WO2025128735A2 - Conjugués anticorps-médicament dirigés contre b7-h4 et leurs méthodes d'utilisation - Google Patents

Conjugués anticorps-médicament dirigés contre b7-h4 et leurs méthodes d'utilisation Download PDF

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WO2025128735A2
WO2025128735A2 PCT/US2024/059633 US2024059633W WO2025128735A2 WO 2025128735 A2 WO2025128735 A2 WO 2025128735A2 US 2024059633 W US2024059633 W US 2024059633W WO 2025128735 A2 WO2025128735 A2 WO 2025128735A2
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seq
antibody
amino acid
acid sequence
sequence according
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WO2025128735A3 (fr
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Daniel P. FITZGERALD
Dallas Benjamin Flies
Solomon Langermann
Kwang Hwa Jung
Hwanhee OH
Chul-Woong CHUNG
Ho Young Song
Myungji KANG
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Ligachem Biosciences Inc
NextCure Inc
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Ligachem Biosciences Inc
NextCure Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • he invention is generally related to the field of antibody-drug conjugates, and more particularly to compositions and methods for targeting tumor cells expressing B7- H4 for elimination with cytotoxin payloads attached to the antibody molecule.
  • B7-H4 (also referred to as VTCN1 or B7x or B7S1) is a member of the B7 family and inhibits T-cell function. It is also upregulated on a variety of tumors and has been proposed to promote tumor growth. High B7-H4 expression is found in numerous tumor tissues providing a correlation of the level of expression on tumor cells with adverse clinical and pathologic features, including tumor aggressiveness.
  • B7-H4 has been associated with decreased inflammatory CD4 + T-cell responses and a correlation between B7-H4-expressing tumor- associated macrophages and FoxP3+ regulatory T cells (Tregs) within the tumor microenvironment. Since B7-H4 is expressed on tumor cells and tumor-associated macrophages in various cancer types, therapeutic blockade of B7-H4 could favorably alter the tumor microenvironment allowing for antigen-specific clearance of tumor cells (Podojil, JR. and Miller, SD, Immunol Rev, 276(l):40-51 (2017)).
  • 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.
  • 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.
  • B7-H4 protein possesses 282 amino acid residues, which have been categorized as including an amino terminal extracellular domain, a large hydrophobic transmembrane domain and a very short intracellular domain (consisting of only 2 amino acid residues). Like other B7 family members, B7-H4 possesses a pair of Ig-like regions in its extracellular domain.
  • the B7-H4 protein has an overall structure of a type I transmembrane protein. The protein has minimal (about 25%) homology with other B7 family members (Zang, X. et al. (2003) “B7x: A Widely Expressed B7 Family Member That Inhibits T Cell Activation," Proc. Natl. Acad. Sci. (USA) 100: 10388-10392).
  • B7-H4 is also expressed on tumor-associated macrophages (TAMs).
  • TAMs inhibit anti -tumor immune responses through the release of humoral mediators and also protect tumors from immune recognition by hampering cell-mediated immune responses through the cell-surface expression of inhibitory molecules such as B7-H4.
  • TAMs derive from resident macrophages or from monocytes recruited by the tumor microenvironment and polarized at the tumor site. Tumor infiltration with TAMs has been associated with poor patient survival and targeting TAMs represents a promising strategy against cancer.
  • B7-H4 is expressed on tumor cells and TAMs in various cancer types, directing therapeutics against B7-H4 could have tremendous synergistic outcomes in favorably altering the tumor micro-environment and eliminating cancer cells.
  • B7-H4 is expressed on tumor cells and TAMs in various cancer types
  • compositions that target B7-H4 expressed on tumor cells and deliver cytotoxic payloads to destroy the tumor cells. Such compositions are useful for the treatment of cancer.
  • compositions and methods of their use for targeting B7-H4 expressing cells and delivering cytotoxic payloads are provided. Such compositions are useful for the treatment of cancers.
  • the compositions are antibodies that specifically bind to B7-H4 and are conjugated to cytotoxic payloads via a linker.
  • the compounds can be antibodies or antigen binding fragments thereof, fusion proteins, aptamers, or agents that specifically bind B7-H4.
  • One embodiment provides a method for treating infection in a subject in need thereof by administering an effective amount of a composition that targets B7-H4 expressed on the surface of a cell and delivers a cytotoxic payload to the cell.
  • the agent is a B7-H4 fusion protein, for example a fusion protein that includes an extracellular domain of B7-H4 or functional variant thereof linked to an immunoglobulin domain.
  • the cancer is characterized by increased expression of B7-H4.
  • the cancer is an ovarian, breast, lung, thyroid, gastrointestinal cancer, acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), endometrial cancer, brain cancer, head and neck cancer, pancreatic cancer, cholangiocarcinomas and bladder cancer.
  • the agent can be administered contemporaneously or in combination with a vaccine or a component thereof.
  • Any of the disclosed methods can include administering to the subject a composition that targets B7-H4 expressed on the surface of a cell and delivers a cytotoxic payload to the cell.
  • One embodiment provides a method for assessing or predicting the efficacy of a treatment using an anti-B7-H4 binding moiety by assaying the cells of a subject in need of treatment to determine whether the cells express B7-H4, binding partners of B7- H4, or both.
  • Exemplary cells to be assayed include but are not limited to cancer cells obtained from the subject.
  • Exemplary cancer cells include but are not limited to, ovarian, breast, lung, thyroid, gastrointestinal cancer, acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), endometrial cancer, brain cancer, head and neck cancer, pancreatic cancer, cholangiocarcinomas (BTC) and bladder cancer.
  • One embodiment provides an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof that has a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:3.
  • One embodiment provides a nucleic acid that encodes a light chain according to SEQ ID NO:3. Another embodiment provides a nucleic acid having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:7.
  • One embodiment provides an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof that has a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:8.
  • One embodiment provides a nucleic acid encoding heavy chain SEQ ID NO:8.
  • Another embodiment provides a nucleic acid having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 12.
  • One embodiment provides an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof that has a light chain containing CDRs according to SEQ ID Nos: 4, 5, and 6.
  • Another embodiment provides an anti-B7-H4 antibody-drug conjugate or an antigen binding fragment thereof having a heavy chain containing CDRs according to SEQ ID Nos: 9, 10, and 11.
  • an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof has a light chain containing CDRs according to SEQ ID Nos: 4, 5, and 6 and a heavy chain containing CDRs according to SEQ ID Nos: 9, 10, and 11.
  • One embodiment provides an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof having a light chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 3 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:8.
  • Another embodiment provides an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof that has a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 13.
  • the antibody-drug conjugate light chain contains CDRs with amino acid sequences according to SEQ ID Nos: 14, 15, and 16.
  • One embodiment provides an antibody-drug conjugate heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:29.
  • Another embodiment provides an antibody-drug conjugate heavy chain containing CDRs with amino acid sequences according to SEQ ID NOs: 9, 11, and 30.
  • One embodiment provides an anti-B7-H4 antibody-drug conjugate, or antigen binding fragment thereof containing light chain CDRs according to SEQ ID Nos: 14, 15, and 16 and heavy chain CDRs according to SEQ ID Nos: 9, 11, and 30.
  • One embodiment provides an anti-B7-H4 antibody-drug conjugate, or antigen binding fragment thereof containing a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 13 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:29.
  • One embodiment provides an anti-B7-H4 antibody-drug conjugate light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:46.
  • Another embodiment provides an anti-B7-H4 antibody-drug conjugate light chain containing CDRs with amino acid sequences according to SEQ ID Nos: 14, 16, and 47.
  • One embodiment provides an antibody-drug conjugate heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:49.
  • Another embodiment provides an antibody-drug conjugate heavy chain containing CDRs with amino acid sequences according to SEQ ID Nos: 9, 11, and 47.
  • One embodiment provides an antibody-drug conjugate, or antigen binding fragment thereof containing light chain CDRs according to SEQ ID Nos: 14, 16, and 47, and heavy chain CDRs according to SEQ ID Nos: 9, 11, and 50.
  • One embodiment provides an anti-B7-H4 antibody-drug conjugate, or an antigen binding fragment thereof containing a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:46 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:49.
  • One embodiment provides an antibody-drug conjugate light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:52.
  • an antibody-drug conjugate light chain contains CDRs with amino acid sequences according to SEQ ID Nos: 53, 54, and 55.
  • One embodiment provides an antibody-drug conjugate heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:65.
  • Another embodiment provides an antibody-drug conjugate heavy chain containing CDRs with amino acid sequences according to SEQ ID Nos: 66, 67, and 68.
  • One embodiment provides an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof containing light chain CDRs according to SEQ ID Nos: 53, 54, and 55, and heavy chain CDRs according to SEQ ID Nos: 66, 67, and 68.
  • One embodiment provides an antibody-drug conjugate, or antigen binding fragment thereof, containing a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 52 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:65.
  • One embodiment provides an antibody-drug conjugate light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:85.
  • Another embodiment provides an antibody-drug conjugate light chain containing CDRs with amino acid sequences according to SEQ ID Nos: 4, 86, and 87.
  • One embodiment provides an antibody-drug conjugate heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:89.
  • One embodiment provides an antibody-drug conjugate heavy chain containing CDRs with amino acid sequences according to SEQ ID Nos: 9, 90, and 91.
  • Another embodiment provides an anti-B7-H4 antibody-drug conjugate, or antigen binding fragment thereof, containing light chain CDRs according to SEQ ID Nos: 4, 86, and 87 and heavy chain CDRs according to SEQ ID Nos: 9, 90, and 91.
  • One embodiment provides an anti-B7-H4 antibody-drug conjugate, or antigen binding fragment thereof, containing a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:85 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 89.
  • One embodiment provides an antibody-drug conjugate light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:93.
  • Another embodiment provides an antibody-drug conjugate light chain containing CDRs with amino acid sequences according to SEQ ID Nos: 94, 95, and 96.
  • One embodiment provides an antibody-drug conjugate heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:98.
  • Another embodiment provides an antibody-drug conjugate heavy chain containing CDRs with amino acid sequences according to SEQ ID Nos: 9, 99, and 100.
  • One embodiment provides an anti-B7-H4 antibody-drug conjugate, or antigen binding fragment thereof, having light chain CDRs according to SEQ ID Nos: 94, 95, and 96, and heavy chain CDRs according to SEQ ID Nos: 9, 99, and 100.
  • One embodiment provides an anti-B7-H4 antibody-drug conjugate, or antigen binding fragment thereof, having a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:93 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:98.
  • One embodiment provides an antibody-drug conjugate light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 102.
  • Another embodiment provides an antibody-drug conjugate light chain containing CDRs with amino acid sequences according to SEQ ID Nos: 103, 104, and 105.
  • One embodiment provides an antibody-drug conjugate heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 107.
  • an antibody-drug conjugate heavy chain containing CDRs with amino acid sequences according to SEQ ID Nos: 9, 108, and 109 contains light chain CDRs according to SEQ ID Nos: 103, 104, and 105 and heavy chain CDRs according to SEQ ID Nos: 9, 108, and 109.
  • One embodiment provides an anti-B7-H4 antibody-drug conjugate, or antigen binding fragment thereof, containing a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 102 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 107.
  • One embodiment provides an antibody-drug conjugate light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 111.
  • Another embodiment provides an antibody-drug conjugate light chain containing CDRs with amino acid sequences according to SEQ ID Nos: 95, 112, and 113.
  • One embodiment provides an antibody-drug conjugate heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:115.
  • One embodiment provides an antibody heavy chain containing CDRs with amino acid sequences according to SEQ ID Nos: 116, 117, and 118.
  • the anti-B7-H4 antibody-drug conjugate, or antigen binding fragment thereof contains light chain CDRs according to SEQ ID Nos: 95, 112, and 113, and heavy chain CDRs according to SEQ ID Nos: 116, 117, and 118.
  • One embodiment provides an anti-B7-H4 antibody-drug conjugate, or antigen binding fragment thereof, containing a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 111 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 115.
  • One embodiment provides an antibody-drug conjugate light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 120.
  • Another embodiment provides an antibody-drug conjugate light chain containing CDRs with amino acid sequences according to SEQ ID Nos: 54, 55, and 121.
  • One embodiment provides an antibody-drug conjugate heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 123.
  • Another embodiment provides an antibody-drug conjugate heavy chain containing CDRs with amino acid sequences according to SEQ ID Nos: 66, 124, and 125.
  • One embodiment provides an anti-B7-H4 antibody-drug conjugate, or antigen binding fragment thereof, containing light chain CDRs according to SEQ ID Nos: 54, 55, and 121 and heavy chain CDRs according to SEQ ID Nos: 66, 124, and 125.
  • One embodiment provides an anti-B7-H4 antibody-drug conjugate, or an antigen binding fragment thereof, containing a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 120 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 123.
  • One embodiment provides an anti-B7-H4 antibody-drug conjugate heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 128 [0074] Another embodiment provides an anti-B7-H4 antibody-drug conjugate heavy chain containing CDRs with amino acid sequences according to SEQ ID Nos: 9, 129, and 130.
  • an anti-B7-H4 antibody-drug conjugate, or antigen binding fragment thereof contains light chain CDRs according to SEQ ID Nos: 14, 15, and 16 and heavy chain CDRs according to SEQ ID Nos: 9, 129, 130.
  • One embodiment provides an anti-B7-H4 antibody-drug conjugate, or antigen binding fragment thereof, containing a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 13 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 128.
  • One embodiment provides an anti-B7-H4 antibody-drug conjugate, or an antigen binding fragment thereof having a light chain with an amino acid sequence according to any one of SEQ ID NO:3, 13, 46, 52, 85, 93, 102, 111, or 120.
  • Another embodiment provides an anti-B7-H4 antibody-drug conjugate, or an antigen binding fragment thereof having a heavy chain with an amino acid sequence according to any one of SEQ ID NO:8, 29, 49, 65, 89, 98, 107, 115, 123, or 128.
  • One embodiment provides an anti-B7-H4 antibody-drug conjugate, or an antigen binding fragment thereof having a light chain with an amino acid sequence according to any one of SEQ ID NOs: 3, 13, 46, 52, 85, 93, 102, 111, or 120, and a heavy chain with an amino acid sequence according to any one of SEQ ID Nos: 8, 29, 49, 65, 89, 98, 107, 115, 123, or 128..
  • an anti-B7-H4 antibody-drug conjugate, or antigen binding fragment thereof having three light chain CDRs with amino acid sequences that are selected from the group consisting of SEQ ID Nos: 4, 5, 6, 14, 15, 16, 47, 53, 54, 55, 86, 87, 94, 95, 96, 103, 104, 105, 112, 113, or 121.
  • One embodiment provides an anti-B7-H4 antibody-drug conjugate, or antigen binding fragment thereof having three heavy chain CDRs with amino acid sequences that are selected from the group consisting of SEQ ID Nos: 9, 10, 11, 30, 50, 66, 67, 68, 90, 91, 99, 100, 108, 109, 116, 117, 118, 124, 125, 129, or 130.
  • One embodiment provides an anti-B7-H4 antibody-drug conjugate, or antigen binding fragment thereof having three light chain CDRs with amino acid sequences that are selected from the group consisting of SEQ ID NOs: 4, 5, 6, 14, 15, 16, 47, 53, 54, 55, 86, 87, 94, 95, 96, 103, 104, 105, 112, 113, and 121, and three heavy chain CDRs with amino acid sequences that are selected from the group consisting of SEQ ID NOs: 9, 10, 11, 30, 50, 66, 67, 68, 90, 91, 99, 100, 108, 109, 116, 117, 118, 124, 125, 129, and 130.
  • Another embodiment provides an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof having a light chain variable domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 19, 20, 21, 22, or 23, and a heavy chain variable domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs:34, 35, 36, or 37.
  • One embodiment provides an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof having a light chain having an amino acid sequence according to any one of SEQ ID NOs: 24, 25, 26, 27, or 28, and a heavy chain having an amino acid sequence according to any one of SEQ ID NOs: 38, 39, 40, or 41.
  • Another embodiment provides an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof having a light chain having an amino acid sequence according to any one of SEQ ID NOs: 24, 25, 26, 27, or 28, and a heavy chain having an amino acid sequence according to any one of SEQ ID NOs: 42, 43, 44, or 45.
  • an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof having a light chain variable domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs:58, 59, 60, or 61, and a heavy chain variable domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 70, 71, 72, 73, or 74.
  • One embodiment provides an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof having a light chain having an amino acid sequence according to any one of SEQ ID NOs: 62, 63, or 64, and a heavy chain having an amino acid sequence according to any one of SEQ ID NOs: 75, 76, 77, 78, or 79.
  • One embodiment provides an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof having a light chain having an amino acid sequence according to any one of SEQ ID NOs: 62, 63, or 64, and a heavy chain having an amino acid sequence according to any one of SEQ ID NOs: 80, 81, 82, 83, or 84.
  • Another embodiment provides an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof having six complementarity determining regions (CDRs), wherein the CDRs include the three light chain CDRs of a polypeptide 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 comprising at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO4, 5, 6, 14, 15, 16, 49, 55, 56, 57, 88, 89, 96, 97, 97, 105, 106, 107, 114, 115, or 123, and the three heavy chain CDRs of a polypeptide selected from the group consisting of SEQ ID NO:9, 10, 11, 31, 52, 68, 69, 70, 92, 93,
  • Another embodiment provides an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof having two light chains and two heavy chains, wherein the two light chains include a polypeptide selected from the group consisting of SEQ ID NO: 24, 25, 26, 27, 28, 62, 63, or 64, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 24, 25, 26, 27, 28, 62, 63, or 64, and the two heavy chains include a polypeptide selected from the group consisting of SEQ ID NO: 38, 39, 40, 41, 75, 76, 77, 78, or 79, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 38, 39, 40, 41, 75, 76, 77, 78, or 79, and wherein the antibody or antigen binding fragment thereof binds to B7-H4.
  • One embodiment provides an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof having two light chains and two heavy chains, wherein the two light chains include a polypeptide selected from the group consisting of SEQ ID NO: 24, 25, 26, 27, 28, 62, 63, or 64, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 24, 25, 26, 27, 28, 62, 63, or 64, and the two heavy chains include a polypeptide selected from the group consisting of SEQ ID NO: 42, 43, 44, 45, 80, 81, 82, 83, or 84, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 42, 43, 44, 45, 80, 81, 82, 83, or 84, and wherein the antibody or antigen binding fragment thereof binds to B7-H4.
  • One embodiment provides an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof having two light chains and two heavy chains, wherein the two light chains include a polypeptide selected from the group consisting of SEQ ID NO: 24, 25, 26, 27, or 28, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 24, 25, 26, 27, or 28, and the two heavy chains include a polypeptide selected from the group consisting of SEQ ID NO: 38, 39, 40, or 41, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 38, 39, 40, or 41, and wherein the antibody or antigen binding fragment thereof binds to B7-H4.
  • the two light chains include a polypeptide selected from the group consisting of SEQ ID NO: 24, 25, 26, 27, or 28, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%
  • Another embodiment provides an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof having two light chains and two heavy chains, wherein the two light chains include a polypeptide selected from the group consisting of SEQ ID NO: 24, 25, 26, 27, or 28, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 24, 25, 26, 27, or 28, and the two heavy chains include a polypeptide selected from the group consisting of SEQ ID NO: 42, 43, 44, or 45, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 42, 43, 44, or 45, and wherein the antibody-drug conjugate or antigen binding fragment thereof binds to B7-H4.
  • the two light chains include a polypeptide selected from the group consisting of SEQ ID NO: 24, 25, 26, 27, or 28, or a variant thereof having at least 50%, 60%, 70%,
  • One embodiment provides an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof having two light chains and two heavy chains, wherein the two light chains include a polypeptide selected from the group consisting of SEQ ID NO: 62, 63, or 64, or a variant thereof comprising at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 62, 63, or 64, and the two heavy chains include a polypeptide selected from the group consisting of SEQ ID NO: 75, 76, 77, 78, or 79, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 75, 76, 77, 78, or 79, and wherein the antibodydrug conjugate or antigen binding fragment thereof binds to B7-H4.
  • Another embodiment provides an anti-B7-H4 antibody-drug conjugate or antigen binding fragment thereof having two light chains and two heavy chains, wherein the two light chains include a polypeptide selected from the group consisting of SEQ ID NO: 62, 63, or 64, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 62, 63, or 64, and the two heavy chains include a polypeptide selected from the group consisting of SEQ ID NO: 80, 81, 82, 83, or 84, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 80, 81, 82, 83, or 84, and wherein the antibody-drug conjugate or antigen binding fragment thereof binds to B7-H4.
  • an anti-B7-H4 antibody-drug conjugate wherein the antibody-drug conjugate comprises an anti-B7-H4 antibody comprising a light chain having an amino acid sequence according to SEQ ID NOs: 63, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 63 and a heavy chain having an amino acid sequence according to SEQ ID NOs: 78, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO:78, a linker and a cytotoxic payload, wherein the linker and payload comprises a structure represented by Compound 1.
  • Another embodiment provides an anti-B7-H4 antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-B7-H4 antibody comprising a light chain having an amino acid sequence according to SEQ ID NOs: 132, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 132 and a heavy chain having an amino acid sequence according to SEQ ID NOs: 133, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 133, a linker and a cytotoxic payload, wherein the linker and payload comprises a structure represented by Compound 1.
  • FIGS 1A-B Anti-tumor activity of B7-H4 antibody drug conjugate (ADC) in a PDX model of triple negative breast cancer.
  • ADC B7-H4 antibody drug conjugate
  • 1 A Mean tumor progression following 3 weekly doses of vehicle control, 3 weekly doses of B7-H4 ADC (1.5 mg/kg), or a single dose of 4.5 mg/kg B7-H4 ADC.
  • IB Growth of individual tumor in each of the 3 treatment groups. Mean tumor volume at endpoint was ⁇ 13mm 3 for the 4.5 mg/kg treatment group and ⁇ 40mm 3 for the 1.5 mg/kg treatment group.
  • FIG. 1 Anti-tumor activity of B7-H4 ADC in an orthotopic xenograft model of HER2+ breast cancer (HCC1569). Mean tumor progression is shown following no treatment or following two doses of B7-H4 ADC at 1.5, 3, or 6 mg/kg (Q21D, Days 14 and 35 post-inoculation, indicated by dashed lines).
  • FIG. 3 Anti-tumor activity of B7-H4 ADC in a transgenic xenograft model of colon cancer (HT29-hB7-H4). Growth of individual tumors is shown following no treatment or following a single dose administration (on Day 8 post-inoculation) of B7- H4 ADC at 1, 3, or 6 mg/kg. 6 of 7 mice in each of the 3 and 6 mg/kg treatment groups achieved complete response (CRs) with no measurable tumor for >3 weeks of consecutive measurements.
  • FIG. 4 Anti-tumor activity of B7-H4 ADC in xenograft models of colon cancer.
  • Four tumor models were analyzed in parallel. These four tumor models were composed (at the time of inoculation, “Day 0”) of a) 100% HT29 cells (B7-H4-negative), b) 25% (transgenic) HT29-hB7-H4 cells mixed with 75% HT29 cells, c) 75% HT29-hB7- H4 cells mixed with 25% HT29 cells, or d) 100% HT29-hB7-H4 cells.
  • B7-H4 ADC treatment had no measurable effect on B7-H4-negative HT29 tumors. 100% of tumors composed of 100% HT29-hB7H4 showed significant regression below baseline measurement. Similarly, 100% of mixed tumors composed of -75% B7-H4+ tumor cells showed significant regression below baseline measurement.
  • FIGS 5A-B Anti-tumor activity of B7-H4 ADC in a xenograft model of transgenic ovarian cancer (OVCAR3-B7-H4-OE).
  • 5A Mean tumor progression is shown following no treatment or following a single dose administration of B7-H4 ADC 6 mg/kg.
  • 5B Regression of tumors following a single dose administration of 1 or 3 mg/kg B7-H4 ADC. 100% complete responses (CR’s) were achieved in all treated tumors at 1 or 3 mg/kg, with no detectable tumor for 2 or more successive measurements.
  • FIGS 6A-B Anti-tumor activity of B7-H4 ADC in a xenograft model of ER/PR+ breast cancer (ZR-75-1).
  • 6A IHC stain for B7-H4 protein expression in a xenograft ZR-75-1 tumor in mice.
  • 6B Tumor progression in sham control mice or following 3 x Q7D doses of 1, 3, or 6 mg/kg B7-H4 ADC (on Days 1, 8, and 15 of treatment, as indicated by dashed lines). Tumor growth inhibition of >80% was achieved at all dose levels.
  • FIG. 7 Comparison of the relative binding of 3 anti-B7-H4 antibodies to recombinant human B7-H4 protein using an ELISA analysis. Relative binding is plotted for B7-H4 MAB (the parent antibody for B7-H4 ADC containing an identical variable domain), B7-H4 ADC (the antibody intermediate for B7-H4 ADC), and clone AZ-E02, an anti-human B7-H4 antibody published by AstraZeneca. Similar binding is seen for B7-H4 MAB and B7-H4 ADC, and comparable or superior binding of B7-H4 ADC compared to AZ-E02 is observed over a concentration gradient of antibody. [00105] Figure 8.
  • B7-H4 MAB the parent antibody for B7-H4 ADC containing an identical variable domain
  • B7-H4 ADC the antibody intermediate for B7-H4 ADC ADC
  • clone AZ-E02 an anti-human B7- H4 antibody published by AstraZeneca. Similar binding is seen for B7-H4 MAB and B7- H4 ADC, and comparable or superior binding of B7-H4 ADC compared to AZ-E02 is seen for each cell line. None of the antibodies show significant binding to B7-H4 negative cells.
  • FIG. 12A-E Comparing internalization of LNCB74 and other anti-B7-H4 antibodies.
  • 12A Flow cytometric measurement of LNCB74 antibody intermediate binding to B7-H4-negative and positive cell lines. Kinetics of internalization of LNCB74 antibody intermediate and comparator B7-H4 antibodies using Fc-glycan-conjugated- pHrodo-labeled antibodies on 12B) SKBR3 cells, 12C) SKBR3 knockout cells, 12D) OVCAR3 cells, 12E) HCC1569 cells. Relative internalization was measured using an Opera Phenix confocal imaging system.
  • FIG. 13 Cytotoxicity assays of LNCB74 in tumor cells from various indications. Dose-dependent killing of B7-H4+ SKBR3 breast cancer cells by LNCB74 in vitro. Low toxicity to a B7-H4- knockout derivative, and sensitivity of SKBR3 cells to free MMAE toxin are shown for reference.
  • FIG. 1 PK analysis (LC/MS/MS) of LNCB74 ADC and Total Antibody in rats following single dose administration at a dose of 3 mg/kg, i.v.
  • a panel of B7-H4-expressing cancer cell lines were assayed for sensitivity. EC50 values for B7-H4 ADC cytotoxicity are listed in Table 1. Where possible, values are shown for B7-H4-negative isogenic cell lines.
  • Serial dilutions of MMAE or B7-H4 ADC were prepared in complete media ranging from 666 nM (100 pg/mL) to 1.76 pM (256 pg/mL). Cells were plated in 96-well plates and incubated in the presence of test articles at 37°C. After 120 h, cell viability was measured using a CellTiter-Glo kit (Promega), according to the manufacturer’s instructions.
  • a molecule is said to be able to “immunospecifically bind” a second molecule if such binding exhibits the specificity and affinity of an antibody to its cognate antigen.
  • Antibodies are said to be capable of immunospecifically binding to a target region or conformation (“epitope”) of an antigen if such binding involves the antigen recognition site of the immunoglobulin molecule.
  • An antibody that immunospecifically binds to a particular antigen may bind to other antigens with lower affinity if the other antigen has some sequence or conformational similarity that is recognized by the antigen recognition site as determined by, e.g., immunoassays, BIACORE® assays, or other assays known in the art, but would not bind to a totally unrelated antigen. In some embodiments, however, antibodies (and their antigen binding fragments) will not cross-react with other antigens. Antibodies may also bind to other molecules in a way that is not immunospecific, such as to FcR receptors, by virtue of binding domains in other regions/domains of the molecule that do not involve the antigen recognition site, such as the Fc region.
  • a molecule is said to “bind” a second molecule if such binding exhibits the specificity and affinity of a receptor to its cognate binding ligand.
  • a molecule can be capable of binding to more than one other molecule.
  • antibody is intended to denote an immunoglobulin molecule that possesses a “variable region” antigen recognition site and include antigen-binding fragments of antibodies.
  • variable region is intended to distinguish such domain of the immunoglobulin from domains that are broadly shared by antibodies (such as an antibody Fc domain).
  • the variable region includes a “hypervariable region” whose residues are responsible for antigen binding.
  • scFv single-chain Fvs
  • sdFv single-chain Fvs
  • intrabodies diabodies, triabodies, tetrabodies, Bis-scFv, minibodies, Fab2, Fab3and anti -idiotypic (anti-Id) antibodies (including, e.g., anti-Id and anti-anti-Id antibodies to antibodies).
  • antibodies include immunoglobulin molecules of any type e.g., IgG, IgE, IgM, IgD, IgA and IgY), class e.g., IgGi, IgG?, IgGs, IgG 4 , IgAi and IgA2) or subclass.
  • the term “antigen binding fragment” of an antibody refers to one or more portions of an antibody that contain the antibody’s Complementarity Determining Regions (“CDRs”) and optionally the framework residues that include the antibody’s “variable region” antigen recognition site and exhibit an ability to immunospecifically bind antigen.
  • CDRs Complementarity Determining Regions
  • Such fragments include Fab', F(ab')2, Fv, single chain (ScFv), and mutants thereof, naturally occurring variants, and fusion proteins including the antibody’s “variable region” antigen recognition site and a heterologous protein e.g., a toxin, an antigen recognition site for a different antigen, an enzyme, a receptor or receptor ligand, etcf
  • fragment refers to a peptide or polypeptide including an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least 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.
  • modulate relates to a capacity to alter an effect, result, or activity (e.g., signal transduction).
  • modulation can be agonistic or antagonistic.
  • Antagonistic modulation can be partial (z.e., attenuating, but not abolishing) or it can completely abolish such activity (e.g., neutralizing).
  • Modulation can include internalization of a receptor following binding of an antibody or a reduction in expression of a receptor on the target cell.
  • Agonistic modulation can enhance or otherwise increase or enhance an activity (e.g., signal transduction).
  • such modulation can alter the nature of the interaction between a ligand and its cognate receptor so as to alter the nature of the elicited signal transduction.
  • the molecules can, by binding to the ligand or receptor, alter the ability of such molecules to bind to other ligands or receptors and thereby alter their overall activity.
  • such modulation will provide at least a 10% change in a measurable immune system activity, at least a 50% change in such activity, or at least a 2-fold, 5-fold, 10-fold, or at least a 100-fold change in such activity.
  • a molecule is said to have substantially the same immunospecificity and/or characteristic as another molecule, if such immunospecificities and characteristics are greater than 60% identical, greater than 70% identical, greater than 75% identical, greater than 80% identical, greater than 85% identical, greater than 90% identical, greater than 95% identical, or greater than 97% identical).
  • the “co-stimulatory” signals encompass positive costimulatory signals (e.g., signals that result in enhancing an activity) and negative costimulatory signals (e.g., signals that result in inhibiting an activity).
  • derivative refers to an antibody or antigen-binding fragment thereof that immunospecifically binds to the same target of a parent or reference antibody but which differs in amino acid sequence from the parent or reference antibody or antigen binding fragment thereof by including one, two, three, four, five or more amino acid substitutions, additions, deletions or modifications relative to the parent or reference antibody or antigen binding fragment thereof.
  • such derivatives will have substantially the same immunospecificity and/or characteristics, or the same immunospecificity and characteristics as the parent or reference antibody or antigen binding fragment thereof.
  • the amino acid substitutions or additions of such derivatives can include naturally occurring (z.e., DNA-encoded) or non-naturally occurring amino acid residues.
  • derivatives encompasses, for example, chimeric or humanized variants, as well as variants having altered CHI, hinge, CH2, CH3 or CH4 regions, so as to form, for example antibodies, etc., having variant Fc regions that exhibit enhanced or impaired effector or binding characteristics.
  • humanized antibody refers to an immunoglobulin including a human framework region and one or more CDR’s from a non-human (usually a mouse or rat) immunoglobulin.
  • the non-human immunoglobulin providing the CDR's is called the “donor” and the human immunoglobulin providing the framework is called the “acceptor.”
  • Constant regions need not be present, but if they are, they should be substantially identical to human immunoglobulin constant regions, i.e., at least about 85- 99%, or about 95% or more identical.
  • all parts of a humanized immunoglobulin, except possibly the CDR’s are substantially identical to corresponding parts of natural human immunoglobulin sequences.
  • valency refers to the number of binding sites available per molecule.
  • immunological humoral (antibody mediated) and/or a cellular (mediated by antigen-specific T cells or their secretion products) response directed against a peptide in a recipient patient.
  • a response can be an active response induced by administration of immunogen or a passive response induced by administration of antibody or primed T-cells.
  • a cellular immune response is elicited by the presentation of polypeptide epitopes in association with Class I or Class II MHC molecules to activate antigen specific CD4 + T helper cells and/or CD8 + cytotoxic T cells.
  • the response may also involve activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia cells, eosinophils, activation or recruitment of neutrophils or other components of innate immunity.
  • the presence of a cell-mediated immunological response can be determined by proliferation assays (CD4 + T cells) or CTL (cytotoxic T lymphocyte) assays.
  • the relative contributions of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be distinguished by separately isolating antibodies and T-cells from an immunized syngeneic animal and measuring protective or therapeutic effect in a second subject.
  • an “immunogenic agent” or “immunogen” is capable of inducing an immunological response against itself on administration to a mammal, optionally in conjunction with an adjuvant.
  • the terms “individual,” “host,” “subject,” and “patient” are used interchangeably herein, and refer to a mammal, including, but not limited to, humans, rodents, such as mice and rats, and other laboratory animals.
  • variant refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide but retains essential properties.
  • a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.
  • a variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions).
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
  • a variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.
  • the hydropathic index of amino acids can be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a polypeptide is generally understood in the art. It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still result in a polypeptide with similar biological activity. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics.
  • the relative hydropathic character of the amino acid determines the secondary structure of the resultant polypeptide, which in turn defines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and cofactors. It is known in the art that an amino acid can be substituted by another amino acid having a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, the substitution of amino acids whose hydropathic indices are within ⁇ 2 is preferred, those within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biological equivalent, and in particular, an immunologically equivalent polypeptide.
  • substitution of amino acids whose hydrophilicity values are within ⁇ 2 is preferred, those within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions that take various foregoing characteristics into consideration are well known to those of skill in the art and include (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gin, His), (Asp: Glu, Cys, Ser), (Gin: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gin), (He: Leu, Vai), (Leu: He, Vai), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Trp: Tyr), (Tyr: Trp, Phe), and (Vai: lie, Leu).
  • Embodiments of this disclosure thus contemplate functional or biological equivalents of a polypeptide as set forth above.
  • embodiments of the polypeptides can include variants having about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the polypeptide of interest.
  • percent (%) sequence identity is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical with the nucleotides or amino acids in a reference nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.
  • % sequence identity of a given nucleotides or amino acids sequence C to, with, or against a given nucleic acid sequence D is calculated as follows:
  • W is the number of nucleotides or amino acids scored as identical matches by the sequence alignment program in that program’s alignment of C and D
  • Z is the total number of nucleotides or amino acids in D. It will be appreciated that where the length of sequence C is not equal to the length of sequence D, the % sequence identity of C to D will not equal the % sequence identity of D to C.
  • the term “pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water and emulsions such as an oil/water or water/oil emulsion, and various types of wetting agents.
  • B7-H4 expression found in numerous tumor tissues, for example, human ovarian cancers, points to a key role for B7-H4 in mediating immune suppression.
  • Tumor-Associated Macrophages (TAMs) expressing B7-H4 have been found to suppress tumor-associated antigen-specific T cell immunity (Kryczek, I. et al. (2006) "B7-H4 Expression Identifies A Novel Suppressive Macrophage Population In Human Ovarian Carcinoma," J. Exp. Med. 203(4):871-881).
  • TAMs Tumor-Associated Macrophages
  • B7-H4 expressed on TAMs is associated with poor patient outcome (Kryczek, I. et al. (2006) "B7-H4 Expression Identifies A Novel Suppressive Macrophage Population In Human Ovarian Carcinoma," J. Exp. Med. 203(4):871-881). Additionally, B7-H4 can be expressed on myeloid derived suppressor cells (MDSCs) where it may exert an immune suppressive effect in viral infection (Garg, A et al. (2017) “Human Immunodeficiency Virus Type-1 Myeloid Derived Suppressor Cells Inhibit Cytomegalovirus Inflammation through Interleukin-27 and B7-H4” Sci. Rep. Mar 24;7:44485).
  • MDSCs myeloid derived suppressor cells
  • B7-H4 expression in the tumor microenvironment was associated with increased infiltration of MDSCs (Vanderstraeten, A. et al. (2014) “Mapping the immunosuppressive environment in uterine tumors: implications for immunotherapy,” Cancer Immunol. Immunother. Jun;63(6):545-57). Therefore, B7-H4 may be expressed on tumor cells, TAM and/or MDSCs where it may exert immune suppressive signaling in cancer.
  • Neutrophils are a major component of the host innate defense against infection and also contribute to autoimmune pathogenesis and chronic inflammation. During infection, neutrophils rapidly migrate to sites of inflammation, become activated, and initiate a cascade of defense mechanisms including phagocytosis, killing, and degradation of microorganisms by antimicrobial and proteolytic proteins, along with the generation of reactive oxygen species. Neutrophils also participate in tissue breakdown, remodeling, wound healing, and modulation of other inflammatory and adaptive immune components. Due to their short life span, neutrophils have to be resupplied continuously during infection and inflammation by expansion from myeloid progenitor cells in the bone marrow.
  • the polypeptides can include an amino acid sequence of full-length B7-H4, or a fragment or variant thereof, or a fusion protein thereof.
  • One embodiment provides human B7-H4 proteins or polypeptides thereof Sequences for human B7-H4 are known in the art. For example, a consensus sequence for B7-H4 is
  • mouse B7-H4 proteins and polypeptides are known in the art.
  • a consensus sequence for mouse B7-H4 is MASLGQIIFWSIINIIIILAGAIALIIGFGISGKHFITVTTFTSAGNIGEDGTLSCTFEP DIKLNGIVIQWLKEGIKGLVHEFKEGKDDLSQQHEMFRGRTAVFADQVVVGNAS LRLKNVQLTDAGTYTCYIRTSKGKGNANLEYKTGAFSMPEINVDYNASSESLRC EAPRWFPQPTVAWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNT YSCMIENDIAKATGDIKVTDSEVKRRSQLQLLNSGPSPCVFSSAFVAGWALLSLS CCLMLR
  • Antibody-drug conjugates of the invention are comprised of an anti-B7-H4 antibody, a cytotoxin and a linker.
  • Antibodies are comprised of light and heavy chains. Each chain is comprised of a variable domain and a constant domain. The constant domain of the light chain can be kappa or lamba sequences as are well known in the art.
  • the heavy chain constant domain is comprised of a first constant domain, a hinge and an Fc domain.
  • Antibodies come in 5 classes, as determined by their sequences. The classes are IgA, IgD, IgE, IgG and IgM.
  • the IgG antibodies comprise 4 subclasses, including IgGl, IgG2, IgG3 and IgG4.
  • the antibody-drug conjugates of the invention are typically of the IgG class and more typically either IgGl or IgG4.
  • the constant domain sequence can be wild type or contain specific mutations to reduce the binding to Fc gamma receptors. These mutations are well known in the art, but include mutating the glycosylation site (e.g., N297Q), mutating the Fc gamma receptor binding site (L234A and L235A or L234F, L235E and P331S).
  • the constant domain sequence can be wild type or contain a specific mutation (S228P) to reduce chain exchange as is well known in the art.
  • One embodiment provides a murine monoclonal antibody or antigen binding fragment thereof that has a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence: DIVMTOSHKFMSTSVGDRVSITCKASODVRTAVAWYOOKPGOSPKLLIYSTSYR YTGVPDRFTGSGSGTEFTFTISSVQAEDLAVYYCOQYYVTPLTFGAGTKLELK (SEQ ID NO:3) that specifically binds to B7-H4.
  • the CDRs are of SEQ ID NO:3 are bolded and underlined and are:
  • Another embodiment provides a nucleic acid that encodes the light chain (SEQ ID NO:3).
  • An exemplary nucleic acid that encodes light chain is GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACA GGGTCAGTATCACCTGCAAGGCCAGTCAGGATGTGAGAACTGCTGTAGCCTG GTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTACTCGACATCCT ACCGGTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGA ATTCACTTTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACT
  • One embodiment provides a murine monoclonal antibody or antigen binding fragment thereof that has a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence: EVOLOQSGTVLARPGASVKMSCKASGYTFTSYWMHWIKORPGOGLEWIGAIYP GNSDTKYNOKFKDKAKLTAVTSASTAYMELSSLTNEDSAVYYCTSTVRNVMD YWGQGTSVTVSS (SEQ ID NO: 8) and specifically binds to B7-H4.
  • One embodiment provides an antibody, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain having CDRs according to SEQ ID Nos: 4, 5, and 6 and specifically binds to B7-H4.
  • One embodiment provides an antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having a heavy chain having CDRs according to SEQ ID Nos: 9, 10, and 11 and specifically binds to B7-H4.
  • Another embodiment provides a nucleic acid that encodes the light chain
  • An exemplary nucleic acid that encodes the light chain is [00180] GAAATCCAGATGACCCAGTCTCCATCCTCTATGTCTGCATCTCTGG GAGACAGAATAACCATCACTTGCCAGGCAACTCAAGACATTGTTAAGAGTTT AAACTGGTATCAACAAAAACCAGGGAAACCCCCTTCATTCCTGATCTATTATA CAGCTCAACTGGCAGAAGGGGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGG
  • One embodiment provides an anti-B7-H4 monoclonal antibody or antigen binding fragment thereof that has a humanized light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with one of the following amino acid sequences:
  • One embodiment provides a murine monoclonal antibody or antigen binding fragment thereof that has a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence: EVQLQQSGTVLARPGASVKMSCKVSGYPFTSYWMHWVKQRPGQGLEWIGAIY PGKSDTEYNPNFKGKAKLTAVTSATTAYMELSSLTNEDSAVYYCTSTWTHYFD YWGQGTTLTVSS (SEQ ID NO:29) and specifically binds to B7-H4.
  • the CDRs are of SEQ ID NO:29 are bolded and underlined and are:
  • Another embodiment provides a nucleic acid encoding heavy chain (SEQ ID NO:29).
  • An exemplary nucleic acid that encodes heavy chain is GAGGTTCAGCTCCAGCAGTCTGGGACTGTTCTGGCAAGGCCTGGGGCTTCAG TGAAGATGTCCTGCAAGGTTTCTGGCTACCCCTTTACCAGCTACTGGATGCAC TGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGCGCTATTTATC CTGGAAAAAGTGACACTGAATACAACCCGAACTTCAAGGGCAAGGCCAAACT GACTGCAGTCACATCTGCCACCACTGCCTACATGGAGCTCAGCAGCCTGACA AATGAGGACTCTGCGGTCTATTACTGTACAAGTACCTGGACCCACTACTTTGA CTACTGGGGCCAAGGCACCACTCTCACAGTCCTCA (SEQ ID NO:31).
  • One embodiment provides a murine monoclonal antibody or antigen binding fragment thereof that has a heavy chain constant domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:
  • SEQ ID NO:32 contains L234A and L235A (AlaAla) to reduce and/or eliminate binding of the
  • underlined and bolded amino acids represent amino acids that differ from the L234F, L235E, P331S (FES) mutant sequence shown below.
  • mutant heavy chain constant domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA
  • One embodiment provides an anti-B7-H4 monoclonal antibody or antigen binding fragment thereof that has a humanized heavy chain variable domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with one of the following amino acid sequences:
  • One embodiment provides an anti-B7-H4 monoclonal antibody or antigen binding fragment thereof that has a humanized heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with one of the following amino acid sequences: Heavy chain B1H1 Variant 1 (AlaAla):
  • One embodiment provides an anti-B7H4 antibody-drug conjugate, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain having CDRs according to SEQ ID Nos: 14, 15, and 16.
  • One embodiment provides an anti-B7H4 antibody-drug conjugate, preferably a monoclonal antibody, or an antigen binding fragment thereof having a heavy chain containing CDRs according to SEQ ID Nos: 9, 11, and 30.
  • One embodiment provides an anti-B7H4 antibody-drug conjugate, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain containing CDRs according to SEQ ID Nos: 14, 15, and 16 and a heavy chain containing CDRs according to SEQ ID Nos: 9, 11, and 30.
  • One embodiment provides an anti-B7H4 antibody-drug conjugate, preferably a monoclonal antibody, or an antigen binding fragment thereof having a light chain at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 13 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:29.
  • Another embodiment provides an anti-B7H4 antibody-drug conjugate preferably a monoclonal antibody, or an antigen binding fragment thereof having a light chain variable domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 19, 20, 21, 22, or 23, and a heavy chain variable domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs:34, 35, 36, or 37. .
  • One embodiment provides an anti-B7H4 antibody-drug conjugate or antigen binding fragment thereof having a light chain having an amino acid sequence according to any one of SEQ ID NOs: 24, 25, 26, 27, or 28, and a heavy chain having an amino acid sequence according to any one of SEQ ID NOs: 38, 39, 40, or 41.
  • Another embodiment provides an anti-B7H4 antibody-drug conjugate or antigen binding fragment thereof having a light chain having an amino acid sequence according to any one of SEQ ID NOs: 24, 25, 26, 27, or 28, and a heavy chain having an amino acid sequence according to any one of SEQ ID NOs: 42, 43, 44, or 45.
  • One embodiment provides an anti-B7H4 antibody-drug conjugate or antigen binding fragment thereof having two light chains and two heavy chains, wherein the two light chains include a polypeptide selected from the group consisting of SEQ ID NO: 24, 25, 26, 27, or 28, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 24, 25, 26, 27, or 28, and the two heavy chains include a polypeptide selected from the group consisting of SEQ ID NO: 38, 39, 40, or 41, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 38, 39, 40, or 41, and wherein the antibody or antigen binding fragment thereof binds to B7-H4.
  • the two light chains include a polypeptide selected from the group consisting of SEQ ID NO: 24, 25, 26, 27, or 28, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%,
  • Another embodiment provides an antibody-drug conjugate or antigen binding fragment thereof having two light chains and two heavy chains, wherein the two light chains include a polypeptide selected from the group consisting of SEQ ID NO: 24, 25, 26, 27, or 28, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 24, 25, 26, 27, or 28, and the two heavy chains include a polypeptide selected from the group consisting of SEQ ID NO: 42, 43, 44, or 45, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 42, 43, 44, or 45, and wherein the antibody or antigen binding fragment thereof binds to B7-H4.
  • the two light chains include a polypeptide selected from the group consisting of SEQ ID NO: 24, 25, 26, 27, or 28, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%,
  • a murine monoclonal antibody is produced by hybridoma clone B1H3 and contains two light chains and two heavy chains and specifically binds B7-H4.
  • i. Light Chain One embodiment provides an anti-B7-H4 murine monoclonal antibody or antigen binding fragment thereof that has a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:
  • the CDRs are of SEQ ID NO:46 are bolded and underlined and are:
  • One embodiment provides an anti-B7-H4 murine monoclonal antibody or antigen binding fragment thereof that has a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:
  • One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain containing CDRs according to SEQ ID Nos: 14, 16, and 47.
  • One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having a light chain at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:46 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:49.
  • Another embodiment provides a nucleic acid that encodes the light chain
  • Humanized B1H10 VL2 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHS GVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTLPWTFGQGTKLEIK (SEQ ID NO:59)
  • One embodiment provides an anti-B7H4 monoclonal antibody or antigen binding fragment thereof that has a humanized light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with one of the following amino acid sequences:
  • Another embodiment provides a nucleic acid encoding heavy chain (SEQ ID NO:65).
  • SEQ ID NO:65 An exemplary nucleic acid that encodes heavy chain is
  • One embodiment provides an anti-B7H4 antibody-drug conjugate or antigenbinding fragment thereof that has a humanized heavy chain variable domain variant having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with one of the following amino acid sequences:
  • One embodiment provides an anti-B7H4 monoclonal antibody or antigen binding fragment thereof that has a humanized heavy chain variant having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the one of the following amino acid sequences:
  • One embodiment provides an anti-B7H4 monoclonal antibody or antigen binding fragment thereof that has a humanized mutant heavy chain variant having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with one of the following amino acid sequences:
  • One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain containing CDRs according to SEQ ID Nos: 53, 54, or 55.
  • One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having a heavy chain containing CDRs according to SEQ ID Nos: 66, 67, or 68.
  • One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain containing CDRs according to SEQ ID Nos: 53, 54, or 55, and a heavy chain containing CDRs according to SEQ ID Nos: 66, 67, or 68.
  • One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having a light chain at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:52 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 65.
  • Another embodiment provides an anti-B7H4 antibody preferably a monoclonal antibody, or an antigen binding fragment thereof having a light chain variable domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs:58, 59, 60, or 61, and a heavy chain variable domain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs:70, 71, 72, 73, or 74.
  • One embodiment provides an anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence according to any one of SEQ ID NOs: 62, 63, or 64, and a heavy chain having an amino acid sequence according to any one of SEQ ID NOs: 75, 76, 77, 78, or 79.
  • Another embodiment provides an anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence according to any one of SEQ ID NOs: 62, 63, or 64, and a heavy chain having an amino acid sequence according to any one of SEQ ID NOs: 80, 81, 82, 83, or 84.
  • One embodiment provides an anti-B7H4 antibody or antigen binding fragment thereof having two light chains and two heavy chains, wherein the two light chains include a polypeptide selected from the group consisting of SEQ ID NO: 62, 63, or 64, or a variant thereof comprising at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 62, 63, or 64, and the two heavy chains include a polypeptide selected from the group consisting of SEQ ID NO: 75, 76, 77, 78, or 79, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 75, 76, 77, 78, or 79.
  • Another embodiment provides an anti-B7H4 antibody or antigen binding fragment thereof having two light chains and two heavy chains, wherein the two light chains include a polypeptide selected from the group consisting of SEQ ID NO: 62, 63, or 64, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 62, 63, or 64, and the two heavy chains include a polypeptide selected from the group consisting of SEQ ID NO: 80, 81, 82, 83, or 84, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 80, 81, 82, 83, or 84.
  • an anti-B7H4 murine monoclonal antibody is produced by hybridoma clone B2E6 and contains two light chains and two heavy chains. i. Light Chain
  • One embodiment provides an anti-B7H4 murine monoclonal antibody or antigen binding fragment thereof that has a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:
  • ETVMTQSHKIMSTSVGDRVTITCKASODVRTAVAWYQOKPGQSPKLLISSASYO YTGVPDRFTGSGSGTDFTFTISSLQAEDLAVYYCHQYYNTPLTFGAGTKLELR (SEQ ID NO: 85).
  • the CDRs are of SEQ ID NO:85 are bolded and underlined and are:
  • Another embodiment provides a nucleic acid that encodes the light chain
  • One embodiment provides an anti-B7H4 murine monoclonal antibody or antigen binding fragment thereof that has a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:
  • the CDRs are of SEQ ID NO:89 are bolded and underlined and are:
  • Another embodiment provides a nucleic acid encoding heavy chain (SEQ ID NO:89).
  • An exemplary nucleic acid that encodes heavy chain is GAGGTTCAGCTCCAGCAGTCTGGGACTGTGCTGGCAAGGCCTGGGGCTTCAG TGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATGCAC TGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGCGCTATTTATC
  • One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain having CDRs according to SEQ ID Nos: 4, 86, and 87
  • One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having a heavy chain having CDRs according to SEQ ID Nos: 9, 90, and 91.
  • One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain containing CDRs according to SEQ ID Nos: 4, 86, and 87, and a heavy chain containing CDRs according to SEQ ID Nos: 9, 90, and 91.
  • One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having a light chain at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:85 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 89.
  • an anti-B7H4 murine monoclonal antibody is produced by hybridoma clone B4B3 and contains two light chains and two heavy chains. i. Light Chain
  • One embodiment provides an anti-B7H4 murine monoclonal antibody or antigen binding fragment thereof that has a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:
  • Another embodiment provides a nucleic acid that encodes the light chain (SEQ ID NO:93).
  • An exemplary nucleic acid that encodes light chain is GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGGTCA AGCCTCCATCTCTTGCAGATCTAGTCAGATCATTGTACATAGTAATGGAAACA CCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATC
  • One embodiment provides an anti-B7H4 murine monoclonal antibody or antigen binding fragment thereof that has a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence: QVQLQQPGAELVKPGASVKLSCKASGYTFISYWMHWVKQRPGQGLEWIGEIDP SDSYTYYNQKFKGKATLTVDKS S ST AYMQLS SLTSEDS AVYYCARRKTWDWY FD VWGAGTT VT VS S (SEQ ID NO:98).
  • Another embodiment provides a nucleic acid encoding heavy chain (SEQ ID NO:98).
  • An exemplary nucleic acid that encodes heavy chain is CAGGTCCAGCTGCAGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAG TGAAGCTGTCCTGCAAGGCTTCTGGATACACCTTCATTAGCTACTGGATGCAC TGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATCGGAGAGATTGATC CTTCTGATAGTTATACTTACTACAATCAAAAGTTCAAGGGCAAGGCCACATTG ACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACAT CTGAGGACTCTGCGGTCTATTACTGTGCAAGAAGGAAAACCTGGGACTGGTA CTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO:101).
  • One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain containing CDRs according to SEQ ID Nos: 94, 95, and 96.
  • One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having a heavy chain containing CDRs according to SEQ ID Nos: 9, 99, and 100.
  • One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain containing CDRs according to SEQ ID Nos: 94, 95, and 96, and a heavy chain containing CDRs according to SEQ ID Nos: 9, 99, and 100.
  • an anti-B7H4 antibody preferably a monoclonal antibody, or an antigen binding fragment thereof having a light chain at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:93 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 98.
  • B4E11 Sequences [00260]
  • an anti-B7H4 murine monoclonal antibody is produced by hybridoma clone B4E11 and contains two light chains and two heavy chains.
  • One embodiment provides an anti-B7H4 murine monoclonal antibody or antigen binding fragment thereof that has a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:
  • Another embodiment provides a nucleic acid that encodes the light chain (SEQ ID NO: 102).
  • SEQ ID NO: 102 An exemplary nucleic acid that encodes light chain is
  • One embodiment provides an anti-B7H4 murine monoclonal antibody or antigen binding fragment thereof that has a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:
  • the CDRs are of SEQ ID NO: 107 are bolded and underlined and are:
  • Another embodiment provides a nucleic acid encoding heavy chain (SEQ ID NO: 107).
  • An exemplary nucleic acid that encodes heavy chain is GAGGTTCAGCTCCAGCAGTCTGGGACTGTGCTGGCAAGGCCTGGGGCTTCAG TGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACCAGCTACTGGATGCAC TGGGTAAAAGAGAGGCCTGGACAGGGTCTGGAATGGATTGGCGCTATTTATC CTGGAGATAGTGATACTAGGTATAATCAGAAGTTCAAGGGCAGGGCCAAACT GACTGCAGTCACATCTGCCAACACTGCCTACATGGAGCTCAGCAGCCTGACA AATGATGACTCTGCGGTCTTCTACTGTACATGTACTACGGCTGGTGTTTTGGA CTACTGGGGTCAAGGAACCTCAGTCACCGTCCTCA (SEQ ID NO: 110).
  • One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain containing CDRs according to SEQ ID Nos: 103, 104, and 105.
  • One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having a heavy chain containing CDRs according to SEQ ID Nos: 9, 108, and 109.
  • One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain containing CDRs according to SEQ ID Nos: 103, 104, and 105, and a heavy chain containing CDRs according to SEQ ID Nos: 9, 108, and 109.
  • One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having a light chain at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 102 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 107.
  • an anti-B7H4 murine monoclonal antibody is produced by hybridoma clone B6C8 and contains two light chains and two heavy chains. i. Light Chain
  • One embodiment provides an anti-B7H4 murine monoclonal antibody or antigen binding fragment thereof that has a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:
  • Another embodiment provides a nucleic acid that encodes the light chain (SEQ ID NO: 111).
  • SEQ ID NO: 111 An exemplary nucleic acid that encodes light chain (SEQ ID NO: 111) is
  • One embodiment provides an anti-B7H4 murine monoclonal antibody or antigen binding fragment thereof that has a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:
  • CDR3 YGLFYGNDGYAMDH (SEQ ID NO: 118) [00281] Another embodiment provides a nucleic acid encoding heavy chain (SEQ ID NO:115).
  • An exemplary nucleic acid that encodes heavy chain is CAGGTCCAACTGCAGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAG TGAAGCTGTCCTGCAAGGCTTCTGGCTACTCTTTCACCAGCTACTGGATGAAC TGGGTGAAGCAGAGGCCTGGACGAGGCCTCGAGTGGATTGGAAGGATTCATC CTTCTGATAGTGAAACTCACTACAATCAAAAGTTCAAGAGCAAGGCCACACT GACTGTAGACAAATCCTCCAGCACAGCCTACATCCAACTCAGCAGCCTGACA TCTGAGGACTCTGCGGTCTATTTTTGTGCAAGATACGGGCTCTTCTATGGTAA CGACGGATATGCTATGGACCACTGGGGTCAAGGAACCTCAG (SEQ ID NO: 119).
  • One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain containing CDRs according to SEQ ID Nos: 97, 116 and 117.
  • One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having a heavy chain containing CDRs according to SEQ ID Nos: 118, 119, and 120.
  • One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain containing CDRs according to SEQ ID Nos 97, 114, and 115, and a heavy chain containing CDRs according to SEQ ID Nos: 118, 119, and 120.
  • One embodiment provides an antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having a light chain at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 113 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 117.
  • an antibody preferably a monoclonal antibody, or an antigen binding fragment thereof having a light chain at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 113 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 117.
  • an anti-B7H4 murine monoclonal antibody is produced by hybridoma clone B9H1 and contains two light chains and two heavy chains. i. Light Chain
  • One embodiment provides an anti-B7H4 murine monoclonal antibody or antigen binding fragment thereof that has a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence: DIQMTQTTSSLSASLGDRVTISCRASODISFYLNWYQQKPDGTVKLLIYYTSRLH SGVPSRFSGSGSGTDYSLTISNLEOEDIATYFCOQGNTLPWTFGGGTKLEIK (SEQ ID NO: 120).
  • Another embodiment provides a nucleic acid that encodes the light chain
  • SEQ ID NO: 120 An exemplary nucleic acid that encodes light chain (SEQ ID NO: 120) is
  • One embodiment provides an anti-B7H4 murine monoclonal antibody or antigen binding fragment thereof that has a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:
  • Another embodiment provides a nucleic acid encoding heavy chain (SEQ ID NO: 1]
  • An exemplary nucleic acid that encodes heavy chain (SEQ ID NO: 125) is
  • One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain containing CDRs according to SEQ ID Nos: 54, 55, and 121.
  • One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having a heavy chain containing CDRs according to SEQ ID Nos: 66, 124, and 125.
  • One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain containing CDRs according to SEQ ID Nos: 54, 55, and 121, and a heavy chain containing CDRs according to SEQ ID Nos: 66, 124, and 125.
  • One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having a light chain at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 120 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 123.
  • an anti-B7H4 murine monoclonal antibody is produced by hybridoma clone B10D7 and contains two light chains and two heavy chains. i. Light Chain
  • EIQMTQSPSSMSASLGDRITITCOATODIVKSLNWYQOKPGKPPSFLIYYTAQLA EGVPSRFSGSGSGSDYSLTISNLESEDFADYYCLQFYEFPPTFGGGTKLEIK (SEQ ID NO: 13).
  • Another embodiment provides a nucleic acid that encodes the light chain (SEQ ID NO: 13).
  • SEQ ID NO: 13 An exemplary nucleic acid that encodes light chain is
  • One embodiment provides an anti-B7H4 murine monoclonal antibody or antigen binding fragment thereof that has a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:
  • Another embodiment provides a nucleic acid encoding heavy chain (SEQ ID NO: 128).
  • An exemplary nucleic acid that encodes heavy chain is GAGGTTCAGCTCCAGCAGTCTGGGACTGTGCTGGCAAGGCCTGGGGCTTCAG TGAAGATGTCCTGCAAGGCTTCTGGCTACCCCTTTACCAGCTACTGGATGCAC TGGGTAAAGCAGAGGCCTGGACAGGGTCTGGAATGGATTGGCGCTATTTATC
  • One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain containing CDRs according to SEQ ID Nos: 14, 15, and 16.
  • One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having a heavy chain containing CDRs according to SEQ ID Nos: 9, 129, and 130.
  • One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or antigen binding fragment thereof that has a light chain containing CDRs according to SEQ ID Nos: 14, 15, and 16 and a heavy chain containing CDRs according to SEQ ID Nos: 9, 129, and 130.
  • One embodiment provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having a light chain at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 13 and a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 128.
  • LNCB74 a monoclonal antibody comprising two light chains and two heavy chains and specifically binds to and is internalized by B7-H4 expressing cells.
  • B7-H4 protein is highly expressed in multiple tumor indications, including breast, ovarian, and endometrial cancers. B7-H4 expression is low and limited in normal healthy human tissues, providing a potential broad therapeutic index for a B7-H4 targeting antibody drug conjugate (ADC).
  • ADC antibody drug conjugate
  • LNCB74 is a B7-H4 antibody conjugated to the microtubule disrupting payload monomethyl auristatin E (MMAE) with a drug-to-antibody ratio of 4 (DAR4).
  • the ADC employs a glucuronidase-cleavable, site-specific linkage conjugated to an engineered cysteine in the antibody light chain via LigaChem Biosciences’ ConjuAllTM technology to increase stability in circulation, improve selective release of payload in tumor cells, and reduce payload release in non-tumor cells.
  • LNCB74 incorporates an Fc mutation to minimize binding and uptake of LNCB74 by Fc receptor expressing immune cells.
  • LNCB74 a monoclonal antibody or antigen binding fragment thereof that has a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:
  • LNCB74 a monoclonal antibody or antigen binding fragment thereof that has a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:
  • One embodiment provides a comparator antibody comprising two light chains and two heavy chains.
  • the Fc portion of this comparator is wild-type, unlike LNCB74 which contains a LAL A mutation which functions to reduce Fc-driven immune cell engagement.
  • One embodiment provides a comparator antibody or antigen binding fragment thereof that has a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:
  • One embodiment provides a comparator antibody or antigen binding fragment thereof that has a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following amino acid sequence:
  • the CDRs of SEQ ID NO: 138 are bolded and underlined and are: CDR1 GSIKSGSYYWG (SEQ ID NO: 139); CDR2 NIYYSGSTYYNPSLRS (SEQ ID NO: 140); and CDR3 AREGSYPNQFDP (SEQ ID NO: 141).
  • the disclosed B7-H4-binding molecules can be antibodies or antigen binding fragments thereof.
  • the disclosed antibodies and antigen binding fragments thereof include whole immunoglobulin (i.e., an intact antibody) of any class, fragments thereof, and synthetic proteins containing at least the antigen binding variable domain of an antibody.
  • the disclosed molecule contains both an antibody light chain as well as at least the variable domain of an antibody heavy chain.
  • such molecules can further include one or more of the CHi, hinge, CEE, CH3, and CH4 regions of the heavy chain (especially, the CHi and hinge regions, or the CHi, hinge and CEE regions, or the CHi, hinge, CEE and CEE regions).
  • the antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgGi, IgG?, IgGs and IgG4.
  • the constant domain is a complement fixing constant domain where it is desired that the antibody exhibit cytotoxic activity, and the class is typically IgGi. In other embodiments, where such cytotoxic activity is not desirable, the constant domain can be of the IgG? or IgG 4 class.
  • the antibody can include sequences from more than one class or isotype, and selecting particular constant domains to optimize desired effector functions is within the ordinary skill in the art.
  • variable domains differ in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen.
  • variable domains of antibodies typically concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable domains.
  • CDRs complementarity determining regions
  • FR framework
  • the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure.
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies.
  • fragments of antibodies which have bioactivity.
  • the fragments whether attached to other sequences or not, include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment.
  • a single chain antibody can be created by fusing together the variable domains of the heavy and light chains using a short peptide linker, thereby reconstituting an antigen binding site on a single molecule.
  • Single-chain antibody variable fragments scFvs
  • the linker is chosen to permit the heavy chain and light chain to bind together in their proper conformational orientation.
  • Divalent single-chain variable fragments can be engineered by linking two scFvs. This can be done by producing a single peptide chain with two VH and two VL regions, yielding tandem scFvs. ScFvs can also be designed with linker peptides that are too short for the two variable regions to fold together (about five amino acids), forcing scFvs to dimerize. This type is known as diabodies. Diabodies have been shown to have dissociation constants up to 40-fold lower than corresponding scFvs, meaning that they have a much higher affinity to their target. Still shorter linkers (one or two amino acids) lead to the formation of trimers (triabodies or tribodies). Tetrabodies have also been produced. They exhibit an even higher affinity to their targets than diabodies.
  • One embodiment provides a monoclonal antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules.
  • Monoclonal antibodies include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity.
  • Antibodies and antigen binding fragments thereof the specifically bind to human B7-H4.
  • One embodiment provides antibodies produced by a hybridoma from the group consisting of B1A1, B1H1, B1H3, B1H10, B2E6, B4B2, B4E11, B6C8, B9H1, and B10D7. a. Antibodies
  • the immunomodulatory agent can be an antibody. Suitable antibodies are known in the art or can be prepared by one of skill in the art. Nucleic acid and polypeptide sequences for B7-H4 are known in the art, and exemplary protein sequences are provided above. The sequences can be used, as discussed in more detail below, by one of skill in the art to prepare an antibody or antigen binding fragment thereof specific for B7-H4. The antibody, or antigen binding fragment, therefore, can be an agonist or antagonist of B7-H4 signaling.
  • the activity (i.e., agonist or antagonist) of an antibody or antigen binding fragment thereof that is specific for B7-H4 can be determined using functional assays that are known in the art, and include the assays discussed below.
  • the assays include determining if the antibody or antigen binding fragment thereof increases (i.e., agonist) or decreases (i.e., antagonist) signaling through B7-H4. Because B7-H4 signal transduction results in a suppressive immune response, agonizing B7-H4 causes a suppressed or reduced immune response. Antagonizing B7-H4 signaling inhibits the immune suppressive response resulting in an overall increase in immune response.
  • the disclosed antibodies and antigen binding fragments thereof immunospecifically bind to B7-H4. In some embodiments, the antibody binds to an extracellular domain of B7-H4.
  • molecules are provided that can immunospecifically bind to B7-H4:
  • VI arrayed on the surface of a live cell, wherein the cell is a tumor cell;
  • VII combinations of I-IV and VI;
  • IX arrayed on the surface of a live myeloid or lymphoid derived cancer cells (AML or ALL) and enhances apoptosis and differentiation resulting in reduced selfrenewal of cancer stem cells.
  • the molecules are capable of inducing antibody dependent cell cytotoxicity (ADCC), complement dependent cytotoxicity (CDC) or cellular apoptosis through other mechanisms, of B7-H4 expressing cell.
  • ADCC antibody dependent cell cytotoxicity
  • CDC complement dependent cytotoxicity
  • cellular apoptosis through other mechanisms, of B7-H4 expressing cell.
  • an antibody or antigen binding fragment thereof that specifically binds to B7-H4
  • purified proteins, polypeptides, fragments, fusions, or epitopes to B7-H4 or polypeptides expressed from nucleic acid sequences thereof can be used.
  • the antibodies or antigen binding fragments thereof can be prepared using any suitable methods known in the art such as those discussed in more detail below. i. Human and Humanized Antibodies
  • non-human antibodies e.g., those derived from mice, rats, or rabbits
  • many non-human antibodies are naturally antigenic in humans, and thus can give rise to undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the methods serves to lessen the chance that an antibody administered to a human will evoke an undesirable immune response.
  • Transgenic animals e.g., mice
  • J(H) antibody heavy chain joining region
  • germ-line mutant mice results in complete inhibition of endogenous antibody production.
  • Transfer of the human germ-line immunoglobulin gene array in such germ -line mutant mice will result in the production of human antibodies upon antigen challenge.
  • the antibodies are generated in other species and “humanized” for administration in humans.
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab’, F(ab’)2, or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient antibody are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementarity determining region
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non- human residues.
  • Humanized antibodies may also contain residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will contain substantially all of at least one, and typically two, variable domains, in which all or substantially all, of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain.
  • Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule. Humanization can be essentially performed by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • a humanized form of a nonhuman antibody is a chimeric antibody or fragment, wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • variable domains both light and heavy
  • the choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important in order to reduce antigenicity.
  • the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences.
  • the human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody.
  • FR human framework
  • Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies.
  • humanized antibodies can be prepared by a process of analysis of the parental sequences and various conceptual humanized products using three dimensional models of the parental and humanized sequences.
  • Three dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art.
  • Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen.
  • FR residues can be selected and combined from the consensus and import sequence so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.
  • the CDR residues are directly and most substantially involved in influencing antigen binding.
  • the antibody can be bound to a substrate or labeled with a detectable moiety or both bound and labeled.
  • detectable moieties contemplated with the present compositions include fluorescent, enzymatic and radioactive markers.
  • a single chain antibody is created by fusing together the variable domains of the heavy and light chains using a short peptide linker, thereby reconstituting an antigen binding site on a single molecule.
  • Single-chain antibody variable fragments scFvs
  • the linker is chosen to permit the heavy chain and light chain to bind together in their proper conformational orientation.
  • Fvs lack the constant regions (Fc) present in the heavy and light chains of the native antibody.
  • In vitro methods are also suitable for preparing monovalent antibodies.
  • Digestion of antibodies to produce fragments thereof, particularly Fab fragments can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment, called the F(ab’)2 fragment, which has two antigen combining sites and is still capable of cross-linking antigen.
  • the Fab fragments produced in the antibody digestion also contain the constant domains of the light chain and the first constant domain of the heavy chain.
  • Fab’ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain domain including one or more cysteines from the antibody hinge region.
  • the F(ab’)2 fragment is a bivalent fragment comprising two Fab’ fragments linked by a disulfide bridge at the hinge region.
  • Fab’-SH is the designation herein for Fab’ in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • Antibody fragments originally were produced as pairs of Fab’ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known. iv. Hybrid Antibodies
  • the antibody can be a hybrid antibody.
  • hybrid antibodies one heavy and light chain pair is homologous to that found in an antibody raised against one epitope, while the other heavy and light chain pair is homologous to a pair found in an antibody raised against another epitope. This results in the property of multi-functional valency, i.e., ability to bind at least two different epitopes simultaneously.
  • Such hybrids can be formed by fusion of hybridomas producing the respective component antibodies, or by recombinant techniques. Such hybrids may, of course, also be formed using chimeric chains. v.
  • the targeting function of the antibody can be used therapeutically by coupling the antibody or a fragment thereof with a therapeutic agent.
  • a therapeutic agent e.g., at least a portion of an immunoglobulin constant region (Fc)
  • Such coupling of the antibody or fragment (e.g., at least a portion of an immunoglobulin constant region (Fc)) with the therapeutic agent can be achieved by making an immunoconjugate or by making a fusion protein, comprising the antibody or antibody fragment and the therapeutic agent.
  • Antibodies can be engineered with Fc variants that extend half-life, e.g., using XtendTM antibody half-life prolongation technology (Xencor, Monrovia, CA). In other embodiments, the half-life of the anti-DNA antibody is decreased to reduce potential side effects.
  • the conjugates disclosed can be used for modifying a given biological response.
  • the drug moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin.
  • An anti-B7H4 antibody or antigen binding fragment thereof having a light chain with an amino acid sequence according to SEQ ID NO:24 and a heavy chain having an amino acid sequence according to SEQ ID NO:39.
  • Proteins and Polypeptides a. Protein and Polypeptide Compositions
  • the immunomodulatory agent can be a protein, polypeptide, or fusion protein.
  • the immunomodulatory agent can be an isolated or recombinant protein or polypeptide, or functional fragment, variant, or fusion protein thereof of B7- H4.
  • the protein or polypeptide, or functional fragment, variant, or fusion protein thereof can be an agonist or an antagonist.
  • an antagonist of B7-H4 is a B7-H4 polypeptide or a fragment or fusion protein thereof that binds to a ligand of B7-H4.
  • the polypeptide can be a soluble fragment, for example the extracellular domain of B7-H4, or a functional fragment thereof, or a fusion protein thereof.
  • a soluble ligand of B7-H4 may serve as an agonist, increasing signal transduction through B7-H4.
  • the activity (i.e., agonist or antagonist) of a protein or polypeptide of B7-H4, or any fragment, variant or fusion protein thereof can be determined using functional assays that are known in the art, and include the assays discussed below.
  • the assays include determining if the protein, polypeptide or fragment, variant or fusion protein thereof increases (i.e., agonist) or decreases (i.e., antagonist) signaling through the B7-H4 receptor.
  • the assay includes determining if the protein, polypeptide or fragment, variant, or fusion protein thereof increases (i.e., agonist) or decreases (i.e., antagonist) the immune response associated with B7-H4.
  • the assays include determining if the protein, polypeptide or fragment, variant, or fusion protein thereof increases (i.e., agonist) or decreases (i.e., antagonist) signaling through B7-H4.
  • the assay includes determining if the protein, polypeptide or fragment, variant, or fusion protein thereof decreases (i.e., agonist) or increases (i.e., antagonist) an immune response negatively regulated by B7-H4.
  • the assay includes determining if the protein, polypeptide or fragment, variant, or fusion protein thereof increases (i.e., antagonist) the apoptosis and differentiation of acute myeloid leukemia cells and acute lymphoblastic leukemia cells resulting in reduced selfrenewal capacity of AML and ALL stem cells.
  • Nucleic acid and polypeptide sequences for B7-H4 are known in the art and exemplary protein and peptide sequences are provided above. The sequences can be used, as discussed in more detail below, by one of skill in the art to prepare any protein or polypeptide of B7-H4, or any fragment, variant, or fusion protein thereof. Generally, the proteins, polypeptides, fragments, variants, and fusions thereof of B7-H4 are expressed from nucleic acids that include sequences that encode a signal sequence. The signal sequence is generally cleaved from the immature polypeptide to produce the mature polypeptide lacking the signal sequence.
  • the signal sequence can be replaced by the signal sequence of another polypeptide using standard molecule biology techniques to affect the expression levels, secretion, solubility, or other property of the polypeptide B7- H4 proteins with and without a signal sequence are disclosed. It is understood that in some cases, the mature protein as it is known or described in the art, i.e., the protein sequence without the signal sequence, is a putative mature protein. During normal cell expression, a signal sequence can be removed by a cellular peptidase to yield a mature protein. The sequence of the mature protein can be determined or confirmed using methods that are known in the art. i. Fragments
  • a fragment of B7-H4 refers to any subset of the polypeptide that is at least one amino acid shorter than full length protein. Useful fragments include those that retain the ability to bind to their natural ligand or ligands.
  • a polypeptide that is a fragment of any full-length B7-H4 typically has at least 20 percent, 30 percent, 40 percent, 50 percent, 60 percent, 70 percent, 80 percent, 90 percent, 95 percent, 98 percent, 99 percent, 100 percent, or even more than 100 percent of the ability to bind its natural ligand respectively as compared to the full-length protein.
  • Fragments of B7-H4 include cell free fragments.
  • Cell free polypeptides can be fragments of full-length, transmembrane, polypeptides that may be shed, secreted or otherwise extracted from the producing cells.
  • Cell free fragments of polypeptides can include some or all of the extracellular domain of the polypeptide and lack some or all of the intracellular and/or transmembrane domains of the full-length protein.
  • polypeptide fragments include the entire extracellular domain of the full- length protein.
  • the cell free fragments of the polypeptides include fragments of the extracellular domain that retain biological activity of full-length protein.
  • the extracellular domain can include 1, 2, 3, 4, or 5 contiguous amino acids from the transmembrane domain, and/or 1, 2, 3, 4, or 5 contiguous amino acids from the signal sequence.
  • the extracellular domain can have 1, 2, 3, 4, 5 or more amino acids removed from the C-terminus, N-terminus, or both.
  • the extracellular domain is the only functional domain of the fragment (e.g., the ligand binding domain).
  • variants of B7-H4, and fragments thereof are also provided.
  • the variant is at least 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, or 99 percent identical to any one of SEQ ID NO: 1.
  • Useful variants include those that increase biological activity, as indicated by any of the assays described herein, or that increase half-life or stability of the protein.
  • the protein and polypeptides of B7-H4, and fragments, variants, and fusion proteins thereof can be engineered to increase biological activity.
  • a B7-H4 polypeptide, protein, or fragment, variant or fusion thereof has been modified with at least one amino acid substitution, deletion, or insertion that increases a function thereof.
  • variant polypeptides can be engineered to have an increased half-life relative to wild type. These variants typically are modified to resist enzymatic degradation. Exemplary modifications include modified amino acid residues and modified peptide bonds that resist enzymatic degradation. Various modifications to achieve this are known in the art. The variants can be modified to adjust for effects of affinity for the receptor on the half-life of proteins, polypeptides, fragments, or fusions thereof at serum and endosomal pH. iii. Fusion Proteins
  • Fusion polypeptides have a first fusion partner including all or a part of a polypeptide B7-H4 fused to a second polypeptide directly or via a linker peptide sequence that is fused to the second polypeptide.
  • the fusion proteins optionally contain a domain that functions to dimerize or multimerize two or more fusion proteins.
  • the peptide/polypeptide linker domain can either be a separate domain, or alternatively can be contained within one of the other domains (first polypeptide or second polypeptide) of the fusion protein.
  • Fusion proteins disclosed herein are of formula I:
  • N represents the N-terminus of the fusion protein
  • C represents the C- terminus of the fusion protein.
  • Ri is a polypeptide or protein of B7-H4 or fragment or variant thereof
  • R2 is an optional peptide/polypeptide linker domain
  • R3 is a second polypeptide.
  • R3 may be a polypeptide or protein of B7-H4, or fragment or variant thereof
  • Ri may be a second polypeptide.
  • the B7-H4 polypeptide is the extracellular domain or a fragment thereof such as the Ig-like C2-domain, or the region framed by the cysteines that form a disulfide bond as discussed above.
  • the fusion protein includes the extracellular domain of B7-H4, or a fragment or variant thereof, fused to an Ig Fc region.
  • Recombinant Ig fusion proteins can be prepared by fusing the coding region of the extracellular domain of an extracellular domain or a fragment or variant thereof to the Fc region of human IgGl, IgG2, IgG3 or IgG4 or mouse IgG2a, or other suitable Ig domain, as described previously (Chapoval, et al., Methods Mol. Med., 45:247-255 (2000)).
  • Modifications may be introduced into the molecule by reacting targeted amino acid residues of the polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Another modification is cyclization of the protein.
  • Examples of chemical derivatives of polypeptides include lysinyl and amino terminal residues derivatized with succinic or other carboxylic acid anhydrides. Derivatization with a cyclic carboxylic anhydride has the effect of reversing the charge of the lysinyl residues.
  • Other suitable reagents for derivatizing amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.
  • Binding properties of the proteins, polypeptides, fragments, variants and fusions thereof are relevant to the dose and dose regimen to be administered.
  • the disclosed the proteins, polypeptides, fragments, variants and fusions thereof have binding properties to a B7-H4 ligand that demonstrate a higher term, or higher percentage, of occupancy of a binding site (e.g., on the ligand) relative to other receptor molecules that bind thereto.
  • the disclosed proteins, polypeptides, fragments, variants and fusions thereof have reduced binding affinity to a B7-H4 ligand relative to wild type protein.
  • An isolated nucleic acid can be, for example, a DNA molecule, provided one of the nucleic acid sequences normally found immediately flanking that DNA molecule in a naturally occurring genome is removed or absent.
  • an isolated nucleic acid includes, without limitation, a DNA molecule that exists as a separate molecule independent of other sequences (e.g., a chemically synthesized nucleic acid, or a cDNA or genomic DNA fragment produced by PCR or restriction endonuclease treatment), as well as recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, lentivirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote.
  • a virus e.g., a retrovirus, lentivirus, adenovirus, or herpes virus
  • 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.
  • an engineered nucleic acid such as a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid.
  • Nucleic acids can be in sense or antisense orientation or can be complementary to a reference sequence encoding a polypeptide or protein of B7-H4.
  • Nucleic acids can be DNA, RNA, or nucleic acid analogs.
  • Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone. Such modification can improve, for example, stability, hybridization, or solubility of nucleic acid. Modifications at the base moiety can include deoxyuridine for deoxythymidine, and 5- methyl-2’-deoxycytidine or 5 -bromo-2’ -deoxy cytidine for deoxycytidine.
  • Modifications of the sugar moiety can include modification of the 2’ hydroxyl of the ribose sugar to form 2’-O-methyl or 2’-O-allyl sugars.
  • the deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six membered, morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained. See, for example, Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev. 7: 187- 195; and Hyrup et al. (1996) Bioorgan. Med. Chem. 4:5-23.
  • Nucleic acids encoding polypeptides can be administered to subjects in need thereof. Nucleic delivery involves introduction of “foreign” nucleic acids into a cell and ultimately, into a live animal. Compositions and methods for delivering nucleic acids to a subject are known in the art (see Understanding Gene Therapy, Lemoine, N.R., ed., BIOS Scientific Publishers, Oxford, 2008).
  • Anti-B7-H4 antibodies may be conjugated to at least one auristatin.
  • Auristatins represent a group of dolastatin analogs that have generally been shown to possess anticancer activity by interfering with microtubule dynamics and GTP hydrolysis, thereby inhibiting cellular division.
  • Auristatin E U.S. Pat. No. 5,635,483
  • Auristatin E is a synthetic analogue of the marine natural product dolastatin 10, a compound that inhibits tubulin polymerization by binding to the same site on tubulin as the anticancer drug vincristine (G. R. Pettit, Prog. Chem. Org. Nat. Prod, 70: 1-79 (1997)).
  • Dolastatin 10, auristatin PE, and auristatin E are linear peptides having four amino acids, three of which are unique to the dolastatin class of compounds.
  • Exemplary embodiments of the auristatin subclass of mitotic inhibitors include, but are not limited to, monomethyl auristatin D (MMAD or auristatin D derivative), monomethyl auristatin E (MMAE or auristatin E derivative), monomethyl auristatin F (MMAF or auristatin F derivative), auristatin F phenylenediamine (AFP), auristatin EB (AEB), auristatin EFP (AEFP), and 5-benzoylvaleric acid-AE ester (AEVB).
  • MMAD or auristatin D derivative monomethyl auristatin E (MMAE or auristatin E derivative
  • MMAF or auristatin F derivative monomethyl auristatin F phenylenediamine (AFP), a
  • anti-B7-H4 antibodies are conjugated to at least one MMAE (mono-methyl auristatin E).
  • MMAE monomethyl auristatin E
  • mAb monoclonal antibody
  • the linker linking MMAE to the anti-B7-H4 antibody is stable in extracellular fluid (i.e., the medium or environment that is external to cells), but is cleaved by cathepsin once the ADC has bound to the specific cancer cell antigen and entered the cancer cell, thus releasing the toxic MMAE and activating the potent anti-mitotic mechanism.
  • the antibody is coupled to a single drug and, therefore, has a DAR of 1.
  • the ADC will have a DAR of 2 to 8, or, alternatively, 2 to 4.
  • the anti-B7-H4 antibodies may be conjugated to at least one maytansinoid to form an ADC.
  • Maytansinoids are potent antitumor agents that were originally isolated from members of the higher plant families Celastraceae, Rhamnaceae and Euphorbiaceae, as well as some species of mosses (Kupchan et al, J. Am. Chem. Soc. 94: 1354-1356 [1972]; Wani et al, J. Chem. Soc. Chem. Commun. 390: [1973]; Powell et al, J. Nat. Prod. 46:660-666 [1983]; Sakai et al, J. Nat. Prod.
  • cytotoxicity of maytansinoids is 1.000-fold greater than conventional chemotherapeutic agents, such as, for example, methotrexate, daunorubicin, and vincristine (see. e.g., U.S. Pat. No. 5,208,020).
  • conventional chemotherapeutic agents such as, for example, methotrexate, daunorubicin, and vincristine (see. e.g., U.S. Pat. No. 5,208,020).
  • Maytansinoids to include maytansine, maytansinol, C-3 esters of maytansinol, and other maytansinol analogues and derivatives (see, e.g., U.S. Pat. Nos. 5,208,020 and 6,441,163, each of which is incorporated by reference herein).
  • C-3 esters of maytansinol can be naturally occurring or synthetically derived.
  • both naturally occurring and synthetic C-3 maytansinol esters can be classified as a C-3 ester with simple carboxylic acids, or a C-3 ester with derivatives of N-methyl-L-alanine, the latter being more cytotoxic than the former.
  • Synthetic maytansinoid analogues are described in, for example, Kupchan et al., J. Med. Chem., 21, 31-37 (1978).
  • Suitable maytansinoids for use in antibody-drug conjugates can be isolated from natural sources, synthetically produced, or semi-synthetically produced. Moreover, the maytansinoid can be modified in any suitable manner, so long as sufficient cytotoxicity is preserved in the ultimate conjugate molecule. In this regard, maytansinoids lack suitable functional groups to which antibodies can be linked. A linking moiety desirably is utilized to link the maytansinoid to the antibody to form the conjugate.
  • maytansinoids include, but are not limited, to DM1 (N 2 '-deacetyl-N 2 '-(3 -mercapto- l-oxopropyl)-maytansine; also referred to as mertansine, drug maytansinoid 1; ImmunoGen, Inc.; see also Chari et al.
  • Ansamitocins are a group of maytansinoid antibiotics that have been isolated from various bacterial sources. These compounds have potent antitumor activities. Representative examples include, but are not limited to ansamitocin Pl, ansamitocin P2, ansamitocin P3, and ansamitocin P4.
  • an anti-B7-H4 antibody is conjugated to at least one DM1. In one embodiment, an anti-B7-H4 antibody is conjugated to at least one DM2. In one embodiment, an anti-B7-H4 antibody is conjugated to at least one DM3. In one embodiment, an anti-B7-H4 antibody is conjugated to at least one DM4.
  • DNA alkylating agent includes a family of DNA alkylating agents including indolino-benzodiazepines (IGNs).
  • IGNs represent a chemical class of cytotoxic molecules with high in vitro potency (IC.sub.50 values in the low pmol/L range) toward cancer cells.
  • Examples of IGN DNA alkylating agents that can be used as a cytotoxic payload in an ADC are described in Miller et al. (2016) Molecular Cancer Therapeutics, 15(8)).
  • the IGN compounds described in Miller et al. bind to the minor groove of DNA followed by covalent reaction of guanine residues with the two imine functionalities in the molecule resulting in cross-linking of DNA.
  • the structure of an exemplary IGN is provided below. d.
  • Other Drugs for Conjugation are described in Miller et al.
  • drugs that may be used in ADCs, i.e., drugs that may be conjugated to the anti-B7-H4 antibodies, are provided below, and include mitotic inhibitors, antitumor antibiotics, immunomodulating agents, gene therapy vectors, alkylating agents, anti angiogenic agents, antimetabolites, boron-containing agents, chemoprotective agents, hormone agents, glucocorticoids, photoactive therapeutic agents, oligonucleotides, radioactive isotopes, radiosensitizers, topoisomerase inhibitors, tyrosine kinase inhibitors, and combinations thereof.
  • mitotic Inhibitors include mitotic inhibitors, antitumor antibiotics, immunomodulating agents, gene therapy vectors, alkylating agents, anti angiogenic agents, antimetabolites, boron-containing agents, chemoprotective agents, hormone agents, glucocorticoids, photoactive therapeutic agents, oligonucleotides, radioactive isotopes, radiosens
  • anti-B7-H4 antibodies may be conjugated to one or more mitotic inhibitor(s) to form an ADC for the treatment of cancer.
  • mitotic inhibitor refers to a cytotoxic and/or therapeutic agent that blocks mitosis or cell division, a biological process particularly important to cancer cells.
  • a mitotic inhibitor disrupts microtubules such that cell division is prevented, often by affecting microtubule polymerization or microtubule depolymerization.
  • an anti-B7-H4 antibody is conjugated to one or more mitotic inhibitor(s) that disrupts microtubule formation by inhibiting tubulin polymerization.
  • the mitotic inhibitor used in the ADCs is IXEMPRA® (ixabepilone).
  • mitotic inhibitors that may be used in the anti-B7-H4 ADCs include dolastatins, e.g., dolastatin 10 and dolastatin 15, and plant alkaloids, e.g., a taxane and vinca alkaloid, e.g., indesine sulfate, vincristine, vinblastine and vinorelbine. Included in the genus of mitotic inhibitors are auristatins and maytansinoids, described above.
  • Anti-B7-H4 antibodies described herein may be conjugated to at least one taxane.
  • taxane refers to the class of antineoplastic agents having a mechanism of microtubule action and having a structure that includes the taxane ring structure and a stereospecific side chain that is required for cytostatic activity. Also included within the term “taxane” are a variety of known derivatives, including both hydrophilic derivatives, and hydrophobic derivatives. Taxane derivatives include, but not limited to, galactose and mannose derivatives described in International Patent Application No.
  • taxanes include, but are not limited to, docetaxel (TAXOTERE®; Sanofi Aventis), paclitaxel (ABRAXANE® or TAXOLTM; Abraxis Oncology), and nanoparticle paclitaxel (ABI-007/ABRAXANE®; Abraxis Bioscience).
  • Anti-B7-H4 antibodies may be conjugated to one or more antitumor antibiotic(s) for the treatment of cancer.
  • antitumor antibiotic means an antineoplastic drug that blocks cell growth by interfering with DNA and is made from a microorganism. Often, antitumor antibiotics either break up DNA strands or slow down or stop DNA synthesis. Examples of antitumor antibiotics that may be included in the anti-B7-H4 ADCs include, but are not limited to, actinomycines (e.g., pyrrolo[2,l-c][l,4]benzodiazepines), anthracyclines, calicheamicins, and duocarmycins.
  • antitumor antibiotics that may be used in the anti- B7-H4 ADCs include bleomycin (BLENOXANETM, Bristol-Myers Squibb), mitomycin, and plicamycin (also known as mithramycin).
  • anti-B7-H4 antibodies may be conjugated to at least one immunomodulating agent.
  • immunomodulating agent refers to an agent that can stimulate or modify an immune response.
  • an immunomodulating agent is an immunostimuator which enhances a subject's immune response.
  • an immunomodulating agent is an immunosuppressant which prevents or decreases a subject's immune response.
  • An immunomodulating agent may modulate myeloid cells (monocytes, macrophages, dendritic cells, megakaryocytes and granulocytes) or lymphoid cells (T cells, B cells and natural killer (NK) cells) and any further differentiated cell thereof.
  • Representative examples include, but are not limited to, bacillus calmette-guerin (BCG) and levamisole (ERGAMISOLTM).
  • BCG Bacillus calmette-guerin
  • ERGAMISOLTM levamisole
  • Other examples of immunomodulating agents that may be used in the ADCs include, but are not limited to, cancer vaccines, and cytokines.
  • cancer vaccine refers to a composition (e.g., a tumor antigen and a cytokine) that elicits a tumor-specific immune response.
  • the response is elicited from the subject's own immune system by administering the cancer vaccine, or, in the case of the instant disclosure, administering an ADC comprising an anti-B7-H4 antibody and a cancer vaccine.
  • the immune response results in the eradication of tumor cells in the body (e.g., primary or metastatic tumor cells).
  • cancer vaccines generally involves the administration of a particular antigen or group of antigens that are, for example, present on the surface a particular cancer cell, or present on the surface of a particular infectious agent shown to facilitate cancer formation.
  • the use of cancer vaccines is for prophylactic purposes, while in other embodiments, the use is for therapeutic purposes.
  • Non-limiting examples of cancer vaccines that may be used in the anti-B7-H4 ADCs include, recombinant bivalent human papillomavirus (HPV) vaccine types 16 and 18 vaccine (CERVARIX®, GlaxoSmithKline), recombinant quadrivalent human papillomavirus (HPV) types 6, 11, 16, and 18 vaccine (GARDASIL®, Merck & Company), and sipuleucel-T (PROVENGE®, Dendreon).
  • the anti-B7-H4 antibody is conjugated to at least one cancer vaccine that is either an immunostimulator or is an immunosuppressant.
  • cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.
  • the disclosure provides an ADC comprising an anti-B7-H4 antibody described herein and a cytokine.
  • the anti-B7-H4 antibodies may be conjugated to at least one colony stimulating factor (CSF).
  • CSFs colony stimulating factors
  • CSFs are growth factors that assist the bone marrow in making red blood cells. Because some cancer treatments (e.g., chemotherapy) can affect white blood cells (which help fight infection), colonystimulating factors may be introduced to help support white blood cell levels and strengthen the immune system. Colony-stimulating factors may also be used following a bone marrow transplant to help the new marrow start producing white blood cells.
  • the anti-B7-H4 antibodies may be conjugated to one or more alkylating agent(s).
  • Alkylating agents are a class of antineoplastic compounds that attaches an alkyl group to DNA.
  • alkylating agents that may be used in the ADCs include, but are not limited to, alkyl sulfonates, ethylenimimes, methylamine derivatives, epoxides, nitrogen mustards, nitrosoureas, triazines and hydrazines.
  • Erlotinib (TARCEVA®), angiostatin, arrestin, endostatin, BAY 12-9566 and w/fluorouracil or doxorubicin, canstatin, carboxyamidotriozole and with paclitaxel, EMD 121974, S-24, vitaxin, dimethylxanthenone acetic acid, IM862, Interleukin- 12, Interleukin-2, NM-3, HuMV833, PTK787, RhuMab, angiozyme (ribozyme), IMC-1C11, Neovastat, marimstat, prinomastat, BMS-275291, COL-3, MM1270, SU101, SU6668, SU11248, SU5416, with paclitaxel, with gemcitabine and cisplatin, and with irinotecan and cisplatin and with radiation, tecogalan, temozolomide and PEG interferon a2b, tetrathiomoly
  • tyrosine kinase inhibitors e.g., erlotinib (TARCEVA®, Genentech, Inc.), imatinib (GLEEVEC®, Novartis Pharmaceutical Corporation), gefitinib (IRESSA®, AstraZeneca Pharmaceuticals), dasatinib (SPRYCEL®, Brystol-Myers Squibb), sunitinib (SUTENT®, Pfizer, Inc.), nilotinib (TASIGNA®, Novartis Pharmaceutical Corporation), lapatinib (TYKERB®, GlaxoSmithKline Pharmaceuticals), sorafenib (NEXAVAR®, Bayer and Onyx), phosphoinositide 3 -kinases (PI3K).
  • erlotinib TARCEVA®, Genentech, Inc.
  • imatinib GLEEVEC®, Novartis Pharmaceutical Corporation
  • gefitinib IRESSA®, AstraZeneca Pharmaceuticals
  • antimetabolies examples include, but are not limited to, a folic acid antagonist (e.g., methotrexate), a pyrimidine antagonist (e.g., 5 -Fluorouracil, Foxuridine, Cytarabine, Capecitabine, and Gemcitabine), a purine antagonist (e.g., 6- Mercaptopurine and 6-Thioguanine) and an adenosine deaminase inhibitor (e.g., Cladribine, Fludarabine, Nelarabine and Pentostatin), as described in more detail below.
  • a folic acid antagonist e.g., methotrexate
  • a pyrimidine antagonist e.g., 5 -Fluorouracil, Foxuridine, Cytarabine, Capecitabine, and Gemcitabine
  • a purine antagonist e.g., 6- Mercaptopurine and 6-Thioguanine
  • an adenosine deaminase inhibitor
  • the anti-B7-H4 antibodies may be conjugated to at least one photoactive therapeutic agent.
  • Photoactive therapeutic agents include compounds that can be deployed to kill treated cells upon exposure to electromagnetic radiation of a particular wavelength. Therapeutically relevant compounds absorb electromagnetic radiation at wavelengths which penetrate tissue.
  • the compound is administered in a non-toxic form that is capable of producing a photochemical effect that is toxic to cells or tissue upon sufficient activation. In other preferred embodiments, these compounds are retained by cancerous tissue and are readily cleared from normal tissues. Non-limiting examples include various chromagens and dyes.
  • Radionuclide Agents Radioactive Isotopes
  • the anti-B7-H4 antibodies may be conjugated to at least one radionuclide agent.
  • Radionuclide agents comprise agents that are characterized by an unstable nucleus that is capable of undergoing radioactive decay.
  • the basis for successful radionuclide treatment depends on sufficient concentration and prolonged retention of the radionuclide by the cancer cell. Other factors to consider include the radionuclide half-life, the energy of the emitted particles, and the maximum range that the emitted particle can travel.
  • the therapeutic agent is a radionuclide selected from the group consisting of n i In, 177 Lu, 212 Bi, 213 Bi, 211 At, 62 Cu, 64 Cu, 67 Cu, 90 Y, 125 I, 131 1, 32 P, 33 P, 47 Sc, m Ag, 67 Ga, 142 Pr, 153 Sm, 161 Th, 166 Dy, 166 Ho, 186 Re, 188 Re, 189 Re, 212 Pb, 223 Ra, 225 Ac, 59 Fe, 75 Se, 77 As, 89 Sr, "Mo, 105 Rh, 109 Pd, 143 Pr, 149 Pm, 169 Er, 194 Ir, 198 Au, 199 Au, and 211 Pb.
  • a radionuclide selected from the group consisting of n i In, 177 Lu, 212 Bi, 213 Bi, 211 At, 62 Cu, 64 Cu, 67 Cu, 90 Y, 125 I, 131 1, 32 P, 33 P, 47 Sc
  • Decay energies of useful alpha-particle-emitting radionuclides are preferably 2,000-10,000 keV, more preferably 3,000-8.000 keV, and most preferably 4,000-7,000 keV. Additional potential radioisotopes of use include n C, 199 Au, 57 Co, 58 Co, 51 Cr, 59 Fe, 75 Se, 2O1 T1, 225 Ac, 76 Br, 169 Yb, and the like. o. Radiosensitizers
  • Radiosensitizers may be activated by electromagnetic radiation of X-rays.
  • Representative examples of X-ray activated radiosensitizers include, but are not limited to, the following: metronidazole, misonidazole, desmethylmisonidazole, pimonidazole, etanidazole, nimorazole, mitomycin C.
  • the anti-B7-H4 antibodies may be conjugated to at least one topoisomerase inhibitor.
  • Topoisomerase inhibitors are chemotherapy agents designed to interfere with the action of topoisomerase enzymes (topoisomerase I and II), which are enzymes that control the changes in DNA structure by catalyzing then breaking and rejoining of the phosphodiester backbone of DNA strands during the normal cell cycle.
  • Representative examples of DNA topoisomerase I inhibitors include, but are not limited to, camptothecins and its derivatives irinotecan (CPT-11, CAMPTOSAR®, Pfizer, Inc.) and topotecan (HYCAMTIN®, GlaxoSmithKline Pharmaceuticals).
  • DNA topoisomerase II inhibitors include, but are not limited to, amsacrine, daunorubicin, doxotrubicin, epipodophyllotoxins, ellipticines, epirubicin, etoposide, razoxane, and teniposide.
  • the anti-B7-H4 antibodies may be conjugated to at least one tyrosine kinase inhibitor.
  • Tyrosine kinases are enzymes within the cell that function to attach phosphate groups to the amino acid tyrosine. By blocking the ability of protein tyrosine kinases to function, tumor growth may be inhibited.
  • Examples of tyrosine kinases that may be used on the ADCs include, but are not limited to, Axitinib, Bosutinib, Cediranib, Dasatinib. Erlotinib, Gefitinib, Imatinib. Lapatinib, Lestaurtinib, Nilotinib. Semaxanib, Sunitinib, and Vandetanib. r. Other Agents
  • Pseudonomas endotoxin Pseudomonas exotoxin (e.g. exotoxin A chain (from Pseudomonas aeruginosa)), restrictocin, ricin A chain, ribonuclease (Rnase), Sapaonaria officinalis inhibitor, saporin, alpha-sarcin.
  • agents include asparaginase (Espar. Lundbeck Inc.), hydroxyurea, levamisole, mitotane (LYSODREN®, Bristol-Myers Squibb), and tretinoin (RENOVA®, Valeant Pharmaceuticals Inc.).
  • anti-B7-H4 antibodies or ADCs are used with any of the foregoing agents in a combination therapy to treat cancer, where the agent is administered prior to, at the same time as, or following administration of the anti-B7-H4 antibody or ADC to the subject.
  • An anti-B7-H4 ADC comprises an anti-B7-H4 antibody and at least one drug(s), whereby the antibody and the at least one drug are conjugated by a linker.
  • linker refers to a chemical moiety that may be bifunctional or multifunctional, and is used to attach an antibody to a drug moiety.
  • a linker may include one conjugating component or may include multiple components.
  • the linker may include a spacer, which is a moiety that extends the drug linkage to avoid, for example, shielding the active site of the antibody or improving the solubility of the ADC.
  • Other examples of components of linkers include a stretcher unit and an amino acid unit.
  • the linker described herein may be cleavable, non-cleavable and hydrophilic or hydrophobic.
  • the cleavable linker is cleavable under intracellular or extracellular conditions, by which an active agent is released from an antibody construct-active agent conjugate in the intracellular environment.
  • the cleavable linker can be cleaved by a cleaving agent present in an intracellular environment (e.g., lysosomes, endosomes, or caveolea).
  • the cleavable linker may be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not being limited to, a lysosomal or endosomal protease.
  • the peptidyl linker has a length of at least two amino acids or a length of at least three amino acids.
  • the peptidyl linker cleavable by an intracellular protease may be, for example, a Val-Cit linker, a Phe-Lys linker (e.g., see U.S. Patent No. 6,214,345, which describes the synthesis of doxorubicin using a Val-Cit linker), or a Vai-Ala linker.
  • the Val-Cit linker or the Val-Ala linker may contain a pentafluorophenyl group and may contain a succinimide group or a maleimide group.
  • Val-Cit linker or the Val-Ala linker may contain a pentafluorophenyl group, may contain a 4-aminobenzoic acid (PAB A) group and a maleimide group, and may contain a PABA group and a succinimide group.
  • PAB A 4-aminobenzoic acid
  • a cleavable linker may be easily hydrolyzed in a pH-sensitive manner, i.e., at certain pH values.
  • the pH-sensitive linker may be hydrolyzed under acidic conditions.
  • acid-labile linkers that can be hydrolyzed in lysosomes (e.g., hydrazone, semicarbazone, thiosemicarbazone, cis-aconic amides, orthoesters, acetals, and ketals) may be used (e.g., see: U.S. Patent NOs. 5,122,368, 5,824,805, and 5,622,929; and Dubowchik and Walker, 1999, Pharm.
  • linkers are relatively stable under neutral pH conditions, such as in blood, but are unstable at pH 5.5, which is the approximate pH of lysosomes, or less than pH 5.0.
  • hydrolysable linkers include thioether linkers (e.g., thioethers attached to a therapeutic agent via an acylhydrazone bond) (e.g., see U.S. Patent No.5, 622, 929).
  • the linker is cleavable under reducing conditions (e.g., a disulfide linker).
  • a disulfide linker including N-succinimidyl-5- acetylthioacetate (SATA), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N- succinimidyl-3-(2-pyridyldithio)butyrate (SPDB), and N-succinimidyl-oxycarbonyl- alpha-methyl-alpha-(2-pyridyl-thio)toluene)- (SMPT), and those that can be formed using SPDB and SMPT (e.g., see: Thorpe et al., 1987, Cancer Res.47: 5924-5931; and U.S. Patent No.4, 880, 935).
  • the linker may be a malonate linker (Johnson et al., 1995, Anticancer Res.15: 1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med- Chem. 3(10): 1299-1304), a 3'-N-amide analogue (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1305-12), a P-glucuronide linker (Jeffery et al., 2006, Bioconjug Chem. 17(3):832- 40), or a P-galactoside linker (Kolodych et al., 2017, Eur J Med Chem. Dec 15;142:376- 382).
  • a malonate linker Johnson et al., 1995, Anticancer Res.15: 1387-93
  • a maleimidobenzoyl linker Liau et al., 1995, Bioorg-Med- Chem. 3(
  • the linker may be a combination of a maleimidocaproyl linker containing a succinimide group and one or more polyethylene glycol molecules.
  • the linker may be a combination of a pentafluorophenyl group and a maleimidocaproyl linker containing one or more polyethylene glycol molecules.
  • the linker may contain a maleimide linked to a polyethylene glycol molecule, wherein the polyethylene glycol allows for more linker flexibility or allows longer linkers to be used.
  • the linker may be a (maleimidocaproyl)-(valine-citrulline)-(para- aminobenzyloxycarbonyl) linker.
  • the linker may be a cleavable linker.
  • the linker may be a protease cleavable linker, an acid-cleavable linker, a disulfide linker, a self-immolative linker or a self-stabilizing linker, a malonate linker, a maleimidobenzoyl linker, a 3'-N-amide analogue, a P- glucuronide linker, or a P-galactoside linker.
  • the protease cleavable linker may include a thiolreactive spacer or a dipeptide, and more specifically, the protease cleavable linker may include a thiol -reactive maleimidocaproyl spacer, a valine-citrulline dipeptide, or a p- amino-benzyloxycarbonyl spacer.
  • the acid-cleavable linker may be a hydrazine linker or a quaternary ammonium linker.
  • Exemplary antibody drug conjugates are disclosed in US 10,583,197, US 9,993,568, US 9,951,072, US 9,919,057, US 9,669,107, US 11,413,353, US 11,173,214, US 11,167,040, US 10,980,890, US 10,583,197, US 10,383,949, US 10,273,235, US 10,183,997, and US 10,118,965, the contents of each of which is fully incorporated by reference herein.
  • a linker covalently attaches an antibody to a drug moiety.
  • An ADC is prepared using a linker having reactive functionality for binding to the antibody and the drug.
  • a cysteine thiol, or an amine, e.g., N-terminus or amino acid side chain such as lysine, of the antibody may form a bond with a functional group of the linker.
  • a linker has a functionality that is capable of reacting with a free cysteine present on an antibody to form a covalent bond.
  • reactive functionalities include maleimide, haloacetamides, a-haloacetyl, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates.
  • a linker has a functionality that is capable of reacting with an electrophilic group present on an antibody.
  • electrophilic groups include, but are not limited to, aldehyde and ketone carbonyl groups.
  • a heteroatom of the reactive functionality of the linker can react with an electrophilic group on an antibody and form a covalent bond to an antibody unit.
  • Nonlimiting exemplary such reactive functionalities include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.
  • an anti-B7-H4 antibody is conjugated to an auristatin, e.g., MMAE, via a linker comprising maleimidocaproyl (“me”), valine citrulline (val-cit or “vc”).
  • Maleimidocaproyl acts as a linker to the anti-B7-H4 antibody and is not cleavable.
  • Val-cit is a dipeptide that is an amino acid unit of the linker and allows for cleavage of the linker by a protease, specifically the protease cathepsin B.
  • the val-cit component of the linker provides a means for releasing the auristatin from the ADC upon exposure to the intracellular environment.
  • p- aminobenzylalcohol acts as a spacer and is self immolative, allowing for the release of the MMAE.
  • an anti-B7-H4 antibody is conjugated to a cytotoxin, via a charged hindered disulfide N-succinimidyl-4-(2-pyridyldithio)butanoate (sSPDB) linker, sSPDB is a cleavable linker that allows the conjugate to be cleaved inside the target cell in the cytosol due to the reducing intracellular environment.
  • sSPDB is a charged hindered disulfide N-succinimidyl-4-(2-pyridyldithio)butanoate
  • an anti-B7-H4 antibody is conjugated to a cytotoxin, via a cleavable peptide linker such as D-Ala-L-dpa.
  • an anti-B7-H4 antibody is conjugated to an IGN via a cleavable peptide linker such as D-Ala-L-dpa.
  • Suitable linkers include, for example, cleavable and non-cleavable linkers.
  • a linker may be a “cleavable linker,” facilitating release of a drug.
  • Nonlimiting exemplary cleavable linkers include acid-labile linkers (e.g., comprising hydrazone), proteasesensitive (e.g., peptidase-sensitive) linkers, photolabile linkers, or disulfide-containing linkers (Chari et al., Cancer Research 52: 127-131 (1992); U.S. Pat. No. 5,208,020).
  • a cleavable linker is typically susceptible to cleavage under intracellular conditions.
  • Suitable cleavable linkers include, for example, a peptide linker cleavable by an intracellular protease, such as lysosomal protease or an endosomal protease.
  • the linker can be a dipeptide linker, such as a valine-citrulline (val-cit) or a phenylalanine-lysine (phe-lys) linker.
  • Linkers are preferably stable extracellularly in a sufficient manner to be therapeutically effective. Before transport or delivery into a cell, the ADC is preferably stable and remains intact, i.e. the antibody remains conjugated to the drug moiety. Linkers that are stable outside the target cell may be cleaved at some efficacious rate once inside the cell. Thus, an effective linker will: (i) maintain the specific binding properties of the antibody; (ii) allow delivery, e.g., intracellular delivery, of the drug moiety; and (iii) maintain the therapeutic effect, e.g., cytotoxic effect, of a drug moiety.
  • the linker is cleavable under intracellular conditions, such that cleavage of the linker sufficiently releases the drug from the antibody in the intracellular environment to be therapeutically effective.
  • the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at certain pH values.
  • the pH-sensitive linker is hydrolyzable under acidic conditions.
  • an acid-labile linker that is hydrolyzable in the lysosome e.g., a hydrazone, semi carb azone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or the like
  • a hydrazone e.g., a hydrazone, semi carb azone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or the like
  • the hydrolyzable linker is a thioether linker (such as, e.g., a thioether attached to the therapeutic agent via an acylhydrazone bond (see, e.g., U.S. Pat. No. 5,622,929).
  • the linker is cleavable under reducing conditions (e.g., a disulfide linker).
  • a disulfide linker e.g., a disulfide linker.
  • disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-5-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2- pyridyldithio)butyrate) and SMPT (N-succinimidyloxycarbonyl-alpha-methyl-alpha-(2- pyridyl-dithio)toluene), SPDB and SMPT.
  • SATA N-succinimidyl-5-acetylthioacetate
  • SPDP N-succinimidyl-3-
  • the linker is cleavable by a cleaving agent, e.g., an enzyme, which is present in the intracellular environment (e.g., within a lysosome or endosome or caveolca).
  • the linker can be, e.g., a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease.
  • the peptidyl linker is at least two amino acids long or at least three amino acids long.
  • Cleaving agents can include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives resulting in the release of active drug inside target cells (see. e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123).
  • Most typical are peptidyl linkers that are cleavable by enzymes that are present in B7-H4-expressing cells. Examples of such linkers are described, e.g., in U.S. Pat. No. 6,214,345, incorporated herein by reference in its entirety and for all purposes.
  • the peptidyl linker cleavable by an intracellular protease is a Val-Cit linker or a Phe-Lys linker (see, e.g., U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin with the val-cit linker).
  • One advantage of using intracellular proteolytic release of the therapeutic agent is that the agent is typically attenuated when conjugated and the serum stabilities of the conjugates are typically high.
  • the linker unit is not cleavable, and the drug is released, for example, by antibody degradation. See U.S. Publication No. 20050238649 incorporated by reference herein in its entirety.
  • An ADC comprising a non-cleavable linker may be designed such that the ADC remains substantially outside the cell and interacts with certain receptors on a target cell surface such that the binding of the ADC initiates (or prevents) a particular cellular signaling pathway.
  • (L-D) is a Linker-Drug moiety
  • the Linker-Drug moiety is made of L- which is a Linker, and -D, which is a drug moiety having, for example, cytostatic, cytotoxic, or otherwise therapeutic activity against a target cell, e.g., a cell expressing B7-H4; and n is an integer from 1 to 20.
  • n ranges from 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or is 1.
  • the -D moieties are the same. In yet another embodiment, the -D moieties are different.
  • a linker component comprises an “amino acid unit.”
  • the amino acid unit allows for cleavage of the linker by a protease, thereby facilitating release of the drug from the immunoconjugate upon exposure to intracellular proteases, such as lysosomal enzymes (Doronina et al. (2003) Nat. Biotechnol. 21 :778-784).
  • Exemplary amino acid units include, but are not limited to, dipeptides, tripeptides, tetrapeptides, and pentapeptides.
  • Exemplary dipeptides include, but are not limited to, valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala- phe); phenylalanine-lysine (fk or phe-lys); phenylalanine-homolysine (phe-homolys); and N-methyl-valine-citrulline (Me-val-cit).
  • Exemplary tripeptides include, but are not limited to, glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly).
  • amino acid unit may comprise amino acid residues that occur naturally and/or minor amino acids and/or non-naturally occurring amino acid analogs, such as citrulline
  • Amino acid units can be designed and optimized for enzymatic cleavage by a particular enzyme, for example, a tumor-associated protease, cathepsin B, C and D, or a plasmin protease.
  • the amino acid unit is valine-citrulline (vc or val-cit).
  • the amino acid unit is phenylalanine-lysine (i.e., fk).
  • the amino acid unit is N-methylvaline-citrulline.
  • the amino acid unit is 5 -aminovaleric acid, homo phenylalanine lysine, tetraisoquinolinecarboxylate lysine, cyclohexylalanine lysine, isonepecotic acid lysine, beta-alanine lysine, glycine serine valine glutamine and isonepecotic acid.
  • ADCs Another approach for the generation of ADCs involves the use of heterobifunctional cross-linkers which link the anti-B7-H4 antibody to the drug moiety.
  • cross-linkers examples include N-succinimidyl 4-(5-nitro-2- pyridyldithio)-pentanoate or the highly water-soluble analog N-sulfosuccinimidyl 4-(5- nitro-2-pyridyldithio)-pentanoate.
  • SPDB N-succinimi
  • the antibodies may be modified with the cross-linkers N-succinimidyl 4-(5-nitro-2-pyridyldithio)-pentanoate, N-sulfosuccinimidyl 4-(5-nitro-2- pyridyldithio)-pentanoate, SPDB, SNPB, SSNPB, SMNP, SCPB, or SSCPB can then react with a small excess of a particular drug that contains a thiol moiety to give excellent yields of an ADC (see also U.S. Pat. No. 6,913,748, incorporated by reference herein).
  • charged linkers are used to conjugate anti-B7-H4 antibodies to drugs to form ADCs.
  • Charged linkers include linkers that become charged after cell processing.
  • the presence of a charged group(s) in the linker of a particular ADC or on the drug after cellular processing provides several advantages, such as (i) greater water solubility of the ADC, (ii) ability to operate at a higher concentration in aqueous solutions, (iii) ability to link a greater number of drug molecules per antibody, potentially resulting in higher potency, (iv) potential for the charged conjugate species to be retained inside the target cell, resulting in higher potency, and (v) improved sensitivity of multidrug resistant cells, which would be unable to export the charged drug species from the cell.
  • the charged or pro-charged cross-linkers are those containing sulfonate, phosphate, carboxyl or quaternary amine substituents that significantly increase the solubility of the ADCs, especially for ADCs with 2 to 20 conjugated drugs.
  • Conjugates prepared from linkers containing a pro-charged moiety would produce one or more charged moieties after the conjugate is metabolized in a cell.
  • linkers that can be used with the compositions and methods include valine-citrulline; maleimidocaproyl; amino benzoic acids; p- aminobenzylcarbamoyl (PAB); lysosomal enzyme-cleavable linkers; maleimidocaproyl- polyethylene glycol (MC(PEG)6-OH); N-methyl-valine citrulline; N-succinimidyl 4-(N- mal eimidom ethyl )cy cl ohexane-1 -carboxylate (SMCC); N-Succinimidyl 4-(2- pyridyldithiojbutanoate (SPDB); and N-Succinimidyl 4-(2-pyridylthio)pentanoate (SPP) (See also US 2011/0076232).
  • MCC mal eimidom ethyl
  • SPDB N-Succinimidyl 4-(2-pyridyldi
  • Another linker for use includes an avidin-biotin linkage to provide an avidin-biotin-containing ADC (See also U.S. Pat. No. 4,676,980, PCT publication Nos. WO 1992/022332 A2, WO 1994/016729A1, WO 1995/015770A 1, WO 1997/031655 A2, WO 1998/035704A1, WO 1999/019500A1, WO2001/09785 A2, WO200 1/090198 Al, W02003/093793A2, W02004/050016A2, W02005/081898A2, W02006/083562A2, W02006/089668A1, W02007/150020A1, WO2008/135237A1, WO2010/111198A1, WO2011/057216A1, WO2011/058321 Al, WO2012/027494 Al, and EP77671B1), wherein some such linkers are resistant to biotinidase cleavage.
  • Additional linkers that may be used include a cohesin/dockerin pair to provide a cohesion-dockerin- containing ADC (See PCT publication Nos. W02008/097866A2, W02008/097870A2, W02008/103947A2, and W02008/103953A2).
  • Additional linkers may contain non-peptide polymers (examples include, but are not limited to, polyethylene glycol, polypropylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, PLA (poly(lactic acid)), PLGA (poly(lactic acid-glycolic acid)), and combinations thereof, wherein a preferred polymer is polyethylene glycol) (See also PCT publication No.
  • maytansinoids comprise a linking moiety that contains a reactive chemical group are C-3 esters of maytansinol and its analogs where the linking moiety contains a disulfide bond, and the chemical reactive group comprises a N-succinimidyl or N-sulfosuccinimidyl ester.
  • the C-3 position having a hydroxyl group, the C-14 position modified with hydroxymethyl, the C-15 position modified with hydroxy and the C-20 position having a hydroxy group are all useful.
  • the linking moiety most preferably is linked to the C-3 position of maytansinol.
  • the conjugation of the drug to the antibody via a linker can be accomplished by any technique known in the art.
  • a number of different reactions are available for covalent attachment of drugs and linkers to antibodies. This may be accomplished by reaction of the amino acid residues of the antibody, including the amine groups of lysine, the free carboxylic acid groups of glutamic and aspartic acid, the sulfhydryl groups of cysteine and the various moieties of the aromatic amino acids.
  • One of the most commonly used non-specific methods of covalent attachment is the carbodiimide reaction to link a carboxy (or amino) group of a compound to amino (or carboxy) groups of the antibody.
  • bifunctional agents such as dialdehydes or imidoesters have been used to link the amino group of a compound to amino groups of an antibody.
  • the Schiff base reaction also involves the periodate oxidation of a drug that contains glycol or hydroxy groups, thus forming an aldehyde which is then reacted with the binding agent. Attachment occurs via formation of a Schiff base with amino groups of the antibody.
  • Isothiocyanates can also be used as coupling agents for covalently attaching drugs to antibodies. Other techniques are known to the skilled artisan and within the scope of the disclosure.
  • an intermediate which is the precursor of the linker, is reacted with the drug under appropriate conditions.
  • reactive groups are used on the drug or the intermediate.
  • the product of the reaction between the drug and the intermediate, or the derivatized drug, is subsequently reacted with the anti- B7-H4 antibody under appropriate conditions.
  • the synthesis and structure of exemplary linkers, stretcher units, amino acid units, self-immolative spacer units are described in U.S. Patent Application Publication Nos. 20030083263, 20050238649 and 20050009751, each if which is incorporated herein by reference.
  • Stability of the ADC may be measured by standard analytical techniques such as mass spectroscopy, HPLC, and the separation/analysis technique LC/MS.
  • the antibody drug conjugate has a structure represented by Formula la or pharmaceutically acceptable salt thereof:
  • Ab is an anti-B7-H4 antibody comprising a light chain having an amino acid sequence according to any one of SEQ ID NOs: 62, 63, or 64, and a heavy chain having an amino acid sequence according to any one of SEQ ID NOs: 75, 76, 77, 78, or 79, each instance of W is -C(O)-, -C(O)NR'-, -C(O)O-, -SO 2 NR'-, -P(O)R"NR'-, -SONR'-, - PO2NR’-, or -NR’C(O)-; each instance of R 1 and R" is independently hydrogen, C1-8 alkyl, C3-8 cycloalkyl, C1-8 alkoxy, C1-8 alkylthio, mono- or di-Ci-8 alkylamino, heteroaryl or aryl; each instance of Z is independently C1-8 alkyl, halogen, cyano, or nitro; each instance of n
  • Y is an alkylene or heteroalkylene, preferably a C1-50 alkylene or C1-50 heteroalkylene, comprising one or more of the following:
  • a heteroarylene e.g., a heteroarylene in the alkylene or heteroalkylene chain
  • n4 is an integer from 1 to 20, preferably 1 to 4.
  • Ab is an anti-B7-H4 antibody comprising a light chain having an amino acid sequence according to any one of SEQ ID NO: 63, and a heavy chain having an amino acid sequence according to any one of SEQ ID NO: 78.
  • W is -C(O)NR'-, further wherein the C is directly bonded to the phenyl ring of Formula la, and NR' is bonded to Y.
  • Y comprises a peptide and the peptide comprises at least one hydrophilic amino acid, preferably an amino acid having a side chain having a moiety that bears a charge at neutral pH in aqueous solution (e.g., an amine, guanidine, or carboxyl moiety), most preferably each amino acid of the peptide is independently selected from alanine, aspartate, asparagine, glutamate, glutamine, glycine, lysine, ornithine, proline, serine, and threonine.
  • Y is covalently bonded to the antibody by a thioether bond, and the thioether bond comprises a sulfur atom of a cysteine of the antibody.
  • Y comprises an oxime and: the oxygen atom of the oxime is on the side of Y that is linked to W and the carbon atom of the oxime is on the side of Y that is linked to Ab; or the carbon atom of the oxime is on the side of Y that is linked to W and the oxygen atom of the oxime is on the side of Y that is linked to Ab.
  • V is a single bond, -O-, -S-, -NR 1 -, -C(O)NR 2 -, -NR 3 C(O)-, -NR 4 SO 2 -, or -SO 2 NR 5 -, preferably -O-;
  • X is -O-, Ci-8 alkylene, or -NR 1 -, preferably -O-;
  • R 1 to R 5 are each independently hydrogen, C1-6 alkyl, C1-6 alkyl C6-20 aryl, or C1-6 alkyl C3-20 heteroaryl;
  • r is an integer from 1 to 10, preferably 2;
  • p is an integer from 0 to 12, preferably 2;
  • q is an integer from 1 to 20, preferably 2, 5, or 11; and
  • w is an integer from 1 to 20, preferably 6 to 20.
  • Y comprises -(CH2CH2O)w-, -O(CH2CH2O) W -, or - (CH2CH 2 O)WCH2-, wherein w is an integer from 1 to 20, preferably 2 to 10.
  • Y comprises a moiety represented by Formula V
  • Y is linear.
  • the term “linear”, when used in the context of variable Y, refers to a unit that couples the antibody to a single therapeutically active agent (e.g., a drug or diagnostic agent) via an unbranched covalent moiety (e.g., a via C1-50 alkylene).
  • a single therapeutically active agent e.g., a drug or diagnostic agent
  • an unbranched covalent moiety e.g., a via C1-50 alkylene.
  • the term “linear”, when used in the context of variable Y, does not preclude substitution (e.g., alkyl, aryl, or heteroaryls) on Y, provided that said substituents are not therapeutically active agents or coupled to further active agents.
  • Y is branched.
  • the term “branched”, when used in the context of variable Y, refers to a unit that couples the antibody to multiple therapeutically active agents (e.g., drugs and/or diagnostic agents) via a covalent moiety.
  • Y may comprise an alkylene that splits at a branching point into multiple alkylene chains, each of which covalently links the antibody to one or more therapeutically active agents (e.g., drugs).
  • Y comprises: i) a branching unit covalently coupled to Ab by a primary linker; ii) a first branch, which couples a first therapeutically active substance, via a first cleavage group, to the branching unit; and iiia) a second branch, which couples a second therapeutically active substance, via a second cleavage group, to the branching unit; or iiib) a second branch which couples an alkyl or heteroalkyl (e.g., a polyethylene glycol monomer or a polyethylene glycol oligomer) to the branching unit.
  • an alkyl or heteroalkyl e.g., a polyethylene glycol monomer or a polyethylene glycol oligomer
  • At least one branching unit has a structure represented R 30 is hydrogen or C1-30 alkyl
  • R 40 is hydrogen or L 5 -COOR 50 ;
  • R 50 is hydrogen or C1-30 alkyl
  • L 2 , L 3 , L 4 , and L 5 are each independently a bond or alkylene.
  • the branching unit is a nitrogen atom. In other embodiments, the branching unit is an amide and the primary linker comprises the carbonyl of the amide. In yet other embodiments, the branching unit is an amide and the secondary linker comprises the carbonyl of the amide. In certain preferred embodiments, the branching unit is lysine.
  • the antibody comprises an amino acid motif recognizable by an isoprenoid transferase at the C-terminus of the antibody, and the thioether bond comprises a sulfur atom of a cysteine of the amino acid motif.
  • the isoprenoid transferase is famesyl protein transferase (FTase) or geranylgeranyl transferase (GGTase).
  • the amino acid motif has a CY1Y1X sequence, further wherein:
  • C is cysteine; each Yi independently is an aliphatic amino acid;
  • X is selected from glutamine, glutamate, serine, cysteine, methionine, alanine, and leucine; and the thioether bond comprises a sulfur atom of a cysteine of the amino acid motif.
  • each Yi is independently selected from alanine, isoleucine, leucine, methionine, and valine.
  • the amino acid motif comprises a CVIM or CVLL.
  • the conjugate comprises at least one of 1 to 20 amino acids between the antibody and the amino acid motif, and at least one of the amino acids is glycine.
  • the amino acid motif has the sequence GGGGGGGCVIM.
  • the antibody conjugate comprises a structure represented by
  • MMAE is monomethyl auri statin E; represents a connection point to Y.
  • glucuronide-based linker connects drug units to an antibody in which its glucuronide unit comprises a glycosidase recognition site that is cleavable by an enzyme having P-glucuronidase activity thereby releasing free drug.
  • Glucuronide-based linkers improve the solubility of ADCs and exhibit sufficient serum stability to provide targeted delivery of a conjugated drug to a targeted cell.
  • Vectors encoding the proteins, polypeptides, fragments, variants and fusions thereof are also provided. Nucleic acids, such as those described above, can be inserted into vectors for expression in cells.
  • a “vector” is a replicon, such as a plasmid, phage, virus or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment.
  • Vectors can be expression vectors.
  • An “expression vector” is a vector that includes one or more expression control sequences, and an “expression control sequence” is a DNA sequence that controls and regulates the transcription and/or translation of another DNA sequence.
  • Nucleic acids in vectors can be operably linked to one or more expression control sequences.
  • “operably linked” means incorporated into a genetic construct so that expression control sequences effectively control expression of a coding sequence of interest.
  • Examples of expression control sequences include promoters, enhancers, and transcription terminating regions.
  • a promoter is an expression control sequence composed of a region of a DNA molecule, typically within 100 nucleotides upstream of the point at which transcription starts (generally near the initiation site for RNA polymerase II). To bring a coding sequence under the control of a promoter, it is necessary to position the translation initiation site of the translational reading frame of the polypeptide between one and about fifty nucleotides downstream of the promoter.
  • Enhancers provide expression specificity in terms of time, location, and level. Unlike promoters, enhancers can function when located at various distances from the transcription site. An enhancer also can be located downstream from the transcription initiation site.
  • a coding sequence is “operably linked” and “under the control” of expression control sequences in a cell when RNA polymerase is able to transcribe the coding sequence into mRNA, which then can be translated into the protein encoded by the coding sequence.
  • Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, tobacco mosaic virus, herpes viruses, cytomegalo virus, retroviruses, vaccinia viruses, adenoviruses, and adeno- associated viruses.
  • Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA), and Invitrogen Life Technologies (Carlsbad, CA).
  • An expression vector can include a tag sequence.
  • Tag sequences are typically expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino terminus. Examples of useful tags include, but are not limited to, green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, FlagTM tag (Kodak, New Haven, CT), maltose E binding protein and protein A.
  • GFP green fluorescent protein
  • GST glutathione S-transferase
  • polyhistidine polyhistidine
  • c-myc hemagglutinin
  • FlagTM tag Kodak, New Haven, CT
  • maltose E binding protein and protein A maltose E binding protein and protein A.
  • a nucleic acid molecule encoding one of the disclosed polypeptides is present in a vector containing nucleic acids that encode one or more domains of an Ig heavy chain constant region, for example, having an amino acid sequence corresponding to the hinge, CH2 and CH3 regions of a human immunoglobulin Cyl chain.
  • Vectors containing nucleic acids to be expressed can be transferred into host cells.
  • the term “host cell” is intended to include prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced.
  • “transformed” and “transfected” encompass the introduction of a nucleic acid molecule (e.g., a vector) into a cell by one of a number of techniques. Although not limited to a particular technique, a number of these techniques are well established within the art.
  • Prokaryotic cells can be transformed with nucleic acids by, for example, electroporation or calcium chloride mediated transformation.
  • Nucleic acids can be transfected into mammalian cells by techniques including, for example, calcium phosphate co-precipitation, DEAE- dextran-mediated transfection, lipofection, electroporation, or microinjection.
  • Host cells e.g., a prokaryotic cell or a eukaryotic cell such as a CHO cell
  • the vectors described can be used to express the proteins, polypeptides, fragments, variants and fusions thereof in cells.
  • An exemplary vector includes, but is not limited to, an adenoviral vector.
  • One approach includes nucleic acid transfer into primary cells in culture followed by autologous transplantation of the ex vivo transformed cells into the host, either systemically or into a particular organ or tissue.
  • Ex vivo methods can include, for example, the steps of harvesting cells from a subject, culturing the cells, transducing them with an expression vector, and maintaining the cells under conditions suitable for expression of the encoded polypeptides. These methods are known in the art of molecular biology.
  • the transduction step can be accomplished by any standard means used for ex vivo gene therapy, including, for example, calcium phosphate, lipofection, electroporation, viral infection, and biolistic gene transfer. Alternatively, liposomes or polymeric microparticles can be used. Cells that have been successfully transduced then can be selected, for example, for expression of the coding sequence or of a drug resistance gene. The cells then can be lethally irradiated (if desired) and injected or implanted into the subject. In one embodiment, expression vectors containing nucleic acids encoding fusion proteins are transfected into cells that are administered to a subject in need thereof.
  • Nucleic acids may also be administered in vivo by viral means.
  • Nucleic acid molecules encoding fusion proteins may be packaged into retrovirus vectors using packaging cell lines that produce replication-defective retroviruses, as is well-known in the art.
  • Other virus vectors may also be used, including recombinant adenoviruses and vaccinia virus, which can be rendered non-replicating.
  • engineered bacteria may be used as vectors.
  • Nucleic acids may also be delivered by other carriers, including liposomes, polymeric micro- and nanoparticles and polycations such as asialoglycoprotein/polylysine.
  • screening assays can include random screening of large libraries of test compounds. Alternatively, the assays may be used to focus on particular classes of compounds suspected of modulating the level of B7-H4. Assays can include determinations of B7-H4 signaling activity, or inhibitory response mediated by B7-H4. Other assays can include determinations of nucleic acid transcription or translation, mRNA levels, mRNA stability, mRNA degradation, transcription rates, and translation rates.
  • compositions including the disclosed immunomodulatory agents are provided.
  • Pharmaceutical compositions containing the immunomodulatory agent can be for administration by parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), transdermal (either passively or using iontophoresis or electroporation), or transmucosal (nasal, vaginal, rectal, or sublingual) routes of administration or using bioerodible inserts and can be formulated in dosage forms appropriate for each route of administration.
  • compositions disclosed herein are administered to a subject in a therapeutically effective amount.
  • effective amount or “therapeutically effective amount” means a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of the disorder being treated or to otherwise provide a desired pharmacologic and/or physiologic effect.
  • the precise dosage will vary according to a variety of factors such as subject-dependent variables (e.g., age, immune system health, etc.), the disease, and the treatment being effected.
  • the immunomodulatory agent is administered locally, for example by injection directly into a site to be treated.
  • the injection causes an increased localized concentration of the immunomodulatory agent composition which is greater than that which can be achieved by systemic administration.
  • the immunomodulatory agent compositions can be combined with a matrix as described above to assist in creating an increased localized concentration of the polypeptide compositions by reducing the passive diffusion of the polypeptides out of the site to be treated.
  • compositions disclosed herein are administered in an aqueous solution, by parenteral injection.
  • the formulation may also be in the form of a suspension or emulsion.
  • pharmaceutical compositions are provided including effective amounts of a peptide or polypeptide, and optionally include pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers.
  • non-aqueous solvents or vehicles examples include propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and com oil, gelatin, and injectable organic esters such as ethyl oleate.
  • the formulations may be lyophilized and redissolved/resuspended immediately before use.
  • the formulation may be sterilized by, for example, filtration through a bacteria retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions.
  • the location of release may be the stomach, the small intestine (the duodenum, the jejunum, or the ileum), or the large intestine.
  • the release will avoid the deleterious effects of the stomach environment, either by protection of the agent (or derivative) or by release of the agent (or derivative) beyond the stomach environment, such as in the intestine.
  • a coating impermeable to at least pH 5.0 is essential.
  • cellulose acetate trimellitate cellulose acetate trimellitate
  • HPMCP 50 hydroxypropylmethylcellulose phthalate
  • HPMCP 55 polyvinyl acetate phthalate
  • PVAP polyvinyl acetate phthalate
  • Eudragit L30DTM AquatericTM
  • CAP cellulose acetate phthalate
  • Eudragit LTM Eudragit STM
  • ShellacTM cellulose acetate trimellitate
  • compositions can be delivered to the lungs while inhaling and traverse across the lung epithelial lining to the blood stream when delivered either as an aerosol or spray dried particles having an aerodynamic diameter of less than about 5 microns.
  • a wide range of mechanical devices designed for pulmonary delivery of therapeutic products can be used, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art.
  • Some specific examples of commercially available devices are the Ultravent nebulizer (Mallinckrodt Inc., St. Louis, Mo.); the Acorn II nebulizer (Marquest Medical Products, Englewood, Colo.); the Ventolin metered dose inhaler (Glaxo Inc., Research Triangle Park, N.C.); and the Spinhaler powder inhaler (Fisons Corp., Bedford, Mass.).
  • Formulations for administration to the mucosa will typically be spray dried drug particles, which may be incorporated into a tablet, gel, capsule, suspension or emulsion. Standard pharmaceutical excipients are available from any formulator.
  • Transdermal formulations may also be prepared. These will typically be ointments, lotions, sprays, or patches, all of which can be prepared using standard technology. Transdermal formulations may require the inclusion of penetration enhancers.
  • Controlled release polymeric devices can be made for long term release systemically following implantation of a polymeric device (rod, cylinder, film, disk) or injection (microparticles).
  • the matrix can be in the form of microparticles such as microspheres, where the agent is dispersed within a solid polymeric matrix or microcapsules, where the core is of a different material than the polymeric shell, and the peptide is dispersed or suspended in the core, which may be liquid or solid in nature.
  • microparticles, microspheres, and microcapsules are used interchangeably.
  • the polymer may be cast as a thin slab or film, ranging from nanometers to four centimeters, a powder produced by grinding or other standard techniques, or even a gel such as a hydrogel.
  • Either non-biodegradable or biodegradable matrices can be used for delivery of fusion polypeptides or nucleic acids encoding the fusion polypeptides, although in some embodiments biodegradable matrices are preferred.
  • These may be natural or synthetic polymers, although synthetic polymers are preferred in some embodiments due to the better characterization of degradation and release profiles.
  • the polymer is selected based on the period over which release is desired. In some cases linear release may be most useful, although in others a pulse release or “bulk release” may provide more effective results.
  • the polymer may be in the form of a hydrogel (typically absorbing up to about 90% by weight of water) and can optionally be crosslinked with multivalent ions or polymers.
  • the matrices can be formed by solvent evaporation, spray drying, solvent extraction and other methods known to those skilled in the art.
  • Bioerodible microspheres can be prepared using any of the methods developed for making microspheres for drug delivery, for example, as described by Mathiowitz and Langer, J. Controlled Release, 5: 13-22 (1987); Mathiowitz, et al., Reactive Polymers, 6:275-283 (1987); and Mathiowitz, et al., J. Appl. Polymer Sci., 35:755-774 (1988).
  • the devices can be formulated for local release to treat the area of implantation or injection - which will typically deliver a dosage that is much less than the dosage for treatment of an entire body - or systemic delivery. These can be implanted or injected subcutaneously, into the muscle, fat, or swallowed.
  • the antibodies can be generated in cell culture, in phage, or in various animals, including but not limited to cows, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, apes. Therefore, in one embodiment, an antibody is a mammalian antibody. Phage techniques can be used to isolate an initial antibody or to generate variants with altered specificity or avidity characteristics. Such techniques are routine and well known in the art. In one embodiment, the antibody is produced by recombinant means known in the art. For example, a recombinant antibody can be produced by transfecting a host cell with a vector comprising a DNA sequence encoding the antibody.
  • One or more vectors can be used to transfect the DNA sequence expressing at least one VL and one VH region in the host cell.
  • Exemplary descriptions of recombinant means of antibody generation and production include Delves, Antibody Production: Essential Techniques (Wiley, 1997); Shephard, et al., Monoclonal Antibodies (Oxford University Press, 2000); Goding, Monoclonal Antibodies: Principles and Practice (Academic Press, 1993); Current Protocols in Immunology (John Wiley & Sons, most recent edition).
  • the disclosed antibodies can be modified by recombinant means to increase greater efficacy of the antibody in mediating the desired function.
  • antibodies can be modified by substitutions using recombinant means.
  • the substitutions will be conservative substitutions.
  • at least one amino acid in the constant region of the antibody can be replaced with a different residue. See, e.g., U.S. Pat. No. 5,624,821, U.S. Pat. No. 6,194,551, Application No. WO 9958572; and Angal, et al., Mol. Immunol. 30: 105-08 (1993).
  • the modification in amino acids includes deletions, additions, and substitutions of amino acids.
  • the antibodies are labeled by joining, either covalently or non- covalently, a substance which provides for a detectable signal.
  • labels and conjugation techniques are known and are reported extensively in both scientific and patent literature. These antibodies can be screened for binding to proteins, polypeptides, or fusion proteins of B7-H4. See, e.g., Antibody Engineering: A Practical Approach (Oxford University Press, 1996).
  • suitable antibodies with the desired biologic activities can be identified using in vitro assays including but not limited to: proliferation, migration, adhesion, soft agar growth, angiogenesis, cell-cell communication, apoptosis, transport, signal transduction, and in vivo assays such as the inhibition of tumor growth.
  • the antibodies provided herein can also be useful in diagnostic applications. As capture or non-neutralizing antibodies, they can be screened for the ability to bind to the specific antigen without inhibiting the receptor-binding or biological activity of the antigen. As neutralizing antibodies, the antibodies can be useful in competitive binding assays.
  • Antibodies that can be used in the disclosed compositions and methods include whole immunoglobulin (i.e., an intact antibody) of any class, fragments thereof, and synthetic proteins containing at least the antigen binding variable domain of an antibody.
  • the variable domains differ in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not usually evenly distributed through the variable domains of antibodies. It is typically concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of the variable domains are called the framework (FR).
  • CDRs complementarity determining regions
  • FR framework
  • variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure.
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies.
  • fragments of antibodies which have bioactivity.
  • the fragments whether attached to other sequences or not, include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment.
  • Techniques can also be adapted for the production of single-chain antibodies specific to an antigenic peptide. Methods for the production of single-chain antibodies are well known to those of skill in the art.
  • a single chain antibody can be created by fusing together the variable domains of the heavy and light chains using a short peptide linker, thereby reconstituting an antigen binding site on a single molecule.
  • Single-chain antibody variable fragments in which the C-terminus of one variable domain is tethered to the N-terminus of the other variable domain via a 15 to 25 amino acid peptide or linker have been developed without significantly disrupting antigen binding or specificity of the binding.
  • the linker is chosen to permit the heavy chain and light chain to bind together in their proper conformational orientation.
  • Divalent single-chain variable fragments can be engineered by linking two scFvs. This can be done by producing a single peptide chain with two VH and two VL regions, yielding tandem scFvs. ScFvs can also be designed with linker peptides that are too short for the two variable regions to fold together (about five amino acids), forcing scFvs to dimerize. This type is known as diabodies. Diabodies have been shown to have dissociation constants up to 40-fold lower than corresponding scFvs, meaning that they have a much higher affinity to their target. Still shorter linkers (one or two amino acids) lead to the formation of trimers (triabodies or tribodies). Tetrabodies have also been produced. They exhibit an even higher affinity to their targets than diabodies.
  • a monoclonal antibody is obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules.
  • Monoclonal antibodies include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity.
  • Monoclonal antibodies can be made using any procedure which produces monoclonal antibodies.
  • a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes may be immunized in vitro.
  • Antibodies may also be made by recombinant DNA methods. DNA encoding the disclosed antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques.
  • Methods of making antibodies using protein chemistry are also known in the art.
  • One method of producing proteins comprising the antibodies is to link two or more peptides or polypeptides together by protein chemistry techniques.
  • peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert - butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc., Foster City, CA).
  • Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert - butyloxycarbonoyl) chemistry Applied Biosystems, Inc., Foster City, CA.
  • a peptide or polypeptide corresponding to the antibody for example, can be synthesized by standard chemical reactions.
  • a peptide or polypeptide can be synthesized and not cleaved from its synthesis resin whereas the other fragment of an antibody can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group which is functionally blocked on the other fragment.
  • peptide condensation reactions these two fragments can be covalently joined via a peptide bond at their carboxyl and amino termini, respectively, to form an antibody, or fragment thereof.
  • the peptide or polypeptide is independently synthesized in vivo as described above. Once isolated, these independent peptides or polypeptides may be linked to form an antibody or antigen binding fragment thereof via similar peptide condensation reactions.
  • enzymatic ligation of cloned or synthetic peptide segments allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides or whole protein domains.
  • native chemical ligation of synthetic peptides can be utilized to synthetically construct large peptides or polypeptides from shorter peptide fragments.
  • This method includes a two-step chemical reaction. The first step is the chemoselective reaction of an unprotected synthetic peptide-alpha- thioester with another unprotected peptide segment containing an amino-terminal Cys residue to give a thioester-linked intermediate as the initial covalent product. Without a change in the reaction conditions, this intermediate undergoes spontaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site.
  • the disclosed proteins, polypeptides, fragments, variants and fusions thereof can be manufactured using conventional techniques that are known in the art.
  • Isolated fusion proteins can be obtained by, for example, chemical synthesis or by recombinant production in a host cell.
  • a nucleic acid containing a nucleotide sequence encoding the protein, polypeptide, fragment, variant or fusion thereof can be used to transform, transduce, or transfect a bacterial or eukaryotic host cell (e.g., an insect, yeast, or mammalian cell).
  • nucleic acid constructs include a regulatory sequence operably linked to a nucleotide sequence encoding the protein, polypeptide, fragment, variant or fusion thereof.
  • Regulatory sequences also referred to herein as expression control sequences
  • Useful prokaryotic and eukaryotic systems for expressing and producing polypeptides are well known in the art include, for example, Escherichia coli strains such as BL-21, and cultured mammalian cells such as CHO cells.
  • viral -based expression systems can be utilized to express fusion proteins.
  • Viral based expression systems are well known in the art and include, but are not limited to, baculoviral, SV40, retroviral, or vaccinia based viral vectors.
  • Mammalian cell lines that stably express proteins, polypeptides, fragments, variants or fusions thereof, can be produced using expression vectors with appropriate control elements and a selectable marker.
  • the eukaryotic expression vectors pCR3.1 (Invitrogen Life Technologies) and p91023(B) are suitable for expression of proteins, polypeptides, fragments, variants or fusions thereof, in, for example, Chinese hamster ovary (CHO) cells, COS-1 cells, human embryonic kidney 293 cells, NIH3T3 cells, BHK21 cells, MDCK cells, and human vascular endothelial cells (HUVEC).
  • Additional suitable expression systems include the GS Gene Expression SystemTM available through Lonza Group Ltd.
  • a protein, polypeptide, fragment, variant or fusion thereof can be produced by (a) ligating amplified sequences into a mammalian expression vector such as pcDNA3 (Invitrogen Life Technologies), and (b) transcribing and translating in vitro using wheat germ extract or rabbit reticulocyte lysate.
  • a mammalian expression vector such as pcDNA3 (Invitrogen Life Technologies)
  • pcDNA3 Invitrogen Life Technologies
  • Proteins, polypeptides, fragments, variants or fusions thereof can be isolated using, for example, chromatographic methods such as affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, DEAE ion exchange, gel filtration, and hydroxylapatite chromatography.
  • Proteins, polypeptides, fragments, variants or fusions thereof can be engineered to contain an additional domain containing amino acid sequence that allows the polypeptides to be captured onto an affinity matrix.
  • an Fc-fusion polypeptide in a cell culture supernatant or a cytoplasmic extract can be isolated using a protein A column.
  • a tag such as c-myc, hemagglutinin, polyhistidine, or FlagTM (Kodak) can be used to aid polypeptide purification.
  • tags can be inserted anywhere within the polypeptide, including at either the carboxyl or amino terminus.
  • Other fusions that can be useful include enzymes that aid in the detection of the polypeptide, such as alkaline phosphatase.
  • Immunoaffinity chromatography also can be used to purify polypeptides. Fusion proteins can additionally be engineered to contain a secretory signal (if there is not a secretory signal already present) that causes the Proteins, polypeptides, fragments, variants or fusions thereof to be secreted by the cells in which it is produced. The secreted Proteins, polypeptides, fragments, variants or fusions thereof can then conveniently be isolated from the cell media.
  • Isolated nucleic acid molecules can be produced by standard techniques, including, without limitation, common molecular cloning and chemical nucleic acid synthesis techniques. For example, polymerase chain reaction (PCR) techniques can be used to obtain an isolated nucleic acid encoding a variant polypeptide. PCR is a technique in which target nucleic acids are enzymatically amplified. Typically, sequence information from the ends of the region of interest or beyond can be employed to design oligonucleotide primers that are identical in sequence to opposite strands of the template to be amplified. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA.
  • PCR polymerase chain reaction
  • Primers typically are 14 to 40 nucleotides in length but can range from 10 nucleotides to hundreds of nucleotides in length.
  • General PCR techniques are described, for example in PCR Primer: A Laboratory Manual, ed. by Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995.
  • reverse transcriptase can be used to synthesize a complementary DNA (cDNA) strand.
  • Ligase chain reaction, strand displacement amplification, self-sustained sequence replication or nucleic acid sequence-based amplification also can be used to obtain isolated nucleic acids. See, for example, Lewis (1992) Genetic Engineering News 12: 1; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87: 1874-1878; and Weiss (1991) Science 254: 1292-1293.
  • Isolated nucleic acids can be chemically synthesized, either as a single nucleic acid molecule or as a series of oligonucleotides (e.g., using phosphoramidite technology for automated DNA synthesis in the 3’ to 5’ direction).
  • oligonucleotides e.g., >100 nucleotides
  • one or more pairs of long oligonucleotides can be synthesized that contain the desired sequence, with each pair containing a short segment of complementarity (e.g., about 15 nucleotides) such that a duplex is formed when the oligonucleotide pair is annealed.
  • Phase III screening Functional assays to confirm that B7-H4 mAbs or combination of mAbs modulate B7-H4 signaling. These assays will utilize cell lines that express endogenous B7-H4, or primary cells such as human monocytes, macrophages and dendritic cell subsets to assess function in the presence of B7-H4 mAbs. Additionally, reporter cells lines may be used to determine if signaling pathways such as NFkB (NFkB reporter) or NF AT (NF AT reporter) are altered following culture with B7-H4 mAbs.
  • NFkB reporter NFkB reporter
  • NF AT reporter NF AT reporter
  • Phase IV screening Functional assays to determine if B7-H4 mAbs are capable of inducing antibody dependent cell cytotoxicity (ADCC), complement dependent cytotoxicity (CDC) or cellular apoptosis through other mechanisms, of B7-H4 expressing cell lines.
  • ADCC antibody dependent cell cytotoxicity
  • CDC complement dependent cytotoxicity
  • B7-H4 mAbs will be tested for the ability to deplete through one of these methods leukemia cell lines, known to express B7-H4 on the cell surface.
  • B7-H4 mAbs may also be engineered to deplete B7-H4 expressing cells and tested as described later in this document through known methods.
  • Phase V screening Functional assays to determine if B7-H4 mAbs are capable of delivering or inducing a negative signal (agonist) via B7-H4 into B7-H4 expressing cells to inhibit cellular function.
  • Cell lines that endogenously express B7-H4, or transfectants of cell lines will be assessed for changes in phenotype and survival following culture with B7-H4 mAbs.
  • reporter cell lines will be used to determine in B7-H4 mAbs modulate positive signaling pathways such as NF-kB (NF-kB reporter) or other known cell signaling reporters. Induction of apoptosis in cell lines will be also be evaluated.
  • Phase II and III assays can be used to predict the concentrations of B7-H4 mAb(s) required to block physiological levels of ligands in vivo.
  • Antagonists or agonists of B7-H4 can be used to modulate immune responses in subjects in need of such treatment.
  • Methods of inducing or enhancing an immune response in a subject include administering a subject an effective amount of immunomodulatory agent, or cells primed ex vivo with the immunomodulatory agent.
  • the immune response can be, for example, a primary immune response to an antigen or an increase effector cell function such as increasing antigen-specific proliferation of T cells, enhancing cytokine production by T cells, stimulating differentiation, or a combination thereof.
  • the agent can increase the development of naive T cells into Th 1, Th 17, Th22, or other cells that secrete, or cause other cells to secrete, inflammatory molecules, including, but not limited to, IL-ip, TNF-a, TGF-beta, IFN-y, IL- 17, IL-6, IL-23, IL-22, IL-21, and MMPs.
  • the agent can reduce or inhibit the activity of Tregs, reduce the production of cytokines such as IL- 10 from Tregs, reduce the differentiation of Tregs, reduce the number of Tregs, reduce the ratio of Tregs within an immune cell population, or reduce the survival of Tregs.
  • the immunomodulatory agent can be administered to a subject in need thereof in an effective amount to overcome T cell exhaustion and/or T cell anergy. Overcoming T cell exhaustion or T cell anergy can be determined by measuring T cell function using known techniques.
  • the methods can be used in vivo or ex vivo as immune response-stimulating therapeutic applications.
  • the agent, or nucleic acid encoding the agent is administered directly to the subject.
  • the agent or nucleic acid encoding the agent is contacted with cells (e.g., immune cells) ex vivo, and the treat cells are administered to the subject (e.g. adoptive transfer).
  • the disclosed immunomodulatory agents can be used for treating a subject having or being predisposed to any disease or disorder to which the subject's immune system mounts an immune response. The agents can enable a more robust immune response to be possible.
  • the disclosed compositions are useful to stimulate or enhance immune responses involving T cells.
  • the immunomodulatory agents utilized for increasing an immune response are typically those that reduce B7-H4 expression, ligand binding, crosslinking, negative signaling, or a combination thereof.
  • the agent can be an antagonist of B7- H4, such as an antagonist (blocking) anti- B7-H4 antibody or antigen binding fragment thereof.
  • the agent can also be a B7-H4 polypeptide, for example, a soluble polypeptide, or fusion protein thereof that can serve as a decoy receptor for one or more B7-H4 ligands or receptors.
  • B7-H4 blockade for example using function blocking anti- B7-H4 antibodies, can be an alternative agent or complementary agent to soluble B7-H4 polypeptides and fusion proteins.
  • B7-H4 blockade is combined with a decoy receptor such as soluble B7-H4 or fusion protein thereof.
  • the combined treatment e.g., B7-H4-Fc and B7-H4 blockade
  • B7-H4-Fc and B7-H4 blockade may be complementary.
  • immune response stimulating therapy (e.g., in the treatment of cancer or infections) includes depletion of B7-H4+ cells.
  • B7-H4 depleting mAbs can be carried out according to known construction and screening methods including those discussed herein. See, for example, Reff, et al, Blood. Vol83, No 2, 1994: pp 435-445, which describes preparation of an anti-CD20 chimeric antibody that binds to human Clq, and mediates complement-dependent cell lysis (CDCC) in the presence of human complement, and anti-body-dependent cellular cytotoxicity (ADCC) with human effector cells.
  • CDC complement-dependent cell lysis
  • ADCC anti-body-dependent cellular cytotoxicity
  • Rituximab destroys B cells and is therefore used to treat diseases which are characterized by overactive, dysfunctional, or excessive numbers of B cells.
  • Other B cell -depleting antibodies include ocrelizumab and ofatumumab.
  • CD3 Abs can preferentially target and deplete activated effector T cells while preserving CD4 + Foxp3 + Tregs.
  • the antibodies transiently deplete T cells although they display no or little complement-dependent and antibody-dependent cellular cytotoxicity. Redirected cell lysis due to the ability to crosslink CD3 molecules expressed by two different cells (cytotoxic CD8+ T cells on one side and other target T cells on the other side) has been shown, however, T cell depletion mostly results from AICD (reviewed in You, Front Immunol. 2015; 6: 242).
  • a subject in need of enhancing their immune response can be administered with an agent that inhibits or blocks B7-H4 suppressive immune response in an amount effective to increase the uptake of antigen by antigen presenting cells (APCs).
  • APCs antigen presenting cells
  • compositions and methods can be used to treat cancer.
  • the agents are used to stimulate or enhance an immune response to cancer in the subject by administering to the subject an amount of an immunomodulatory agent that reduces B7-H4 expression, ligand binding, crosslinking, negative signaling, or a combination thereof
  • Cancer cells acquire a characteristic set of functional capabilities during their development, albeit through various mechanisms. Such capabilities include evading apoptosis, self-sufficiency in growth signals, insensitivity to anti -growth signals, tissue invasion/metastasis, limitless replicative potential, and sustained angiogenesis.
  • cancer cell is meant to encompass both pre-malignant and malignant cancer cells.
  • cancer refers to a benign tumor, which has remained localized. In other embodiments, cancer refers to a malignant tumor, which has invaded and destroyed neighboring body structures and spread to distant sites. In yet other embodiments, the cancer is associated with a specific cancer antigen (e.g., pan-carcinoma antigen (KS 1/4), ovarian carcinoma antigen (CA125), prostate specific antigen (PSA), carcinoembryonic antigen (CEA), CD 19, CD20, HER2/neu, etc.).
  • KS 1/4 pan-carcinoma antigen
  • CA125 ovarian carcinoma antigen
  • PSA prostate specific antigen
  • CEA carcinoembryonic antigen
  • CD 19, CD20, HER2/neu etc.
  • carcinoma including that of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, head and neck, thyroid and skin; including squamous cell carcinoma; hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Berketts lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; other tumors, including melanoma, seminoma, tetratocarcinoma
  • Cancers caused by aberrations in apoptosis can also be treated by the disclosed methods and compositions.
  • Such cancers may include, but are not limited to, follicular lymphomas, carcinomas with p53 mutations, hormone dependent tumors of the breast, prostate and ovary, and precancerous lesions such as familial adenomatous polyposis, and myelodysplastic syndromes.
  • malignancy or dysproliferative changes (such as metaplasias and dysplasias), or hyperproliferative disorders, are treated or prevented by the methods and compositions in the ovary, bladder, breast, colon, lung, skin, pancreas, or uterus.
  • sarcoma, melanoma, or leukemia is treated or prevented by the methods and compositions.
  • compositions and methods are particularly useful for the treatment of cancers that are associated with cells that express abnormally high levels of B7-H4.
  • leukemias including, but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia leukemias and myelodysplastic syndrome, chronic leukemias such as but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as, but not limited to, Hodgkin's disease or non-Hodgkin's disease lymphomas (e.g., diffuse anaplastic lymphoma kinase (ALK) negative, large B-cell lymphoma (DLBCL); diffuse anaplastic lymphoma kinase (ALK) negative, large B-cell lymphoma (DLBCL); diffuse anaplastic lymphoma kinase (
  • cancers include myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma and papillary adenocarcinomas (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia and Murphy et al., 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books U.S.A., Inc., United States of America).
  • compositions below are to be understood as exemplary compositions related to the present disclosure. Such are not intended to be limiting of the scope of the present disclosure.
  • compositions described herein can be administered to a subject in need thereof, either alone or in combination with a pharmaceutically acceptable excipient and/or carrier, in an amount sufficient to induce an appropriate anti-tumor response.
  • Administration can include injection, infusion, other methods disclosed herein, and other methods known in the art.
  • Administration includes but is not limited to intravenous, intramuscular, subcutaneous, and the like.
  • the response can comprise, without limitation, specific immune response, non-specific immune response, both specific and non-specific response, innate response, primary immune response, adaptive immunity, secondary immune response, memory immune response, immune cell activation, immune cell proliferation, immune cell differentiation, and cytokine expression.
  • the invention provides a method of treating cancer in a mammal by administering to a mammal an effective amount of an antibody-drug conjugate.
  • Effective amounts of the antibody-drug conjugate can be determined by one of skill in the art with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (i.e. subject). It can generally be stated that the antibody-drug conjugate can be administered simultaneously or separately from other agents subject to the same or different dosing and timing regimens, as described herein. The antibody-drug conjugates may also be administered multiple times at these dosages.
  • the antibody-drug conjugates can be administered by using infusion techniques that are commonly known in immunotherapy (Rosenberg, et al., New Eng.
  • the optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.
  • An effective amount for a particular patient may vary depending on factors such as the condition being treated, the overall health of the patient, the route and dose of administration and the severity of side effects. Guidance for methods of treatment and diagnosis is available (Maynard, et al., Interpharm Press, 1996; Dent, Urch Publ., 2001).
  • an effective amount of the antibody-drug conjugates described herein may be given in one dose, but is not restricted to one dose.
  • the administration can be two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more, administrations of the compositions.
  • the administrations can be spaced by time intervals of one minute, two minutes, three, four, five, six, seven, eight, nine, ten, or more minutes, by intervals of about one hour, two hours, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, and so on.
  • the term "about” means plus or minus any time interval within 30 minutes.
  • the administrations can also be spaced by time intervals of one day, two days, three days, four days, five days, six days, seven days, eight days, nine days, ten days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, and combinations thereof.
  • the disclosure is not limited to dosing intervals that are spaced equally in time, but encompass doses at non-equal intervals, such as a priming schedule consisting of administration at 1 day, 4 days, 7 days, and 25 days, just to provide a non-limiting example.
  • the antibody-drug conjugates of the present invention can be administered in a dose, or dosages, where each dose comprises about O. lmg, 0.5mg, Img, 2, mg, 3 mg, 4, mg, 5mg, lOmg, 15mg, 20mg, 30mg, 40mg, 50mg, 60mg, 70mg, 80mg, 90mg, lOOmg, 200mg, 250mg, 300mg, 350mg, 400mg, 500mg, 600mg, and the like.
  • the antibody-drug conjugates of the present invention can be administered in a dose, or dosages, where each dose is dependent on subject body weight.
  • a dose, or dosages can be administered at about O. lmg/kg, about 0.5 mg/kg, about 1 mg/kg, at about 2mg/kg, about 4mg/kg, about 6mg/kg, about 8mg/kg, about lOmg/kg, about 12mg/kg, and the like.
  • Various compositions disclosed herein can be administered at different dosages.
  • a dosing schedule of, for example, once/week, twice/week, three times/week, four times/week, five times/week, six times/week, seven times/week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, and the like, is available for the antibody-drug conjugates disclosed herein.
  • the dosing schedules encompass dosing for a total period of time of, for example, one week, two weeks, three weeks, four weeks, five weeks, six weeks, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, and twelve months.
  • similar benefit-risk profiles can be seen for two different dosing schedules.
  • a dosing schedule of 400mg once every six weeks may have a similar benefit-risk profile in a subject undergoing a dosing schedule of 200mg once every 3 weeks.
  • cycles of the above dosing schedules can be repeated about, e.g., every seven days; every 14 days; every 21 days; every 28 days; every 35 days; 42 days; every 49 days; every 56 days; every 63 days; every 70 days; and the like.
  • An interval of non-dosing can occur between a cycle, where the interval can be about, e.g., seven days; 14 days; 21 days; 28 days; 35 days; 42 days; 49 days; 56 days; 63 days; 70 days; and the like.
  • Dosing schedules of the present disclosure can be related to cycles.
  • a dosing schedule for an antibody-drug conjugate disclosed herein may be designed such that doses are given on certain days of a cycle.
  • doses may be administered on days 1, 15, and 29 of a repeating 42-day cycle.
  • cycle may repeat until a subject exhibits adverse side effects to doses.
  • a cycle can additionally repeat until a subject is sufficiently cured of a disease.
  • the term "about” means plus or minus one day, plus or minus two days, plus or minus three days, plus or minus four days, plus or minus five days, plus or minus six days, or plus or minus seven days.
  • An effective amount of a therapeutic agent is one that will decrease or ameliorate the symptoms normally by at least 10%, more normally by at least 20%, most normally by at least 30%, typically by at least 40%, more typically by at least 50%, most typically by at least 60%, often by at least 70%, more often by at least 80%, and most often by at least 90%, conventionally by at least 95%, more conventionally by at least 99%, and most conventionally by at least 99.9%.
  • Administration of doses, or dosages, of antibody-drug conjugates disclosed herein can be subject to change.
  • the dosing and timing regimens may be changed according to factors known in the art.
  • administration of dosages of the antibody-drug conjugates may be delayed allowing for resolution of any observed toxi cities.
  • Administration may resume if no medical condition or other circumstance exists that would make the participant unsuitable for further treatment.
  • B7-H4 inhibits the growth of bone marrow-derived neutrophil progenitors, suggesting an inhibitory function of B7-H4 in neutrophil expansion (Zhu, G. et al., Blood, 113: 1759-1767 (2009)).
  • the infection or disease can be caused by a bacterium, virus, protozoan, helminth, or other microbial pathogen that enters intracellularly and is attacked, i.e., by cytotoxic T lymphocytes.
  • the infection or disease can be acute or chronic.
  • An acute infection is typically an infection of short duration.
  • immune cells begin expressing immunomodulatory receptors. Accordingly, in some embodiments, the method includes increasing an immune stimulatory response against an acute infection.
  • the infection can be caused by, for example, but not limited to Candida albicans, Listeria monocytogenes, Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria meningitidis, Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa or Mycobacterium.
  • the disclosed compositions are used to treat chronic infections, for example infections in which T cell exhaustion or T cell anergy has occurred causing the infection to remain with the host over a prolonged period of time.
  • Exemplary infections to be treated are chronic infections caused by a hepatitis virus, a human immunodeficiency virus (HIV), a human T-lymphotrophic virus (HTLV), a herpes virus, an Epstein-Barr virus, or a human papilloma virus.
  • HIV human immunodeficiency virus
  • HTLV human T-lymphotrophic virus
  • herpes virus an Epstein-Barr virus
  • Epstein-Barr virus Epstein-Barr virus
  • compositions can be administered for the treatment of 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), and common cold (e.g., human rhinovirus) and other viral infections, caused by, for example, HTLV, hepatitis virus, respiratory syncytial virus, vaccinia virus, and rabies virus.
  • immunodeficiency e.g., HIV
  • papilloma e.g., HPV
  • herpes e.g., HSV
  • encephalitis e.g., influenza virus A
  • common cold e.g., human rhinovirus
  • the molecules can be administered topically to treat viral skin diseases such as herpes lesions or shingles, or genital warts.
  • the molecules can also be administered systemically to treat systemic viral diseases, including, but not limited to, AIDS, influenza, the common cold, or encephalitis.
  • Representative infections that can be treated include but are not limited to infections cause by microorganisms including, but not limited to, Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio, Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium, Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus, Hemophilus influenza type B (HIB), Hyphomicrobium, Legionella, Leptspirosis, Listeria, Meningococcus A, B and C, Methanobacterium, Micrococcus, Myobacterium, Mycoplasma, Myxococcus, Neisseria, Nitrobacter, Oscillatoria, Prochloron, Proteus, Pseudomonas, Phodospirillum
  • microorganisms that can be treated using the disclosed compositions and methods include, bacteria, such as those of 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.), Genus Mucor ales (mucor, absidia, rhizophus), Sporothrix (schenkii), Blastomyces (dermatitidis), Paracoccidioides (brasiliensis), Coccidioides (immitis) and Histoplasma (capsulatuma), Entamoeba, histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba sp., Giardia lambia
  • Methods of reducing or inhibiting an immune response in a subject include administering a subject an effective amount of immunomodulatory agent, or cells primed ex vivo with the immunomodulatory agent.
  • the immune response can be, for example, a primary immune response to an antigen or an increase effector cell function such as increasing antigen-specific proliferation of T cells, enhancing cytokine production by T cells, stimulating differentiation, or a combination thereof.
  • the agent reduces T cell proliferation, T cell cytokine production, T cell differentiation, or a combination thereof.
  • the agent can reduce the development of naive T cells into Thl, Thl7, Th22, or other cells that secrete, or cause other cells to secrete, inflammatory molecules, including, but not limited to, IL-ip, TNF-a, TGF-beta, IFN-y, IL- 17, IL-6, IL-23, IL-22, IL-21, and MMPs.
  • the agent can increase or promote the activity of Tregs, increase the production of cytokines such as IL-10 from Tregs, increase the differentiation of Tregs, increase the number of Tregs, increase the ratio of Tregs within an immune cell population, or increase the survival of Tregs.
  • the methods can be used in vivo or ex vivo as immune response-inhibiting therapeutic applications.
  • the agent, or nucleic acid encoding the agent is administered directly to the subject.
  • the agent or nucleic acid encoding the agent is contacted with cells (e.g., immune cells) ex vivo, and the treat cells are administered to the subject (e.g. adoptive transfer).
  • the disclosed immunomodulatory agents can be used for treating a subject having or being predisposed to any disease or disorder to which the subject's immune system mounts an overactive or inappropriate immune response.
  • the agents can enable a less robust immune response to be possible.
  • the disclosed compositions are useful to reduce or inhibit immune responses involving T cells.
  • the immunomodulatory agents utilized for reducing an immune response are typically those that increase B7-H4 expression, ligand binding, crosslinking, negative signaling, or a combination thereof.
  • the agent can be an agonist of B7-H4, such as an agonist (stimulating) anti- B7-H4 antibody or antigen binding fragment thereof.
  • compositions and methods can be used to treat inflammation.
  • the agents are used to reduce or inhibit an immune response in the subject by administering to the subject an amount of an immunomodulatory agent that increases B7- H4 expression, ligand binding, crosslinking, negative signaling, or a combination thereof.
  • the method can reduce or more symptoms of inflammation.
  • inflammation can be acute, chronic, or persistent inflammation.
  • the immunomodulatory agents slow down the immune system.
  • agent can be used to control hyper-inflammatory response causing damage healthy tissues.
  • the agents are administered to a subject undergoing a hyper-inflammatory response. In such cases, controlling excessive immune responses can be beneficial to the subject.
  • Inflammatory and Autoimmune Diseases/disorders [00620] Agents that increase B7-H4 expression, ligand binding, crosslinking, negative signaling, or a combination thereof can also be used to treat inflammatory or autoimmune diseases and disorders.
  • 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 sprue-dermatitis, chronic fatigue syndrome immune deficiency, syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, cicatricial pemphigoid, cold agglutinin disease, Crest syndrome, Crohn’s disease, Dego’s disease, dermatomyositis, dermatomyositis - juvenile, discoid lupus, essential mixed
  • the inflammation or autoimmune disease is caused by a pathogen, or is the result of an infection.
  • the disclosed immunomodulatory agents can be administered to a subject in need thereof alone or in combination with one or more additional therapeutic agents.
  • the immunomodulatory agent and the additional therapeutic agent are administered separately, but simultaneously.
  • the immunomodulatory agent and the additional therapeutic agent can also be administered as part of the same composition.
  • the immunomodulatory agent and the second therapeutic agent are administered separately and at different times, but as part of the same treatment regime.
  • the subject can be administered a first therapeutic agent 1, 2, 3, 4, 5, 6, or more hours, or 1, 2, 3, 4, 5, 6, 7, or more days before administration of a second therapeutic agent.
  • the subject can be administered one or more doses of the first agent every 1, 2, 3, 4, 5, 6 7, 14, 21, 28, 35, or 48 days prior to a first administration of second agent.
  • the immunomodulatory agent can be the first or the second therapeutic agent.
  • the immunomodulatory agent and the additional therapeutic agent can be administered as part of a therapeutic regimen. For example, if a first therapeutic agent can be administered to a subject every fourth day, the second therapeutic agent can be administered on the first, second, third, or fourth day, or combinations thereof. The first therapeutic agent or second therapeutic agent may be repeatedly administered throughout the entire treatment regimen.
  • Exemplary molecules include, but are not limited to, cytokines, chemotherapeutic agents, radionuclides, other immunotherapeutics, enzymes, antibiotics, antivirals (especially protease inhibitors alone or in combination with nucleosides for treatment of HIV or Hepatitis B or C), anti-parasites (helminths, protozoans), growth factors, growth inhibitors, hormones, hormone antagonists, antibodies and bioactive fragments thereof (including humanized, single chain, and chimeric antibodies), antigen and vaccine formulations (including adjuvants), peptide drugs, anti-inflammatories, ligands that bind to Toll-Like Receptors (including but not limited to CpG oligonucleotides) to activate the innate immune system, molecules that mobilize and optimize the adaptive immune system, other molecules that activate or up-regulate the action of cytotoxic T lymphocytes, natural killer cells and helper T-cells, and other molecules that deactivate or down-regulate
  • the additional therapeutic agents are selected based on the condition, disorder or disease to be treated.
  • the immunomodulatory agent can be coadministered with one or more additional agents that function to enhance or promote an immune response or reduce or inhibit an immune response.
  • a B7-H4 immunomodulatory agent can be used in a preventive or prophylactic role in the treatment and prevention of disease as discussed above, and also in the context of severe trauma injuries like major burn, open bone fracture, accidental amputation or other wounds. Therefore, the B7-H4 immunomodulatory agents can be administered to the subject in combination with an antimicrobial such as an antibiotic, an antifungal, an antiviral, an antiparasitics, or essential oil.
  • an antimicrobial such as an antibiotic, an antifungal, an antiviral, an antiparasitics, or essential oil.
  • the subject is administered the B7-H4 immunomodulatory agent and/or the antimicrobial at time of admission to the hospital to prevent further bacterial, fungal or viral complications.
  • the antibiotic can target pathogens and the B7-H4 immunomodulatory agent can stimulate the immune system to provide an enhanced response to treat or prevent further infection or disease.
  • the B7-H4 immunomodulatory agents can be combined with one or more chemotherapeutic agents and pro-apoptotic agents.
  • Representative chemotherapeutic agents include, but are not limited to amsacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide, irinotecan, leucovorin, liposomal doxorubicin, liposomal daunorubicin, lomustine, melphalan, mercaptopur
  • B7-H4 immunomodulatory agents are coadministered with a PD-1 antagonist.
  • Programmed Death-1 (PD-1) is a member of the CD28 family of receptors that delivers a negative immune response when induced on T cells.
  • Contact between PD-1 and one of its ligands (B7-H1 or B7-DC) induces an inhibitory response that decreases T cell multiplication and/or the strength and/or duration of a T cell response.
  • Suitable PD-1 antagonists are described in U.S. Patent Nos.
  • 8,114,845, 8,609,089, and 8,709,416, which are specifically incorporated by reference herein in their entities, and include compounds or agents that either bind to and block a ligand of PD-1 to interfere with or inhibit the binding of the ligand to the PD-1 receptor, or bind directly to and block the PD-1 receptor without inducing inhibitory signal transduction through the PD-1 receptor.
  • the PD-1 receptor antagonist binds directly to the PD- 1 receptor without triggering inhibitory signal transduction and also binds to a ligand of the PD-1 receptor to reduce or inhibit the ligand from triggering signal transduction through the PD-1 receptor.
  • PD-1 signaling is driven by binding to a PD-1 ligand (such as B7-H1 or B7-DC) in close proximity to a peptide antigen presented by major histocompatibility complex (MHC) (see, for example, Freeman, Proc. Natl. Acad. Sci. U. S. A, 105: 10275-10276 (2008)). Therefore, proteins, antibodies or small molecules that prevent co-ligation of PD-1 and TCR on the T cell membrane are also useful PD-1 antagonists.
  • MHC major histocompatibility complex
  • the PD-1 receptor antagonists are small molecule antagonists or antibodies that reduce or interfere with PD-1 receptor signal transduction by binding to ligands of PD-1 or to PD-1 itself, especially where co-ligation of PD-1 with TCR does not follow such binding, thereby not triggering inhibitory signal transduction through the PD-1 receptor.
  • PD-1 antagonists contemplated by the methods of this invention include antibodies that bind to PD-1 or ligands of PD-1, and other antibodies.
  • Suitable anti -PD-1 antibodies include, but are not limited to, those described in the following publications:
  • anti-B7-Hl antibodies include, but are not limited to, those described in the following publications:
  • PCT/US06/022423 (WO/2006/133396, pub. 14 December 2006)
  • PCT/US07/088851 (WO/2008/083174, pub. 10 July 2008)
  • US 2006/0110383 (pub. 25 May 2006)
  • exemplary PD-1 receptor antagonists include, but are not limited to B7-DC polypeptides, including homologs and variants of these, as well as active fragments of any of the foregoing, and fusion proteins that incorporate any of these.
  • the fusion protein includes the soluble portion of B7-DC coupled to the Fc portion of an antibody, such as human IgG, and does not incorporate all or part of the transmembrane portion of human B7-DC.
  • PD-1 antagonists include those that bind to the ligands of the PD-1 receptor. These include the PD-1 receptor protein, or soluble fragments thereof, which can bind to the PD-1 ligands, such as B7-H1 or B7-DC, and prevent binding to the endogenous PD-1 receptor, thereby preventing inhibitory signal transduction. B7-H1 has also been shown to bind the protein B7.1 (Butte et al., Immunity, Vol. 27, pp. 111-122, (2007)).
  • Such fragments also include the soluble ECD portion of the PD-1 protein that includes mutations, such as the A99L mutation, that increases binding to the natural ligands (Molnar et al., PNAS, 105: 10483-10488 (2008)).
  • B7-1 or soluble fragments thereof which can bind to the B7-H1 ligand and prevent binding to the endogenous PD-1 receptor, thereby preventing inhibitory signal transduction, are also useful.
  • PD -1 and B7-H1 anti-sense nucleic acids can also be PD-1 antagonists.
  • Such anti-sense molecules prevent expression of PD-1 on T cells as well as production of T cell ligands, such as B7-H1, PD- L1 and/or PD-L2.
  • T cell ligands such as B7-H1, PD- L1 and/or PD-L2.
  • siRNA for example, of about 21 nucleotides in length, which is specific for the gene encoding PD-1, or encoding a PD-1 ligand, and which oligonucleotides can be readily purchased commercially
  • carriers such as polyethyleneimine (see Cubillos-Ruiz et al., J. Clin. Invest.
  • CTLA4 antagonists Other molecules useful in mediating the effects of T cells in an immune response are also contemplated as additional therapeutic agents.
  • the molecule is an antagonist of CTLA4, for example an antagonistic anti-CTLA4 antibody.
  • An example of an anti-CTLA4 antibody contemplated for use in the methods of the invention includes an antibody as described in PCT/US2006/043690 (Fischkoff et al., WO/2007/056539).
  • an anti-CTLA4 antibody useful in the methods of the invention are Ipilimumab, a human anti-CTLA4 antibody, administered at a dose of, for example, about 10 mg/kg, and Tremelimumab a human anti-CTLA4 antibody, administered at a dose of, for example, about 15 mg/kg. See also Sammartino, et al., Clinical Kidney Journal, 3(2): 135-137 (2010), published online December 2009.
  • the antagonist is a small molecule.
  • a series of small organic compounds have been shown to bind to the B7-1 ligand to prevent binding to CTLA4 (see Erbe et al., J. Biol. Chem., 277:7363-7368 (2002). Such small organics could be administered alone or together with an anti-CTLA4 antibody to reduce inhibitory signal transduction of T cells.

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Abstract

L'invention concerne des compositions et des méthodes d'utilisation de celles-ci permettant d'éliminer des cellules tumorales qui expriment B7-H4. De telles compositions comprennent des conjugués anticorps-médicament anti-B7-H4.
PCT/US2024/059633 2023-12-11 2024-12-11 Conjugués anticorps-médicament dirigés contre b7-h4 et leurs méthodes d'utilisation Pending WO2025128735A2 (fr)

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