therapeutic agent to the subject.

[0130] 18. A pharmaceutical composition comprising an effective amount of an immunomodulator that increases B7-H4 expression, ligand binding, cross-linking, negative signaling, or a combination thereof, selected from the group consisting of a function-activating anti-B7-H4 antibody, to attenuate an immune response in a subject in need thereof.

[0131] 19. A pharmaceutical composition according to embodiment 18, wherein the immune response is a primary immune response to an antigen or enhances effector cell function, such as increasing antigen-specific proliferation of T cells, enhancing cytokine production by T cells, stimulating differentiation, or a combination thereof.

[0132] 20. A method of attenuating an immune response in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of embodiment 18.

[0133] 21. The method of embodiment 20, wherein the subject suffers from inflammation.

[0134] 22. A method according to embodiment 20, wherein the subject suffers from an autoimmune disease.

[0135] 23. The method of embodiment 22, wherein the autoimmune disease is rheumatoid arthritis.

[0136] 24. The method of any one of embodiments 20-23, wherein the subject or disease or condition is characterized by reduced expression of B7-H4.

[0137] 25. The method of any one of embodiments 20-23, further comprising administering a second therapeutic agent to the subject.

[0138] 26. A composition or method according to any of the preceding embodiments, wherein the B7-H4 is expressed on tumor cells.

[0139] 27. A molecule comprising an antigen-binding fragment of an anti-human B7-H4 antibody, wherein the molecule immunospecifically binds to human B7-H4.

[0140] 28. A molecule according to embodiment 27, wherein the molecule immunospecifically binds to human B7-H4: (I) is arranged on the surface of cells; (II) is arranged on the surface of living cells at endogenous concentrations; (III) is arranged on the surface of living cells and modulates the binding between B7-H4 and its cellular receptor; (IV) is arranged on the surface of living cells and inhibits immunosuppression produced by tumor-associated macrophages; (V) is arranged on the surface of living cells and modulates the activity of tumor-associated macrophages; (VI) is arranged on the surface of living tumor cells and inhibits tumor-mediated inhibition; or (VII) is arranged on the surface of living tumor cells and causes tumor-specific cell lysis.

[0141] 29. A molecule according to embodiment 27, wherein the molecule is capable of internalization into the cell and is capable of mediating the death of the cell.

[0142] 30. The molecule of embodiment 28, wherein the living cell is a tumor cell, a pathogen-infected cell, or a macrophage.

[0143] 31. A molecule according to embodiment 27, wherein the molecule is a detectable label or comprises a binding toxin, drug, receptor, enzyme, receptor ligand.

[0144] 32. A molecule according to embodiment 27, wherein the molecule is an anti-B7H4 antibody, and wherein the antibody is: (I) a monoclonal antibody, a chimeric antibody or a humanized antibody; or (II) a bispecific, trispecific or multispecific antibody.

[0145] 33. The molecule of embodiment 27, wherein the molecule is an IgG1 or IgG4 antibody.

[0146] 34. A molecule according to embodiment 27, wherein the molecule has ADCC activity and is capable of directing tumor or TAM killing activity and / or inhibiting TAM or tumor-mediated inhibition.

[0147] 35. The molecule of embodiment 27, wherein the molecule is a bispecific, trispecific or multispecific antibody capable of binding to B7-H4 and different molecules on the same cell.

[0148] 36. A pharmaceutical composition for treating cancer or an infectious disease, comprising a therapeutically effective amount or a prophylactically effective amount of a molecule as described in embodiment 27, and a physiologically acceptable carrier or excipient, wherein the molecule antagonizes B7-H4-mediated inhibition to upregulate an immune response.

[0149] 37. A method for enhancing or inducing an immune response in a subject in need thereof, comprising: administering an effective amount of the pharmaceutical composition as described in embodiment 36.

[0150] 38. A monoclonal antibody or antigen-binding fragment thereof having a light chain that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:3.

[0151] 39. A nucleic acid encoding the light chain as set forth in SEQ ID NO: 3.

[0152] 40. A nucleic acid having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:7.

[0153] 41. An anti-B7H4 monoclonal antibody or antigen-binding fragment thereof having a heavy chain that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:8.

[0154] 42. A nucleic acid encoding the heavy chain as set forth in SEQ ID NO:8.

[0155] 43. A nucleic acid having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:12.

[0156] 44. An anti-B7H4 antibody or antigen-binding fragment thereof having a light chain comprising CDRs as listed in SEQ ID NOs: 4, 5 and 6.

[0157] 45. An anti-B7H4 antibody or antigen-binding fragment thereof having a heavy chain comprising CDRs as listed in SEQ ID NOs: 9, 10 and 11.

[0158] 46. ​​An anti-B7H4 antibody or antigen-binding fragment thereof having a light chain comprising CDRs as listed in SEQ ID NOs: 4, 5 and 6 and a heavy chain comprising CDRs as listed in SEQ ID NOs: 9, 10 and 11.

[0159] 47. An anti-B7H4 antibody or antigen-binding fragment thereof having a light chain that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:3 and a heavy chain that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:8.

[0160] 48. An anti-B7H4 monoclonal antibody or antigen-binding fragment thereof having a light chain that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:13.

[0161] 49. An anti-B7H4 antibody light chain comprising CDRs with amino acid sequences as listed in SEQ ID NOs: 14, 15 and 16.

[0162] 50. An anti-B7H4 antibody heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:29.

[0163] 51. An anti-B7H4 antibody heavy chain comprising CDRs with amino acid sequences as listed in SEQ ID NOs: 9, 11 and 30.

[0164] 52. An anti-B7H4 monoclonal antibody or an antigen-binding fragment thereof, comprising the light chain CDRs listed in SEQ ID NOs: 14, 15 and 16 and the heavy chain CDRs listed in SEQ ID NOs: 9, 11 and 30.

[0165] 53. An anti-B7H4 antibody or antigen-binding fragment thereof, comprising a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:13 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:29.

[0166] 54. An anti-B7H4 antibody light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:46.

[0167] 55. An anti-B7H4 antibody light chain comprising CDRs with amino acid sequences as listed in SEQ ID NOs: 14, 16 and 47.

[0168] 56. An anti-B7H4 antibody heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:49.

[0169] 57. An anti-B7H4 antibody heavy chain comprising CDRs with amino acid sequences as listed in SEQ ID NOs: 9, 11 and 50.

[0170] 58. An anti-B7H4 antibody or antigen-binding fragment thereof, comprising the light chain CDRs listed in SEQ ID NOs: 14, 47 and 16 and the heavy chain CDRs listed in SEQ ID NOs: 9, 11 and 50.

[0171] 59. An anti-B7H4 antibody or antigen-binding fragment thereof, comprising a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:46 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:49.

[0172] 60. An anti-B7H4 antibody light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:52.

[0173] 61. An anti-B7H4 antibody light chain comprising CDRs with amino acid sequences as listed in SEQ ID NOs: 53, 54 and 55.

[0174] 62. An anti-B7H4 antibody heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:65.

[0175] 63. An anti-B7H4 antibody heavy chain comprising CDRs with amino acid sequences as listed in SEQ ID NOs: 66, 67 and 68.

[0176] 64. An anti-B7H4 antibody or antigen-binding fragment thereof comprising the light chain CDRs listed in SEQ ID NOs: 53, 54 and 55 and the heavy chain CDRs listed in SEQ ID NOs: 66, 67 and 68.

[0177] 65. An anti-B7H4 antibody or antigen-binding fragment thereof, comprising a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:52 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:65.

[0178] 66. An antibody light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:85.

[0179] 67. An antibody light chain comprising CDRs with amino acid sequences as listed in SEQ ID NOs: 4, 86 and 87.

[0180] 68. An antibody heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:89.

[0181] 69. An antibody heavy chain comprising CDRs with amino acid sequences as listed in SEQ ID NOs: 9, 90 and 91.

[0182] 70. An anti-B7H4 antibody or antigen-binding fragment thereof comprising the light chain CDRs listed in SEQ ID NOs: 4, 86 and 87 and the heavy chain CDRs listed in SEQ ID NOs: 9, 90 and 91.

[0183] 71. An anti-B7H4 antibody or antigen-binding fragment thereof, comprising a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:85 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:89.

[0184] 72. An antibody light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:93.

[0185] 73. An antibody light chain comprising CDRs with amino acid sequences as listed in SEQ ID NOs: 94, 95 and 96.

[0186] 74. An antibody heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:98.

[0187] 75. An antibody heavy chain comprising CDRs with amino acid sequences as listed in SEQ ID NOs: 9, 99 and 100.

[0188] 76. An anti-B7H4 antibody or antigen-binding fragment thereof comprising the light chain CDRs listed in SEQ ID NOs: 94, 95 and 96 and the heavy chain CDRs listed in SEQ ID NOs: 9, 99 and 100.

[0189] 77. An anti-B7H4 antibody or antigen-binding fragment thereof, comprising a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:93 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:98.

[0190] 78. An antibody light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:102.

[0191] 79. An antibody light chain comprising CDRs whose amino acid sequences are listed in SEQ ID NOs: 103, 104 and 105.

[0192] 80. An antibody heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:107.

[0193] 81. An antibody heavy chain comprising CDRs with amino acid sequences as listed in SEQ ID NOs: 9, 108 and 109.

[0194] 82. An anti-B7H4 antibody or antigen-binding fragment thereof comprising the light chain CDRs listed in SEQ ID NOs: 103, 104 and 105 and the heavy chain CDRs listed in SEQ ID NOs: 9, 108 and 109.

[0195] 83. An anti-B7H4 antibody or antigen-binding fragment thereof, comprising a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:102 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:107.

[0196] 84. An antibody light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:111.

[0197] 85. An antibody light chain comprising CDRs having amino acid sequences as listed in SEQ ID NOs: 95, 112 and 113.

[0198] 86. An antibody heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:115.

[0199] 87. An antibody heavy chain comprising CDRs with amino acid sequences as listed in SEQ ID NOs: 116, 117 and 118.

[0200] 88. An anti-B7H4 antibody or antigen-binding fragment thereof comprising the light chain CDRs listed in SEQ ID NOs: 95, 112 and 113 and the heavy chain CDRs listed in SEQ ID NOs: 116, 117 and 118.

[0201] 89. An anti-B7H4 antibody or antigen-binding fragment thereof, comprising a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:111 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:115.

[0202] 90. An anti-B7H4 antibody light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:120.

[0203] 91. An anti-B7H4 antibody light chain comprising CDRs with amino acid sequences as listed in SEQ ID NOs: 54, 55 and 121.

[0204] 92. An anti-B7H4 antibody heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:123.

[0205] 93. An anti-B7H4 antibody heavy chain comprising CDRs with amino acid sequences as listed in SEQ ID NOs: 66, 124 and 125.

[0206] 94. An anti-B7H4 antibody or antigen-binding fragment thereof comprising the light chain CDRs listed in SEQ ID NOs: 54, 55 and 121 and the heavy chain CDRs listed in SEQ ID NOs: 66, 124 and 125.

[0207] 95. An anti-B7H4 antibody or antigen-binding fragment thereof, comprising a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:120 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:123.

[0208] 96. An anti-B7H4 antibody heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:128.

[0209] 97. An anti-B7H4 antibody heavy chain comprising CDRs with amino acid sequences as listed in SEQ ID NOs: 9, 129 and 130.

[0210] 98. An anti-B7H4 antibody or antigen-binding fragment thereof comprising the light chain CDRs listed in SEQ ID NOs: 14, 15 and 16 and the heavy chain CDRs listed in SEQ ID NOs: 9, 129 and 130.

[0211] 99. An anti-B7H4 antibody or antigen-binding fragment thereof, comprising a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:13 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:128.

[0212] 100. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in any one of SEQ ID NO: 3, 13, 46, 52, 85, 93, 102, 111 or 120.

[0213] 101. An anti-B7H4 antibody or antigen-binding fragment thereof, comprising a heavy chain having an amino acid sequence as set forth in any one of SEQ ID NO: 8, 29, 49, 65, 89, 98, 107, 115, 123 or 128.

[0214] 102. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in any one of SEQ ID NOs: 3, 13, 46, 52, 85, 93, 102, 111 or 120 and a heavy chain having an amino acid sequence as set forth in any one of SEQ ID NOs: 8, 29, 49, 65, 89, 98, 107, 115, 123 or 128.

[0215] 103. An anti-B7H4 antibody or antigen-binding fragment thereof, comprising three light chain CDRs whose amino acid sequences are selected from the group consisting of SEQ ID NO: 4, 5, 6, 14, 15, 16, 47, 53, 54, 55, 86, 87, 94, 95, 96, 103, 104, 105, 112, 113 or 121.

[0216] 104. An anti-B7H4 antibody or antigen-binding fragment thereof, comprising three heavy chain CDRs whose amino acid sequences are selected from the group consisting of SEQ ID NO: 9, 10, 11, 30, 50, 66, 67, 68, 90, 91, 99, 100, 108, 109, 116, 117, 118, 124, 125, 129 or 130.

[0217] 105. An anti-B7H4 antibody or antigen-binding fragment thereof, comprising three light chain CDRs whose amino acid sequences are selected from the group consisting of SEQ ID NOs: 4, 5, 6, 14, 15, 16, 47, 53, 54, 55, 86, 87, 94, 95, 96, 103, 104, 105, 112, 113 and 121 and three heavy chain CDRs whose amino acid sequences are selected from the group consisting of SEQ ID NOs: 9, 10, 11, 30, 50, 66, 67, 68, 90, 91, 99, 100, 108, 109, 116, 117, 118, 124, 125, 129 and 130.

[0218] 106. An anti-B7H4 antibody or antigen-binding fragment thereof comprising six complementarity determining regions (CDRs),

[0219] wherein the CDRs comprise three light chain CDRs of a polypeptide selected from the group consisting of SEQ ID NO: 4, 5, 6, 14, 15, 16, 49, 55, 56, 57, 88, 89, 96, 97, 97, 105, 106, 107, 114, 115 or 123, or variants thereof comprising at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 4, 5, 6, 14, 15, 16, 49, 55, 56, 57, 88, 89, 96, 97, 97, 105, 106, 107, 114, 115 or 123,

[0220] and three heavy chain CDRs of a polypeptide selected from the group consisting of SEQ ID NO: 9, 10, 11, 31, 52, 68, 69, 70, 92, 93, 101, 102, 118, 119, 120, 126, 127, 131 or 132, or a variant thereof comprising at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 9, 10, 11, 31, 52, 68, 69, 70, 92, 93, 101, 102, 118, 119, 120, 126, 127, 131 or 132; and

[0221] wherein the antibody or antigen-binding fragment thereof binds to B7-H4.

[0222] 107. An anti-B7H4 antibody or antigen-binding fragment thereof, comprising a light chain variable domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to any one of SEQ ID NO: 19, 20, 21, 22 or 23 and a heavy chain variable domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to any one of SEQ ID NO: 34, 35, 36 or 37.

[0223] 108. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in any one of SEQ ID NOs: 24, 25, 26, 27 or 28 and a heavy chain having an amino acid sequence as set forth in any one of SEQ ID NOs: 38, 39, 40 or 41.

[0224] 109. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in any one of SEQ ID NOs: 24, 25, 26, 27 or 28 and a heavy chain having an amino acid sequence as set forth in any one of SEQ ID NOs: 42, 43, 44 or 45.

[0225] 110. An anti-B7H4 antibody or antigen-binding fragment thereof, comprising a light chain variable domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to any one of SEQ ID NOs:58, 59, 60 or 61 and a heavy chain variable domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to any one of SEQ ID NOs:70, 71, 72, 73 or 74.

[0226] 111. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in any one of SEQ ID NOs: 62, 63 or 64 and a heavy chain having an amino acid sequence as set forth in any one of SEQ ID NOs: 75, 76, 77, 78 or 79.

[0227] 112. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in any one of SEQ ID NOs: 62, 63 or 64 and a heavy chain having an amino acid sequence as set forth in any one of SEQ ID NOs: 80, 81, 82, 83 or 84.

[0228] 113. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising two light chains and two heavy chains,

[0229] wherein the two light chains comprise a polypeptide selected from the group consisting of SEQ ID NO: 24, 25, 26, 27 or 28 or a variant thereof comprising at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 24, 25, 26, 27 or 28,

[0230] and the two heavy chains comprise a polypeptide selected from the group consisting of SEQ ID NO: 38, 39, 40 or 41, or a variant thereof comprising at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity with SEQ ID NO: 38, 39, 40 or 41, and

[0231] wherein the antibody or antigen-binding fragment thereof binds to B7-H4.

[0232] 114. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising two light chains and two heavy chains,

[0233] wherein the two light chains comprise a polypeptide selected from the group consisting of SEQ ID NO: 24, 25, 26, 27 or 28 or a variant thereof comprising at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 24, 25, 26, 27 or 28,

[0234] and the two heavy chains comprise a polypeptide selected from the group consisting of SEQ ID NO:42, 43, 44 or 45, or a variant thereof comprising at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity with SEQ ID NO:42, 43, 44 or 45, and

[0235] wherein the antibody or antigen-binding fragment thereof binds to B7-H4.

[0236] 115. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising two light chains and two heavy chains,

[0237] wherein the two light chains comprise a polypeptide selected from the group consisting of SEQ ID NO: 62, 63 or 64 or a variant thereof comprising at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 62, 63 or 64,

[0238] and the two heavy chains comprise a polypeptide selected from the group consisting of SEQ ID NO: 75, 76, 77, 78 or 79, or a variant thereof comprising at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 75, 76, 77, 78 or 79, and

[0239] wherein the antibody or antigen-binding fragment thereof binds to B7-H4.

[0240] 116. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising two light chains and two heavy chains,

[0241] wherein the two light chains comprise a polypeptide selected from the group consisting of SEQ ID NO: 62, 63 or 64 or a variant thereof comprising at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 62, 63 or 64,

[0242] and the two heavy chains comprise a polypeptide selected from the group consisting of SEQ ID NO: 80, 81, 82, 83 or 84, or a variant thereof comprising at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity with SEQ ID NO: 80, 81, 82, 83 or 84, and

[0243] wherein the antibody or antigen-binding fragment thereof binds to B7-H4.

[0244] 117. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:24 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:38.

[0245] 118. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:24 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:39.

[0246] 119. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:24 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:40.

[0247] 120. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:24 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:41.

[0248] 121. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:24 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:42.

[0249] 122. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:24 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:43.

[0250] 123. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:24 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:44.

[0251] 124. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:24 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:45.

[0252] 125. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:25 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:38.

[0253] 126. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:25 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:39.

[0254] 127. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:25 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:40.

[0255] 128. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:25 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:41.

[0256] 129. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:25 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:42.

[0257] 130. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:25 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:43.

[0258] 131. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:25 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:44.

[0259] 132. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:25 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:45.

[0260] 133. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:26 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:38.

[0261] 134. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:26 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:39.

[0262] 135. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:26 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:40.

[0263] 136. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:26 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:41.

[0264] 137. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:26 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:42.

[0265] 138. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:26 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:43.

[0266] 139. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:26 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:44.

[0267] 140. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:26 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:45.

[0268] 141. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:27 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:38.

[0269] 142. An anti-B7H4 antibody or antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:27 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:39.

[0270] 143. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:27 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:40.

[0271] 144. An anti-B7H4 antibody or antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:27 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:41.

[0272] 145. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:27 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:42.

[0273] 146. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:27 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:43.

[0274] 147. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:27 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:44.

[0275] 148. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:27 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:45.

[0276] 149. An anti-B7-H4 antibody or antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:28 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:38.

[0277] 150. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:28 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:39.

[0278] 151. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:28 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:40.

[0279] 152. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:28 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:41.

[0280] 153. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:28 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:42.

[0281] 154. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:28 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:43.

[0282] 155. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:28 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:44.

[0283] 156. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:28 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:45.

[0284] 157. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:62 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:75.

[0285] 158. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:62 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:76.

[0286] 159. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:62 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:77.

[0287] 160. An anti-B7H4 antibody or antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:62 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:78.

[0288] 161. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:62 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:79.

[0289] 162. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:62 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:80.

[0290] 163. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:62 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:81.

[0291] 164. An anti-B7H4 antibody or antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:62 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:82.

[0292] 165. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:62 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:83.

[0293] 166. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:62 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:84.

[0294] 167. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:63 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:75.

[0295] 168. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:63 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:76.

[0296] 169. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:63 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:77.

[0297] 170. An anti-B7H4 antibody or antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:63 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:78.

[0298] 171. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:63 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:79.

[0299] 172. An anti-B7H4 antibody or antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:63 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:80.

[0300] 173. An anti-B7H4 antibody or antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:63 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:81.

[0301] 174. An anti-B7H4 antibody or antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:63 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:82.

[0302] 175. An anti-B7H4 antibody or antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:63 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:83.

[0303] 176. An anti-B7H4 antibody or antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:63 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:84.

[0304] 177. An anti-B7H4 antibody or antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:64 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:75.

[0305] 178. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:64 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:76.

[0306] 179. An anti-B7H4 antibody or antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:64 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:77.

[0307] 180. An anti-B-H4 antibody or antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:64 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:78.

[0308] 181. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:64 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:79.

[0309] 182. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:64 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:80.

[0310] 183. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:64 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:81.

[0311] 184. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:64 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:82.

[0312] 185. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:64 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:83.

[0313] 186. An anti-B7H4 antibody or an antigen-binding fragment thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:64 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:84. DETAILED DESCRIPTION

[0314] I. Definitions

[0315] As used herein, molecules are said to "immunospecifically bind" to each other if the binding between them demonstrates the antibody's specificity and affinity for its cognate antigen. If such binding involves the antigen recognition site of the immunoglobulin molecule, the antibody is said to be able to immunospecifically bind to a target region or conformation ("epitope") of the antigen. Antibodies that immunospecifically bind to a particular antigen may bind to other antigens with lower affinity if the other antigens have certain sequence or conformational similarities, which can be determined by immunoassays, The antibody may be identified by the antigen recognition site, but will not bind to completely unrelated antigens. However, in some embodiments, the antibody (and its antigen-binding fragment) will not cross-react with other antigens. The antibody may also bind to other molecules in a non-immune specific manner, such as by binding to domains in other regions / domains of molecules not involved in the antigen recognition site (e.g., Fc region) and binding to FcR receptors.

[0316] As used herein, molecules are said to be capable of "physiologically specific binding" to one another if the binding between the molecules exhibits the specificity and affinity of a receptor for its cognate binding ligand. A molecule is capable of physiologically specific binding to one or more other molecules.

[0317] As used herein, the term "antibody" refers to an immunoglobulin molecule having a "variable region" antigen recognition site, including an antigen-binding fragment of an antibody. The term "variable region" is intended to distinguish such domains of the immunoglobulin from domains widely shared by antibodies (e.g., antibody Fc domains). The variable region includes a "hypervariable region" whose residues are responsible for antigen binding. The hypervariable region includes amino acid residues from the "complementarity determining region" ("CDR") (i.e., generally around residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain, and residues 27-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or those residues from the "hypervariable region loops" (i.e., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain, and residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917). "Framework region" ("FR") residues are those variable domain residues other than the hypervariable region residues as defined herein.The term "antibody" includes monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, and cammerized antibodies (see, e.g., Muyldermans et al., 2001, Trends Biochem. Sci. 26:230; Nuttall et al., 2000, Cur. Pharm. Biotech. 1:253; Reichmann and Muyldermans, 1999, J. Immunol. Meth. 231:25; International Publication Nos. WO 94 / 04678 and WO 94 / 25591; US ​​Patent No. 6,005,079), single-chain Fv (scFv) (see, e.g., Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp.269-315 (1994)), single-chain antibodies, disulfide-linked Fv (sdFv), intracellular antibodies, double antibodies, three antibodies, four antibodies, Bis-scFv, mini antibodies, Fab2, Fab3 and anti-idiotype (anti-ID) antibodies (including, for example, anti-ID and anti-anti-ID antibodies to antibodies). In particular, such antibodies include immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), any class (e.g., IgGi, IgG2, IgG3, IgG4, IgAi and IgA2) or subclass.

[0318] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more portions of an antibody, which contains the complementarity determining region ("CDR") of the antibody and optionally the framework residues of the "variable region" antigen recognition site of the antibody, and exhibits the ability to immunospecifically bind to an antigen. Such fragments include Fab', F(ab')2, Fv, single chain (ScFv) and mutants and natural variants thereof, as well as fusion proteins including the "variable region" antigen recognition site of the antibody and a heterologous protein (e.g., toxins, antigen recognition sites of different antigens, enzymes, receptors or receptor ligands, etc.).

[0319] As used herein, the term "fragment" refers to a peptide or polypeptide comprising an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues.

[0320] As used herein, the term "modulation" refers to the ability to change an effect, result, or activity (e.g., signal transduction). Such modulation can be agonistic or antagonistic. Antagonistic modulation can be partial (i.e., attenuated but not eliminated) or can completely eliminate such activity (e.g., neutralization). Modulation can include internalization of receptors after antibody binding or reduction of receptor expression on target cells. Agonistic modulation can enhance or otherwise increase or enhance activity (e.g., signal transduction). In further embodiments, such modulation can change the nature of the interaction between a ligand and its cognate receptor, thereby changing the nature of the signal transduction induced. For example, a molecule can change the ability of such a molecule to bind to other ligands or receptors by binding to a ligand or receptor, thereby changing its overall activity. In some embodiments, such modulation will cause a measurable immune system activity to change by at least 10%, such activity to change by at least 50%, or at least 2-fold, 5-fold, 10-fold, or at least 100-fold.

[0321] When used in the context of a binding effect or an exhibited effect, the term "substantially" is intended to mean that the observed effect is physiologically or therapeutically relevant. Thus, for example, a molecule is capable of substantially blocking the activity of a ligand or receptor, provided that the degree of blocking is physiologically or therapeutically relevant (e.g., if such degree is greater than 60% complete blocking, greater than 70% complete blocking, greater than 75% complete blocking, greater than 80% complete blocking, greater than 85% complete blocking, greater than 90% complete blocking, greater than 95% complete blocking, or greater than 97% complete blocking). Similarly, different molecules are considered to have substantially the same immunospecificity and / or characteristics if the identity of the immunospecificity and characteristics between them is greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 97%.

[0322] As used herein, "co-stimulatory" signals include positive co-stimulatory signals (eg, signals that result in an enhancement of activity) and negative co-stimulatory signals (eg, signals that result in an inhibition of activity).

[0323] The term "derivative" refers to an antibody or antigen-binding fragment thereof that immunospecifically binds to the same target of a parent or reference antibody, but differs from the amino acid sequence of the parent or reference antibody or its antigen-binding fragment by one, two, three, four, five or more amino acid substitutions, additions, deletions or modifications relative to the parent or reference antibody or its antigen-binding fragment. In some embodiments, such derivatives will have substantially the same immunospecificity and / or characteristics, or have the same immunospecificity and characteristics as the parent or reference antibody or its antigen-binding fragment. The amino acid substitutions or additions of such derivatives may include naturally occurring (i.e., DNA-encoded) or non-naturally occurring amino acid residues. The term "derivative" includes, for example, chimeric or humanized variants, as well as variants having variant CHI regions, hinge regions, CH2 regions, CH3 regions or CH4 regions, thereby forming, for example, antibodies having variant Fc regions (exhibiting enhanced or reduced effector or binding properties), etc.

[0324] As used herein, a "chimeric antibody" is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules, for example, an antibody having variable regions derived from a non-human antibody and human immunoglobulin constant regions.

[0325] As used herein, the term "humanized antibody" refers to an immunoglobulin, including a human framework region and one or more CDRs derived from a non-human (usually mouse or rat) immunoglobulin. The non-human immunoglobulin providing the CDR is referred to as a "donor", and the human immunoglobulin providing the framework is referred to as an "acceptor". The constant region does not have to be present, but if present, they should be substantially identical to the human immunoglobulin constant region, that is, the identity is at least about 85-99%, or about 95% or higher. Therefore, all parts of the humanized immunoglobulin (except for possible CDRs) are substantially identical to the corresponding parts of the natural human immunoglobulin sequence. A humanized antibody is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. For example, a humanized antibody will not include a typical chimeric antibody, because, for example, the entire variable region of a chimeric antibody is non-human.

[0326] The term "endogenous concentration" refers to the level at which a molecule is naturally expressed (ie, in the absence of an expression vector or recombinant promoter) by a cell, which may be a normal cell, a cancer cell, or an infected cell.

[0327] As used herein, the words "treat" and "therapeutic use" refer to the elimination, alleviation or amelioration of one or more symptoms of a disease or condition. As used herein, a "therapeutically effective amount" refers to the amount of a therapeutic agent sufficient to mediate the clinically relevant elimination, alleviation or amelioration 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 subject. A therapeutically effective amount may refer to an amount of a therapeutic agent sufficient to delay or minimize the onset of a disease, for example, to delay or minimize the spread of cancer. A therapeutically effective amount may also refer to an amount of a therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease.

[0328] As used herein, the term "prophylactic agent" refers to an agent that can be used to prevent a disease or condition before any symptoms of such disease or condition are detected. A "prophylactically effective" amount refers to an amount of a prophylactic agent sufficient to mediate such prevention. A prophylactically effective amount may also refer to an amount of a prophylactic agent that provides a prophylactic benefit in the prevention of a disease.

[0329] As used herein, the term "cancer" refers to a neoplasm or tumor caused by uncontrolled abnormal cell growth. As used herein, cancer clearly includes leukemia and lymphoma. The term "cancer" refers to a disease involving cells that may metastasize to distal sites and exhibit phenotypic traits different from non-cancerous cells, for example, forming colonies in a three-dimensional matrix (e.g., soft agar), or forming tubular networks or reticular matrices in a three-dimensional basement membrane or extracellular matrix preparation. Non-cancerous cells will not form colonies in soft agar and will not form spherical structures in a three-dimensional basement membrane or extracellular matrix preparation.

[0330] As used herein, "immune cell" refers to any cell derived from hematopoietic origin, including but not limited to T cells, B cells, monocytes, dendritic cells, and macrophages.

[0331] As used herein, "inflammatory molecules" refer to molecules that cause an inflammatory response, including but not limited to cytokines and metalloproteinases, for example, including 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 MMP.

[0332] As used herein, "valency" refers to the number of available binding sites per molecule.

[0333] As used herein, words such as "immunology" or "immune" response refer to the formation of beneficial humoral (mediated by antibodies) and / or cellular (mediated by antigen-specific T cells or their secretory products) responses to peptides in the recipient patient. Such responses can be active responses induced by administering immunogens, or passive responses induced by administering antibodies or induced T cells. The appearance of polypeptide epitopes associated with class I or class II MHC molecules triggers a cellular immune response to activate antigen-specific CD4+T helper cells and / or CD8+ cytotoxic T cells. Such responses may also involve activation or recruitment of other components of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia, eosinophils, neutrophils or innate immunity. The presence of a cell-mediated immune response can be determined by cell proliferation assays (CD4+T cells) or CTL (cytotoxic T lymphocyte) assays. By isolating antibodies and T cells from an immunized syngeneic animal and measuring the protection or therapeutic effect in another subject, the relative contribution of humoral and cellular responses to the protection or therapeutic effect of the immunogen can be distinguished.

[0334] An "immunogenic agent" or "immunogen" is capable of inducing an immune response against itself when administered to a mammal, optionally in combination with an adjuvant.

[0335] As used herein, the terms "individual," "host," "subject," and "patient" are used interchangeably herein and refer to mammals, including but not limited to humans, rodents (eg, mice and rats), and other laboratory animals.

[0336] As used herein, the term "polypeptide" refers to an amino acid chain of any length, whether or not modified (e.g., phosphorylated or glycosylated). The term "polypeptide" includes proteins and fragments thereof. Polypeptides can be "exogenous," meaning that they are "heterologous," i.e., foreign to the host cell utilized, such as human polypeptides produced by bacterial cells. Polypeptides are disclosed herein as sequences of amino acid residues. These sequences are written from left to right in the direction from the amino terminus to the carboxyl terminus. According to standard nomenclature, amino acid residue sequences are represented by three-letter or single-letter codes as follows: 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).

[0337] 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. Typical variants of a polypeptide differ from other reference polypeptides in terms of amino acid sequence. Typically, the differences are limited, so the sequences of the reference polypeptide and the variant are very similar overall and identical in many regions. Variants and reference polypeptides may differ in amino acid sequence due to one or more modifications (e.g., substitutions, additions and / or deletions). The substituted or inserted amino acid residues may or may not be amino acid residues encoded by the genetic code. Variants of polypeptides may be naturally occurring, such as allelic variants, or may be variants that are not known to be naturally occurring.

[0338] The structures of the polypeptides of the present disclosure can be modified and altered and still obtain molecules with characteristics similar to the polypeptides (e.g., conservative amino acid substitutions). For example, certain amino acids can be replaced with other amino acids in the sequence without significant loss of activity. Because the biological functional activity of a polypeptide is defined by the interactive ability and properties of the polypeptide, certain amino acid sequence substitutions can be made in a polypeptide sequence and still obtain a polypeptide with similar properties.

[0339] In making such changes, the hydropathic index of the amino acids may be considered. The importance of the hydropathic index of amino acids in conferring interactive biological function to polypeptides is generally known in the art. It is well known that certain amino acids can be substituted with other amino acids having a similar hydropathic index or score and still produce a polypeptide with similar biological activity. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics. 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); glutamate (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0340] It is believed that the relative hydrophilicity of an amino acid determines the secondary structure of the resulting polypeptide, which in turn defines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens and cofactors. It is known in the art that amino acids can be substituted with other amino acids having a similar hydropathic index and still obtain functionally equivalent polypeptides. In such variations, it is preferred to use amino acids with a hydropathic index within ±2, particularly preferably to use amino acids with a hydropathic index within ±1, and more particularly preferably to use amino acids with a hydropathic index within ±0.5.

[0341] Similar amino acid substitutions may also be used based on hydrophilicity, particularly where the resulting biologically functional equivalent polypeptides or peptides are intended for use in immunological embodiments. The following hydrophilicity values ​​are assigned to 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); tryptophan (-3.4). It will be appreciated that amino acids can be substituted with other amino acids having similar hydrophilicity values ​​and still obtain biologically equivalent, particularly immunologically equivalent polypeptides. In such changes, it is preferred to use amino acids with hydrophilicity values ​​within ±2, particularly preferred to use amino acids with hydrophilicity values ​​within ±1, and even more particularly preferred to use amino acids with hydrophilicity values ​​within ±0.5.

[0342] As described above, amino acid substitutions are generally based on the relative similarity of the amino acid side chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into account various of the aforementioned properties are well known to those of skill in the art and include (original residue: exemplary substitutions): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gln, His), (Asp: Glu, Cys, Ser), (Gln: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gln), (He: Leu, Val), (Leu: Ile, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Trp: Tyr), (Tyr: Trp, Phe) and (Val: Ile, Leu). Therefore, embodiments of the present disclosure contemplate functional or biological equivalents of the polypeptides described above. In particular, embodiments of the polypeptides may include variants having about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the polypeptide of interest.

[0343] The term "percentage (%) of sequence identity" is defined as follows: the percentage of nucleotides or amino acids in a candidate sequence that are identical to those in a reference nucleic acid sequence, after alignment of the sequences and introduction of spaces (if necessary) to achieve maximum percentage of sequence identity. Sequence alignment to determine the percentage of sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR). Suitable parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences, can be determined by known methods.

[0344] For the purposes of this article, the % sequence identity of a given nucleotide or amino acid sequence C to, with, or to a given nucleic acid sequence D (alternatively expressed as: a given sequence C to, with, or to a given sequence D has or contains a specific % sequence identity) is calculated as follows:

[0345] 100 times the fraction W / Z,

[0346] Wherein, W refers to the number of nucleotides or amino acids scored as identical matches by the sequence alignment program in the alignment of C and D, and Z refers to the total number of nucleotides or amino acids in D. It should be understood that the length of sequence C is not equal to the length of sequence D, and the % sequence identity from C to D is not equal to the % sequence identity from D to C.

[0347] As used herein, the term "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutical carriers, such as phosphate-buffered saline, water, and emulsions (eg, oil / water or water / oil emulsions), and various types of wetting agents.

[0348] II. Composition

[0349] The high B7-H4 expression levels found in many tumor tissues (e.g., human ovarian cancer) suggest that B7-H4 plays a key role in mediating immunosuppression. Similarly, it has been found that tumor-associated macrophages (TAMs) expressing B7-H4 can suppress tumor-associated antigen-specific T cell immunity (Kryczek, I. et al. (2006) "B7-H4 Expression Identifies A Novel Suppressive Macrophage Population In Human Ovarian Carcinoma," J. Exp. Med. 203 (4): 871-881). The intensity of B7-H4 expression in TAMs is significantly correlated with the number of regulatory T cells in tumors. Moreover, B7-H4 expressed on TAMs is associated with poor patient prognosis (Kryczek, I. et al. (2006) "B7-H4 Expression Identifies A Novel Suppressive Macrophage Population In Human Ovarian Carcinoma," J. Exp. Med. 203 (4): 871-881). In addition, B7-H4 can be expressed in myeloid-derived suppressor cells (MDSC), where it may play an immunosuppressive role in viral infection (Garg, A et al. (2017) "Human Immunodeficiency Virus Type-1 Myeloid Derived Suppressor Cells Inhibit Cytomegalovirus Inflammation through Interleukin-27 and B7-H4" Sci. Rep. Mar 24; 7: 44485). However, a study on uterine cancer showed that the expression of B7-H4 in the tumor microenvironment was associated with increased MDSC infiltration (Vanderstraeten, A. et al. (2014) "Mapping the immunosuppressive environment inuterine tumors: implications for immunotherapy," Cancer Immunol. Immunother. Jun; 63 (6): 545-57). Therefore, B7-H4 may be expressed in tumor cells TAM and / or MDSC, where it may play a role in immunosuppressive signaling in cancer.

[0350] Neutrophils are a major component of the host's innate 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 series of defense mechanisms, including phagocytosis, killing, and degradation of microorganisms by antimicrobial proteins and proteolytic proteins, accompanied by the generation of reactive oxygen species. Neutrophils are also involved in the regulation of tissue decomposition, remodeling, wound healing, and other inflammatory and adaptive immune components. Due to the short life span of neutrophils, neutrophils must be continuously replenished during infection and inflammation by the expansion of myeloid progenitor cells in the bone marrow. In vitro B7-H4 inhibits the growth of bone marrow-derived neutrophil progenitor cells, indicating that B7-H4 has an inhibitory function in neutrophil expansion (Zhu, G. et al., Blood, 113: 1759-1767 (2009)). Therefore, a composition that can regulate B7-H4 signal transduction is provided.

[0351] A.B7-H4 sequence

[0352] One embodiment provides a B7-H4 polypeptide. The polypeptide may include the amino acid sequence of the full-length B7-H4, or a fragment or variant thereof, or a fusion protein thereof.

[0353] One embodiment provides a human B7-H4 protein or polypeptide thereof. The sequence of human B7-H4 is known in the art. For example, a consensus sequence of B7-H4 is

[0354]

[0355] (SEQ ID NO: 1, Q7Z7D3 (VTCN1_HUMAN)), wherein amino acids 1-27 are signal sequences (the underlined portion is based on U.S. Patent Publication No. 2016 / 0039905, the entire contents of which are incorporated herein by reference). Sica et al. indicate that amino acids 1-20 constitute a signal sequence. UniProtKB accession number Q7Z7D3 indicates that the extracellular domain comprises amino acids 25-259.

[0356] Another embodiment provides mouse B7-H4 proteins and polypeptides. The sequence of mouse B7-H4 is known in the art. For example, a consensus sequence of mouse B7-H4 is

[0357] MASLGQIIFWSIINIIIILAGAIALIIGFGISGKHFITVTTFTSAGNIGEDGTLSCTFEPDIKLNGIVIQWLKEGIKGLVHEFKEGKDDLSQQHEMFRGRTAVFADQVWGNASLRLKNVQLTDAGTYTCYIRTSKGKGNA NLEYKTGAFSMPEINVDYNASSESLRCEAPRWFPQPTVASQVDQGANFSEVSNTSFELNSENVTMKWSVLYNVTINNTYSCMIENDIAKATGDIKVTDSEVKRRSQLQLLNSGPPSPCVFSSAFVAGWALLSLLSCCLMLR

[0362] (SEQ ID NO: 2, Q7TSP5 (VTCN1_MOUSE), the entire contents of which are incorporated herein by reference.)

[0363] 1. Anti-B7-H4 antibody sequence

[0364] a.B1A1 sequence

[0365] One embodiment provides a murine monoclonal antibody produced by hybridoma clone B1A1, which comprises two light chains and two heavy chains and specifically binds to B7-H4.

[0366] i. Light chain

[0367] One embodiment provides a murine monoclonal antibody or antigen-binding fragment thereof having a light chain that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to the following amino acid sequence:

[0368]

[0369] And specifically binds to B7-H4.

[0370] The CDRs are listed in SEQ ID NO:3, shown in bold and underlined, respectively:

[0371]

[0372] as well as

[0373]

[0374] Another embodiment provides a nucleic acid encoding a light chain (SEQ ID NO: 3). An exemplary nucleic acid encoding a light chain (SEQ ID NO: 3) is

[0375] GACATTTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGTATCACCTGCAAGGCCAGTCAGGATGTGAGAACTGCTGTAGCCTGGTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTACTCGACATCCTACCGG TACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGAATTCACTTTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAATATTATGTTACTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA(SEQ ID NO:7).

[0381] ii. Heavy chain

[0382] One embodiment provides a murine monoclonal antibody or antigen-binding fragment thereof having a heavy chain that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to the following amino acid sequence:

[0383]

[0384] And specifically binds to B7-H4.

[0385] The CDRs are listed in SEQ ID NO:8, shown in bold and underlined, respectively:

[0386]

[0387] as well as

[0388]

[0389] Another embodiment provides a nucleic acid encoding a heavy chain (SEQ ID NO: 8). An exemplary nucleic acid encoding a heavy chain (SEQ ID NO: 8) is

[0390] GAGGTTCAGCTCCAGCAGTCTGGGACTGT GCTGGCAAGGCCTGGGGCTTC AGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACCAGCTACTGGATGCACTGGATAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGCGCTATTTATCCTGGAAATAGTGATACTAAATACAACCAGAAGTTCAAGGACAAGG CCAAACTGACTGCAGTCACATCTGCCAGCACTGCCTACATGGAGCTCAGCAGCCTGACAAATGAGGACTCTGCGGTCTATTACTGTACATCTACGGTACGGAATGTTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA(SEQ ID NO:12).

[0397] One embodiment provides an antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain containing CDRs as set forth in SEQ ID NOs: 4, 5, and 6, and specifically binding to B7-H4.

[0398] One embodiment provides an antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a heavy chain comprising CDRs as set forth in SEQ ID NOs: 9, 10, and 11, and specifically binding to B7-H4.

[0399] One embodiment provides an antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain containing CDRs as listed in SEQ ID NOs: 4, 5 and 6 and a heavy chain containing CDRs as listed in SEQ ID NOs: 9, 10 and 11, and specifically binds to B7-H4.

[0400] One embodiment provides an antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:3 and a heavy chain that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:8, and specifically binds to B7-H4.

[0401] b.B1H1 sequence

[0402] In one embodiment, a murine monoclonal antibody is produced by hybridoma clone B1H1 and contains two light chains and two heavy chains.

[0403] i. Light chain

[0404] One embodiment provides a murine monoclonal antibody or antigen-binding fragment thereof having a light chain that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to the following amino acid sequence:

[0405]

[0406] The CDRs are listed in SEQ ID NO: 13, shown in bold and underlined, respectively:

[0407]

[0408] as well as

[0409]

[0410] Another embodiment provides a nucleic acid encoding a light chain (SEQ ID NO: 13).

[0411] An exemplary nucleic acid encoding a light chain (SEQ ID NO: 13) is

[0412] GAAATCCAGATGACCCAGTCTCCATCCTCTATGTCTGCATCTCTGGGAGACAGAATAACCATCACTTGCCAGGCAACTCAAGACATTGTTAAGAGTTTAAACTGGTATCAACAAAAACCAGGGAAACCCCCTTCATTCCTGATCTATTATACAGCTCAACTG GCAGAAGGGGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGGTCAGACTATTCTCTGACAATCAGCAACCTGGAGTCTGAAGATTTTGCAGACTATTACTGTCTACAGTTTTATGAGTTTCCTCCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA(SEQ ID NO:17).

[0413] One embodiment provides a monoclonal antibody or antigen-binding fragment thereof having a light chain constant domain that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to the following amino acid sequence:

[0414] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 18), and specifically binds B7-H4.

[0415] ii. Humanized light chain

[0416] One embodiment provides an anti-B7-H4 monoclonal antibody or antigen-binding fragment thereof having a humanized light chain variable domain that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to one of the following amino acid sequences:

[0417] Humanized B1H1 VL1:

[0418] DIQMTQSPSSSLSASVGDRVTITCQATQDIVKSLNWYQQKPGKPPKFLIYYTAQLAEGVPSRFSGSGSGTDYTLTIS SLQSEDFATYYCLQFYEFPPTFGGGTKVEIK(SEQ ID NO:19)

[0419] Humanized B1H1 VL2:

[0420] DIQMTQSPSSSLSASVGDRVTITCQATQDIVKSLNWYQQKPGKPPKFLIYYTAQLAEGVPSRFSGSGSGTDYTLTIS SLQPEDFATYYCLQFYEFPPTFGGGTKVEIK(SEQ ID NO:20)

[0421] Humanized B1H1 VL3:

[0422] DIQMTQSPSSSLSASVGDRVTITCQATQDIVKSLNWYQQKPGKAPKFLIYYTAQLAEGVPSRFSGSGSGTDYTLTIS SLQPEDFATYYCLQFYEFPPTFGGGTKVEIK(SEQ ID NO:2l)

[0423] Humanized B1H1 VL4:

[0424] DIQMTQSPSSSLSASVGDRVTITCQATQDIVKSLNWYQQKPGKPPKFLIYYTAQLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQFYEFPPTFGGGTKVEIK(SEQ ID NO:22)

[0425] Humanized B1H1 VL5:

[0426] DIQMTQ SPSSLSASVGDRVTITCQATQDIVKSLNWYQQKPGKPPKFAIYYTAQLAEGVPSRFSGSGSGTDYTLTIS SLQPEDFATYYCLQFYEFPPTFGGGTKVEIK(SEQ ID NO:23)

[0427] One embodiment provides an anti-B7-H4 monoclonal antibody or antigen-binding fragment thereof having a humanized light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to one of the following amino acid sequences:

[0428] Full-length B1H1 light chain variant 1:

[0429] DIQMTQSPSSSLSASVGDRVTITCQATQDIVKSLNWYQQKPGKPPKFLIYYTAQLAEGVPSRFSGSGSGTDYTLTISSLQSEDFATYYCLQFYEFPPTFGGGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:24)

[0433] Full-length B1H1 light chain variant 2:

[0434] DIQMTQSPSSLSASVGDRVTITCQATQDIVKSLNWYQQKPGKPPKFLIYYTAQLAEGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQFYEFPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC(SEQID NO:25)

[0438] Full-length B1H1 light chain variant 3:

[0439] DIQMTQSPSSLSASVGDRVTITCQATQDIVKSLNWYQQKPGKAPKFLIYYTAQLAEGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQFYEFPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHK VY ACE VTHQGL S SP VTKSFNRGEC(SEQ ID NO:26)

[0440] Full-length B1H1 light chain variant 4:

[0441] DIQMTQSPSSLSASVGDRVTITCQATQDIVKSLNWYQQKPGKPPKFLIYYTAQLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQFYEFPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKAD YEKHKVYACEVTHQGL SSPVTKSFNRGEC(SEQID NO:27)

[0442] Full-length B1H1 light chain variant 5:

[0443] DIQMTQSPSSSLSASVGDRVTITCQATQDIVKSLNWYQQKPGKPPKFLIYYTAQLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQFYEFPPTFGG GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC(SEQID NO:28)

[0444] iii. Heavy chain

[0445] One embodiment provides a murine monoclonal antibody or antigen-binding fragment thereof having a heavy chain that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to the following amino acid sequence:

[0446] EVOLOOSGTVLARPGASVKMSCKVSGYPFT SYWMHW VKORPGOGLEWI GAIYPGKSDTEYNPNFKGK AKLTAVTSATTAYMELSSLTNEDSAVYYCTS TWTHYFDY WGOGTTLTVSS (SEQ ID NO: 29), and specifically binds to B7-H4.

[0447] The CDRs are listed in SEQ ID NO:29, shown in bold and underlined, respectively:

[0448] CDR1 SYWMH (SEQ ID NO: 9);

[0449] CDR2 AIYPGKSDTEYNPNFKG (SEQ ID NO: 30); and

[0450] CDR3 TVRNVMDY (SEQ ID NO:11).

[0451] Another embodiment provides a nucleic acid encoding a heavy chain (SEQ ID NO: 29). An exemplary nucleic acid encoding a heavy chain (SEQ ID NO: 29) is

[0452] GAGGTTCAGCTCCAGCAGTCTGGGACTGTTCTGGCAAGGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGTTTCTGGCTACCCCTTTACCAGCTACTGGATGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGCGCTATTTATCCTGGAAAAAGTGACACTGAA TACAACCCGAACTTCAAGGGCAAGGCCAAACTGACTGCAGTCACATCTGCCACCACTGCCTACATGGAGCTCAGCAGCCTGACAAATGAGGACTCTGCGGTCTATTACTGTACAAGTACCTGGACCCACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA(SEQ ID NO:31).

[0453] One embodiment provides a murine monoclonal antibody or antigen-binding fragment thereof having a heavy chain constant domain that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to the following amino acid sequence:

[0454] ASTKGPSVFPLA SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:32), and specifically binds B7-H4.

[0455] Amino acids shown in bold and underlined represent amino acids that differ from the mutant sequence.

[0456] Another embodiment provides a mutant heavy chain constant domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to the following amino acid sequence:

[0457] ASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:33)

[0458] Amino acids shown in bold and underlined represent amino acids that differ from the wild-type sequence.

[0459] iv. Humanized heavy chain

[0460] One embodiment provides an anti-B7-H4 monoclonal antibody or antigen-binding fragment thereof having a humanized heavy chain variable domain that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to one of the following amino acid sequences:

[0461] Humanized B1H1 VH1:

[0462] EVQLVQSGAEVKKPGASVKVSCKVSGYPFTSYWMHWVRQAPGQGLEWIGAIYPGKSDTEYAPKFQGRVTLTADTSSTAYMELSSLRSEDTAVYYCTSTWTHYFDYWGQGTTVTVSS(SEQ ID NO:34)

[0463] Humanized B1H1 VH2:

[0464] QVQLVQSGAEVKKPGASVKVSCKVSGYPFTSYWMHWVRQAPGQGLEWMGAIYPGKSDTEYAQKFQGRVTLTADTSSTAYMELSSLRSEDTAVYYCTSTWTHYFDYWGQGTTVTVSS(SEQ ID NO:35)

[0465] Humanized B1H1 VH3:

[0466] QVQLVQSGAEVKKPGASVKVSCKVSGYPFTSYWMHWVRQAPGQGLEWMGAIYPGKSDTEYAQKFQGRVTLTADTSSTAYMELSSLRSEDTATYYCTSTWTHYFDYWGQGTTVTVSS(SEQ ID NO:36)

[0467] Humanized B1H1 VH4:

[0468] QVQLVQSGAEVKKPGASVKVSCKVSGYPFTSYYMHWVRQAPGQGLEWMGAIYPGKSDTEYAQKFQGRVTLTADTSSTAYMELSSLRSEDTATYYCTSTWTHYFDYWGQGTTVTVSS(SEQ ID NO:37)

[0469] One embodiment provides an anti-B7-H4 monoclonal antibody or antigen-binding fragment thereof having a humanized heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to one of the following amino acid sequences:

[0470] Heavy Chain B1H1 Variant 1:

[0471] EVQLVQSGAEVKKPGASVKVSCKVSGYPFTSYWMHWVRQAPGQGLEWIGAIYPGKSDTEYAPKFQGRVTLTADTSTSTAYMELSSLRSEDTAVYYCTSTWTHYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:38)

[0472] Heavy chain B1H1 variant 2:

[0473] QVQLVQSGAEVKKPGASVKVSCKVSGYPFTSYWMHWVRQAPGQGLEWMGAIYPGKSDTEYAQKFQGRVTLTADTSTSTAYMELSSLRSEDTAVYYCTSTWTHYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:39)

[0474] Heavy chain B1H1 variant 3:

[0475] QVQLVQSGAEVKKPGASVKVSCKVSGYPFTSYWMHWVRQAPGQGLEWMGAIYPGKSDTEYAQKFQGRVTLTADTSTSTAYMELSSLRSEDTATYYCTSTWTHYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:40)

[0476] Heavy chain B1H1 variant 4:

[0477] QVQLVQSGAEVKKPGASVKVSCKVSGYPFTSYYMHWVRQAPGQGLEWMGAIYPGKSDTEYAQKFQGRVTLTADTSSTAYMELSSLRSEDTATYYCTSTWTHYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:41)

[0478] In another embodiment, the monoclonal antibody or antigen-binding fragment thereof has a humanized heavy chain that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to one of the following amino acid sequences:

[0479] Heavy chain B1H1 mutant variant 1:

[0480] EVQLVQSGAEVKKPGASVKVSCKVSGYPFTSYWMHWVRQAPGQGLEWIGAIYPGKSDTEYAPKFQGRVTLTADTSTSTAYMELSSLRSEDTAVYYCTSTWTHYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPLPEEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:42);

[0481] Heavy chain B1H1 mutant variant 2:

[0482] QVQLVQSGAEVKKPGASVKVSCKVSGYPFTSYWMFIWVRQAPGQGLEWMGAIYPGKSDTEYAQKFQGRVTLTADTSTSTAYMELSSLRSEDTAVYYCTSTWMYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKV SNKALPLPEEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:43);

[0483] Heavy chain B1H1 mutant variant 3:

[0484] QVQLVQSGAEVKKPGASVKVSCKVSGYPFTSYWMFIWVRQAPGQGLEWMGAIYPGKSDTEYAQKFQGRVTLTADTSTSTAYMELSSLRSEDTATYYCTSTWTHYFDYWGQGTTYTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKV SNKALPLPEEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:44); and

[0485] Heavy chain B1H1 mutant variant 4:

[0486] QVQLVQSGAEVKKPGASVKVSCKVSGVPFTSYYMHWVRQAPGQGLEWMGAIYPGKSDTEYAQKFQGRVTLTADTSSTAYMELSSLRSEDTATYYCTSTWTHYFDYWGQGTTVTSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSWT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKV SNKALPLPEEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 45).

[0487] One embodiment provides an anti-B7-H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain comprising CDRs as listed in SEQ ID NOs: 14, 15 and 16.

[0488] One embodiment provides an anti-B7-H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a heavy chain containing CDRs as listed in SEQ ID NOs: 9, 11 and 30.

[0489] One embodiment provides an anti-B7-H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain containing CDRs as listed in SEQ ID NOs: 14, 15 and 16 and a heavy chain containing CDRs as listed in SEQ ID NOs: 9, 11 and 30.

[0490] One embodiment provides an anti-B7-H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:13 and a heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:29.

[0491] Another embodiment provides an anti-B7-H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain variable domain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to any one of SEQ ID NOs: 19, 20, 21, 22 or 23 and a heavy chain variable domain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to any one of SEQ ID NOs: 34, 35, 36 or 37.

[0492] One embodiment provides an anti-B7-H4 antibody or an antigen-binding fragment thereof having a light chain with an amino acid sequence as set forth in any one of SEQ ID NOs: 24, 25, 26, 27 or 28 and a heavy chain with an amino acid sequence as set forth in any one of SEQ ID NOs: 38, 39, 40 or 41.

[0493] Another embodiment provides an anti-B7-H4 antibody or an antigen-binding fragment thereof having a light chain with an amino acid sequence as set forth in any one of SEQ ID NOs: 24, 25, 26, 27 or 28 and a heavy chain with an amino acid sequence as set forth in any one of SEQ ID NOs: 42, 43, 44 or 45.

[0494] One embodiment provides an anti-B7-H4 antibody or an antigen-binding fragment thereof having two light chains and two heavy chains, wherein the two light chains comprise a polypeptide selected from the group consisting of SEQ ID NO: 24, 25, 26, 27 or 28 or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 24, 25, 26, 27 or 28, and the two heavy chains comprise a polypeptide selected from the group consisting of SEQ ID NO: 38, 39, 40 or 41 or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 38, 39, 40 or 41, and wherein the antibody or antigen-binding fragment thereof binds to B7-H4.

[0495] Another embodiment provides an antibody or an antigen-binding fragment thereof having two light chains and two heavy chains, wherein the two light chains comprise a polypeptide selected from the group consisting of SEQ ID NO: 24, 25, 26, 27 or 28, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 24, 25, 26, 27 or 28, and the two heavy chains comprise a polypeptide selected from the group consisting of SEQ ID NO: 42, 43, 44 or 45, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 42, 43, 44 or 45, and wherein the antibody or antigen-binding fragment thereof binds to B7-H4.

[0496] c.B1H3 sequence

[0497] In one embodiment, a murine monoclonal antibody is produced by hybridoma clone B1H3, contains two light chains and two heavy chains, and specifically binds to B7-H4.

[0498] i. Light chain

[0499] One embodiment provides an anti-B7-H4 murine monoclonal antibody or antigen-binding fragment thereof having a light chain that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to the following amino acid sequence:

[0500]

[0501] The CDRs are listed in SEQ ID NO:46, shown in bold and underlined, respectively:

[0502]

[0503] CDR2 YYTTQLAE (SEQ ID NO:47); and

[0504] CDR3 LQFYEFPPT (SEQ ID NO:16).

[0505] Another embodiment provides a nucleic acid encoding a light chain (SEQ ID NO: 46).

[0506] An exemplary nucleic acid encoding a light chain (SEQ ID NO: 46) is

[0507] GAAATCCAGATGACCCAGTCTCCATCCTCTATGTCTGCATCTCTGGGAGACACAATAACCATCACTTGCCAGGCAACTCAAGACATTGTTAAGAGTTTAAACTGGTATCAACAAAAACCAGGGAAACCCCCTTCATTCCTGATCTATTATACAACTCAACTG GCAGAAGGGGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGGTCAGACTATTCTCTGACAATCAGCAACCTGGACTCTGAAGATTTTGCAGACTATTACTGTCTACAGTTTTATGAGTTTCCTCCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA(SEQ ID NO:48).

[0508] ii. Heavy chain

[0509] One embodiment provides an anti-B7-H4 murine monoclonal antibody or antigen-binding fragment thereof having a heavy chain that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to the following amino acid sequence:

[0510] EVOLOOSGTVLARPGASVKMSCKASGYTFS SYWMH WVKORPGOGLEWI GAIYPGKS DTSYNOKFOG KAKLTAVTSASTAEMELTSLTNEDSAVYYCTS TWTHYFDY WGOGTTL TVSS (SEQ ID NO: 49).

[0511] The CDRs are listed in SEQ ID NO:49, shown in bold and underlined, respectively:

[0512] CDR1 SYWMH (SEQ ID NO: 9);

[0513] CDR2 AIYPGKSDTEYNPNFKG (SEQ ID NO: 50); and

[0514] CDR3 TVRNVMDY (SEQ ID NO:11).

[0515] Another embodiment provides a nucleic acid encoding the heavy chain (SEQ ID NO: 49).

[0516] An exemplary nucleic acid encoding the heavy chain (SEQ ID NO: 49) is

[0517] GAGGTTCAGCTCCAGCAGTCTGGGACTGTTCTGGCAAGGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTTCCAGCTACTGGATGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGCGCTATTTATCCTGGAAAAAGTGATACTAGC TACAACCAGAAGTTCCAGGGCAAGGCCAAACTGACTGCAGTCACATCTGCCAGCACTGCCTTCATGGAGCTCACCAGCCTGACAAATGAGGACTCTGCGGTCTATTACTGTACAAGTACCTGGACCCACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA(SEQ ID NO:51).

[0518] One embodiment provides an anti-B7-H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain containing CDRs as listed in SEQ ID NOs: 14, 16 and 47.

[0519] One embodiment provides an anti-B7-H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a heavy chain containing CDRs as listed in SEQ ID NOs: 9, 11 and 50.

[0520] One embodiment provides an anti-B7-H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain containing CDRs as listed in SEQ ID NOs: 14, 16 and 47 and a heavy chain containing CDRs as listed in SEQ ID NOs: 9, 11 and 50.

[0521] One embodiment provides an anti-B7-H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:46 and a heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:49.

[0522] d.B1H10 sequence

[0523] In one embodiment, the anti-B7-H4 murine monoclonal antibody is produced by hybridoma clone B1H10 and contains two light chains and two heavy chains.

[0524] i. Light chain

[0525] One embodiment provides an anti-B7-H4 murine monoclonal antibody or antigen-binding fragment thereof having a light chain that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to the following amino acid sequence:

[0526] DIOMTOTT SSL S ASLGDRVTISCRASODISNYLNW YOOKPDGTIKLLIY YTSRLHS GVPSRFSGSGSGSDYSLTISNLEOEDIATYEC OOGNTLPWT FGGGTKLEFK (SEQ ID NO: 52). The CDRs are as listed in SEQ ID NO: 52, shown in bold and underlined, respectively:

[0527] CDR1 RASQDISNYLN (SEQ ID NO:53);

[0528] CDR2 YTSRLHS (SEQ ID NO:54); and

[0529] CDR3 QQGNTLPWT (SEQ ID N0 55).

[0530] Another embodiment provides a nucleic acid encoding a light chain (SEQ ID NO: 52).

[0531] An exemplary nucleic acid encoding a light chain (SEQ ID NO: 52) is

[0532] GATATCCAGATGACACAAACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGAC ATTAGCAATTATTTAAACTGGT ATCAGC AGAAACCAGATGGAACTATTAAACTCCTGATCTATTACACATCAAGATTACATTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGATCAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGGT GGACGTTCGGTGGAGGCACCAAGCTGGAATTCAAA (SEQ ID NO: 56).

[0533] One embodiment provides an anti-B7-H4 monoclonal antibody or antigen-binding fragment thereof having a light chain constant domain that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to the following amino acid sequence:

[0534] DIQMTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKTIKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTLPWTFGQGTKLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:57)

[0535] ii. Humanized B1H10 light chain

[0536] In another embodiment, the anti-B7-H4 monoclonal antibody or antigen-binding fragment thereof has a humanized light chain variable domain that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to one of the following amino acid sequences:

[0537] Humanized B1H10 VL1:

[0538] DIQMTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKTIKLLIYYTSRLHSGVPSR FSGSGSGTDYTLTISSLQPEDFATYFCQQGNTLPWTFGQGTKLEIK(SEQ ID NO:58)

[0539] Humanized B1H10 VL2:

[0540] DIQMTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTLPWTFGQGTKLEIK(SEQ ID NO:59)

[0541] Humanized B1H10 VL3:

[0542] DIQMTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGQTLPWTFGQGTKLEIK(SEQ ID NO:60)

[0543] Humanized B1H10 VL4:

[0544] DIQMTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGSTLPWTFGQGTKLEIK(SEQ ID NO:61)

[0545] One embodiment provides an anti-B7-H4 monoclonal antibody or antigen-binding fragment thereof having a humanized light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to one of the following amino acid sequences:

[0546] Humanized B1H10 light chain variant 1:

[0547] DIQMTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTLPWTFGQ GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC(SEQID NO:62)

[0548] Humanized B1H10 light chain variant 2:

[0549] DIQMTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGQTLPWTFGQGTKLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:63)

[0550] Humanized B1H10 light chain variant 3:

[0551] DIQMTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGSTLPWTFGQGTKLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:64)

[0552] iii. Heavy chain

[0553] One embodiment provides an anti-B7-H4 murine monoclonal antibody or antigen-binding fragment thereof having a heavy chain that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to the following amino acid sequence:

[0554] EVOLOOSGPELVKPGASVKMSCKASGYTFT DYYMN WVROSHGKSLEWI GRVNPSNGGTNYNOKFKG KATLTVDKSLSTAYMOLSSLTSEDSAVYYCA RRHNYADF WGOGTTLTV SS (SEQ ID NO: 65).

[0555] The CDRs are listed in SEQ ID NO:65, shown in bold and underlined, respectively:

[0556] CDR1 DYYMN (SEQ ID NO: 66);

[0557] CDR2 RVNPSNGGTNYNQKFKG (SEQ ID NO:67); and

[0558] CDR3 RHNYADF (SEQ ID NO: 68).

[0559] Another embodiment provides a nucleic acid encoding a heavy chain (SEQ ID NO: 65). An exemplary nucleic acid encoding a heavy chain (SEQ ID NO: 65) is

[0560] GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGTAAGGCTTCTGGATACACATTCACTGACTACATGAACTGGGTGAGGCAGAGTCATGGAAAGAGCCTTGAGTGGATTGGACGTGTTAATCCTAGCAATGGTGGTACTAA CTACAACCAGAAATTCAAGGGCAAGGCCACATTGACAGTAGACAAATCCCTCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGACGACATAACTACGCAGACTTCTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA(SEQ ID NO:69).

[0561] iv. Humanized B1H10 heavy chain

[0562] One embodiment provides an anti-B7-H4 monoclonal antibody or antigen-binding fragment thereof having a humanized heavy chain variable domain variant having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to one of the following amino acid sequences:

[0563] Humanized B1H10 VH1:

[0564] EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWIGRVNPSNGGTNYAQKFQGRVTLTVDKSTSTAYMELSSLRSEDTAVYYCARRHNYADFWGQGTTVTVSS(SEQ ID NO:70)

[0565] Humanized B1H10 VH2:

[0566] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRVNPANGGTNYAQKFQGRVTLTVDTSTSTAYMELSSLRSEDTAVYYCARRHNYADFWGQGTTVTVSS(SEQ ID NO:7l)

[0567] Humanized B1H10 VH3:

[0568] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRVNPSSGGTNYAQKFQGRVTLTVDTSTSTAYMELSSLRSEDTAVYYCARRHNYADFWGQGTTVTVSS(SEQ ID NO:72)

[0569] Humanized B1H10 VH4:

[0570] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRVNPSNGGTNYAQKFQGRVTLTVDTSKSTAYMELSSLRSEDTAVYYCARRHNYADFWGQGTTVTVSS(SEQ ID NO:73)

[0571] Humanized B1H10 VH5:

[0572] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRVNPSSGGTNYAQKFQGRVTLTVDTSKSTAYMELSSLRSEDTAVYYCARRHNYADFWGQGTTVTVSS(SEQ ID NO:74)

[0573] One embodiment provides an anti-B7-H4 monoclonal antibody or antigen-binding fragment thereof having a humanized heavy chain variant having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to one of the following amino acid sequences:

[0574] Humanized B1H10 heavy chain variant 1:

[0575] EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWIGRVNPSNGGTNYAQKFQGRVTLTVDKSTSTAYMELSSLRSEDTAVYYCARRHNYADFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:75)

[0576] Humanized B1H10 heavy chain variant 2:

[0577] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRVNPANGGTNYAQKFQGRVTLTVDTSTSTAYMELSSLRSEDTAVYYCARRHNYADFWGQGTTVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:76)

[0578] Humanized B1H10 heavy chain variant 3:

[0579] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRVNPSSGGTNYAQKFQGRVTLTVDTSTSTAYMELSSLRSEDTAVYYCARRHNYADFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:77)

[0580] Humanized B1H10 heavy chain variant 4:

[0581] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRVNPSNGGTNYAQKFQGRVTLTVDTSKSTAYMELSSLRSEDTAVYYCARRHNYADFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:78)

[0582] Humanized B1H10 heavy chain variant 5:

[0583] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRVNPSSGGTNYAQKFQGRVTLTVDTSKSTAYMELSSLRSEDTAVYYCARRHNYADFWGQGTTVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:79)

[0584] One embodiment provides an anti-B7-H4 monoclonal antibody or antigen-binding fragment thereof having a humanized mutant heavy chain variant having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to one of the following amino acid sequences:

[0585] Humanized mutant B1H10 heavy chain variant 1:

[0586] EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWIGRVNPSNGGTNYAQKFQGRVTLTVDKSTSTAYMELSSLRSEDTAVYYCARRHNYADFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPLPEEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:80)

[0587] Humanized mutant B1H10 heavy chain variant 2:

[0588] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRVNPANGGTNYAQKFQGRVTLTVDTSTSTAYMELSSLRSEDTAVYYCARRHNYADFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPLPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:8l)

[0589] Humanized mutant B1H10 heavy chain variant 3:

[0590] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRVNPSSGGTNYAQKFQGRVTLTVDTSTSTAYMELSSLRSEDTAVYYCARRHNYADFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:82)

[0591] Humanized mutant B1H10 heavy chain variant 4:

[0592] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRVNPSNGGTNYAQKFQGRVTLTVDTSKSTAYMELSSLRSEDTAVYYCARRHNYADFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:83)

[0593] Humanized mutant B1H10 heavy chain variant 5:

[0594] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRVNPSSGGTNYAQKFQGRVTLTVDTSKSTAYMELSSLRSEDTAVYYCARRHNYADFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPLPEEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:84)

[0595] One embodiment provides an anti-B7-H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain containing CDRs as listed in SEQ ID NO: 53, 54 or 55.

[0596] One embodiment provides an anti-B7-H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a heavy chain containing CDRs as listed in SEQ ID NO:66, 67 or 68.

[0597] One embodiment provides an anti-B7-H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain containing CDRs as listed in SEQ ID NO:53, 54 or 55 and a heavy chain containing CDRs as listed in SEQ ID NO:66, 67 or 68.

[0598] One embodiment provides an anti-B7-H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:52 and a heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:65.

[0599] Another embodiment provides an anti-B7-H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain variable domain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to any one of SEQ ID NOs:58, 59, 60 or 61 and a heavy chain variable domain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to any one of SEQ ID NOs:70, 71, 72, 73 or 74.

[0600] One embodiment provides an anti-B7-H4 antibody or an antigen-binding fragment thereof having a light chain with an amino acid sequence as set forth in any one of SEQ ID NOs: 62, 63, or 64 and a heavy chain with an amino acid sequence as set forth in any one of SEQ ID NOs: 75, 76, 77, 78, or 79.

[0601] Another embodiment provides an anti-B7-H4 antibody or an antigen-binding fragment thereof having a light chain with an amino acid sequence as set forth in any one of SEQ ID NOs: 62, 63, or 64 and a heavy chain with an amino acid sequence as set forth in any one of SEQ ID NOs: 80, 81, 82, 83, or 84.

[0602] One embodiment provides an anti-B7-H4 antibody or an antigen-binding fragment thereof having two light chains and two heavy chains, wherein the two light chains comprise a polypeptide selected from the group consisting of SEQ ID NO: 62, 63 or 64 or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or higher sequence identity with SEQ ID NO: 62, 63 or 64; and the two heavy chains comprise a polypeptide selected from the group consisting of SEQ ID NO: 75, 76, 77, 78 or 79 or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or higher sequence identity with SEQ ID NO: 75, 76, 77, 78 or 79.

[0603] Another embodiment provides an anti-B7-H4 antibody or an antigen-binding fragment thereof having two light chains and two heavy chains, wherein the two light chains comprise a polypeptide selected from the group consisting of SEQ ID NO: 62, 63 or 64 or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or higher sequence identity with SEQ ID NO: 62, 63 or 64; and the two heavy chains comprise a polypeptide selected from the group consisting of SEQ ID NO: 80, 81, 82, 83 or 84 or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or higher sequence identity with SEQ ID NO: 80, 81, 82, 83 or 84.

[0604] e.B2E6 sequence

[0605] In one embodiment, the anti-B7-H4 murine monoclonal antibody is produced by hybridoma clone B2E6 and contains two light chains and two heavy chains.

[0606] i. Light chain

[0607] One embodiment provides an anti-B7-H4 murine monoclonal antibody or antigen-binding fragment thereof having a light chain that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to the following amino acid sequence:

[0608]

[0609] The CDRs are listed in SEQ ID NO:85, shown in bold and underlined, respectively:

[0610]

[0611] CDR2 SASYQYT (SEQ ID NO: 86); and

[0612] CDR3HQYYNTPLT (SEQ ID NO:87).

[0613] Another embodiment provides a nucleic acid encoding a light chain (SEQ ID NO: 85).

[0614] An exemplary nucleic acid encoding a light chain (SEQ ID NO: 85) is

[0615] GAAACTGTGATGACCCAGTCTCACAAAATCATGTCCACTTCAGTAGGAGACAGGGTCACCATCACCTGCAAGGCCAGTCAGGATGTGAGAACTGCTGTGGCCTGGTATCAACAGAAACCAGGACAATCTCCTAAATTACTAATTTCCTCGGCATCCTACCAA TACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCATCAGCAGTTTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCATCAGTATTATAATACTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAGA(SEQ ID NO:88).

[0616] ii. Heavy chain

[0617] One embodiment provides an anti-B7-H4 murine monoclonal antibody or antigen-binding fragment thereof having a heavy chain that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to the following amino acid sequence:

[0618] EVOLOOSGTVLARPGASVKMSCKASGYTFT SYWMH WVKORPGOGLEWI GAIYPGKSDTTYNOKFEG KAKLTAVTSDSTAYMDLSSSLTNEDSAVYYCTS SVRNAMDY WGOGTSVTVSS (SEQ ID NO: 89).

[0619] The CDRs are listed in SEQ ID NO:89, shown in bold and underlined, respectively:

[0620] CDR1 SYWMH (SEQ ID NO: 9);

[0621] CDR2 AIYPGKSDTT YNQKFE G (SEQ ID NO: 90); and

[0622] CDR3 SVRNAMDY (SEQ ID NO:91).

[0623] Another embodiment provides a nucleic acid encoding a heavy chain (SEQ ID NO: 89). An exemplary nucleic acid encoding a heavy chain (SEQ ID NO: 89) is

[0624] GAGGTTCAGCTCCAGCAGTCTGGGACTGT GCTGGCAAGGCCTGGGGCTTC AGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGCGCTATTTATCCTGGAAAAAGTGATACTACCTACAACCAGAAGTTCGAGGGCAAGG CCAAACTGACTGCAGTCACATCTGACAGCACAGCCTACATGGATCTCAGTAGCCTGACAAATGAGGACTCTGCGGTCTATTACTGTACATCTTCGGTTCGGAATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA(SEQ ID NO:92).

[0625] One embodiment provides an anti-B7-H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain comprising CDRs as listed in SEQ ID NOs: 4, 86 and 87.

[0626] One embodiment provides an anti-B7-H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a heavy chain containing CDRs as listed in SEQ ID NOs: 9, 90 and 91.

[0627] One embodiment provides an anti-B7-H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain containing CDRs as listed in SEQ ID NOs: 4, 86 and 87 and a heavy chain containing CDRs as listed in SEQ ID NOs: 9, 90 and 91.

[0628] One embodiment provides an anti-B7-H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:85 and a heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:89.

[0629] f.B4B3 sequence

[0630] In one embodiment, the anti-B7-H4 murine monoclonal antibody is produced by hybridoma clone B4B3 and contains two light chains and two heavy chains.

[0631] i. Light chain

[0632] One embodiment provides an anti-B7-H4 murine monoclonal antibody or antigen-binding fragment thereof having a light chain that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to the following amino acid sequence:

[0633] DVLMTOTPLSLPVSLGGOASISC RSSOIIVHSNGNTYLEW YLOKPGOSPKLLI YKVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC FOGSHVPWT FGGGTKLEIK (SEQ ID NO:93)

[0634] The CDRs are listed in SEQ ID NO:93, shown in bold and underlined, respectively:

[0635]

[0636] as well as

[0637]

[0638] Another embodiment provides a nucleic acid encoding a light chain (SEQ ID NO: 93).

[0639] An exemplary nucleic acid encoding a light chain (SEQ ID NO: 93) is

[0640] GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGGTCAAGCCTCCATCTCTTGCAGATCTAGTCAGATCATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTC CAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA(SEQ ID NO:97).

[0641] ii. Heavy chain

[0642] One embodiment provides an anti-B7-H4 murine monoclonal antibody or antigen-binding fragment thereof having a heavy chain that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to the following amino acid sequence:

[0643]

[0644] The CDRs are listed in SEQ ID NO:98, shown in bold and underlined, respectively:

[0645]

[0646] as well as

[0647]

[0648] Another embodiment provides a nucleic acid encoding a heavy chain (SEQ ID NO: 98). An exemplary nucleic acid encoding a heavy chain (SEQ ID NO: 98) is

[0649] CAGGTCCAGCTGCAGCAGCCTGGGGCTGAACTGGT GAAGCCTGGGGCTTC AGTGAAGCTGTCCTGCAAGGCTTCTGGATACACCTTCATTAGCTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATCGGAGAGATTGATCCTTCTGATAGTTATACTTACTACAATCAAAAGTTCAAGGGCAAGGCCA CATTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGAAGGAAAACCTGGGACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA(SEQ ID NO:101).

[0650] One embodiment provides an anti-B7-H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain comprising CDRs as listed in SEQ ID NOs: 94, 95 and 96.

[0651] One embodiment provides an anti-B7-H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a heavy chain containing CDRs as listed in SEQ ID NOs: 9, 99 and 100.

[0652] One embodiment provides an anti-B7-H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain containing CDRs as listed in SEQ ID NOs:94, 95 and 96 and a heavy chain containing CDRs as listed in SEQ ID NOs:9, 99 and 100.

[0653] One embodiment provides an anti-B7-H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:93 and a heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:98.

[0654] g.B4E11 sequence

[0655] In one embodiment, the anti-B7-H4 murine monoclonal antibody is produced by hybridoma clone B4E11 and contains two light chains and two heavy chains.

[0656] i. Light chain

[0657] One embodiment provides an anti-B7-H4 murine monoclonal antibody or antigen-binding fragment thereof having a light chain that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to the following amino acid sequence:

[0658]

[0659] The CDRs are listed in SEQ ID NO: 102, shown in bold and underlined, respectively:

[0660] CDR1 KASQDVSTAVA (SEQ ID NO:103);

[0661] CDR2 SASYRYT (SEQ ID NO: 104); and

[0662]

[0663] Another embodiment provides a nucleic acid encoding a light chain (SEQ ID NO: 102).

[0664] An exemplary nucleic acid encoding a light chain (SEQ ID NO: 102) is

[0665] GACATTTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCACTATCACCTGCAAGGCCAGTCAGGATGTGAGTACTGCTGTAGCCTGGTATCAACAGAAACCAGGACAGTCTCCTAAACTACTGATTTCCTCGGCATCCTACCG GTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAACATTATAGTACTCCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAGA(SEQ ID NO:106).

[0666] ii. Heavy chain

[0667] One embodiment provides an anti-B7-H4 murine monoclonal antibody or antigen-binding fragment thereof having a heavy chain that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to the following amino acid sequence:

[0668] EVOLOOSGTVLARPGASVKMSCKASGYTFT SYWMH WYKERPGOGLEWI GAIYPGDSDTRYNOKFKG RAKLTAVTSANTAYMELSSLTNDDSAVFYCTC TTAGVLDY WGOGTSVTVSS (SEQ ID NO: 107).

[0669] The CDRs are listed in SEQ ID NO: 107, shown in bold and underlined, respectively:

[0670]

[0671] as well as

[0672]

[0673] Another embodiment provides a nucleic acid encoding a heavy chain (SEQ ID NO: 107). An exemplary nucleic acid encoding a heavy chain (SEQ ID NO: 107) is

[0674] GAGGTTC AGCTCC AGCAGTCTGGGACTGT GCTGGCAAGGCCTGGGGCTTC AGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACCAGCTACTGGATGCACTGGGTAAAAGAGAGGCCTGGAC AGGGTCTGGAATGGATTGGCGCTATTTATCCTGGAGAT AGTGATACTAGGTATAATCAGAAGTT CAAGGGC AGGGCCAAACTGACTGC AGTCACATCTGCCAACACTGCCTACATGGAGCTCAGCAGCCTGACAAATGATGACTCTGCGGTCTTCTACTGTACATGTACTACGGCTGGTGTTTTGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 110).

[0675] One embodiment provides an anti-B7-H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain containing CDRs as listed in SEQ ID NOs: 103, 104 and 105.

[0676] One embodiment provides an anti-B7-H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a heavy chain containing CDRs as listed in SEQ ID NOs: 9, 108 and 109.

[0677] One embodiment provides an anti-B7-H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain containing CDRs as listed in SEQ ID NOs: 103, 104 and 105 and a heavy chain containing CDRs as listed in SEQ ID NOs: 9, 108 and 109.

[0678] One embodiment provides an anti-B7-H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:102 and a heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:107.

[0679] h.B6C8 sequence

[0680] In one embodiment, the anti-B7-H4 murine monoclonal antibody is produced by hybridoma clone B6C8 and contains two light chains and two heavy chains.

[0681] i. Light chain

[0682] One embodiment provides an anti-B7-H4 murine monoclonal antibody or antigen-binding fragment thereof having a light chain that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to the following amino acid sequence:

[0683]

[0684] The CDRs are listed in SEQ ID NO: 111, shown in bold and underlined, respectively:

[0685] CDR1 TSSQSIVHGNGNTYLE (SEQ ID NO:112);

[0686] CDR2 KVSNRFS (SEQ ID NO:95); and

[0687]

[0688] Another embodiment provides a nucleic acid encoding a light chain (SEQ ID NO: 111).

[0689] An exemplary nucleic acid encoding a light chain (SEQ ID NO: 111) is

[0690] GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAGGCCTCCATCTCTTGCACATCTAGTCAGAGCATTGTACATGGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTC CAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA(SEQ ID NO:114).

[0691] ii. Heavy chain

[0692] One embodiment provides an anti-B7-H4 murine monoclonal antibody or antigen-binding fragment thereof having a heavy chain that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to the following amino acid sequence:

[0693]

[0694] The CDRs are listed in SEQ ID NO: 115, shown in bold and underlined, respectively:

[0695]

[0696] as well as

[0697]

[0698] Another embodiment provides a nucleic acid encoding a heavy chain (SEQ ID NO: 115). An exemplary nucleic acid encoding a heavy chain (SEQ ID NO: 115) is

[0699] CAGGTCC AACTGCAGCAGCCTGGGGCTGAACTGGT GAAGCCTGGGGCTTC AGTGAAGCTGTCCTGCAAGGCTTCTGGCTACTCTTTCACCAGCTACTGGATGAACTGGGTGAAGCAGAGGCCTGGACGAGGCCTCGAGTGGATTGGAAGGATTC ATCCTTCTGAT AGTGAAACTCACTACAATCAAAAGTTCAAGAGCAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATCCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTTTTGTGCAAGATACGGGCTCTTCTATGGTAACGACGGATATGCTATGGACCACTGGGGTCAAGGAACCTCAG (SEQ ID NO: 119).

[0700] One embodiment provides an anti-B7-H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain containing CDRs as listed in SEQ ID NOs: 97, 116 and 117.

[0701] One embodiment provides an anti-B7-H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a heavy chain containing CDRs as listed in SEQ ID NOs: 118, 119 and 120.

[0702] One embodiment provides an anti-B7-H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain containing CDRs as listed in SEQ ID NOs: 97, 114 and 115 and a heavy chain containing CDRs as listed in SEQ ID NOs: 118, 119 and 120.

[0703] One embodiment provides an antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:113 and a heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:117.

[0704] i.B9H1 sequence

[0705] In one embodiment, the anti-B7-H4 murine monoclonal antibody is produced by hybridoma clone B9H1 and contains two light chains and two heavy chains.

[0706] i. Light chain

[0707] One embodiment provides an anti-B7-H4 murine monoclonal antibody or antigen-binding fragment thereof having a light chain that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to the following amino acid sequence:

[0708] DIOMTOTT SSLSASLGDRVTISC RASODISF YLNW YOOKPDGT VKLLIY YTSRLHS GVPSRFSGSGSGTDYSLTISNLEOEDIATYE COOGNTLPWTF GGGTKLEIK (SEQ ID NO: 120). The CDRs are as listed in SEQ ID NO: 120, shown in bold and underlined, respectively:

[0709] CDR1 RASQDISFYLN (SEQ ID NO:121);

[0710] CDR2 YTSRLHS (SEQ ID NO:54); and

[0711] CDR3 QQGNTLPWT (SEQ ID N0 55).

[0712] Another embodiment provides a nucleic acid encoding a light chain (SEQ ID NO: 120).

[0713] An exemplary nucleic acid encoding a light chain (SEQ ID NO: 120) is

[0714] GATATCCAGATGACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGCTTTTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAAGATTA CACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACACTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA(SEQ ID NO:122).

[0715] ii. Heavy chain

[0716] One embodiment provides an anti-B7-H4 murine monoclonal antibody or antigen-binding fragment thereof having a heavy chain that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to the following amino acid sequence:

[0717] EVOLOOSGPELVKPGASVKMSCKASGYTFT DYYMN WVKOSHGKSLEWI GRVNPSNGGTSYNOKFKGK ATLTVDKSL SAAYMOLN SLTSEP SAVYY C ARRHNYPD YW GOGTTLTVSS (SEQ ID NO: 123).

[0718] The CDRs are listed in SEQ ID NO: 123, shown in bold and underlined, respectively:

[0719] CDR1 DYYMN (SEQ ID NO: 66);

[0720] CDR2 RVNPSN GGTS YNQKFKG (SEQ ID NO: 124); and

[0721] CDR3 RHNYPDY (SEQ ID NO: 125).

[0722] Another embodiment provides a nucleic acid encoding a heavy chain (SEQ ID NO: 125). An exemplary nucleic acid encoding a heavy chain (SEQ ID NO: 125) is

[0723] GAGGTCCAGCTGCAACAGTCTGGACCTGAACTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGTAAGGCTTCTGGATACACATTCACTGACTACATGAACTGGGTGAAGCAGAGTC ATGGAAAGAGCCTTGAGTGGATTGGACGT GTTAATCCTAGCAATGGT GGTACTAGCTACAACCAGAAGTTC AAGGGC AAGGCC AC ATTGAC AGTAGACAAATCCCTCAGCGCAGCCTATATGCAGCTCAACAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGAAGGCATAACTACCCTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO: 126).

[0724] One embodiment provides an anti-B7-H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain comprising CDRs as listed in SEQ ID NOs: 54, 55, and 121.

[0725] One embodiment provides an anti-B7-H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a heavy chain containing CDRs as listed in SEQ ID NOs: 66, 124 and 125.

[0726] One embodiment provides an anti-B7-H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain containing CDRs as listed in SEQ ID NOs: 54, 55 and 121 and a heavy chain containing CDRs as listed in SEQ ID NOs: 66, 124 and 125.

[0727] One embodiment provides an anti-B7-H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:120 and a heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:123.

[0728] j.B10D7 sequence

[0729] In one embodiment, the anti-B7-H4 murine monoclonal antibody is produced by hybridoma clone B10D7 and contains two light chains and two heavy chains.

[0730] i. Light chain

[0731] One embodiment provides an anti-B7-H4 monoclonal antibody or antigen-binding fragment thereof having a light chain that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to the following amino acid sequence:

[0732] EIOMTO SPSSMS ASLGDRITITCOATODIVKSLNW YOOKPGKPP SFLIYYTAOLAEGVP SRFSGSGSGSDYSLTISNLESEDFADYY CLOF YEFPPTF GGGTKLEIK (SEQ ID NO: 13).

[0733] The CDRs are listed in SEQ ID NO: 13, shown in bold and underlined, respectively:

[0734] CDR1 QATQDIVKSLN(SEQ ID NO:14);

[0735] CDR2 YTAQLAE (SEQ ID NO: 15); and

[0736] CDR3 LQFYEFPPT (SEQ ID NO:16).

[0737] Another embodiment provides a nucleic acid encoding a light chain (SEQ ID NO: 13).

[0738] An exemplary nucleic acid encoding a light chain (SEQ ID NO: 13) is

[0739] GAAATCCAGATGACCCAGTCTCCATCCTCTATGTCTGCATCTCTGGGAGACAGAATAACCATCACTTGCCAGGCAACTCAAGACATTGTTAAGAGTTTAAACTGGTATCAACAAAAACCAGGGAAACCCCCTTCATTCCTGATCTATTATACAGCTCAACTG GCAGAAGGGGTCCCGTCAAGGTTCAGTGGCAGTGGGTCTGGGTCAGACTATTCTCTGACAATCAGCAACCTGGAGTCTGAAGATTTTGCAGACTATTACTGTCTACAGTTTTATGAGTTTCCTCCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAA(SEQ ID NO:127).

[0740] ii. Heavy chain

[0741] One embodiment provides an anti-B7-H4 murine monoclonal antibody or antigen-binding fragment thereof having a heavy chain that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to the following amino acid sequence:

[0742] EVOLOOSGTVLARPGASVKMSCKASGYPFT SYWMHW VKORPGOGLEWI GAIYPGNSD TRYNPNFKG KANLTAVTSATTAYMELSSLTNEESAVYYCTS TWTHYFDY WGOGTTLT VSS (SEQ ID NO: 128).

[0743] The CDRs are listed in SEQ ID NO: 128, shown in bold and underlined, respectively:

[0744] CDR1 SYWMH (SEQ ID NO: 9);

[0745] as well as

[0746]

[0747] Another embodiment provides a nucleic acid encoding a heavy chain (SEQ ID NO: 128). An exemplary nucleic acid encoding a heavy chain (SEQ ID NO: 130) is

[0748] GAGGTTC AGCTCC AGCAGTCTGGGACTGT GCTGGC AAGGCCTGGGGCTTC AGTGAAGATGTCCTGCAAGGCTTCTGGCTACCCCTTTACCAGCTACTGGATGCACTGGGTAAAGCAGAGGCCTGGACAGGGTTCTGGAATGGATTGGCGCTATTTATCCTGGAAATAGTGATACTAGGTACAACCCGAATTTCAAGGGCAAGG CCAACCTGACTGCAGTCACATCTGCCACCACTGCCTACATGGAGCTCAGCAGCCTGACAAATGAGGAATCTGCGGTCTATTACTGTACAAGTACCTGGACCCACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA(SEQ ID NO:131).

[0749] One embodiment provides an anti-B7-H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain comprising CDRs as listed in SEQ ID NOs: 14, 15 and 16.

[0750] One embodiment provides an anti-B7-H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a heavy chain containing CDRs as listed in SEQ ID NOs: 9, 129 and 130.

[0751] One embodiment provides an anti-B7-H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain containing CDRs as listed in SEQ ID NOs: 14, 15 and 16 and a heavy chain containing CDRs as listed in SEQ ID NOs: 9, 129 and 130.

[0752] One embodiment provides an anti-B7-H4 antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, having a light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:13 and a heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:128.

[0753] 2. Antibody Composition

[0754] The disclosed B7-H4 binding molecules can be antibodies or antigen-binding fragments thereof. The disclosed antibodies and antigen-binding fragments thereof include any type of complete immunoglobulin (i.e., full antibodies), fragments thereof, and synthetic proteins containing at least the antigen-binding variable domains of antibodies. In some embodiments, the disclosed molecules contain both antibody light chains and at least the variable domains of antibody heavy chains. In other embodiments, such molecules may further include one or more of the CHi, hinge, CH2, CH3, and CH4 regions of the heavy chain (particularly CHi and hinge regions, or CHi, hinge and CH2 regions, or CHi, hinge, CH2, and CH3 regions). The antibody can be selected from any type of immunoglobulin, including IgM, IgG, IgD, IgA, and IgE, and any subtype, including IgG1, IgG2, IgG3, and IgG4. In some embodiments, the constant domain is a complement fixing constant domain, wherein the antibody is expected to exhibit cytotoxic activity, and the type is typically IgGi. In other embodiments, when such cytotoxic activity is not expected, the constant domain may be of IgG2 or IgG4 type. Antibodies may comprise sequences from more than one class or subclass, and selection of particular constant domains to optimize desired effector functions is within the ordinary skill in the art.

[0755] The variable domains differ in sequence between antibodies and are used for the binding and specificity of each particular antibody for its particular antigen. However, the variability is usually not evenly distributed in the variable domains of antibodies. It is usually concentrated in three segments called "complementarity determining regions (CDRs)" or "hypervariable regions" in the light and heavy chain variable domains. The more conserved parts of the variable domains are called "frameworks (FRs)". The variable domains of native heavy and light chains each contain four FR regions, mainly in a β-folded configuration, connected by three CDRs, wherein the three CDRs form a loop connecting the β-folded configuration and in some cases form part of the β-folded configuration. The CDRs in each chain are tightly bound together by the FR regions and, together with the CDRs in the other chain, contribute to the formation of the antigen binding site of the antibody.

[0756] Also disclosed are antibody fragments having biological activity. These fragments, whether or not linked to other sequences, include insertions, deletions, substitutions or other selected modifications of specific regions or specific amino acid residues, provided that the activity of the fragment is not significantly altered or impaired compared to the unmodified antibody or antibody fragment.

[0757] The technology can also be adapted to produce specific single chain antibodies against B7-H4. Methods for producing single chain antibodies are well known to those skilled in the art. Single chain antibodies can be created by fusing the variable domains of the heavy and light chains together using a short peptide linker, thereby reconstructing the antigen binding site on a single molecule. Single chain antibody variable fragments (scFv) have been developed in which the C-terminus of one variable domain is tethered to the N-terminus of the other variable domain by a 15 to 25 amino acid peptide or linker without significantly disrupting antigen binding or specificity of binding. The linker is selected to allow the heavy and light chains to be bound together in their proper conformational orientation.

[0758] A divalent single-chain variable fragment (di-scFv) can be engineered by connecting two scFvs. This is done by generating a single peptide chain with two VH regions and two VL regions, resulting in a tandem scFv. ScFv can also be designed with a connecting peptide that is too short for the two variable regions to fold together (about five amino acids), forcing the scFv to dimerize. This type is called a "diabody". Studies have shown that the dissociation constant of diabodies is up to 40 times lower than that of the corresponding scFv, which means that they have a higher affinity for their targets. But shorter linkers (one or two amino acids) still lead to the formation of trimers (tribodies). Tetrabodies have also been produced. Compared to diabodies, they have a higher affinity for their targets.

[0759] One embodiment provides a monoclonal antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small fraction of the antibody molecules. Monoclonal antibodies include "chimeric" antibodies, in which a portion of the heavy and / or light chains are identical or homologous to corresponding sequences of antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remaining antibody chains are identical or homologous to corresponding sequences of antibodies derived from other species or belonging to other antibody classes or subclasses, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity.

[0760] One embodiment provides antibodies and antigen-binding fragments thereof that specifically bind to human B7-H4.

[0761] One embodiment provides an antibody produced by a hybridoma from the group consisting of B1A1, B1H1, B1H3, B1H10, B2E6, B4B2, B4E11, B6C8, B9H1, and B10D7.

[0762] a. Antibodies

[0763] The immunomodulator can be an antibody. Suitable antibodies are known in the art or can be prepared by one 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. As discussed in more detail below, one skilled in the art can use the sequences to prepare specific antibodies or antigen-binding fragments thereof against B7-H4. Thus, the antibody or antigen-binding fragment thereof can be an agonist or antagonist of B7-H4 signaling.

[0764] The activity (i.e., agonist or antagonist) of a specific antibody or antigen binding fragment thereof against B7-H4 can be determined using functional assays known in the art, including the following assays. Typically, the assay involves determining whether the antibody or antigen binding fragment thereof enhances (i.e., agonist) or attenuates (i.e., antagonist) signaling through B7-H4. Because B7-H4 signaling results in an inhibitory immune response, agonizing B7-H4 results in an inhibited or attenuated immune response. Antagonizing B7-H4 signaling will inhibit the immunosuppressive response, resulting in an overall enhanced immune response.

[0765] In some embodiments, the disclosed antibodies and antigen-binding fragments thereof immunospecifically bind to B7-H4. In some embodiments, the antibodies bind to the extracellular domain of B7-H4.

[0766] For example, molecules that immunospecifically bind to B7-H4 are provided:

[0767] (I) arranged on the surface of cells (especially living cells);

[0768] (II) arrayed on the surface of cells (especially living cells) at endogenous concentrations;

[0769] (III) arrayed on the surface of living cells and modulating the binding between B7-H4 and its ligands;

[0770] (IV) arrayed on the surface of living cells and attenuating or inhibiting the immunosuppression produced by B7-H4;

[0771] (V) arrayed on the surface of living cells and inducing or enhancing immunosuppression produced by B7-H4;

[0772] (VI) arrayed on the surface of a living cell, wherein the cell is a tumor cell;

[0773] (VII) a combination of I-IV and VI;

[0774] (VIII) a combination of I-III and V-IV; and

[0775] (IX) is present on the surface of living myeloid or lymphoid cancer cells (AML or ALL) and promotes apoptosis and differentiation, leading to a decrease in the self-renewal of cancer stem cells.

[0776] In some embodiments, the molecules are capable of inducing antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), or apoptosis of cells expressing B7-H4 by other mechanisms.

[0777] To prepare antibodies or antigen-binding fragments thereof that can specifically bind to B7-H4, purified B7-H4 proteins, polypeptides, fragments, fusions or epitopes or polypeptides expressed by their nucleic acid sequences can be used. The antibodies or antigen-binding fragments thereof can be prepared using any suitable method known in the art, such as the methods discussed in more detail below.

[0778] i. Human antibodies and humanized antibodies

[0779] Many non-human antibodies (e.g., antibodies derived from mice, rats, or rabbits) are naturally antigenic in humans and therefore can cause an adverse immune response when administered to humans. Therefore, the use of human or humanized antibodies in these methods can reduce the likelihood that antibodies administered to humans will cause an adverse immune response.

[0780] In the absence of endogenous immunoglobulin production, transgenic animals (e.g., mice) that are capable of producing fully human antibodies upon immunization can be used. For example, it has been shown that homozygous deletion of the antibody heavy chain joining region (J(H)) gene in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. If the human germline immunoglobulin gene array is transplanted into such germline mutant mice, human antibodies will be produced upon antigen challenge.

[0781] Alternatively, antibodies can be produced in other species and "humanized" for use in humans. Humanized non-human (e.g., mouse) antibodies are in the form of chimeric immunoglobulins, immunoglobulin chains or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequences derived from non-human immunoglobulins. Humanized antibodies include human immunoglobulins (receptor antibodies), wherein residues from the complementary determining regions (CDRs) of the receptor antibodies are replaced by residues from CDRs of non-human species (donor antibodies), such as mice, rats or rabbits with the desired specificity, affinity and ability. In some cases, the Fv framework residues of human immunoglobulins are replaced by corresponding non-human residues. Humanized antibodies may also contain residues that are neither present in the receptor antibody nor in the imported CDR or framework sequences. In general, humanized antibodies will contain substantially all of at least one (and usually two) variable domains, wherein all or substantially all of the CDR regions correspond to the CDR regions of non-human immunoglobulins, and all or substantially all of the FR regions are FR regions of human immunoglobulin consensus sequences. In the best case, humanized antibodies will also contain at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a human immunoglobulin.

[0782] The method of humanizing non-human antibodies is well known in the art. Generally speaking, humanized antibodies have one or more amino acid residues introduced from non-human sources. These non-human amino acid residues are generally referred to as "import" residues, which are usually taken from "import" variable domains. Antibody humanization technology generally involves using recombinant DNA technology to manipulate the DNA sequence of one or more polypeptide chains encoding antibody molecules. Humanization can be implemented by replacing rodent CDR or CDR sequence with corresponding human antibody sequence. Therefore, the humanized form of non-human antibodies (or their fragments) is a chimeric antibody or fragment, wherein substantially less than a complete human variable domain has been replaced by the corresponding sequence from non-human species. In practice, humanized antibodies are usually human antibodies, in which some CDR residues and possibly some FR residues are replaced by residues from similar sites in rodent antibodies.

[0783] Selecting people's light chain and heavy chain variable domains to prepare humanized antibodies is very important for reducing antigenicity. According to the "best fit" method, the variable domain sequences of rodent antibodies are screened with the entire library of known people's variable domain sequences. Then, the human sequence closest to the rodent sequence is used as the human skeleton (FR) of humanized antibodies. Another method uses a specific skeleton derived from the consensus sequence of all human antibodies of a specific light chain or heavy chain subgroup. The same skeleton can be used for several different humanized antibodies.

[0784] More importantly, the antibody is humanized while retaining high affinity and other favorable biological properties to the antigen. In order to achieve this goal, the three-dimensional model of the parental and humanized sequences can be used to prepare humanized antibodies through the analytical process of the parental sequence and various conceptual humanized products. The three-dimensional immunoglobulin model is generally available and familiar to those skilled in the art. Computer programs can be used to illustrate and display the possible three-dimensional conformational structure of the selected candidate immunoglobulin sequence. By checking the displayed content, the effect that the residue may play in the function of the candidate immunoglobulin sequence can be analyzed, that is, the residues that affect the candidate immunoglobulin and its antigen binding ability can be analyzed. In this way, FR residues can be selected from the common and imported sequences and combined to obtain the desired antibody characteristics, such as an increase in affinity to the target antigen. In general, the CDR residues directly and most of them actually affect antigen binding.

[0785] The antibody may be conjugated to a substrate or labeled with a detectable moiety, or both. Detectable moieties contemplated by the present compositions include fluorescent labels, enzyme labels, and radioactive labels.

[0786] ii. Single-chain antibody

[0787] Methods for producing single chain antibodies are well known to those skilled in the art. Single chain antibodies are created by fusing the variable domains of the heavy and light chains together using a short peptide linker, thereby reconstructing the antigen binding site on a single molecule. Single chain antibody variable fragments (scFv) have been developed in which the C-terminus of one variable domain is tethered to the N-terminus of the other variable domain by a 15 to 25 amino acid peptide or linker without significantly disrupting antigen binding or specificity of binding. The linker is selected to allow the heavy and light chains to be bound together in their proper conformational orientation. These Fvs lack the constant region (Fc) present in the natural antibody heavy and light chains.

[0788] iii. Monovalent Antibodies

[0789] In vitro methods are also suitable for preparing monovalent antibodies. Antibodies can be digested using conventional techniques known in the art to produce fragments thereof, particularly Fab fragments. For example, papain can be used for digestion. Papain digestion of antibodies usually produces two identical antigen-binding fragments called "Fab fragments", each of which has a single antigen-binding site and a residual Fc fragment. Pepsin treatment produces a fragment called "F(ab')2 fragment", which has two antigen-binding sites and is still able to cross-link with antigen.

[0790] The Fab fragment produced in the antibody digestion also contains the constant domain of the light chain and the first constant domain of the heavy chain. The difference between the Fab' fragment and the Fab fragment is that some residues are added to the carboxyl end of the heavy chain domain, including one or more cysteines from the hinge region of the antibody. The F(ab')2 fragment is a bivalent fragment, comprising two Fab' fragments connected by a disulfide bond in the hinge region. Fab'-SH is the name of Fab' herein, wherein the cysteine ​​residues of the constant domains carry free thiol groups. Antibody fragments are initially generated as paired Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0791] iv. Hybrid Antibodies

[0792] The antibody can be a hybrid antibody. In a hybrid antibody, one heavy chain and light chain pair is homologous to the heavy chain and light chain pair found in an antibody for one epitope, while the other heavy chain and light chain pair is homologous to the heavy chain and light chain pair found in an antibody for another epitope. This results in the property of multifunctional valence, i.e. the ability to simultaneously bind at least two different epitopes. Such hybrids can be formed by fusion of the hybridomas that produce the respective component antibodies or by recombinant technology. Such hybrids can certainly also be formed using chimeric chains.

[0793] v. Conjugates or fusions of antibody fragments

[0794] By coupling the antibody or its fragment to a therapeutic agent, the targeting function of the antibody can be used therapeutically. Such coupling of the antibody or fragment (e.g., at least a portion of an immunoglobulin constant region (Fc)) to the therapeutic agent can be achieved by preparing an immunoconjugate or fusion protein (comprising the antibody or antibody fragment and the therapeutic agent).

[0795] Such conjugation of the antibody or fragment to the therapeutic agent can be achieved by preparing an immunoconjugate or fusion protein, or by linking the antibody or fragment to a nucleic acid such as siRNA, comprising the antibody or antibody fragment and the therapeutic agent.

[0796] In some embodiments, the antibody is modified to alter its half-life. In some embodiments, it may be desirable to increase the half-life of the antibody so that it remains in the circulation or at the site of treatment for a longer period of time. For example, it may be desirable to maintain the titer of the antibody in the circulation or at the site of treatment for a longer period of time. Antibodies may be engineered using Fc variants that extend their half-life, for example, using Xtend TMAntibody half-life extension technology (Xencor, Monrovia, California). In other embodiments, the half-life of the anti-DNA antibody is reduced to reduce potential side effects. The disclosed conjugates can be used to modify a given biological response. The drug moiety should not be construed as limited to traditional chemotherapeutic agents. For example, the drug moiety can be a protein or polypeptide having a desired biological activity. For example, such proteins can include toxins such as abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin.

[0797] vi. Exemplary B7H4 Antibodies

[0798] Exemplary B7H4 antibodies or antigen-binding fragments are disclosed herein. The antibody may include one or more heavy chains and one or more light chains of mouse anti-human B7-H4 antibody B1H1 or B1H10. In some embodiments, the B7H45 antibody includes part or all of the light chain CDRs, the entire light chain variable region, part or all of the heavy chain CDRs, the entire heavy chain variable region, or a combination thereof of any mouse anti-human B7H4 antibody B1H1 or B1H10. Exemplary combinations are disclosed below.

[0799] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:24 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:38.

[0800] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:24 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:39.

[0801] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:24 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:40.

[0802] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:24 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:41.

[0803] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:24 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:42.

[0804] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:24 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:43.

[0805] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:24 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:44.

[0806] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:24 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:45.

[0807] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:25 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:38.

[0808] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:25 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:39.

[0809] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:25 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:40.

[0810] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:25 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:41.

[0811] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:25 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:42.

[0812] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:25 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:43.

[0813] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:25 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:44.

[0814] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:25 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:45.

[0815] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:26 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:38.

[0816] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:26 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:39.

[0817] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:26 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:40.

[0818] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:26 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:41.

[0819] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:26 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:42.

[0820] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:26 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:43.

[0821] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:26 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:44.

[0822] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:26 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:45.

[0823] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:27 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:38.

[0824] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:27 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:39.

[0825] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:27 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:40.

[0826] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:27 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:41.

[0827] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:27 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:42.

[0828] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:27 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:43.

[0829] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:27 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:44.

[0830] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:27 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:45.

[0831] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:28 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:38.

[0832] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:28 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:39.

[0833] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:28 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:40.

[0834] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:28 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:41.

[0835] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:28 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:42.

[0836] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:28 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:43.

[0837] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:28 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:44.

[0838] An anti-B7H4 antibody or an antigen-binding fragment thereof has a light chain with an amino acid sequence as set forth in SEQ ID NO:28 and a heavy chain with an amino acid sequence as set forth in SEQ ID NO:45.

[0839] 2. Proteins and peptides

[0840] a. Protein and Peptide Compositions

[0841] The immunomodulator can be a protein, a polypeptide or a fusion protein. For example, the immunomodulator can be an isolated or recombinant protein or polypeptide of B7-H4, or a functional fragment, variant or fusion protein thereof.

[0842] The protein or polypeptide, or a functional fragment, variant or fusion protein thereof can be an agonist or antagonist. For example, in some embodiments, an antagonist of B7-H4 is a B7-H4 polypeptide or a fragment or fusion protein thereof that binds to a ligand of B7-H4. The polypeptide can be a soluble fragment, such as an extracellular domain of B7-H4, or a functional fragment thereof, or a fusion protein thereof. In some embodiments, a soluble ligand of B7-H4 can be used as an agonist to enhance signal transduction through B7-H4.

[0843] The activity (i.e., agonist or antagonist) of a protein or polypeptide of B7-H4, or any fragment, variant, or fusion protein thereof, can be determined using functional assays known in the art, including the following assays. Typically, the assay comprises determining whether the protein, polypeptide, or fragment, variant, or fusion protein thereof enhances (i.e., agonist) or attenuates (i.e., antagonist) signaling through a B7-H4 receptor. In some embodiments, the assay comprises determining whether the protein, polypeptide, or fragment, variant, or fusion protein thereof enhances (i.e., agonist) or attenuates (i.e., antagonist) an immune response associated with B7-H4. Typically, the assay comprises determining whether the protein, polypeptide, or fragment, variant, or fusion protein thereof enhances (i.e., agonist) or attenuates (i.e., antagonist) signaling through B7-H4. In some embodiments, the assay comprises determining whether the protein, polypeptide, or fragment, variant, or fusion protein thereof attenuates (i.e., agonist) or enhances (i.e., antagonist) an immune response negatively regulated by B7-H4. In some embodiments, the assay comprises determining whether the protein, polypeptide, or fragment, variant, or fusion protein thereof enhances (i.e., antagonists) apoptosis and differentiation of acute myeloid leukemia cells and acute lymphoblastic leukemia cells, thereby resulting in a reduction in the self-renewal capacity of AML and ALL stem cells.

[0844] The nucleic acid and polypeptide sequences of B7-H4 are known in the art, and exemplary protein and peptide sequences are provided above. As discussed in more detail below, one skilled in the art can use the sequences to prepare any protein or polypeptide of B7-H4, or any fragment, variant or fusion protein thereof. Typically, the protein, polypeptide, or fragment, variant and fusion of B7-H4 are expressed by a nucleic acid, and such nucleic acid comprises a sequence encoding a signal sequence. The signal sequence is typically cleaved from an immature polypeptide to produce a mature polypeptide lacking the signal sequence. By standard molecular biology techniques, the signal sequence can be replaced with the signal sequence of another polypeptide to affect the expression level, secretion, solubility or other properties of the polypeptide B7-H4 protein (with and without a signal sequence). It should be understood that in some cases, a mature protein, i.e., a protein sequence without a signal sequence, as known or described in the art, is a recognized mature protein. During normal cell expression, the signal sequence can be removed by a cellular peptidase to produce a mature protein. The sequence of the mature protein can be determined or confirmed using methods known in the art.

[0845] i. Fragment

[0846] As used herein, a B7-H4 fragment refers to any portion of a polypeptide that is at least one amino acid shorter than the full-length protein. Useful fragments include those that retain the ability to bind to one or more of its natural ligands. The ability of a polypeptide that is any full-length B7-H4 fragment to bind to its natural ligand is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 100% or even greater than 100% compared to the full-length protein.

[0847] B7-H4 fragments include cell-free fragments. Cell-free polypeptides can be fragments of full-length transmembrane polypeptides that can be shed, secreted or otherwise extracted from production cells. Cell-free fragments of polypeptides can include part or all of the extracellular domain of the polypeptide, while lacking part or all of the intracellular and / or transmembrane domains of the full-length protein. In one embodiment, the polypeptide fragment includes the entire extracellular domain of the full-length protein. In other embodiments, the cell-free fragment of the polypeptide includes a fragment of the extracellular domain that retains the biological activity of the full-length protein. The extracellular domain can include 1, 2, 3, 4 or 5 adjacent amino acids from the transmembrane domain, and / or 1, 2, 3, 4 or 5 adjacent amino acids from the signal sequence. Alternatively, the extracellular domain can have 1, 2, 3, 4, 5 or more amino acids removed from the C-terminus, the N-terminus or both. In some embodiments, the extracellular domain is the only functional domain (e.g., a ligand binding domain) of the fragment.

[0848] ii. Variants

[0849] Variants of B7-H4 and fragments thereof are also provided. In some embodiments, the variant is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 1. Useful variants include variants that increase biological activity, or variants that increase protein half-life or stability, as shown in any of the assays described herein. B7-H4 proteins and polypeptides, and fragments, variants and fusion proteins thereof, can be engineered to increase biological activity. For example, in some embodiments, the B7-H4 polypeptide, protein, or fragment, variant or fusion thereof has been modified to have at least one amino acid substitution, deletion or insertion that can enhance its function.

[0850] Finally, the variant polypeptide can be engineered to increase its half-life relative to the wild type. These variants are often modified to resist enzymatic degradation. Exemplary modifications include amino acid residue modifications and peptide bond modifications that resist enzymatic degradation. Various modifications to achieve this purpose are known in the art. The variant can be modified to adjust the effect of receptor affinity on the half-life of its protein, polypeptide, fragment or fusion at serum and endosomal pH.

[0851] iii. Fusion protein

[0852] The fusion polypeptide has a first fusion partner, including all or part of a polypeptide B7-H4 fused directly to a second polypeptide or fused to a second polypeptide through a connecting peptide sequence fused to the second polypeptide. The fusion protein optionally contains a domain, the function of which is to dimerize or polymerize two or more fusion proteins. The peptide / polypeptide joint domain can be a separate domain, or can be included in one of the other domains (the first polypeptide or the second polypeptide) of the fusion protein. Similarly, the domain whose function is to dimerize or polymerize the fusion protein can be a separate domain, or can be included in one of the other domains (the first polypeptide, the second polypeptide or the peptide / polypeptide joint domain) of the fusion protein. In one embodiment, the dimerization / multimerization domain and the peptide / polypeptide joint domain are identical.

[0853] The fusion protein disclosed herein has a molecular structural formula I:

[0854] N-R1-R2-R3-C

[0855] Wherein, "N" represents the N-terminus of the fusion protein, and "C" represents the C-terminus of the fusion protein. In some embodiments, "Ri" 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 can be a polypeptide or protein of B7-H4 or a fragment or variant thereof, and Ri can be a second polypeptide. In some embodiments, the B7-H4 polypeptide is an extracellular domain or a fragment thereof, such as an Ig-like C2 domain, or a region composed of cysteines that form disulfide bonds as described above.

[0856] Dimerization or multimerization can occur between or among two or more fusion proteins through the dimerization or multimerization domain. Alternatively, dimerization or multimerization of the fusion protein can occur by chemical cross-linking. The dimer or multimer formed can be a homodimer / homomultimer or a heterodimer / heteromultimer.

[0857] In some embodiments, the fusion protein includes the extracellular domain of B7-H4 or a fragment or variant thereof fused to an Ig Fc region. As previously described, recombinant Ig fusion proteins can be prepared by fusing the coding region of the extracellular domain of the extracellular domain or a fragment or variant thereof to the Fc region of human IgG1, IgG2, IgG3 or IgG4 or mouse IgG2a or other suitable Ig domains (Chapoval, et al., Methods Mol. Med., 45: 247-255 (2000)).

[0858] iv. Peptide modification

[0859] The polypeptides and fusion proteins can be modified by chemical moieties that may be present in the polypeptides in a normal cell environment, such as phosphorylation, methylation, amidation, sulfation, acylation, glycosylation, ubiquitination, and ubiquitination. Fusion proteins can also be modified with markers that can directly or indirectly provide a detectable signal, including but not limited to radioisotopes and fluorescent compounds.

[0860] The polypeptides and fusion proteins can also be modified by chemical moieties that are not normally added to polypeptides in a cellular environment. For example, the disclosed fusion proteins can also be modified by covalent attachment of polymer chains, including but not limited to polyethylene glycol polymer (PEG) chains (i.e., PEGylation). Macromolecules combined with PEG have recently become an effective strategy to change the pharmacokinetic (PK) characteristics of various drugs to improve their therapeutic potential. PEG coupling increases the retention of drugs in the circulation by preventing enzymatic digestion, slowing renal filtration, and reducing the generation of neutralizing antibodies. In addition, PEG conjugates can be used for the multimerization of the fusion protein.

[0861] Modifications can be introduced into the molecule by reacting targeted amino acid residues of the polypeptide with an organic derivatizing agent capable of reacting with selected side chains or terminal residues.Another modification is to cyclize the protein.

[0862] Examples of chemical derivatives of the polypeptide include lysyl and amino terminal residues derivatized with succinic anhydride or other carboxylic anhydrides. Derivatization with cyclic carboxylic anhydrides has the effect of reversing the charge of the lysyl residues. Other suitable reagents for derivatizing amino-containing residues include imino esters, such as methyl pyridoxal imine; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzene sulfonic acid; O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed glyoxylate reactions. Carboxyl side groups, aspartyl or glutamyl groups can be selectively modified by reaction with a carbodiimide (R—N═C═N—R′), such as 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. In addition, aspartyl and glutamyl residues can be converted to asparaginyl and glutaminyl residues by reaction with ammonia. The fusion protein may also include one or more D-amino acids in place of one or more L-amino acids.

[0863] v. Modified binding properties

[0864] The binding properties of the proteins, polypeptides, or fragments, variants and fusions thereof are related to the dose and dosing regimen to be administered. In one embodiment, the disclosed proteins, polypeptides, or fragments, variants and fusions thereof have the property of binding to the B7-H4 ligand, occupying the binding site (e.g., on the ligand) for a longer time or a higher percentage of occupancy relative to other receptor molecules bound thereto. In other embodiments, the disclosed proteins, polypeptides, or fragments, variants and fusions thereof have decreased binding affinity for the B7-H4 ligand relative to the wild-type protein.

[0865] In some embodiments, the protein, polypeptide, or fragment, variant, and fusion thereof has a relatively high affinity for the B7-H4 ligand and may therefore have a relatively slow dissociation rate. In other embodiments, the protein, polypeptide, or fragment, variant, and fusion thereof is administered intermittently over a period of days, weeks, or months to attenuate the immune response and allow the immune response to recover before the next administration, which can be used to alter the immune response without completely turning the immune response on or off, thereby avoiding long-term side effects.

[0866] 3. Isolated Nucleic Acid Molecules

[0867] Disclosed herein are nucleic acid sequences encoding the separation of the protein, polypeptide, or fragments, variants, and fusions thereof. As used herein, "isolated nucleic acid" refers to nucleic acids separated from other nucleic acid molecules present in a mammalian genome, including nucleic acids that are typically located on one or both sides of a nucleic acid in a mammalian genome. As used herein, the term "isolated" with respect to nucleic acids also includes combinations with any non-naturally produced nucleic acid sequences, because such non-naturally produced sequences do not exist in nature, and there are no closely adjacent sequences in the naturally produced genome.

[0868] For example, an isolated nucleic acid can be a DNA molecule, provided that one of the nucleic acid sequences that is normally adjacent to the DNA molecule in a naturally occurring genome has been removed or is absent. Thus, 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 a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, a lentivirus, an adenovirus, or a herpes virus), or the genomic DNA of a prokaryotic or eukaryotic organism. In addition, an isolated nucleic acid can include an engineered nucleic acid, such as a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid. Nucleic acids that are present among hundreds to millions of other nucleic acids in, for example, a cDNA library or a genomic library, or a gel slice containing a restriction digest of genomic DNA are not considered isolated nucleic acids.

[0869] The nucleic acid encoding the protein, polypeptide, or its fragment, variant, and fusion can be optimized to express in the selected expression host. Codons can be replaced with alternative codons encoding the same amino acids to account for differences in codon usage between the mammal producing the nucleic acid sequence and the expression host. In this way, the nucleic acid can be synthesized using codons preferred by the expression host.

[0870] Nucleic acid can be in sense or antisense direction, or can be complementary to a reference sequence of a polypeptide or protein encoding B7-H4. Nucleic acid can be DNA, RNA or nucleic acid analogs. Nucleic acid analogs can be modified at the base moiety, sugar moiety or phosphate backbone. Such modification can improve, for example, the stability, hybridization or solubility of the nucleic acid. The base moiety modification can include deoxyuridine (for deoxythymidine), and 5-methyl-2'-deoxycytidine or 5-bromo-2'-deoxycytidine (for deoxycytidine). The sugar moiety modification can include modification of the 2' hydroxyl of the ribose to form 2'-O-methyl sugar or 2'-O-allyl sugar. The deoxyribose phosphate backbone can be modified to generate a morpholino nucleic acid, wherein each base moiety is connected to a six-membered morpholino ring, or to generate a peptide nucleic acid, wherein the deoxyphosphate backbone is replaced by a pseudopeptide backbone and four bases are retained. See, for example, Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev. 7:187-195; and Hyrup et al. (1996) Bioorgan. Med. Chem. 4:5-23. In addition, the deoxyphosphate backbone can be replaced by, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoramidite, or an alkylphosphotriester backbone.

[0871] Nucleic acids encoding polypeptides can be administered to subjects in need thereof. Administration of nucleic acids involves the introduction of "exogenous" nucleic acids into cells and ultimately into living animals. Compositions and methods for administering nucleic acids to subjects are known in the art (see Understanding Gene Therapy, Lemoine, NR, ed., BIOS Scientific Publishers, Oxford, 2008).

[0872] 4. Vectors and Host Cells

[0873] Also provided are vectors encoding the proteins, polypeptides, or fragments, variants and fusions thereof. A nucleic acid as described above can be inserted into a vector to be expressed in a cell. As used herein, a "vector" is a replicon, such as a plasmid, phage, virus or cosmid, into which another DNA segment can be inserted to cause replication of the inserted segment. A vector can be an expression vector. An "expression vector" is a vector comprising one or more expression control sequences, which are DNA sequences that control and regulate the transcription and / or translation of another DNA sequence.

[0874] The nucleic acid in the vector can be operably connected to one or more expression control sequences. As used herein, "operably connected" refers to being incorporated into a genetic construct so that the expression control sequence can effectively control the expression of the target coding sequence. Examples of expression control sequences include promoters, enhancers, and transcription termination regions. A promoter is an expression control sequence consisting of a region of a DNA molecule, usually within 100 nucleotides upstream of the transcription start point (usually near the start site of RNA polymerase II). In order for the promoter to control the coding sequence, the translation start site of the translation reading frame of the polypeptide must be positioned between one to about fifty nucleotides downstream of the promoter. In terms of time, position, and level, enhancers have expression specificity. Unlike promoters, enhancers can function at different distances from the transcription site. Enhancers can also be located downstream of the transcription start site. A coding sequence can be "operably connected" and "controlled by" an expression control sequence in a cell, but the condition is that RNA polymerase is able to transcribe the coding sequence into mRNA, which can then be translated into a protein encoded by the coding sequence.

[0875] Suitable expression vectors include, but are not limited to, plasmids and viral vectors derived from, for example, bacteriophage, baculovirus, tobacco mosaic virus, herpes virus, cytomegalovirus, retrovirus, vaccinia virus, adenovirus, and adeno-associated virus, etc. Many vectors and expression systems are commercially available from companies such as Novagen (Madison, Wisconsin), Clontech (Palo Alto, California), Stratagene (La Jolla, California), and Invitrogen Life Technologies (Carlsbad, California).

[0876] The expression vector may include a tag sequence. The tag sequence is typically expressed as a fusion with the encoded polypeptide. Such tags may be inserted anywhere within the polypeptide, including at the carboxyl or amino terminus. Examples of useful tags include, but are not limited to, green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, Flag TM Tag (Kodak, New Haven, Connecticut), 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 an Ig heavy chain constant region, e.g., a hinge region, a Cyl chain corresponding to a human immunoglobulin. H Zone 2 and C H Amino acid sequence of region 3.

[0877] The vector containing the nucleic acid to be expressed can be transferred into a host cell. The term "host cell" is intended to include prokaryotic cells and eukaryotic cells, wherein a recombinant expression vector can be introduced. As used herein, "conversion" and "transfection" include introducing a nucleic acid molecule (e.g., a vector) into a cell by one of a variety of techniques. Although not limited to a particular technique, there are many very mature techniques in this area. Prokaryotic cells can be transformed with nucleic acids by techniques such as electroporation or calcium chloride-mediated transformation. Nucleic acids can be transfected into mammalian cells by techniques such as 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 generate proteins, polypeptides, or fragments, variants and fusions described herein.

[0878] The vector can be used to express the protein, polypeptide, or its fragment, variant and fusion in the cell. Exemplary vectors include but are not limited to adenoviral vectors. One method includes transferring the nucleic acid to primary cells in culture, and then autotransplanting the transformed cells in vitro into the host, which can be systemically transplanted or transplanted into a specific organ or tissue. The in vitro method can include, for example, the following steps: obtaining cells from the subject, culturing the cells, transducing the cells with an expression vector, and maintaining the cells under conditions suitable for expressing the encoded polypeptide. These methods are known in the field of molecular biology. The transduction step can be completed by any standard method for in vitro gene therapy, including, for example, calcium phosphate, lipofection, electroporation, viral infection and gene gun gene transfer. Alternatively, liposomes or polymer particles can be used. Cells that have been successfully transduced can then be selected, for example, for expressing the coding sequence or drug resistance gene. The cells can then be irradiated to a lethal dose (if necessary) and then injected or implanted into the subject. In one embodiment, an expression vector containing a nucleic acid encoding a fusion protein is transfected into a cell, and the cell is administered to a subject in need thereof.

[0879] In vivo nucleic acid therapy can be achieved by directly transferring functionally active DNA into mammalian somatic tissues or organs in vivo. For example, the nucleic acid encoding the polypeptide disclosed herein can be directly administered to lymphoid tissue. Alternatively, lymphoid tissue-specific targeting can be achieved using lymphoid tissue-specific transcription regulators (TREs), such as B lymphocytes, T lymphocytes, or dendritic cell-specific TREs. Lymphoid tissue-specific TREs are known in the art.

[0880] Nucleic acids can also be administered in vivo by viral means. As is well known in the art, nucleic acid molecules encoding fusion proteins can be packaged into retroviral vectors by using packaging cell lines that produce replication-defective retroviruses. Other viral vectors can also be used, including recombinant adenoviruses and vaccinia viruses that may not replicate. In addition to naked DNA or RNA or viral vectors, engineered bacteria can also be used as vectors.

[0881] Nucleic acids can also be administered via other carriers, including liposomes, polymeric microparticles and nanoparticles, and polycations, such as asialoglycoprotein / polylysine, and the like.

[0882] In addition to viral and vector-mediated gene transfer in vivo, physical means well known in the art can also be used to directly transfer DNA, including administration of plasmid DNA and particle bombardment-mediated gene transfer.

[0883] 5. Small molecules

[0884] The immunomodulator can be a small molecule. Small molecule agonists and antagonists of B7-H4 are known in the art or can be identified using routine screening methods.

[0885] In some embodiments, the screening assays may include random screening of large libraries of test compounds. Alternatively, the assays may be used to focus on determining specific classes of compounds suspected of modulating B7-H4 levels. Assays may include assays for B7-H4 signaling activity or inhibitory responses mediated by B7-H4. Other assays may include assays for nucleic acid transcription or translation, mRNA levels, mRNA stability, mRNA degradation, transcription rates, and translation rates.

[0886] C. Pharmaceutical Compositions

[0887] Pharmaceutical compositions comprising the disclosed immunomodulators are provided. Pharmaceutical compositions containing the immunomodulators can be administered parenterally (intramuscularly, intraperitoneally, intravenously (IV), or subcutaneously), transdermally (passively or using iontophoresis or electroporation), or transmucosally (nasal, vaginal, rectal, or sublingual) or using bioerodible inserts, and can be formulated to suit each route of administration.

[0888] In some in vivo methods, the compositions disclosed herein are administered to a subject in a therapeutically effective amount. As used herein, the term "effective amount" or "therapeutically effective amount" refers to a dose sufficient to treat, suppress or alleviate one or more symptoms of the disease being treated or otherwise provide a desired pharmacological and / or physiological effect. The exact dose will vary according to a variety of factors, such as the variables (e.g., age, immune system health, etc.) of the subject, the disease, and ongoing treatment.

[0889] For the disclosed immunomodulators, as further research is conducted, information about appropriate dosage levels for treating various diseases in different patients will emerge, and the ordinary technician will consider the treatment background, age and overall health of the recipient to determine the appropriate dosage. The selected dosage depends on the desired therapeutic effect, the route of administration and the desired duration of treatment. For the disclosed immunomodulators, a dosage level of 0.001 to 20 mg / kg body weight / day is generally administered to mammals. In general, for intravenous injection or infusion, the dosage may be lower.

[0890] In certain embodiments, the immunomodulator is administered topically, for example by direct injection into the area to be treated. Typically, the injection results in an increase in the local concentration of the immunomodulator composition, which is higher than that achievable by systemic administration. As described above, the immunomodulator composition can be used in conjunction with a matrix to help increase the local concentration of the polypeptide composition by reducing passive diffusion of the polypeptide beyond the area to be treated.

[0891] 1. Preparations for parenteral administration

[0892] In some embodiments, the compositions disclosed herein, including compositions containing peptides and polypeptides, are administered in the form of aqueous solutions by parenteral injection. The preparation can also be in the form of a suspension or emulsion. A pharmaceutical composition is generally provided, including an effective amount of peptides or polypeptides, and optionally including a pharmaceutically acceptable diluent, preservative, solubilizer, emulsifier, adjuvant and / or carrier. Such compositions optionally include one or more of the following: diluent, sterile water, buffered saline with various buffer contents (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; and additives, such as detergents and solubilizers (e.g., Tween 20 (polysorbate-20), Tween 80 (polysorbate-80)), antioxidants (e.g., ascorbic acid, sodium pyrosulfite) and preservatives (e.g., Thimersol, benzyl alcohol) and fillers (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles include propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil and corn oil), gelatin, and injectable organic esters (e.g., ethyl oleate). The formulation may be lyophilized and can be re-dissolved / resuspended prior to use. The formulation may be sterilized, for example, by filtering through a bacteria-retaining filter, incorporating a sterilizing agent into the composition, irradiating the composition, or heating the composition.

[0893] 2. Oral administration preparations

[0894] In an embodiment, the composition is formulated to be suitable for oral administration. For a general description of oral solid dosage forms, see Chapter 89 of Remington's Pharmaceutical Sciences, 18th edition, 1990 (Mack Publishing Co. Easton Pa. 18042). Solid dosage forms include tablets, capsules, pills, lozenges, cachets, pellets, powders or granules, or the material is integrated into a microparticle preparation of a polymer (such as polylactic acid, polyglycolic acid, etc.), or into a liposome. Such compositions may affect the physical state, stability, in vivo release rate and in vivo clearance rate of the disclosed substance. See, for example, Remington's Pharmaceutical Sciences, 18th edition (1990, Mack Publishing Co., Easton, Pa. 18042), pages 1435-1712, which are incorporated herein by reference. The composition can be prepared in liquid form, or in dry powder (e.g., lyophilized) form. Liposomes or protein-like encapsulation can be used to prepare the composition. Liposome encapsulation can be used, and liposomes can be derivatized with a variety of different polymers (e.g., U.S. Pat. No. 5,013,556). See also Marshall, K. In: Modem Pharmaceutics Edited by GS Banker and CT Rhodes Chapter 10, 1979. Typically, the formulation will include the peptide (or its chemically modified form) and an inert ingredient that protects the peptide in the gastric environment and releases the biologically active substance in the intestine.

[0895] The agent may be chemically modified to make the oral administration of the derivative effective. Generally, the chemical modification contemplated is the attachment of at least one moiety to the component molecule itself, wherein the moiety can be absorbed from the stomach or intestine into the bloodstream, or directly into the intestinal mucosa. It is also desirable to increase the overall stability of one or more of the components and increase the in vivo circulation time. Polyethylene glycolation (PEGylation) is a typical chemical modification for pharmaceutical use. Other moieties that may be used include propylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, polyproline, poly-1,3-dioxolane and poly-1,3,6-trioxocane (see, e.g., 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).

[0896] Another embodiment provides liquid dosage forms for oral administration, including pharmaceutically acceptable emulsions, solutions, suspensions and syrups, which may contain other components, including inert diluents; adjuvants (such as wetting agents, emulsifiers and suspending agents, etc.); and sweeteners, flavoring agents and fragrances.

[0897] Controlled release oral formulations may be an ideal dosage form. The agent may be incorporated into an inert matrix that allows release of the agent by diffusion or leaching mechanisms (e.g., gums). Slowly degrading matrices may also be incorporated into the formulation. Another form of controlled release is based on the oral osmotic pump (OROS) therapeutic system (Alza Corp.), that is, the drug is encapsulated in a semipermeable membrane that allows water to enter and push the drug out through a small opening due to osmotic action.

[0898] For oral formulations, the site of drug release may be the stomach, small intestine (duodenum, jejunum or ileum) or large intestine. In some embodiments, the release of the agent can avoid the harmful effects of the gastric environment by protecting the agent (or derivative) or by releasing the agent (or derivative) outside the gastric environment (e.g., in the intestine, etc.). In order to ensure complete resistance to gastric juice, an agent coating that is impermeable at least under pH 5.0 conditions is essential. Examples of more common inert ingredients used as enteric coatings are: cellulose acetate trimellitate (CAT), hydroxypropyl methylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D TM 、Aquateric TM 、Cellulose acetate phthalate (CAP), Eudragit L TM 、Eudragit S TM and Shellac TM These coatings can be used as hybrid films.

[0899] 3. Topical drug delivery preparations

[0900] The disclosed immunomodulators can be administered topically. Although topical administration to the lungs, nose, oral (sublingual, buccal), vaginal or rectal mucosa is particularly effective, for most peptide preparations, topical administration is not effective.

[0901] When the composition is delivered in the form of aerosol or spray-dried particles having an aerodynamic diameter of less than about 5 μm, the composition can be delivered to the lungs and traverse the lung epithelial lining to the bloodstream upon inhalation.

[0902] A variety of mechanical devices designed for pulmonary administration of therapeutic products may be used, including but not limited to nebulizers, metered dose inhalers, and dry powder inhalers, all of which are familiar to those skilled in the art. Some specific examples of commercially available devices are: Ultravent nebulizer (Mallinckrodt Inc., St. Louis, Missouri); Acorn II nebulizer (Marquest Medical Product, Englewood, Colorado); Ventolin metered dose inhaler (Glaxo Inc., Research Triangle Park, North Carolina); and Spinhaler dry powder inhaler (Fisons Corp., Bedford, Massachusetts). Nektar, Alkermes, and Mannkind have all been approved for use in inhalable insulin powder formulations or are in clinical trials applying this technology to the formulations described herein.

[0903] Transmucosal formulations are usually spray-dried drug particles that can be incorporated into tablets, gels, capsules, suspensions, or emulsions. Standard pharmaceutical excipients are available from any formulator.

[0904] Transdermal preparations can also be prepared. These transdermal preparations are typically ointments, lotions, sprays or patches, all of which can be prepared using standard techniques. Transdermal preparations may require the addition of a penetration enhancer.

[0905] 4. Controlled Release Polymer Matrix

[0906] The immunomodulators disclosed herein can also be administered in the form of controlled release formulations. Controlled release polymer devices can be manufactured to release the drug systemically for a long time after implantation of the polymer device (rods, cylinders, films, discs) or injection (microparticles). The matrix can be in the form of microparticles such as microspheres, wherein the immunomodulator is dispersed in a solid polymer matrix or microcapsules, and the microparticle core is made of a different material from the polymer shell, and the peptide is dispersed or suspended in the microparticle core, which can be liquid or solid in nature. Unless specifically defined herein, microparticles, microspheres and microcapsules are used interchangeably. In addition, the polymer can be cast into thin sheets or films ranging from nanometers to four centimeters, or made into dry powders by grinding or other standard techniques, or even made into gels, such as hydrogels.

[0907] Either non-biodegradable or biodegradable matrices can be used to deliver fusion polypeptides or nucleic acids encoding the fusion polypeptides, but in some embodiments biodegradable matrices are preferred. These matrices can be natural polymers or synthetic polymers, but in some embodiments synthetic polymers are preferred due to better degradation and release characteristics. The polymer can be selected based on the desired release cycle. In some cases, linear release may be most useful, but in other cases, pulse release or "batch release" may provide more effective results. The polymer can be in the form of a hydrogel (usually absorbing up to about 90% water (by weight)) and optionally cross-linked with multivalent ions or polymers.

[0908] The matrix can be prepared by solvent evaporation, spray drying, solvent extraction, and other methods known to those skilled in the art. Bioerodible microspheres can be prepared by any method developed for making drug delivery microspheres, for example, as 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).

[0909] The devices can be formulated for local release of drugs to treat the site of implantation or injection, delivering doses that are generally much smaller than those for systemic treatment or systemic administration. These devices can be implanted subcutaneously or injected into muscle and fat or swallowed.

[0910] III. Production Method

[0911] A. Antibody Preparation Methods

[0912] The antibodies can be prepared in cell culture, phage or various animals, including but not limited to cattle, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, and apes. Therefore, in one embodiment, the antibodies are mammalian antibodies. Phage technology can be used to isolate initial antibodies or to prepare variants with variant specificity or affinity characteristics. This technology is routine and well known in the art. In one embodiment, the antibodies are produced by recombinant methods known in the art. For example, a recombinant antibody can be prepared by transfecting a host cell with a vector containing a DNA sequence encoding the antibody. One or more vectors can be used to transfect the DNA sequence expressing at least one VL region and one VH region in the host cell. Exemplary descriptions of recombinant methods for antibody preparation and production include Delves, Antibody Production:Essential Techniques (Wiley, 1997); Shephard, et al. Monoclonal Antibodies (Oxford University Press, 2000); Goding, MonoclonalAntibodies: Principles and Practice (Academic Press, 1993); Current Protocols in Immunology (John Wiley & Sons, latest edition).

[0913] The disclosed antibodies can be modified by recombinant means to improve the effectiveness of the antibodies in mediating the desired function. Therefore, within the scope of the present invention, the antibodies can be modified by substitution using recombinant means. Typically, the substitutions are conservative substitutions. For example, at least one amino acid in the constant region of the antibody can be replaced by a different residue. See, for example, U.S. Pat. No. 5,624,821, U.S. Pat. No. 6,194,551, Application No. WO 9958572; and Angal, et al., Mol. Immunol. 30:105-08 (1993). Amino acid modifications include amino acid deletions, additions, and substitutions. In some cases, such changes can be made to reduce adverse activities, such as complement-dependent cytotoxicity. Typically, the antibodies can be labeled by covalent or non-covalent attachment (a substance that provides a detectable signal). Various labels and binding techniques are known and widely reported in the scientific and patent literature. Antibodies that can bind to B7-H4 proteins, polypeptides, or fusion proteins can be screened. See, for example, Antibody Engineering:A Practical Approach (Oxford University Press, 1996).

[0914] For example, suitable antibodies with the desired biological activity can be identified using in vitro assays, including but not limited to in vivo assays such as proliferation, migration, adhesion, soft agar growth, angiogenesis, cell-to-cell communication, apoptosis, transport, signal transduction, and inhibition of tumor growth. The antibodies provided herein can also be used for diagnostic applications. As capture or non-neutralizing antibodies, they can be screened for their ability to bind to a specific antigen without inhibiting the receptor binding or biological activity of the antigen. As neutralizing antibodies, they can be used in competitive binding assays.

[0915] Antibodies that can be used in the disclosed compositions and methods include any class of complete immunoglobulins (i.e., complete antibodies), fragments thereof, and synthetic proteins containing at least the antigen-binding variable domains of antibodies. The variable domains between different antibodies differ in sequence and are used to achieve specific binding of each specific antibody to its specific antigen. However, variability is usually not evenly distributed in the variable domains of antibodies. It is usually concentrated in three segments called "complementarity determining regions (CDRs)" or "hypervariable regions" in the light chain and heavy chain variable domains. The more conserved parts of the variable domains are called "frameworks (FRs)". The variable domains of natural heavy and light chains each contain four FR regions, mainly in a β-folded configuration, connected by three CDRs, wherein the three CDRs form a loop connecting the β-folded configuration, and in some cases form a part of the β-folded configuration. The CDRs in each chain are tightly bound together by the FR region, and together with the CDRs in the other chain, promote the formation of the antigen binding site of the antibody.

[0916] Also disclosed are antibody fragments having biological activity. These fragments, whether or not linked to other sequences, include insertions, deletions, substitutions or other selected modifications of specific regions or specific amino acid residues, provided that the activity of the fragment is not significantly altered or impaired compared to the unmodified antibody or antibody fragment.

[0917] The technology can also be adapted to produce specific single chain antibodies against antigenic peptides. Methods for producing single chain antibodies are well known to those skilled in the art. Single chain antibodies can be created by fusing the variable domains of the heavy and light chains together using a short peptide linker, thereby reconstructing the antigen binding site on a single molecule. Single chain antibody variable fragments (scFv) have been developed in which the C-terminus of one variable domain is tethered to the N-terminus of the other variable domain by a 15 to 25 amino acid peptide or linker without significantly disrupting antigen binding or specificity of binding. The linker is selected to allow the heavy and light chains to be combined in their proper conformational orientation.

[0918] A divalent single-chain variable fragment (di-scFv) can be engineered by connecting two scFvs. This is done by generating a single peptide chain with two VH regions and two VL regions, resulting in a tandem scFv. ScFv can also be designed with a connecting peptide that is too short for the two variable regions to fold together (about five amino acids), forcing the scFv to dimerize. This type is called a "diabody". Studies have shown that the dissociation constant of diabodies is up to 40 times lower than that of the corresponding scFv, which means that they have a higher affinity for their targets. But shorter linkers (one or two amino acids) still lead to the formation of trimers (tribodies). Tetrabodies have also been produced. Compared to diabodies, they have a higher affinity for their targets.

[0919] Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small fraction of antibody molecules. Monoclonal antibodies include "chimeric" antibodies, in which a portion of the heavy and / or light chains are identical or homologous to corresponding sequences of antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remaining antibody chains are identical or homologous to corresponding sequences of antibodies derived from other species or belonging to other antibody classes or subclasses, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity.

[0920] Monoclonal antibodies can be prepared using any procedure that can produce monoclonal antibodies. In the hybridoma method, mice or other suitable host animals are usually immunized with an immunizing agent to stimulate lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.

[0921] Antibodies can also be prepared by recombinant DNA methods. DNA encoding the disclosed antibodies can be easily separated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding murine antibody heavy and light chains). Antibody or active antibody fragment libraries can also be generated and screened using phage display technology.

[0922] Methods for preparing antibodies using protein chemistry are also known in the art. One method of producing a protein comprising the antibody is to link two or more peptides or polypeptides together using protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using chemicals such as Fmoc (9-fluorenylmethylcarbonyl) or Boc (tert-butyloxycarbonyl) and currently available laboratory equipment. (Applied Biosystems, Inc., Foster City, California). For example, peptides or polypeptides corresponding to the antibody can be synthesized by standard chemical reactions, which can be easily understood by those skilled in the art. For example, a peptide or polypeptide is synthesized but not cleaved from its synthetic resin, while another fragment of the antibody is synthesized and then cleaved from the resin, thereby exposing the functionally blocked end groups on the other fragment. Through a peptide condensation reaction, the two fragments can be covalently linked by peptide bonds at their carboxyl and amino ends, respectively, to form an antibody or fragment thereof. Alternatively, as described above, the peptide or polypeptide is independently synthesized in vivo. Once separated, these independent peptides or polypeptides can be connected by similar peptide condensation reactions to form an antibody or its antigen-binding fragment.

[0923] For example, enzymatic ligation of cloned or synthetic peptides allows the ligation of relatively short peptides to produce larger peptides, polypeptides, or whole protein domains. Alternatively, native chemical ligation of synthetic peptides can be used to synthetically construct large peptides or polypeptides from short peptides. This method involves a two-step chemical reaction. The first step is to chemically react an unprotected synthetic peptide-α-thioester with another unprotected peptide containing an amino-terminal cysteine ​​residue to obtain a thioester ligation intermediate as the initial covalent product. Without changing the reaction conditions, this intermediate will undergo a rapid spontaneous intramolecular reaction to form a native peptide bond at the ligation site.

[0924] B. Protein Production Methods

[0925] The disclosed protein, polypeptide, or its fragment, variant, and fusion can all be produced using conventional techniques known in the art. For example, the isolated fusion protein can be obtained by chemical synthesis or recombinant production in a host cell. For recombinant production of a protein, polypeptide, or its fragment, variant, or fusion, a nucleic acid containing a nucleotide sequence encoding the protein, polypeptide, or its fragment, variant, or fusion can be used to transform, transduce, or transfect bacteria or eukaryotic host cells (e.g., insects, yeast, or mammalian cells). In general, a nucleic acid construct includes a regulatory sequence operably linked to a nucleotide sequence encoding the protein, polypeptide, or its fragment, variant, or fusion. Regulatory sequences (also referred to herein as "expression control sequences") do not usually encode gene products, but affect the expression of the nucleic acid sequences operably linked thereto.

[0926] Efficient prokaryotic and eukaryotic systems for expressing and producing polypeptides are well known in the art and include E. coli strains, such as BL-21, and cultured mammalian cells, such as CHO cells.

[0927] In eukaryotic host cells, many viral vector expression systems can be used to express fusion proteins. Viral vector expression systems are well known in the art and include, but are not limited to, viral vectors based on baculovirus, SV40, retrovirus or vaccinia virus.

[0928] Mammalian cell lines that can stably express proteins, polypeptides, or fragments, variants, or fusions thereof can be produced using expression vectors with appropriate regulatory factors and selection markers. For example, the eukaryotic expression vectors pCR3.1 (Invitrogen Life Technologies) and p91023 (B) (see Wong et al. (1985) Science 228: 810-815) are suitable for expressing proteins, polypeptides, or fragments, variants, or fusions thereof in, for example, Chinese hamster ovary (CHO) cells, COS-1 cells, human embryonic kidney 293 cells, NIH3T3 cells, BHK21 cells, MDCK cells, and human vascular endothelial cells (HUVEC). Other suitable expression systems include the GS Gene Expression System available from Lonza Group Ltd. TM .

[0929] After introduction of the expression vector by electroporation, lipofection, calcium phosphate or calcium chloride coprecipitation, DEAE dextran or other suitable transfection methods, stable cell lines can be selected (e.g., by metabolic selection or antibiotic resistance to G418, kanamycin or hygromycin). The transfected cells can be cultured to express the polypeptide of interest, and the polypeptide can be recovered from, for example, the cell culture supernatant or from lysed cells. In addition, proteins, polypeptides, or fragments, variants or fusions thereof can be produced by (a) ligating the amplified sequence into a mammalian expression vector such as pcDNA3 (Invitrogen Life Technologies), and (b) transcription and translation in vitro using wheat germ extract or rabbit reticulocyte lysate.

[0930] For example, chromatography such as affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, DEAE ion exchange, gel chromatography, and hydroxyapatite chromatography can be used to separate proteins, polypeptides, or fragments, variants, or fusions thereof. In some embodiments, proteins, polypeptides, or fragments, variants, or fusions thereof can be engineered to contain additional domains containing amino acid sequences that can capture the polypeptide to an affinity matrix. For example, a protein A chromatography column can be used to separate Fc fusion polypeptides in cell culture supernatants or cytoplasmic extracts. In addition, c-myc, hemagglutinin, polyhistidine, or Flag can be used to separate Fc fusion polypeptides. TM (Kodak) and other tags support polypeptide purification. Such tags can be inserted anywhere in the polypeptide, including at the carboxyl or amino terminus. Other useful fusions include enzymes that help detect polypeptides, such as alkaline phosphatase. Immunoaffinity chromatography can also be used to purify polypeptides. In addition, the fusion protein can be engineered to contain a secretion signal that can promote the production of cell secreted proteins, polypeptides, fragments, or fragments, variants or fusions thereof (if there is no secretion signal). The secreted protein, polypeptide, or fragment, variant or fusion can then be simply separated from the cell culture medium.

[0931] C. Methods for producing isolated nucleic acid molecules

[0932] Isolated nucleic acid molecules can be produced by standard techniques, including but not limited to ordinary molecular cloning and chemical nucleic acid synthesis techniques. For example, polymerase chain reaction (PCR) technology can be used to obtain isolated nucleic acids encoding variant polypeptides. PCR is a technique for enzymatic amplification of target nucleic acids. Typically, sequence information from the end or outside of the target region can be used to design oligonucleotide primers that have the same sequence as the opposite strand of the template to be amplified. PCR can be used to amplify specific sequences from DNA and RNA, including sequences from total genomic DNA or total cellular RNA. Primers are typically 14 to 40 nucleotides in length, but can range in length from 10 nucleotides to hundreds of nucleotides. For example, in Dieffenbach & Dveksler, PCR Primer Conventional PCR techniques are described in: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1995. When RNA is used as a template source, reverse transcriptase can be used to synthesize a complementary DNA (cDNA) strand. Ligase chain reaction, strand displacement amplification, self-sustaining sequence replication or nucleic acid sequence amplification can also be used to obtain isolated nucleic acids. See, for example, Lewis (1992) Genetic Engineering News 12:1; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878; and Weiss (1991) Science 254:1292-1293.

[0933] Isolated nucleic acids can be synthesized chemically (e.g., automated DNA synthesis in the 3' to 5' direction using the phosphoramidite technique) 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, with each pair of long sequences containing complementary short segments (e.g., about 15 nucleotides) so that duplexes are formed when the oligonucleotide pairs are annealed. DNA polymerase can be used to extend the oligonucleotides so that each oligonucleotide pair produces a single double-stranded nucleic acid molecule, which is then ligated into a vector. Isolated nucleic acids can also be obtained by mutation. Nucleic acids encoding proteins can be mutated using standard techniques, including oligonucleotide-directed mutagenesis and / or site-directed mutagenesis by PCR. See Ausubel et al., Short Protocols in Molecular Biology , Chapter 8, Green Publishing Associates and John Wiley&Sons, 1992.

[0934] IV. Assays and Antibody Screening

[0935] Antibody screening assays include:

[0936] 1. Comparative analysis of the ligand binding affinity of B7-H4-Fc and B7-H4.

[0937] 2. Functional assay to confirm that B7-H4-Fc blocks signaling in B7-H4 expressing cells.

[0938] Reporter cells can be used for these assays, or alternatively primary B7-H4+ cells can be utilized.

[0939] High affinity mAbs against B7-H4 can be generated using proprietary immunization techniques using either B7-H4-deficient ("knockout") mice or wild-type mice. The autoimmunity-prone mouse NZB / WF1 can be used to generate mAbs that overcome "tolerance."

[0940] 1. Phase I Screening: mAbs that bind to cell lines transfected to express cell surface B7-H4. In addition, mAbs should have the ability to bind to B7-H4 endogenously expressed on the surface of a subset of primary human cells. These mAbs should be highly specific for B7-H4. Antibodies can be screened using an ELISA with purified B7-H4 protein to detect anti-B7-H4 antibodies.

[0941] 2. Phase II screening: B7-H4-specific mAbs should be able to block the binding of B7-H4 to its ligands and / or target cells.

[0942] 3. Phase III Screening: Functional assays to confirm that B7-H4 mAbs or mAb combinations modulate B7-H4 signaling. These assays will utilize cell lines or primary cells that express endogenous B7-H4, such as human monocytes, macrophages, and dendritic cell subsets, to assess function in the presence of B7-H4 mAbs. In addition, reporter cells can be used to determine whether signaling pathways (e.g., NFkB (NFkB reporter) or NFAT (NFAT reporter)) are altered after incubation with B7-H4 mAbs.

[0943] 4. Phase IV Screening: Functional assays to determine whether B7-H4 mAbs are able to induce antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), or apoptosis of B7-H4-expressing cell lines by other mechanisms. In particular, the ability of B7-H4 mAbs to deplete leukemia cell lines (known to express B7-H4 on the cell surface) is tested by one of these methods. B7-H4 mAbs can also be engineered to deplete B7-H4-expressing cells and tested by known methods as described later herein.

[0944] 5. Phase V Screening: Functional assays to determine whether B7-H4 mAbs are able to transmit or induce negative signals (agonists) through B7-H4 into B7-H4 expressing cells to inhibit cell function. Cell lines or cell line transfectants that endogenously express B7-H4 will be evaluated for changes in phenotype and viability after culture with B7-H4 mAbs. In other assays, reporter cell lines will be used to determine modulation of positive signaling pathways in B7-H4 mAbs, such as NF-kB (NF-kB reporter gene) or other known cell signaling reporter genes. Apoptosis induction in cell lines will also be evaluated.

[0945] Phase II and III assays can be used to predict the concentration of B7-H4 mAb required to block physiological levels of ligand in vivo.

[0946] V. How to use

[0947] B7-H4 antagonists or agonists can be used to modulate the immune response in a subject in need of such treatment.

[0948] Exemplary methods are discussed in detail below.

[0949] A. Stimulation of immune response

[0950] 1. Treatment strategy

[0951] A method for inducing or enhancing an immune response in a subject is provided. In general, the method includes administering an effective amount of an immunomodulator to a subject, or using the immunomodulator to activate immune cells in vitro. For example, the immune response may be a primary immune response to an antigen or enhance effector cell function, such as increasing the antigen-specific proliferation of T cells, enhancing the production of cytokines caused by T cells, stimulating differentiation, or a combination thereof. In some embodiments, the immunomodulator may increase the development of Th1, Th17, Th22 or other cells that secrete inflammatory molecules or promote other cells to secrete inflammatory molecules, and the inflammatory molecules include but are not limited to IL-1β, TNF-α, TGF-β, IFN-γ, IL-17, IL-6, IL-23, IL-22, IL-21 and MMP. In some embodiments, the immunomodulator may reduce or inhibit the activity of regulatory T cells, reduce the production of cytokines (e.g., IL-10) by regulatory T cells, reduce regulatory T cell differentiation, reduce the number of regulatory T cells, reduce the proportion of regulatory T cells in an immune cell population, or reduce the survival rate of regulatory T cells. The immunomodulator may be administered to a subject in need thereof in an effective amount to overcome T cell exhaustion and / or T cell anergy. The efficacy of overcoming T cell exhaustion or T cell anergy may be determined by measuring T cell function using known techniques.

[0952] The method can be used as an immune response stimulation treatment in vivo or in vitro. Therefore, in some embodiments, the immunomodulator or the nucleic acid encoding the immunomodulator is directly administered to the subject. In some embodiments, the immunomodulator or the nucleic acid encoding the immunomodulator is contacted with cells (e.g., immune cells) in vitro, and the therapeutic cells are administered to the subject (e.g., adoptive transfer). In general, the disclosed immunomodulator can be used to treat subjects suffering from or susceptible to any disease or disorder that easily causes the immune system to produce an immune response. The immunomodulator can enhance the immune response. The disclosed composition is conducive to stimulating or enhancing the immune response of related T cells.

[0953] The immunomodulator for enhancing the immune response is typically an immunomodulator that reduces B7-H4 expression, ligand binding, cross-linking, negative signaling, or a combination thereof. For example, the immunomodulator can be a B7-H4 antagonist, such as an antagonist that (blocks) an anti-B7-H4 antibody or an antigen-binding fragment thereof. The immunomodulator can also be a B7-H4 polypeptide, such as a soluble polypeptide, or a fusion protein thereof, which can be used as a decoy receptor for one or more B7-H4 ligands or receptors.

[0954] B7-H4 blockers, such as the use of function-blocking anti-B7-H4 antibodies, can be alternatives or complements to soluble B7-H4 polypeptides and fusion proteins. For example, in some embodiments, a B7-H4 blocker is used in combination with a decoy receptor (e.g., soluble B7-H4 or fusion protein). Combination therapies (e.g., B7-H4-Fc and a B7-H4 blocker) can be complementary.

[0955] In some embodiments, immune response stimulation therapy (eg, in the treatment of cancer or infection) comprises depletion of B7-H4+ cells.

[0956] The development and identification of B7-H4 consuming mAbs can be carried out according to known construction and screening methods (including the methods discussed herein). See, for example, Reff, et al., Blood. Vol83, No 2, 1994: pp 435-445, which describes the preparation of anti-CD20 chimeric antibodies that can bind to human C1q and mediate complement-dependent cell lysis (CDCC) in the presence of human complement, as well as antibody-dependent cellular cytotoxicity (ADCC) using human effector cells. Rituximab can destroy B cells and is therefore used to treat diseases characterized by overactive, dysfunctional or excessive numbers of B cells. Other B cell depletion antibodies include Orelizumab and Ofatumumab. In another example, CD3Ab can preferentially target and deplete activated effector T cells while retaining CD4+Foxp3+ regulatory T cells. Although it has no or little complement-dependent and antibody-dependent cellular toxicity, the antibody temporarily depletes T cells. However, the results of redirected cell lysis due to the ability to cross-link CD3 molecules expressed by two different cells, one cytotoxic CD8+ T cells and the other other targeted T cells, suggest that T cell depletion is mainly caused by AICD (for a review, see You, Front Immunol. 2015; 6: 242).

[0957] An agent that inhibits or blocks the B7-H4 inhibitory immune response may be administered to a subject in need of an enhanced immune response in an amount effective to increase the uptake of the antigen by antigen presenting cells (APCs).

[0958] 2. Subjects to be treated

[0959] a. Cancer treatment

[0960] The disclosed compositions and methods can be used to treat cancer. In general, a subject's immune response to cancer can be stimulated or enhanced by administering to the subject an amount of an immunomodulator that can reduce B7-H4 expression, ligand binding, cross-linking, negative signaling, or a combination thereof. Cancer cells acquire a series of characteristic functional capabilities during their development, but these capabilities are achieved through various mechanisms. These capabilities include avoiding apoptosis, self-sufficiency in growth signals, insensitivity to anti-growth signals, tissue infiltration / metastasis, unlimited replication potential, and sustained angiogenesis. The term "cancer cell" encompasses precancerous cancer cells and malignant cancer cells. In some embodiments, "cancer" refers to a benign tumor that remains locally. In other embodiments, "cancer" refers to a malignant tumor that has invaded and destroyed adjacent body structures and spread to distal sites. In other embodiments, 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), CD 19, CD20, HER2 / nerve sheath, etc.).

[0961] The methods and compositions disclosed herein are useful for treating or preventing a variety of cancers or other abnormal proliferative diseases, including, but not limited to, the following: carcinomas, 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; squamous cell carcinoma; hematopoietic tumors of the lymphatic system, including leukemias, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burkitt's lymphoma; hematopoietic tumors of the myeloid system, including acute and chronic myeloid leukemia; Myeloid leukemia and promyelocytic leukemia; mesenchymal tumors, including fibrosarcoma and rhabdomyosarcoma; other tumors, including melanoma, seminoma, teratoma, neuroblastoma and glioma; central and peripheral nervous system tumors, including astrocytoma, neuroblastoma, glioma and schwannoma; mesenchymal tumors, including fibrosarcoma, rhabdomyosarcoma and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid carcinoma and teratoma.

[0962] Cancers caused by abnormal apoptosis can also be treated by the disclosed methods and compositions. Such cancers include, but are not limited to, follicular lymphoma, cancers with p53 mutations, hormone-dependent tumors such as breast cancer, prostate cancer, and ovarian cancer, as well as precancerous lesions such as familial adenomatous polyposis and myelodysplastic syndrome.

[0963] In specific embodiments, malignant or dysplastic changes (e.g., metaplasia and dysplasia) or hyperproliferative diseases in the ovary, bladder, breast, colon, lung, skin, pancreas, or uterus are treated or prevented by the methods and compositions. In other specific embodiments, sarcomas, melanomas, or leukemias are treated or prevented by the methods and compositions.

[0964] The disclosed compositions and methods are particularly useful for treating cancers associated with cells that express abnormally high levels of B7-H4.

[0965] Specific cancers and related diseases that can be treated or prevented by the methods and compositions disclosed herein include, but are not limited to: leukemias, including, but not limited to, acute leukemias, acute lymphocytic leukemias, acute granulocytic leukemias (e.g., myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroid leukemia, and myelodysplastic syndrome), chronic leukemias (e.g., but not limited to, chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia); polycythemia vera; lymphomas, such as, but not limited to, Hodgkin's lymphoma or non-Hodgkin's lymphoma (e.g., diffuse anaplastic lymphoma kinase (ALK) negative, large B-cell lymphoma (DL-CL), and non-Hodgkin's lymphoma (NHL), including, but not limited to, ALK-negative, ALK-negative, non-Hodgkin's lymphoma (NHL), and non-Hodgkin's lymphoma (NHL). BCL); diffuse anaplastic lymphoma kinase (ALK) positive, large B-cell lymphoma (DLBCL); anaplastic lymphoma kinase (ALK) positive, ALK+ anaplastic large cell lymphoma (ALCL), acute myeloid lymphoma (AML); multiple myeloma, such as but not limited to smoldering multiple myeloma, non-secretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedullary plasmacytoma; Waldenstrom macroglobulinemia; undefined monoclonal gammopathy; benign monoclonal gammopathy; heavy chain disease; bone and connective tissue sarcomas, such as but not limited to osteosarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, bone meningioma, soft tissue sarcoma, angiosarcoma (angioendothelioma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, schwannoma, rhabdomyosarcoma, synovial sarcoma; brain tumors, including but not limited to glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, non-glioma, acoustic neuroma, craniopharyngioma, medulloblastoma, meningioma, perineum, fibroblastoma, primary brain lymphoma; breast cancer, including but not limited to adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular carcinoma, papillary breast cancer, Paget's disease and inflammatory breast cancer; adrenal cancer, including but not limited to pheochromocytoma and adrenocortical carcinoma; thyroid cancer, such as, but not limited to, papillary or follicular thyroid cancer, medullary thyroid cancer, and anaplastic thyroid cancer; pancreatic cancer, including, but not limited to, islet cell tumors; gastrinomas, glucagonomas, vasodilatory peptide tumors, somatostatin-secreting tumors, and carcinoid or islet cell tumors; pituitary cancer, including, but not limited to, Cushing's disease, prolactin-secreting tumors, acromegaly, and diabetes insipidus; eye cancer, including, but not limited to, eye melanomas, such as iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancer, including, but not limited to, squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancer, including, but not limited to, squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancer, 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 cancer, borderline tumors, germ cell tumors and stromal tumors; esophageal cancer, including but not limited to squamous cell carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma and oat cell (small cell) carcinoma; gastric cancer, including but not limited to adenocarcinoma, fungal (polypoid) carcinoma, ulcerative carcinoma, superficial spreading carcinoma, diffuse spreading carcinoma, malignant lymphoma, liposarcoma, fibrosarcoma and carcinosarcoma; colon cancer; rectal cancer; liver cancer, including but not limited to hepatocellular carcinoma and hepatoblastoma, gallbladder cancer, including but not limited to adenocarcinoma; bile duct cancer, including but not limited to papillary carcinoma, nodular carcinoma and diffuse carcinoma; 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; testicular cancer, including but not limited to Germinoma, seminoma, anaplastic cell tumor, classical (typical) cell tumor, seminoma, non-seminoma, embryonal 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, mucoepidermoid carcinoma and adenoid cystic carcinoma; pharyngeal cancer, including but not limited to squamous cell carcinoma and verrucous carcinoma; skin cancer, including but not limited to basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, tonsillar malignant melanoma, acral lentiginous melanoma; kidney cancer, including but not limited to renal cell carcinoma, adenocarcinoma, nephritis, fibrosarcoma, transitional cell carcinoma (renal pelvis and / or uterus); Wilms' tumor; bladder cancer, including but not limited to transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, carcinosarcoma. In addition, cancer includes myxosarcoma, osteosarcoma, endotheliosarcoma, lymphangioendothelioma, mesothelioma, synovioma, hemangioblastoma, epithelial cancer, cystadenocarcinoma, bronchial carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma and papillary adenocarcinoma (for a review of such diseases, see Fishman et al., 1985, Medicine, 2d Ed., JB Lippincott Co., Philadelphia and Murphy et al., 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books USA, Inc. USA). ;

[0966] b. Treatment of infection

[0967] The disclosed compositions and methods can be used to treat infections and infectious diseases. Generally, an immune response to an infectious agent in a subject can be stimulated or enhanced by administering to the subject an amount of an immunomodulator that can reduce B7-H4 expression, ligand binding, cross-linking, negative signaling, or a combination thereof. The methods can alleviate one or more symptoms of infection.

[0968] Neutrophils are a major component of the host's innate 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 series of defense mechanisms, including phagocytosis, killing, and degradation of microorganisms by antimicrobial proteins and proteolytic proteins, accompanied by the generation of reactive oxygen species. Neutrophils are also involved in the regulation of tissue decomposition, remodeling, wound healing, and other inflammatory and adaptive immune components. Due to the short life span of neutrophils, neutrophils must be continuously replenished during infection and inflammation by the expansion of myeloid progenitor cells in the bone marrow. In vitro B7-H4 inhibits the growth of bone marrow-derived neutrophil progenitor cells, indicating that B7-H4 has an inhibitory function in neutrophil expansion (Zhu, G. et al., Blood, 113: 1759-1767 (2009)).

[0969] One embodiment provides a method for treating an infection in a subject in need thereof by administering an effective amount of an agent that inhibits or blocks a B7-H4 inhibitory immune response, which increases neutrophils and / or enhances innate immunity.

[0970] Infection or disease can be caused by bacteria, viruses, protozoa, helminths, or other microbial pathogens that enter cells and are attacked (ie, by cytotoxic T lymphocytes).

[0971] Infection or disease can be acute or chronic. Acute infection is usually a short-term infection. During acute microbial infection, immune cells begin to express immunomodulatory receptors. Therefore, in some embodiments, the method includes enhancing the immune stimulation response against acute infection.

[0972] The infection may be caused by bacteria or fungi such as, but not limited to, Candida albicans, Listeria monocytogenes, Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria meningitidis, Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, or Mycobacterium.

[0973] In some embodiments, the disclosed compositions are used to treat chronic infections, such as infections in which T cell exhaustion or T cell anergy has occurred, causing the infection to persist in the host for a longer period of time.

[0974] Exemplary infections to be treated are chronic infections caused by hepatitis viruses, human immunodeficiency virus (HIV), human T-cell lymphotrophic virus (HTLV), herpes virus, Epstein-Barr virus, or human papilloma virus.

[0975] Since viral infections are primarily cleared by T cells, enhancing T cell activity will have a therapeutic effect in cases where faster or thorough clearance of infectious viral agents is beneficial to animals or human subjects. Therefore, the disclosed compositions can be used to treat local or systemic viral infections, including but not limited to immunodeficiency (e.g., HIV), papilloma (e.g., HPV), herpes (e.g., HSV), encephalitis, influenza (e.g., human influenza virus A), 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 topically applied to treat viral skin diseases, such as herpes lesions or herpes zoster or genital warts. The molecules can also be systemically applied to treat systemic viral diseases, including but not limited to AIDS, influenza, common cold, or encephalitis.

[0976] Representative infections that can be treated include, but are not limited to, infections caused by microorganisms, including, but not limited to, Actinomycetes, Anabaena, Bacillus, Bacteroides, Bdellovibrio, Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobacter, Chromobacterium, Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Helicobacter, Haemophilus, Haemophilus influenzae type B (HIB), Mycobacterium, Legionella, Leptospira, Listeria, A, B and C meningococci, Methanobacterium, Micrococcus, Mycobacterium, Mycoplasma, Myxococcus, Neisseria, Bacteria, Nitrobacter, Oscillatoria, Prototheca, Proteus, Pseudomonas, Rhodospirilla, Rickettsia, Salmonella, Shigella, Helicobacter, Spirochete, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus, Pyroplasma, Thiobacillus and Treponema, Vibrio, Yersinia, Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia rickettsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis and Schistosoma mansoni.

[0977] Other microorganisms that can be treated using the disclosed compositions and methods include bacteria, such as Klebsiella, Serratia, Pasteurella; pathogens associated with cholera, tetanus, botulism, anthrax, plague, and Lyme disease; or fungal or parasitic pathogens, such as Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus, Aspergillus (fumigatus, niger, etc.), Mucor (Mucor, Absidia, Rhizopus), Sporothrix (Sporothrix), Blastomyces (Blastomyces dermatitidis), Paracoccidioides (Nocardia brasiliensis) , Coccidioides (coccidioides) and Histoplasma (capsule), Entamoeba, Amoeba dysenteriae, Balantidium coli, Naegleria fowleri, Acanthamoeba, Flagellates, Cryptosporidium, Pneumocystis carinii, Plasmodium vivax, Babesia volei, Trypanosoma brucei, Trypanosoma cruzi, Toxoplasma, etc., Sporothrix, Blastomyces, Paracoccidioides, Coccidioides, Histoplasma, Entamoeba, Amoeba dysenteriae, Balantidium coli, Biflagellate Amoeba, Acanthamoeba, Giardia, Cryptosporidium, Pneumocystis, Plasmodium, Babesia or Trypanosoma, etc.

[0978] B. Suppression of immune response

[0979] 1. Treatment strategy

[0980] A method for inducing or suppressing an immune response in a subject is provided. In general, the method includes administering an effective amount of an immunomodulator to the subject, or using the immunomodulator to activate immune cells in vitro. For example, the immune response can be a primary immune response to an antigen or enhance effector cell function, such as increasing the antigen-specific proliferation of T cells, enhancing the production of cytokines caused by T cells, stimulating differentiation, or a combination thereof. Therefore, in some embodiments, the immunomodulator reduces T cell proliferation, T cell cytokine production, T cell differentiation, or a combination thereof. In some embodiments, the immunomodulator can reduce the development of initial T cells to secrete inflammatory molecules or promote other cells to secrete inflammatory molecules Th1, Th17, Th22 or other cells, the inflammatory molecules include but are not limited to IL-1β, TNF-α, TGF-β, IFN-γ, IL-17, IL-6, IL-23, IL-22, IL-21 and MMP. In some embodiments, the immunomodulator can enhance or promote the activity of regulatory T cells, increase the production of cytokines (e.g., IL-10) by regulatory T cells, increase the differentiation of regulatory T cells, increase the number of regulatory T cells, increase the proportion of regulatory T cells within an immune cell population, or increase the survival rate of regulatory T cells.

[0981] The method can be used as an immune response suppression therapy in vivo or in vitro. Therefore, in some embodiments, the immunomodulator or the nucleic acid encoding the immunomodulator is directly administered to the subject. In some embodiments, the immunomodulator or the nucleic acid encoding the immunomodulator is contacted with cells (e.g., immune cells) in vitro, and the therapeutic cells are administered to the subject (e.g., adoptive transfer). In general, the disclosed immunomodulator can be used to treat subjects suffering from or susceptible to any disease or disorder that easily causes the immune system to produce an overactive or inappropriate immune response. The immunomodulator can weaken the immune response. The disclosed composition is conducive to reducing or suppressing the immune response of related T cells.

[0982] The immunomodulator for attenuating an immune response is typically an immunomodulator that reduces B7-H4 expression, ligand binding, cross-linking, negative signaling, or a combination thereof. For example, the immunomodulator can be a B7-H4 agonist, such as an agonist of an anti-B7-H4 antibody or an antigen-binding fragment thereof.

[0983] a. Inflammatory response

[0984] The disclosed compositions and methods can be used to treat inflammation. In general, the immune response of a subject can be reduced or inhibited by administering to the subject an amount of an immunomodulator that increases B7-H4 expression, ligand binding, cross-linking, negative signaling, or a combination thereof. The methods can alleviate one or more symptoms of inflammation. Inflammation can be acute, chronic, or persistent.

[0985] In some embodiments, the immunomodulator slows down the immune system response. For example, an immunomodulator can be used to control an excessive inflammatory response that causes damage to healthy tissue. Therefore, in some embodiments, the immunomodulator is administered to a subject who has experienced an excessive inflammatory response. In this case, controlling the excessive immune response may be beneficial to the subject.

[0986] b. Inflammatory and autoimmune diseases / disorders

[0987] Immunomodulators that increase B7-H4 expression, ligand binding, cross-linking, negative signaling, or a combination 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), Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac dermatitis, chronic fatigue syndrome immunodeficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, cicatricial pemphigoid, cold agglutinin disease, acrosclerosis syndrome, Crohn's disease, DiGao's disease, dermatomyositis, juvenile dermatomyositis, discoid lupus, essential cryoglobulinemia, fibromyalgia-fibromyositis, Grave's disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, insulin-dependent diabetes mellitus (type I), juvenile arthritis, Meniere's syndrome, juvenile mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyrenal 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, myotonia solium, Takayasu arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis.

[0988] In some embodiments, the inflammatory or autoimmune disease is caused by a pathogen, or is caused by an infection.

[0989] VI. Combination therapy

[0990] The disclosed immunomodulators can be administered to a subject in need thereof alone or in combination with one or more other therapeutic agents. In some embodiments, the immunomodulator is administered separately from the other therapeutic agent but simultaneously. The immunomodulator and the other therapeutic agent can also be administered as part of the same composition. In other embodiments, the immunomodulator is administered separately from the second therapeutic agent and at different times, but as part of the same treatment regimen.

[0991] The first therapeutic agent may be administered to the subject 1, 2, 3, 4, 5, 6 or more hours, or 1, 2, 3, 4, 5, 6, 7 or more days prior to administration of the second therapeutic agent. In some embodiments, one or more doses of the first therapeutic agent may be administered to the subject every 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35 or 48 days prior to the first administration of the second therapeutic agent. The immunomodulator may be the first or second therapeutic agent.

[0992] The immunomodulator and the other therapeutic agent can be administered as part of a treatment regimen. For example, if the first therapeutic agent can be administered to the subject every four days, the second therapeutic agent can be administered on the first, second, third, or fourth day, or a combination thereof. The first therapeutic agent or the second therapeutic agent can be administered repeatedly throughout the treatment regimen.

[0993] Exemplary molecules include, but are not limited to, cytokines, chemotherapeutic agents, radionuclides, other immunotherapeutic agents, enzymes, antibiotics, antiviral agents (particularly protease inhibitors used alone or in combination with nucleosides to treat HIV or hepatitis B or C), antiparasitic agents (helminths, protozoa), growth factors, growth inhibitory agents, hormones, hormone antagonists, antibodies and biologically active fragments thereof (including humanized, single chain and chimeric antibodies), antigens and vaccine formulations (including adjuvants), polypeptide drugs, anti-inflammatory drugs, ligands that bind to Toll-like receptors (including but not limited to CpG oligodeoxynucleotides) to activate the innate immune system, molecules that activate 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 inhibitory or regulatory T cells.

[0994] The other therapeutic agent is selected according to the condition, disorder or disease to be treated. For example, the immunomodulator can be co-administered with one or more other therapeutic agents that can enhance or promote an immune response, or reduce or inhibit an immune response.

[0995] A. Enhance immune response

[0996] 1. Antimicrobial drugs

[0997] For example, a B7-H4 immunomodulator can be used to treat and prevent the above diseases, and can also be used to treat severe traumatic injuries, such as severe burns, open fractures, accidental amputations or other trauma. Therefore, the B7-H4 immunomodulator can be administered to a subject in combination with an antimicrobial agent such as an antibiotic, antifungal agent, antiviral agent, antiparasitic agent or essential oil.

[0998] In some embodiments, the B7-H4 immunomodulator and / or the antimicrobial agent is administered to the subject upon admission to the hospital to prevent further bacterial, fungal or viral complications. Antibiotics can target pathogens, while the B7-H4 immunomodulator can stimulate the immune system to enhance the immune response, thereby treating or preventing further infection or disease.

[0999] 2. Chemotherapeutic agents

[1000] The B7-H4 immunomodulator can be used in combination with one or more chemotherapeutic agents and pro-apoptotic agents. Representative chemotherapeutic agents include, but are not limited to, amsacrine, mycobacterium, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, cleritase, cyclophosphamide, cytarabine, dacarbazine, actinomycin, daunorubicin, docetaxel, docerubicin, adriamycin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, The invention relates to apoptosis-promoting agents, for example, leucovorin, liposome doxorubicin, liposome daunorubicin, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozotocin, eufodine, temozolomide, teniposide, thiotepa, thioguanine, topotecan, susulfan, vinblastine, vincristine, vindesine, vinorelbine, or a combination thereof. Representative pro-apoptotic agents include, but are not limited to, fludarabine, staurosporine, cycloheximide, actinomycin D, lactosylceramide, 15d-PGJ (2), and a combination thereof.

[1001] 3. Other immunomodulators

[1002] a. PD-1 antagonists

[1003] In some embodiments, the B7-H4 immunomodulator is co-administered with a PD-1 antagonist. Programmed death factor-1 (PD-1) is a member of the CD28 receptor family that, when induced on T cells, can produce a negative immune response. Contact between PD-1 and one of its ligands (B7-H1 or B7-DC) induces an inhibitory response, thereby reducing T cell proliferation and / or the intensity and / or duration of T cell responses. Suitable PD-1 antagonists are described in U.S. Pat. Nos. 8,114,845, 8,609,089, and 8,709,416, the entire contents of which are incorporated herein by reference, and include compounds or antagonists that bind to and block PD-1 ligands that interfere with or inhibit binding of the PD-1 ligand to the PD-1 receptor, or directly bind to and block the PD-1 receptor without inducing inhibitory signal transduction through the PD-1 receptor.

[1004] In some embodiments, the PD-1 receptor antagonist can directly bind to the PD-1 receptor without triggering inhibitory signal transduction, and can also bind to the ligand of the PD-1 receptor to reduce or inhibit the ligand triggering signal transduction through the PD-1 receptor. By reducing the number of ligands that bind to the PD-1 receptor and trigger inhibitory signal transduction, a small number of cells will be weakened by the negative signal transmitted by the PD-1 signal transduction, thereby enhancing the immune response.

[1005] It is believed that PD-1 signaling is driven by binding to PD-1 ligands (e.g., B7-H1 or B7-DC) in close proximity 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 PD-1 and TCR from being co-attached on the T cell membrane are also useful PD-1 antagonists.

[1006] In some embodiments, the PD-1 receptor antagonist is a small molecule antagonist or antibody, which can reduce or interfere with PD-1 receptor signal transduction by binding to PD-1 ligand or to PD-1 itself, particularly in cases where the co-ligation of PD-1 with TCR does not follow such a binding pattern, and thus does not trigger inhibitory signal transduction through the PD-1 receptor.

[1007] Other PD-1 antagonists contemplated by the methods of the present invention include antibodies and other antibodies that bind to PD-1 or PD-1 ligands.

[1008] Suitable anti-PD-1 antibodies include, but are not limited to, those described in the following publications:

[1009] PCT / IL03 / 00425 (Hardy et al., WO / 2003 / 099196)

[1010] PCT / JP2006 / 309606 (Korman et al., WO / 2006 / 121168)

[1011] PCT / US2008 / 008925 (Li et al., WO / 2009 / 014708)

[1012] PCT / JP03 / 08420 (Honjo et al., WO / 2004 / 004771)

[1013] PCT / JP04 / 00549 (Honjo et al., WO / 2004 / 072286)

[1014] PCT / IB2003 / 006304 (Collins et al., WO / 2004 / 056875)

[1015] PCT / US2007 / 08885 1 (Ahmed et al., WO / 2008 / 083 174)

[1016] PCT / US2006 / 026046 (Korman et al., WO / 2007 / 005874)

[1017] PCT / US2008 / 084923 (Terrett et al., WO / 2009 / 073533)

[1018] Berger et al., Clin. Cancer Res., 14:30443051 (2008).

[1019] Specific examples of anti-PD-1 antibodies include the antibody described in Kosak, US20070166281 (published on July 19, 2007), paragraph 42), which is a human anti-PD-1 antibody and is administered at a dose of 3 mg / kg in some embodiments.

[1020] Exemplary anti-B7-H1 antibodies include, but are not limited to, those described in the following publications:

[1021] PCT / US06 / 022423 (WO / 2006 / 133396, publication date: December 14, 2006)

[1022] PCT / US07 / 088851 (WO / 2008 / 083174, published on July 10, 2008)

[1023] US2006 / 0110383 (publication date: May 25, 2006)

[1024] Specific examples of the anti-B7-H1 antibody include the antibody described in (WO / 2007 / 005874, published on January 11, 2007), which is a human anti-B7-H1 antibody.

[1025] Other anti-PD-1 and anti-B7-H1 antibodies are disclosed in 2014 / 0044738, the entire contents of which are incorporated herein by reference.

[1026] For anti-B7-DC antibodies, see U.S. Published Application Nos. 7,411,051, 7,052,694, 7,390,888, and 2006 / 0099203.

[1027] Other exemplary PD-1 receptor antagonists include, but are not limited to, B7-DC polypeptides, including homologues and variants thereof, and any of the above-mentioned active fragments and fusion proteins incorporated into any of the above-mentioned. In some embodiments, the fusion protein includes a B7-DC soluble portion coupled to an antibody (e.g., human IgG) Fc portion, and does not incorporate all or part of the transmembrane portion of human B7-DC.

[1028] The PD-1 antagonist may also be a fragment of mammalian B7-H1, such as from a mouse or primate (e.g., human), wherein the fragment binds and blocks PD-1 but does not result in inhibitory signal transduction through PD-1. The fragment may also be part of a fusion protein, such as an Ig fusion protein.

[1029] Other effective polypeptide PD-1 antagonists include polypeptides that bind to PD-1 receptor ligands. The polypeptide includes a PD-1 receptor protein or a soluble fragment thereof that can bind to a PD-1 ligand (e.g., B7-H1 or B7-DC) and prevent binding to an endogenous PD-1 receptor, thereby preventing inhibition of signal transduction. Studies have shown that B7-H1 can bind to B7.1 protein (Butte et al., Immunity, Vol. 27, pp. 111-122, (2007)). Such fragments also include soluble ECD portions of PD-1 proteins, including mutations that increase binding to natural ligands, such as A99L mutations (Molnar et al., PNAS, 105: 10483-10488 (2008)). B7-1 or a soluble fragment thereof that can bind to a PD-1 ligand and prevent binding to an endogenous PD-1 receptor, thereby preventing inhibition of signal transduction, is also useful.

[1030] DNA and RNA PD-1 and B7-H1 antisense nucleic acids and siRNA molecules can also be used as PD-1 antagonists. Such antisense molecules prevent the expression of PD-1 on T cells and the production of T cell ligands, such as B7-H1, PD-L1 and / or PD-L2. For example, siRNA (e.g., about 21 nucleotides in length, which is specific to genes encoding PD-1 or encoding PD-1 ligands, and its oligonucleotides are easily purchased) complexed with carriers such as polyethyleneimine (see Cubillos-Ruiz et al., J. Clin. Invest. 119 (8): 2231-2244 (2009)) is easily absorbed by cells expressing PD-1 and PD-1 ligands and reduces the expression of these receptors and ligands to reduce inhibitory signal transduction in T cells, thereby activating T cells.

[1031] b. CTLA4 antagonists

[1032] Other molecules that are beneficial in mediating T cell effects in immune responses may also be used as other therapeutic agents. In some embodiments, the molecule is a CTLA4 antagonist, such as an antagonist anti-CTLA4 antibody. Examples of anti-CTLA4 antibodies contemplated for use in the methods of the present invention include antibodies described in PCT / US2006 / 043690 (Fischkoff et al., WO / 2007 / 056539).

[1033] Anti-PD-1, anti-B7-H1 and anti-CTLA4 antibody dosages are known in the art and may also 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. Doses suitable for human subjects may be between 5 and 15 mg / kg, with 10 mg / kg of antibody (e.g., human anti-PD-1 antibody) being a specific embodiment.

[1034] Specific examples of anti-CTLA4 antibodies that can be used in the methods of the present invention include ipilimumab (a human anti-CTLA4 antibody, for example, which can be administered at a dose of about 10 mg / kg) and tremelimumab (a human anti-CTLA4 antibody, for example, which can be administered at a dose of about 15 mg / kg). See also Sammartino, et al., Clinical Kidney Journal, 3(2):135-137 (2010), published online in December 2009.

[1035] In other embodiments, the antagonists are all small molecules. Studies have shown that a series of small molecule organic compounds can bind to B7-1 ligands to prevent binding to CTLA4 (see Erbe et al., J. Biol. Chem., 277: 7363-7368 (2002). Such small molecule organic compounds can be administered alone or together with anti-CTLA4 antibodies to reduce inhibitory signal transduction of T cells.

[1036] 4. Enhancer

[1037] In some embodiments, the additional therapeutic agent includes an enhancer. The enhancer acts to enhance the efficacy of the immune response upregulator by more than one mechanism, but the exact mechanism of action is not necessary for the broad practice of the invention.

[1038] In some embodiments, the enhancer is cyclophosphamide. or ) is an oxazaphosphorus drug, and its analogs include ifosfamide (IFO, Ifex), perfosfamide, clofosfamide (trofosfamide; Ixoten) and pharmaceutically acceptable salts, solvates, prodrugs and metabolites thereof (U.S. Patent Application No. 20070202077, the entire contents of which are incorporated herein by reference). Ifosfamide It is a structural analog of cyclophosphamide, and its mechanism of action is believed to be the same or substantially similar to that of cyclophosphamide. Perphosphamide (4-hydroperoxycyclophosphamide) and triphosphamide are also alkylating agents, structurally related to cyclophosphamide. For example, perphosphamide can alkylate DNA, thereby inhibiting DNA replication and RNA and protein synthesis. New oxazaphosphorus derivatives have been designed and evaluated in an attempt to enhance selectivity and response and reduce 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 derivatives include mafosfamide (NSC 345842), glucamide ammonium phosphamide (D19575, β-D-glucosyl isophosphamide mustard), S-(-)-bromophosphamide (CBM-11), NSC 612567 (aldophosphamide perhydrothiazine) and NSC 613060 (aldophosphamide thiazole). Mafosfamide, an oxazaphosphorus analog, is a chemically stable 4-hydroxy-CPA 4-thioethane sulfonate. Glucamide is an IFO derivative in which isophosphamide mustard (an alkylated metabolite of IFO) is glycosidically linked to a β-D-glucose molecule. For a description of other cyclophosphamide analogs, see U.S. Pat. No. 5,190,929, entitled "Cyclophosphamide analogs useful as anti-tumor agents," the entire contents of which are incorporated herein by reference.

[1039] Although CTX itself is nontoxic, some of its metabolites are cytotoxic alkylating agents that induce DNA cross-linking and, at higher doses, strand breaks. Many cells are resistant to CTX because they express high levels of the detoxifying enzyme aldehyde dehydrogenase (ALDH). CTX targets proliferating lymphocytes because lymphocytes (but not hematopoietic stem cells) express only low levels of ALDH, and circulating cells are the most sensitive to DNA alkylating agents.

[1040] Low doses of CTX (<200 mg / kg) can have immunostimulatory effects, including stimulating antitumor immune responses in human and mouse cancer models (Brode & Cooke Crit Rev. Immunol. 28: 109-126 (2008)). These low doses are subtherapeutic and have no direct antitumor activity. In contrast, high doses of CTX inhibit antitumor responses. There are several mechanisms that can explain the role of CTX in enhancing antitumor immune responses: (a) depletion of CD4+CD25+FoxP3+ regulatory T cells (especially proliferative regulatory T cells, which may be particularly suppressive); (b) depletion of B lymphocytes; (c) induction of nitric oxide (NO), thereby inhibiting tumor cell growth; (d) activation and expansion of CD1 1b+Gr-1+MDSC. These primary effects have many secondary effects; for example, after depletion of regulatory T cells, macrophages produce more IFN-γ and less IL-10. Our results suggest that CTX induces type IIFN expression and promotes homeostatic proliferation of lymphocytes.

[1041] Regulatory T cell depletion is often cited as a mechanism by which CTX enhances anti-tumor immune responses. This conclusion is based in part on the results of adoptive transfer experiments. In the AB1-HA tumor model, the cure rate of CTX treatment performed on day 9 was 75%. At day 12, the transfer of purified regulatory T cells 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+ regulatory T cells after CTX pretreatment eliminated the vaccine treatment response (Taieb, JJ Immunol. 176: 2722-2729 (2006)).

[1042] A large number of human clinical trials have shown that low-dose CTX is a safe, well-tolerated and effective enhancer of anti-tumor immune responses (Bas, & Mastrangelo Cancer Immunol. Immunother. 47: 1-12 (1998)).

[1043] The optimal CTX dose for enhancing anti-tumor immune responses is a subtherapeutic dose that reduces total T cell counts by lowering regulatory T cell levels below the normal range (see Machiels et al., Cancer Res. 61:3689-3697 (2001)).

[1044] In human clinical trials using CTX as an immunopotentiator, a dose of 300 mg / m2 is generally used. For an average male (6 feet, 170 pounds (78 kg), with a body surface area of ​​1.98 m2), a 300 mg / m2 dose is 8 mg / kg or 624 mg total protein. In a mouse cancer model, efficacy was observed in a dose range of 15-150 mg / kg, which was related to the 0.45-4.5 mg total protein content in 30 g mice (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)).

[1045] For large mammals, such as primates (e.g., human patients), the above mg / m2 doses can be used, but unit doses administered at limited time intervals can also be used. Such unit doses can be administered daily for a limited period of time, 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, which are all factors specifically considered by the present invention. The same regimen can be applied to other enhancers described herein.

[1046] In other embodiments, the enhancing agent is an agent that can reduce the activity and / or number of regulatory T lymphocytes (T-regs), such as sunitinib. Anti-TGFp or imatinib The treatment regimen may also include the administration of an adjuvant.

[1047] Useful enhancers also include mitotic inhibitors such as paclitaxel, aromatase inhibitors (e.g., letrozole) and angiogenesis inhibitors (VEGF inhibitors such as Avastin, VEGF-trap) (see, e.g., 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.), anthracyclines, oxaliplatin, doxorubicin, TLR4 antagonists, and IL-18 antagonists.

[1048] B. Weakened immune response

[1049] 1. Immunosuppressants

[1050] In some embodiments, an immune response or inflammatory / autoimmune disease / disorder is treated by administering to a subject a B7-H4 immunomodulator and a second modulator that 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 TNFR-Ig ), TNF-α blockers (e.g., etanercept, infliximab, certolizumab, and adalimumab), cyclophosphamide (CTX) (i.e., Revimmune TM ), methotrexate (MTX) (i.e., ), Belimumab (ie, ) or other immunosuppressive drugs (e.g., cyclosporin A, FK506 compounds, rapamycin compounds or steroids), antiproliferative agents, cytotoxic agents, or other compounds that may contribute to immunosuppression.

[1051] The therapeutic agent can be a CTLA-4 fusion protein, such as CTLA-4-Ig (abatacept). The CTLA-4-Ig fusion protein competes with the co-stimulatory receptor CD28 on T cells and binds to CD80 / CD86 (B7-1 / B7-2) on antigen presenting cells, thereby inhibiting T cell activation. In another embodiment, the therapeutic agent is a CTLA-4-Ig fusion protein named "Belacept". Belatacept contains two amino acid substitutions (L104E and A29Y) that can significantly increase the affinity for CD86 in vivo. In another embodiment, the therapeutic agent is Maxy-4.

[1052] In another embodiment, the therapeutic agent is cyclophosphamide (CTX). Revimmune TM Cyclophosphamide (CTX), also known as cyclophosphamide, is a nitrogen mustard alkylating agent from the oxazoline group. It can be used to treat various types of cancer and some autoimmune diseases. Cyclophosphamide (CTX) is the drug of choice for the treatment of diffuse proliferative glomerulonephritis in patients with renal lupus.

[1053] The therapeutic agent may be administered to a patient in need thereof in an amount effective to reduce anti-double-stranded DNA (anti-ds DNA) autoantibody levels in the blood or serum and / or to reduce proteinuria.

[1054] In another embodiment, the therapeutic agent can increase the amount of adenosine in serum, see, e.g., WO 08 / 147482. For example, the second therapeutic agent can be CD73-Ig, recombinant CD73, or another agent that increases CD73 expression (e.g., a cytokine or a monoclonal antibody or a small molecule), see, e.g., WO 04 / 084933. In another embodiment, the therapeutic agent is interferon-β.

[1055] The therapeutic agent can be a small molecule that inhibits or reduces differentiation, proliferation, activity and / or cytokine production and / or secretion by Th1, Th17, Th22 and / or other cells that secrete or promote other cells to secrete inflammatory molecules (including 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 MMP).

[1056] In another embodiment, the therapeutic agent is a small molecule that interacts with regulatory T cells, enhances T cell activity, promotes or enhances IL-10 secretion by regulatory T cells, increases the number of regulatory T cells, improves the suppressive capacity of regulatory T cells, or a combination thereof.

[1057] In some embodiments, the composition is capable of increasing T cell activity or production. Exemplary regulatory T cell enhancers include, but are not limited to, glucocorticoids, fluticasone...

Claims

1. A pharmaceutical composition comprising an effective amount of an immunomodulator that reduces B7-H4 expression, ligand binding, cross-linking, negative signaling, or a combination thereof, wherein the immunomodulator is selected from the group consisting of: (i) a soluble B7-H4 polypeptide or fusion protein, (ii) function-blocking anti-B7-H4 antibodies, (iii) antibodies that deplete B7-H4 positive cells, and (iv) any combination thereof; To enhance the immune response of a subject in need thereof.

2. The pharmaceutical composition according to claim 1, wherein the immunomodulator is a B7-H4 fusion protein.

3. The pharmaceutical composition of claim 2, wherein the B7-H4 fusion protein comprises an extracellular domain of B7-H4 connected to an immunoglobulin domain for a functional variant thereof.

4. The pharmaceutical composition according to claim 3, wherein the B7-H4 fusion protein comprises the amino acid sequence SEQ ID NO: 1 or a fragment thereof.

5. The pharmaceutical composition according to claim 1, wherein the immunomodulator is a B7-H4 fusion protein.

6. The pharmaceutical composition according to claim 5, wherein the B7-H4 fusion protein comprises an extracellular domain of B7-H4 or a functional variant thereof connected to an immunoglobulin domain.

7. The pharmaceutical composition according to claim 6, wherein the B7-H4 fusion protein comprises the amino acid sequence SEQ ID NO: 1 or a fragment thereof.

8. The pharmaceutical composition according to claim 1, wherein the immunomodulator is a soluble B7-H4 protein or a functional fragment or variant thereof.

9. The pharmaceutical composition according to claim 8, wherein the B7-H4 protein consists of the extracellular domain of B7-H4 or a functional fragment or variant thereof.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the immune response is a primary immune response to an antigen or enhances effector cell function, such as increasing antigen-specific proliferation of T cells, enhancing cytokine production by T cells, stimulating differentiation, or a combination thereof.

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