Molecules that act specifically in tissues where cell death is observed

Abstract

The object of the present invention is to provide a molecule that acts specifically in the tissue that is required to be treated or targeted, such as diseased tissue, abnormal tissue, etc., but does not act or acts poorly in healthy or normal tissue, which makes it possible to provide a molecule useful for medicine with reduced side effects while providing significant therapeutic or preventive effects. Provided is a molecule that binds to a component or a part thereof (e.g., filaments or histones that form the cytoskeleton or nucleoskeletons) of a cell that is exposed to the extracellular environment due to cell death, and acts specifically in a tissue where cell death is observed, such as diseased tissue, abnormal tissue, etc. More specifically, provided is a molecule that forms a complex with the component or a part thereof by direct or indirect binding of one or more of the molecules to one or more of the membrane proteins in the cell, thereby providing signaling or blocking in the cell in the tissue. TIFF2024504903000006.tif111128

Description

[Technical field]

[0001] The present invention relates to molecules that act specifically in tissues where cell death is observed through binding to components constituting cells or parts thereof that are exposed to the extracellular environment upon cell death, and that confer a signal or block in cells in tissues such as immune cells or diseased cells; said molecules for use as drugs; use of said molecules in the preparation of drugs; pharmaceutical compositions comprising said molecules; methods for conferring a signal or block in cells in tissues; polynucleotides encoding said molecules; vectors comprising said polynucleotides; cells harboring said vectors, and the like.

[0002] More specifically, the present invention relates to antigen-binding molecules comprising a first moiety that binds to a first antigen, which is a damage-associated molecular pattern (DAMP), and a second moiety that binds to an antigen different from the first antigen, and medical uses thereof. [Background technology]

[0003] Cancer therapies that have local (site-specific) activity mainly in cancer tissues have been developed by utilizing the characteristics of the cancer cells themselves and the tumor microenvironment (TME). Some antibody therapeutics such as Herceptin and cetuximab target cancer antigens that are mainly expressed in cancer cells and exert cytotoxic activity mainly against tumor cells through ADCC, signal inhibition, etc. A method for delivering physiologically active ligands such as cytokines to solid tumors by immunocytokines containing ligands fused with antibody molecules that bind to cancer antigens highly expressed on cancer cells is also known.

[0004] TME is known to be a hypoxic and nutrient-poor state that leads to cell death, especially in solid cancers, and is therefore found to be an environment rich in dead cells. There are many molecules that are released or exposed to the cell surface when cells die, and are enriched in cancer but not in normal tissue. Some of the molecules on dead cells are recognized by receptors expressed in phagocytes such as dendritic cells or macrophages, and then dead cells are eliminated (removed) by phagocytes. For example, Clec9A is known to be expressed in a subset of dendritic cells and binds to dead cells and also to F-actin ectopically exposed on the cell surface of dead cells (Patent Documents 1 and 2).

[0005] There are many therapies that regulate cell functions, such as T cell activation, B cell activation, cell death induction, and signal inhibition. As reported, many antibodies targeting costimulatory molecules are in clinical development in cancer (Non-Patent Document 1). For example, utomirumab, an anti-CD137 agonist antibody, and CDX-1140, an anti-CD40 agonist antibody, are currently in clinical development. These molecules are expected to show antitumor efficacy by enhancing T cell activation directly or indirectly through dendritic cells, enhancing the tumoricidal activity of macrophages, etc. In early clinical trials, these antibodies have shown some clinical responses.

[0006] However, one of the concerns of current molecules that regulate cell function is systemic activity. These antibodies can bind to target molecules anywhere in the body, including normal tissues, and can cause toxicity. For example, anti-CD137 antibodies (BMS) cause liver toxicity.

[0007] As one of the possible strategies to overcome systemic toxicity, bispecific antibodies that target tumor antigens and antigens that regulate immune cell function have been evaluated. Anti-CD137 and tumor antigen binding bispecific antibodies showed lower toxicity compared with anti-CD137 monospecific antibodies (Non-Patent Document 2). However, in some cases, loss of tumor antigens in cancer tissues has been reported, which may cause the loss of anti-tumor effect of tumor antigen-specific antibodies. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] WO 2013 / 053008 A2 [Patent Document 2] JP 2011-519959 A [Non-patent literature]

[0009] [Non-Patent Document 1] Patrick et al., Nature Reviews Drug Discovery, Vol.17, p.509-527, 2018 [Non-Patent Document 2] Miguel Gaspar et al, Cancer Immunol Res. 2020 Jun;8(6):781-793 Summary of the Invention [Problem to be solved by the invention]

[0010] It is an object of the present invention to provide a strategy to overcome the systemic toxicity caused by therapeutic agents targeting one or more antigens expressed in diseased tissues such as cancer, and to conditionally regulate cellular function in the unique environment in diseased tissues such as the TME. Furthermore, it is an object of the present invention to provide molecules that act specifically in the tissues sought to be treated or targeted, such as diseased tissues, abnormal tissues, etc., but do not or act poorly in healthy or normal tissues, making it possible to provide molecules useful in medicines with reduced side effects while providing significant therapeutic or prophylactic effects.

[0011] In particular, it is an object of the present invention to provide antigen-binding molecules that are capable of eliciting an immune response in diseased tissue, but which elicit no or poor immune response in healthy or normal tissue. [Means for solving the problem]

[0012] The present invention provides a molecule that acts specifically in tissues where cell death is observed, such as diseased tissues, abnormal tissues, etc., by binding to a component or a part of a cell (e.g., filaments or histones forming the cytoskeleton or nucleoskeleton) that is specifically observed in diseased tissues (e.g., TME) that is exposed to the extracellular environment due to cell death, and at the same time binding to a target molecule (e.g., membrane protein) on or in a cell (e.g., immune cells, diseased cells, such as tumor cells, autoreactive cells, and virus-infected cells). More specifically, a molecule is provided that enables bridging between a component or a part of a cell that is exposed to the extracellular environment due to cell death and a target molecule on or in a cell, such as an immune cell or diseased cell (e.g., cancer cell, autoreactive cell, and virus-infected cell), and providing signaling or blocking in the cell in the tissue. More specifically, by providing an increase in the cross-linking by multiple molecules between a component or a part of a cell that is exposed to the extracellular environment due to cell death and a target molecule on or in a cell, such as an immune cell or diseased cell, an increase in signaling or blocking via a target molecule in the cell is achieved.

[0013] In particular, as described and outlined in more detail below and in the Examples, the present invention provides antigen-binding molecules that bind to antigens found outside cells, particularly in the extracellular space of diseased tissues, such as cancer tissues and tissues affected by autoimmune diseases. Without being bound by any theory, it is believed that these antigens are present outside cells due to the cell damage and destruction that occurs in these diseased tissues. By being able to bind to such antigens, the antigen-binding molecules of the present invention induce an immune response that is specific to the diseased tissue.

[0014] In particular, the following embodiments are part of the present invention, and the skilled reader will understand that the specific embodiments included below are intended independently as specific embodiments for each and every general embodiment set forth below.

[0015] Furthermore, the present disclosure provides the following aspects. [A1] A molecule comprising a first portion that binds to a first antigen and a second portion that binds to a second antigen, The first antigen is a component or part of the cytoskeleton, cell membrane, or organelle, or a cytoplasmic protein, or a metabolic product that is exposed to the extracellular environment upon cell death; and The second antigen is a different antigen from the first antigen. molecule. [A2] The molecule of [A1], wherein the cell death is cell death in an affected tissue. [A3] The molecule of [A1] or [A2], wherein the first antigen is a filament, a histone, or a nucleosome containing a histone, which forms the cytoskeleton or the nucleoskeleton. [A4] The molecule of [A3], wherein the first antigen is an intermediate filament. [A5] The molecule of [A4], wherein the first antigen is F-actin. [A6] Any one of the molecules [A1] to [A5], wherein the first antigen is an antigen formed by multimerization of multiple molecules. [A7] A molecule of [A1] or [A2], wherein the first antigen is phosphatidylserine, which constitutes a cell membrane. [A8] Any one of the molecules [A1] to [A7], wherein the cell death is necrosis, apoptosis, autophagic cell death, or accidental cell death. [A9] The second antigen is any one of the molecules [A1] to [A8], which is a membrane protein involved in signal transduction in a cell. [A10] The molecule of [A9], wherein the cell is an immune cell. [A11] The molecule of [A10], wherein the immune cell is a T cell, a killer cell, a helper T cell, a regulatory T cell, a B cell, a memory B cell, a NK cell, a NKT cell, a dendritic cell, a macrophage, an eosinophil, a neutrophil cell, or a basophil. [A12] The molecule of [A9], wherein the cell is a tumor cell or an autoreactive cell. [A13] Any one of the molecules [A9] to [A12], characterized in that a complex formed by the binding of one or more of the molecules to a first antigen binds to one or more of the membrane proteins via the second portion, thereby conferring signal transduction or blocking via the membrane protein in a cell. [A14] The molecule according to [A13], which confers signal transduction via a membrane protein in a cell. [A15] The molecule according to [A13], characterized in that the molecule provides signal blocking via a membrane protein in a cell. [A16] Any one of the molecules of [A9] to [A15], wherein the membrane protein is a membrane protein in a cell membrane or a membrane protein of an endosome. [A17] Any one of the molecules of [A11] to [A16], wherein the membrane protein is a membrane protein in the cell membrane of a T cell selected from the group consisting of a T cell receptor, CD3, a costimulatory molecule, and a costimulatory molecule; or a membrane protein in the cell membrane of a cell other than a T cell that binds to a costimulatory molecule or a costimulatory molecule in the cell membrane of a T cell. [A18] The molecule of [A17], wherein the costimulatory molecule is CD28 (TP44), CD278 (ICOS), CD27 (TNFRSF-7), CD30, CD134 (OX40), CD357 (GITR). Tim-2, CD28H (TMIGD2), CD137 (4-1BB, TNFRSF-9), CD226 (DNAM-1), or CD244 (2B4). [A19] The molecule of [A17], wherein the co-inhibitory molecule is CD279 (PD-1), CD152 (CTLA-4), TIGIT, BTLA, CD366 (Tim-3), CD96, CD112R, or CD200R. [A20] The molecule of [A17], wherein the membrane protein that binds to a costimulatory or costimulatory molecule is CD274 (PD-L1), CD273 (PD-L2), CD80, CD86, CD276 (ICOSL), CD276 (VICN1), VISTA, HHLA2, a member of the butyrophilin family, CD270 (HVEM), CD137L (TNFSF-9), CD252 (OX40L), CD70 (TNFSF-7), CD40 (TNFRSF-7), GIRL (TNFSF-18), CD155 (PVR, NECL5), CD112 (NECTIN-2), CD200 (MOX1), CD48 (BCM-1), or Gal-9. [A21] Any one of the molecules [A1] to [A20], wherein the first portion is an antibody variable region or a single domain antibody (VHH). [A22] Any one of the molecules [A1] to [A20], wherein the first antigen is F-actin and the first portion is a molecule that binds to F-actin or a portion thereof. [A23] The molecule of [A22], wherein the molecule that binds to F-actin or a portion thereof is the extracellular domain of Clec9A or a portion thereof. [A24] Any one of the molecules of [A9] to [A23], wherein the second portion is an antibody variable region or a single domain antibody (VHH), or a ligand. [A25] The first antigen is F-actin; the second antigen is a membrane protein involved in signal transduction in the cell; the second portion is one or both of the Fab regions of an antibody variable region that contains two Fab regions, which binds to a second antigen; and The first portion is a molecule that binds to F-actin or a portion thereof, which is linked via a linker or directly to the C-terminus of the Fc region connected to the antibody variable region; One of the molecules [A1] to [A9]. [A26] The molecule of [A25], wherein the cell is an immune cell. [A27] The molecule of [A26], wherein the immune cell is a T cell, a killer cell, a helper T cell, a regulatory T cell, a B cell, a memory B cell, a NK cell, a NKT cell, a dendritic cell, a macrophage, an eosinophil, a neutrophil cell, or a basophil. [A28] The molecule of [A25], wherein the cell is a tumor cell or an autoreactive cell. [A29] Any one of the molecules [A25] to [A28], characterized in that a complex formed by the binding of one or more of the molecules to F-actin binds to one or more membrane proteins via a second portion, thereby providing a signal transduction or blocking via the membrane protein in a cell. [A30] The molecule according to [A29], which confers signal transduction via a membrane protein in a cell. [A31] The molecule according to [A29], which provides signal blocking via a membrane protein in a cell. [A32] Any one of the molecules [A25] to [A31], wherein the membrane protein is a membrane protein in a cell membrane or a membrane protein of an endosome. [A33] Any one of the molecules [A27] to [A32], wherein the membrane protein is a membrane protein in the cell membrane of a T cell selected from the group consisting of a T cell receptor, CD3, a costimulatory molecule, and a costimulatory molecule; or a membrane protein in the cell membrane of a cell other than a T cell that binds to a costimulatory molecule or a costimulatory molecule in the cell membrane of a T cell. [A34] The molecule of [A33], wherein the costimulatory molecule is CD28 (TP44), CD278 (ICOS), CD27 (TNFRSF-7), CD30, CD134 (OX40), CD357 (GITR). Tim-2, CD28H (TMIGD2), CD137 (4-1BB, TNFRSF-9), CD226 (DNAM-1), or CD244 (2B4). [A35] The molecule of [A33], wherein the co-inhibitory molecule is CD279 (PD-1), CD152 (CTLA-4), TIGIT, BTLA, CD366 (Tim-3), CD96, CD112R, or CD200R. [A36] The molecule of [A33], wherein the membrane protein that binds to a costimulatory or costimulatory molecule is CD274 (PD-L1), CD273 (PD-L2), CD80, CD86, CD276 (ICOSL), CD276 (VICN1), VISTA, HHLA2, a member of the butyrophilin family, CD270 (HVEM), CD137L (TNFSF-9), CD252 (OX40L), CD70 (TNFSF-7), CD40 (TNFRSF-7), GIRL (TNFSF-18), CD155 (PVR, NECL5), CD112 (NECTIN-2), CD200 (MOX1), CD48 (BCM-1), or Gal-9. [A37] Any one of the molecules [A25] to [A36], wherein the first portion is an antibody variable region or a single domain antibody (VHH). [A38] Any one of the molecules [A25] to [A37], wherein the molecule that binds to F-actin or a portion thereof is the extracellular domain of Clec9A or a portion thereof. [A39] Any one of the molecules of [A25] to [A38], wherein the second portion is an antibody variable region or a single domain antibody (VHH), or a ligand. [A40] A molecule selected from any one of [A1] to [A20], which is a compound comprising a first portion and a second portion. [A41] The molecule of [A40], wherein the compound binds to a first antigen and a second antigen upon receipt of light radiation.

[0016] A pharmaceutical composition comprising any one of the molecules [B1][A1] to [A41]. [B2] A pharmaceutical composition of [B1] for imparting signal transduction or blocking in a cell. [B3] A pharmaceutical composition of [B1] or [B2] for imparting signaling or blocking in immune cells. [B4] A pharmaceutical composition according to any one of [B1] to [B3] for activating or inhibiting immunity. [B5] Any one of the pharmaceutical compositions of [B1] to [B4] for treating or preventing cancer or an autoimmune disease. [B6] A molecule of any one of [A1] to [A41] for use as a drug. [B7] A molecule of [B6] to confer signaling or blocking in a cell. [B8] A molecule of [B6] or [B7] to confer signaling or blocking in immune cells. [B9] Any one of the molecules [B6] to [B8] for activating or inhibiting the immune system. [B10] Any one of the molecules [B5] to [B9] for treating or preventing cancer or an autoimmune disease. [B11] Use of any one of the molecules [A1] to [A41] in the preparation of a drug. [B12] The use of [B11], wherein the agent is an agent for imparting or blocking signaling in a cell. [B13] The use of [B11] or [B12], wherein the agent is an agent for imparting a signal transduction or blockage in an immune cell. [B14] Use of any one of [B11] to [B13], wherein the drug is a drug for activating or inhibiting immunity. [B15] Use of any one of [B11] to [B14], wherein the drug is a drug for treating or preventing cancer or an autoimmune disease. [B16] A method for inducing or blocking signaling in a cell, comprising the step of administering to a subject any one of the molecules of [A1] to [A41]. A method for activating or inhibiting immunity, comprising the step of administering to a subject any one of the molecules of [B17][A1] to [A41]. [B18] Any one of the methods [B15] to [B17], wherein the cell is an immune cell, a cancer cell, or an autoreactive cell. A method for treating or preventing cancer or an autoimmune disease, comprising the step of administering to a subject any one of the molecules of [B19][A1] to [A41]. [B20] [A1] to [A39], the method for producing any one of the molecules. A polynucleotide encoding any one of the molecules [B21][A1] to [A39]. A polynucleotide, particularly DNA, encoding any one of the molecules [B22][A1] to [A39]. [B23] A vector comprising the polynucleotide of [B22]. A vector comprising a DNA sequence encoding an antigen-binding molecule as defined in any one of [B24] [A1] to [A39], in particular for recombinant protein expression. A host cell carrying the [B25][B23] or [B24] vector.

[0017] In addition, the present disclosure also specifically provides the following aspects [C1] to [C15]. [C1] A molecule comprising a first portion that binds to a first antigen and a second portion that binds to a second antigen, The first antigen is a component or part of the cytoskeleton, cell membrane, or organelle, or a cytoplasmic protein, or a metabolic product that is exposed to the extracellular environment upon cell death; and The second antigen is a different antigen from the first antigen. molecule. [C2] The molecule of [C1], wherein the cell death is cell death in an affected tissue. [C3] A molecule of [C1] or [C2], wherein the first antigen is a filament, a histone, or a histone-containing nucleosome that forms the cytoskeleton or nucleoskeleton. [C4] The first antigen is an intermediate filament molecule, [C3]. [C5] The molecule of [C4] in which the first antigen is F-actin. [C6] A molecule selected from [C1] to [C5], wherein the first antigen is an antigen formed by multimerization of multiple molecules. [C7] Any one of the molecules [C1] to [C6], wherein the cell death is necrosis, apoptosis, autophagic cell death, or accidental cell death. [C8] Any one of the molecules [C1] to [C7], wherein the second antigen is a membrane protein involved in signal transduction in a cell. [C9] The molecule of [C8], wherein the cell is an immune cell. [C10] Any one of the molecules [C1] to [C9], wherein the first portion is an antibody variable region or a single domain antibody (VHH). [C11] Any one of the molecules [C1] to [C9], wherein the first antigen is F-actin and the first portion is a molecule that binds to F-actin or a portion thereof. [C12] the first antigen is F-actin; the second antigen is a membrane protein involved in signal transduction in the cell; the second portion is one or both of the Fab regions of an antibody variable region that contains two Fab regions, which binds to a second antigen; and The first portion is a molecule that binds to F-actin or a portion thereof, which is linked via a linker or directly to the C-terminus of the Fc region connected to the antibody variable region; One of the molecules [C1] to [C9]. A pharmaceutical composition comprising any one of the molecules [C13][C1] to [C12]. [C14] Use of any one of the molecules [C1] to [C12] in the preparation of a drug. A method for producing any one of the molecules [C15][C1] to [C12].

[0018] In particular, the present disclosure further provides the following aspects [D1] to [D44]. [D1] (1) at least one first moiety that binds to a first antigen, and (2) at least one second moiety that binds to a second antigen; An antigen-binding molecule comprising: the first antigen is a damage-associated molecular pattern (DAMP); and The second antigen is a different antigen from the first antigen. Antigen-binding molecules. [D1.1] (1) at least one first moiety that binds to a first antigen, and (2) at least one second moiety that binds to a second antigen; An antigen-binding molecule comprising: the first antigen is a tissue-derived antigen characteristic of a tissue condition, in particular, the antigen is exposed to the extracellular environment, e.g., a damage-associated molecular pattern (DAMP); and the second antigen is different from the first antigen, Antigen-binding molecules. [D2] The antigen-binding molecule of [D1] or [D1.1], wherein the second antigen is a membrane protein of an immune cell. [D3] An antigen-binding molecule of any one of [D1] and [D2], wherein the molecular pattern (DAMP) is exposed to the extracellular environment due to cell damage, particularly cell death. [D4] The molecule, (A) the cell damage can provide cell contact and / or a signal, the contact and / or signal involving the second antigen; and / or (B) comprising the first portion at the Fc terminus of the antigen-binding molecule; and / or (C) comprising the second portion in an antigen-binding region, preferably an F(ab')2 region, of the antigen-binding molecule; and / or (D) comprises at least one first portion and at least one second portion, in particular, said antigen-binding molecule comprises one first portion and one second portion, more particularly, said antigen-binding molecule comprises at least two, preferably two first portions, and at least two, preferably two second portions; and / or (E) the compound is a compound capable of binding to the first antigen and the second antigen upon light irradiation; Any one of the antigen-binding molecules [D1] to [D3]. [D5] The cell damage is (i) including, in particular, being, cell death; and / or (ii) occurs in diseased tissues, particularly selected from tissues affected by a condition or disease, in particular by cancer or an autoimmune disease, in particular in the tumor microenvironment (TME); and / or (iii) caused by any one of the following: necrosis, apoptosis, autophagic cell death, and accidental cell death; and / or (iv) cell death selected from the group consisting of necrosis, apoptosis, autophagic cell death, and accidental cell death; Any one of the antigen-binding molecules [D1] to [D4]. [D6] The first antigen is (i) selected from the group consisting of a cytoskeletal component or a portion thereof, a cell membrane component or a portion thereof, an organelle component or a portion thereof, a cytoplasmic protein or a portion thereof, and a metabolite; and / or (ii) located on any one selected from the group consisting of filaments (particularly intermediate filaments), nucleosomes, the cytoskeleton, and / or the nucleoskeletal system; and / or (iii) capable of multimerization of multiple molecules; and / or (iv) present in a multimeric molecule; In particular, the first antigen is (i*) selected from the group consisting of cytoskeletal components or parts thereof, cell membrane components or parts thereof, organelle components or parts thereof, cytoplasmic proteins or parts thereof, and metabolites; and / or (ii*) Located on any one selected from the group consisting of filaments (particularly intermediate filaments), nucleosomes, cytoskeleton, and / or nuclear skeleton; Any one of the antigen-binding molecules [D1] to [D5]. [D7] The first antigen is (A) Actin, specifically F-actin, or (B) heat shock proteins, particularly selected from HSP90 and GRP78, among which HSP90; (C) Phosphatidylserine (PS), specifically phosphatidylserine that is part of the cell membrane; (D) histones or components thereof; (E) Histone deacetylase complex subunits, specifically SAP130; (F) HMGN proteins, in particular selected from HMGB1 and HMGN1; (G) hepatoma-derived growth factor; (H)BCL-2, (I) Calreticulin, and (J) Cyclophilin A is selected from the group consisting of In particular, the first antigen is selected from the group consisting of F-actin, GRP78, phosphatidylserine, HSP90, and SAP130; Among them, the first antigen is selected from the group consisting of F-actin, phosphatidylserine, and HSP90; In particular, the first antigen is selected from the group consisting of F-actin and HSP90; Preferably, the first antigen is F-actin. Any one of the antigen-binding molecules [D1] to [D6]. [D8] The first part is (A) an Ig-type binding moiety; or (B) A binding polypeptide, particularly a non-Ig type binding moiety. Any one of the antigen-binding molecules, [D1] to [D7], selected from the group consisting of: [D9] The first part is (A) a moiety capable of binding to actin, particularly F-actin; (B) a moiety capable of binding to a heat shock protein, in particular selected from HSP90 and GRP78; (C) a moiety capable of binding to phosphatidylserine (PS), specifically phosphatidylserine that is part of the cell membrane; (D) a moiety capable of binding to a histone or a component thereof; (E) A portion capable of binding to a histone deacetylase complex subunit, specifically SAP130; (F) a moiety capable of binding to an HMGN protein, in particular selected from HMGB1 and HMGN1; (G) a moiety capable of binding to hepatoma-derived growth factor; (H) a moiety capable of binding to BCL-2; (I) a moiety capable of binding to calreticulin, and (J) A moiety capable of binding to cyclophilin A is selected from the group consisting of In particular, the first moiety is selected from the group consisting of a moiety capable of binding to F-actin, a moiety capable of binding to GRP78, a moiety capable of binding to phosphatidylserine, a moiety capable of binding to HSP90, and a moiety capable of binding to SAP130; Among others, the first moiety is selected from the group consisting of a moiety capable of binding to F-actin, a moiety capable of binding to phosphatidylserine, and a moiety capable of binding to HSP90; In particular, the first moiety is selected from the group consisting of a moiety capable of binding to F-actin and a moiety capable of binding to HSP90; Preferably, the first moiety is a moiety capable of binding to F-actin. Any one of the antigen-binding molecules [D1] to [D8]. [D10] (A) the moiety capable of binding to F-actin is a binding polypeptide, preferably a Clec9A polypeptide, and / or (B) the moiety capable of binding to GRP78 is an Ig-type binding moiety, preferably an Ig-type binding moiety derived from the antibody GA20 or Mab159; and / or (C) the moiety capable of binding to phosphatidylserine is an Ig-type binding moiety, preferably an Ig-type binding moiety derived from the antibody 3G4, and / or (D) the moiety capable of binding to HSP90 is an Ig-type binding moiety, preferably an Ig-type binding moiety derived from the antibody 1.5.1 or 6H8; and / or (E) the moiety capable of binding to SAP130 is a binding polypeptide, preferably an mClec4e polypeptide; In particular, the Ig-type binding moiety is selected from the group consisting of a single domain antibody (VHH), a combination of an antibody heavy chain variable (VH) region and an antibody light chain variable (VL) region, a single domain antibody (sdAb), a single chain Fv (scFv), a single chain antibody, an Fv, a single chain Fv2 (scFv2), an Fab, and an F(ab')2; Among others, the Ig-type binding moiety is selected from the group consisting of VHH, scFv, scFv2, Fab, and F(ab')2, [D9] Antigen-binding molecule. [D11] (A) when the first antigen is actin, preferably F-actin, the first moiety is a Clec9A polypeptide; or (B) if the first antigen is GRP78, phosphatidylserine, or HSP90, the first moiety is an Ig-type binding moiety; or (C) when the first antigen is SAP130, the first moiety is a mClec4e polypeptide; Any one of the antigen-binding molecules [D1] to [D9]. [D12] The antigen-binding molecule of any one of [D1] to [D11], wherein when the first antigen is actin or SAP130, the first binding moiety is an Ig-type binding moiety. [D13] The second antigen is a membrane protein of an immune cell, particularly a membrane protein in the cell membrane of a T cell selected from the group consisting of a T cell receptor, CD3, CD137, CD40, CTLA4, a costimulatory molecule, or a costimulatory molecule; Among them, the second antigen is a membrane protein involved in signal transduction in cells, preferably a signal transduction receptor. Any one of the antigen-binding molecules [D1] to [D12]. [D14] The antigen-binding molecule of any one of [D1] to [D13], wherein the second portion is an Ig-type binding portion. [D15] The second antigen is (A) Involved in signal transduction in cells; especially, (A-1) the second antigen is a membrane protein, and / or (A-2) the second antigen is a signal transduction receptor, and / or (A-3) the cell is (i) an immune cell (among which the immune cell is selected from the group consisting of T cells, killer cells, helper T cells, regulatory T cells, B cells, memory B cells, NK cells, NKT cells, dendritic cells, macrophages, eosinophils, neutrophil cells, and basophils), (ii) a tumor cell, or (iii) an autoreactive cell; and / or (B) an immune cell receptor antigen, Any one of the antigen-binding molecules [D1] to [D14]. [D16] The second antigen is (A) Endosomal membrane proteins, (B) Membrane proteins in the plasma membrane of T cells. In particular, a membrane protein in the plasma membrane of a T cell selected from the group consisting of a T cell receptor, CD3, CD137, CD40, CTLA4, a costimulatory molecule, or a costimulatory molecule; (Membrane proteins capable of binding to costimulatory or co-inhibitory molecules are selected from among CD274 (PD-L1), CD273 (PD-L2), CD80, CD86, CD276 (ICOSL), CD276 (VICN1), VISTA, HHLA2, members of the butyrophilin family, CD270 (HVEM), CD137L (TNFSF-9), CD252 (OX40L), CD70 (TNFSF-7), CD40 (TNFRSF-7), GIRL (TNFSF-18), CD155 (PVR, NECL5), CD112 (NECTIN-2), CD200 (MOX1), CD48 (BCM-1), and Gal-9). (C) a membrane protein in the plasma membrane of a cell other than a T cell, capable of binding to a costimulatory or costimulatory molecule in the plasma membrane of a T cell, (In particular, the costimulatory molecule is selected from the group consisting of CD28 (TP44), CD278 (ICOS), CD27 (TNFRSF-7), CD30, CD134 (OX40), CD357 (GITR). Tim-2, CD28H (TMIGD2), CD137 (4-1BB, TNFRSF-9), CD226 (DNAM-1), and CD244 (2B4), and / or the co-inhibitory molecule is selected from the group consisting of CD279 (PD-1), CD152 (CTLA-4), TIGIT, BTLA, CD366 (Tim-3), CD96, CD112R, or CD200R, and / or Among others, the second antigen is selected from the group consisting of CD3, CD137, CD40, and CTLA4; In particular, the second antigen is selected from the group consisting of CD3 and CD137; Preferably, the second antigen is CD3. Any one of the antigen-binding molecules [D1] to [D15]. [D17] The second part is (A) A moiety capable of binding to an endosomal membrane protein; (B) a portion capable of binding to a membrane protein in the plasma membrane of a T cell, in particular a membrane protein in the plasma membrane of a T cell selected from the group consisting of a T cell receptor, CD3, CD137, CD40, CTLA4, a costimulatory molecule, or a costimulatory molecule; (Membrane proteins capable of binding to costimulatory or co-inhibitory molecules are selected from among CD274 (PD-L1), CD273 (PD-L2), CD80, CD86, CD276 (ICOSL), CD276 (VICN1), VISTA, HHLA2, members of the butyrophilin family, CD270 (HVEM), CD137L (TNFSF-9), CD252 (OX40L), CD70 (TNFSF-7), CD40 (TNFRSF-7), GIRL (TNFSF-18), CD155 (PVR, NECL5), CD112 (NECTIN-2), CD200 (MOX1), CD48 (BCM-1), and Gal-9). And (C) a moiety capable of binding to a membrane protein in the cell membrane of a cell other than a T cell, which is capable of binding to a costimulatory or co-inhibitory molecule in the cell membrane of a T cell, (In particular, the costimulatory molecule is selected from the group consisting of CD28 (TP44), CD278 (ICOS), CD27 (TNFRSF-7), CD30, CD134 (OX40), CD357 (GITR). Tim-2, CD28H (TMIGD2), CD137 (4-1BB, TNFRSF-9), CD226 (DNAM-1), and CD244 (2B4), and / or the co-inhibitory molecule is selected from the group consisting of CD279 (PD-1), CD152 (CTLA-4), TIGIT, BTLA, CD366 (Tim-3), CD96, CD112R, or CD200R.) is selected from the group consisting of Among others, the second portion is selected from the group consisting of a portion capable of binding to CD3, a portion capable of binding to CD137, a portion capable of binding to CD40, and a portion capable of binding to CTLA4; In particular, the second moiety is selected from the group consisting of a moiety capable of binding to CD3 and a moiety capable of binding to CD137; Preferably, the second moiety is a moiety capable of binding to CD3. Any one of the antigen-binding molecules [D1] to [D16]. [D18] (A) the moiety capable of binding to CD3 is an Ig-type binding moiety, and / or (B) the moiety capable of binding to CD137 is an Ig-type binding moiety; and / or (C) the moiety capable of binding to CD40 is an Ig-type binding moiety; and / or (D) the moiety capable of binding to CTLA4 is an Ig-type binding moiety; In particular, the Ig-type binding moiety is selected from the group consisting of a single domain antibody (VHH), a combination of an antibody heavy chain variable (VH) region and an antibody light chain variable (VL) region, a single domain antibody (sdAb), a single chain Fv (scFv), a single chain antibody, an Fv, a single chain Fv2 (scFv2), an Fab, and an F(ab')2; Among others, the Ig-type binding moiety is selected from the group consisting of VHH, scFv, scFv2, Fab, and F(ab')2, [D17] Antigen-binding molecule. [D19] The molecule is characterized in that a complex formed by binding of one or more of the molecules to a first antigen can bind to more than one membrane protein via a second moiety; especially, (i) a complex formed by binding of one or more of said molecules to a first antigen is capable of binding to one or more of the membrane proteins via a second moiety such that it confers signaling in a cell via said one or more membrane proteins; or (ii) a complex formed by binding of one or more of said molecules to a first antigen is capable of binding to one or more of the membrane proteins via a second moiety such that it provides signal blocking in a cell via said one or more membrane proteins; Any one of the antigen-binding molecules [D1] to [D18]. [D20] The molecule is (A) a polypeptide complex, optionally a fusion protein; (B) a polynucleotide (preferably composed of two entities, preferably connected by a linker), and (C) a medium-sized chemical compound (preferably composed of two entities, preferably connected by a linker); (D) a small chemical compound (preferably composed of two entities, preferably connected by a linker) is selected from the group consisting of especially, (A-1) the molecule is a polypeptide complex; In particular, the molecule is an antibody or an antibody fragment thereof, In particular, the molecule is an antibody, Preferably, the antibody is an IgG type antibody and / or a bispecific antibody; or (A-2) the molecule is a polypeptide complex that is a fusion protein; Among others, the molecule is an antibody, or an antibody fragment thereof, which is a fusion protein, In particular, the molecule is an antibody that is a fusion protein, Preferably, the antibody is (a) an IgG type antibody having at least one binding polypeptide, preferably two binding polypeptides, fused to its Fc domain, and / or (b) a bispecific antibody having at least one binding polypeptide, preferably two binding polypeptides, fused to its Fc domain, Any one of the antigen-binding molecules [D1] to [D19]. [D21] The antigen-binding molecule of any one of [D1] to [D20], wherein the molecule is an antibody. [D22] The antigen-binding molecule of [D20] or [D21], wherein the molecule is an IgG antibody. [D23] The antibody is capable of binding to a membrane protein of an immune cell and further comprises at least one binding polypeptide, preferably linked to an Fc region; In particular, the binding polypeptide is selected from a Clec9A polypeptide and a mClec4e polypeptide; Among others, the antibody is an anti-CD3 antibody comprising a Clec9A polypeptide; an anti-CD137 antibody comprising a Clec9A polypeptide; an anti-CD3 antibody comprising a mClec4e polypeptide, and Anti-CD137 Antibodies Containing mClec4e Polypeptides Selected from the group consisting of: Any one of the antigen-binding molecules [D20] to [D22]. [D24] The antibody is a bispecific antibody, In particular, the antigen-binding molecule is a bispecific antibody against a membrane protein of an immune cell and against a DAMP, Among others, the antibody is bispecific anti-HSP90 anti-CD3 antibody, bispecific anti-HSP90 anti-CD3 antibody, bispecific anti-GRP78 anti-CD3 antibody, bispecific anti-GRP78 anti-CD137 antibody, bispecific anti-phosphatidylserine anti-CD3 antibodies, and Bispecific anti-phosphatidylserine anti-CD137 antibody Selected from the group consisting of: Any one of the antigen-binding molecules [D20] to [D23]. [D25] (i-1) the first moiety is an Ig-type binding moiety, or (i-2) the first portion is a binding polypeptide; and / or (ii) the second portion is an Ig-type binding portion; especially, (a) the first portion is an Ig-type binding moiety selected from the group consisting of a single domain antibody (VHH), a combination of an antibody heavy chain variable (VH) region and an antibody light chain variable (VL) region, a single domain antibody (sdAb), a single chain Fv (scFv), a single chain antibody, an Fv, a single chain Fv2 (scFv2), an Fab, and an F(ab')2; and / or (b) the second portion is an Ig-type binding moiety selected from the group consisting of a single domain antibody (VHH), a combination of an antibody heavy chain variable (VH) region and an antibody light chain variable (VL) region, a single domain antibody (sdAb), a single chain Fv (scFv), a single chain antibody, Fv, a single chain Fv2 (scFv2), Fab, and F(ab')2; and / or (c) the binding polypeptide is a non-Ig type binding moiety; Preferably, (a*) the first portion is a binding domain selected from the group consisting of VHH, scFv, scFv2, Fab and F(ab')2; and / or (b*) the second portion is a binding domain selected from the group consisting of VHH, scFv, scFv2, Fab and F(ab')2; and / or (c*) the binding polypeptide is a non-Ig binding moiety, in particular a non-Ig binding moiety attached to the Fc domain of the antigen-binding molecule; Any one of the antigen-binding molecules [D1] to [D24]. [D26] The molecule is (A) An antibody capable of binding to F-actin and an antigen involved in signal transduction in cells; (B) an antibody capable of binding to F-actin and an immune cell receptor; or (C) an antibody capable of binding to HSP90 and an antigen involved in signal transduction in a cell; (D) an antibody capable of binding to HSP90 and an immune cell receptor; or (E) an antibody capable of binding to GRP78 and an antigen involved in signal transduction in a cell; (F) an antibody capable of binding to GRP78 and an immune cell receptor; or (G) an antibody capable of binding to phosphatidylserine and an antigen involved in signal transduction in cells; (H) an antibody capable of binding to phosphatidylserine and an immune cell receptor; (I) an antibody capable of binding to SAP130 and an antigen involved in signal transduction in a cell; and (J) Antibodies capable of binding to SAP130 and immune cell receptors is selected from the group consisting of Preferably, the molecule is (A-1) an antibody capable of binding to F-actin and CD3; (A-2) An antibody capable of binding to F-actin and CD137; (C-1) an antibody capable of binding to HSP90 and CD3; (C-2) an antibody capable of binding to HSP90 and CD137; (E-1) an antibody capable of binding to GRP78 and CD3; (E-2) an antibody capable of binding to GRP78 and CD137; (G-1) an antibody capable of binding to phosphatidylserine and CD3; (G-2) an antibody capable of binding to phosphatidylserine and CD137; (I-1) an antibody capable of binding to SAP130 and CD3; and (I-2) Antibody capable of binding to SAP130 and CD137 Selected from the group consisting of: Any one of the antigen-binding molecules [D20] to [D25]. [D27] The molecule is (A) an antibody comprising two first moieties capable of binding to F-actin, among others being a binding polypeptide, preferably a Clec9A polypeptide, attached to the Fc domain of the antibody, and two second moieties involved in signal transduction in a cell, among others being Ig-type binding moieties; (B) an antibody comprising two first moieties capable of binding to F-actin, among others, which are binding polypeptides, preferably Clec9A polypeptides, attached to the Fc domain of the antibody, and two second moieties which are immune cell receptor antigens, among others, Ig type binding moieties; (C) an antibody comprising two first moieties capable of binding to HSP90, among them Ig-type binding moieties, and two second moieties involved in signal transduction in a cell, among them Ig-type binding moieties; (D) an antibody comprising two first moieties capable of binding to HSP90, among which Ig-type binding moieties, and two second moieties which are immune cell receptor antigens, among which Ig-type binding moieties; (E) an antibody comprising two first moieties capable of binding to GRP78, among them Ig-type binding moieties, and two second moieties involved in signal transduction in a cell, among them Ig-type binding moieties; (F) an antibody comprising two first moieties capable of binding to GRP78, among which are Ig-type binding moieties, and two second moieties which are immune cell receptor antigens, among which are Ig-type binding moieties; (G) An antibody comprising two first moieties capable of binding to phosphatidylserine, among them Ig-type binding moieties, and two second moieties involved in signal transduction in cells, among them Ig-type binding moieties; (H) An antibody comprising two first moieties capable of binding to phosphatidylserine, among which Ig-type binding moieties, and two second moieties which are immune cell receptor antigens, among which Ig-type binding moieties; (I) an antibody comprising two first moieties capable of binding to SAP130, preferably a binding polypeptide, preferably an mClec4e polypeptide, attached to the Fc domain of the antibody, among others, and two second moieties involved in signal transduction in cells, among others Ig-type binding moieties; (J) An antibody comprising two first moieties capable of binding to SAP130, preferably a binding polypeptide attached to the Fc domain of the antibody, preferably an mClec4e polypeptide, among others, and two second moieties which are immune cell receptor antigens, among others Ig type binding moieties. is selected from the group consisting of Preferably, the molecule is (A1) An antibody comprising two first moieties capable of binding to F-actin, preferably a binding polypeptide attached to the Fc domain of the antibody, preferably a Clec9A polypeptide, among others, and two second moieties capable of binding to CD3, among others Ig-type binding moieties; (A2) An antibody comprising two first moieties capable of binding to F-actin, preferably a binding polypeptide attached to the Fc domain of the antibody, preferably a Clec9A polypeptide, among others, and two second moieties capable of binding to CD137, among others Ig-type binding moieties; (C1) An antibody comprising two first moieties capable of binding to HSP90, among which Ig-type binding moieties, and two second moieties capable of binding to CD3, among which Ig-type binding moieties; (C2) An antibody comprising two first moieties capable of binding to HSP90, among which Ig-type binding moieties, and two second moieties capable of binding to CD137, among which Ig-type binding moieties; (E1) An antibody comprising two first moieties capable of binding to GRP78, among which Ig-type binding moieties, and two second moieties capable of binding to CD3, among which Ig-type binding moieties; (E2) an antibody comprising two first moieties capable of binding to GRP78, among which Ig-type binding moieties, and two second moieties capable of binding to CD137, among which Ig-type binding moieties; (G1) An antibody comprising two first moieties capable of binding to phosphatidylserine, among others Ig-type binding moieties, and two second moieties capable of binding to CD3, among others Ig-type binding moieties; (G2) An antibody comprising two first moieties capable of binding to phosphatidylserine, in particular Ig-type binding moieties, and two second moieties capable of binding to CD137, among others Ig-type binding moieties; (I1) An antibody comprising two first moieties capable of binding to SAP130, preferably a binding polypeptide attached to the Fc domain of the antibody, preferably an mClec4e polypeptide, among others, and two second moieties capable of binding to CD3, among others Ig-type binding moieties; (I2) An antibody comprising two first moieties capable of binding to SAP130, preferably a binding polypeptide attached to the Fc domain of the antibody, preferably a mClec4e polypeptide, and two second moieties capable of binding to CD137, preferably Ig-type binding moieties. Selected from the group consisting of: Any one of the antigen-binding molecules [D20] to [D26]. [D28] A pharmaceutical composition comprising any one of the antigen-binding molecules of [D1] to [D27]. [D29] (i) medicines, and / or (ii) a method for treating a medical condition, particularly a medical condition involving cell death, and / or (iii) a method of treating a medical condition in a subject, the method comprising contacting the antigen-binding molecule with damaged cells; (iv) a method for treating a medical condition in a subject, comprising contacting the antigen-binding molecule with a damage-associated molecular pattern (DAMP) in a patient, in particular further comprising contacting the antigen-binding molecule with the second antigen; (v) a method of treating a medical condition in a subject, the method comprising administering the antigen-binding molecule to a patient suffering from a condition involving cell damage, in particular cell death; (Among other methods, the method includes contacting a first and a second antigen with a plurality of antigen-binding molecules in close proximity.) The antigen-binding molecule or pharmaceutical composition according to any one of [D1] to [D28] for use in a method for treating a disease in which the antigen-binding molecule or pharmaceutical composition is administered orally. [D30] The antigen-binding molecule or pharmaceutical composition according to any one of [D1] to [D29] for use in medicine. [D31] An antigen-binding molecule or pharmaceutical composition according to any of [D1] to [D27] for use in a method for treating or preventing a medical condition. [D32] The antigen-binding molecule or pharmaceutical composition for use in [D31], wherein the medical condition is selected from the group consisting of cancer and immune, preferably autoimmune, diseases. [D33] (i) the method comprises a step of administering to a patient an antigen-binding molecule according to any one of [D1] to [D27], and / or (ii) the method is a method for activating or inhibiting an immune response, and / or (iii) the method is for conferring a signaling or blocking effect in a cell; especially, (a) the method includes a step of contacting the antigen-binding molecule with a damage-associated molecular pattern (DAMP) that is exposed to the extracellular environment due to cell damage, wherein the DAMP is selected from the group consisting of F-actin, GRP78, phosphatidylserine, HSP90, and SAP130; and / or (b) the method comprises the step of contacting the antigen-binding molecule with a cell selected from among (ai) an immune cell (wherein the immune cell is selected from the group consisting of a T cell, a killer cell, a helper T cell, a regulatory T cell, a B cell, a memory B cell, a NK cell, a NKT cell, a dendritic cell, a macrophage, an eosinophil, a neutrophil cell, and a basophil), (a-ii) a tumor cell, and (a-iii) an autoreactive cell; and / or (c) the antigen-binding molecule comprises at least one, preferably two, Clec9A polypeptides, and among these, the antigen-binding molecule further comprises (c-1) at least two, preferably two moieties involved in signal transduction in a cell, or (c-2) at least two, preferably two moieties that are immune cell receptor antigens; The antigen-binding molecule or pharmaceutical composition for use according to any one of [D29] to [D32]. [D34] The method is for treating or preventing cancer or an immune (preferably autoimmune) disease, Among others, the method is for treating or preventing cancer, In particular, the method is for treating cancer. The antigen-binding molecule or pharmaceutical composition for use according to any one of [D18] and [D19]. [D35] A polynucleotide encoding the antigen-binding molecule according to any one of [D1] to [D27]. [D36] A polynucleotide, particularly DNA, encoding any one of the molecules [D1] to [D27]. A vector comprising the polynucleotide of [D37], [D35] or [D36]. [D38] A vector, particularly for recombinant protein expression, comprising a DNA sequence encoding an antigen-binding molecule as defined in any one of [D1] to [D27]. A host cell comprising the vector of [D39], [D37] or [D38]. [D40] A host cell comprising the vector of [D37] or [D38], particularly for recombinant protein expression of an antigen-binding molecule encoded by the vector. [D41] Use of at least one first portion and at least one second portion for the production of an antigen-binding molecule comprising at least one first portion and at least one second portion, comprising: (1) at least one first portion binds to a first antigen; and (2) at least one second moiety binds to a second antigen; the first antigen is a damage-associated molecular pattern (DAMP); and The second antigen is different from the first antigen, preferably the second antigen is a membrane protein of an immune cell. use. [D42] Use of an antigen-binding molecule for targeting cells in a tissue in which cell death is to be observed or detected, (1) at least one first portion binds to a first antigen; and (2) at least one second moiety binds to a second antigen; the first antigen is a damage-associated molecular pattern (DAMP); and The second antigen is different from the first antigen, preferably the second antigen is a membrane protein of an immune cell. use. [D43] Use of an antigen-binding molecule for inducing or blocking signal transduction in cells in a tissue in which cell death is observed, (1) at least one first portion binds to a first antigen; and (2) at least one second moiety binds to a second antigen; the first antigen is a damage-associated molecular pattern (DAMP); and The second antigen is different from the first antigen, preferably the second antigen is a membrane protein of an immune cell. use. [D44] A kit for producing a molecule that specifically acts in a tissue in which cell death is observed, the molecule comprising: (1) at least one first moiety that binds to a first antigen; and (2) at least one second moiety that binds to a second antigen; Includes; the first antigen is a damage-associated molecular pattern (DAMP); and the second antigen is different from the first antigen, preferably the second antigen is a membrane protein of an immune cell, The kit comprises: at least one first container containing at least a first portion; and At least one second container containing at least the second portion. Including the kit.

[0019] In addition, the present disclosure also specifically provides the following aspects [E1] to [E32]. [E1] (1) a first moiety that binds to a first antigen selected from the group consisting of actin, heat shock proteins, phosphatidylserine, histones, histone deacetylase complex subunits, HMGN proteins, hepatoma-derived growth factor, BCL-2, calreticulin, and cyclophilin A; and (2) a second portion that binds to a second antigen that is distinct from the first antigen and is selected from a membrane protein on a T cell or an antigen-presenting cell (APC); An antigen-binding molecule comprising: [E2] The antigen-binding molecule of [E1], wherein the actin is F-actin. [E3] The antigen-binding molecule of [E1], wherein the heat shock protein is HSP90, GRP78, HSP70, HSP60, HSP72, or GP96. [E4] The antigen-binding molecule of [E3], wherein the heat shock protein is HSP90 or GRP78. [E5] The antigen-binding molecule of [E1], wherein the phosphatidylserine is a part of a cell membrane. [E6] The antigen-binding molecule of [E1], wherein the histone deacetylase complex subunit is SAP130. [E7] The antigen-binding molecule of [E1], wherein the HMGN protein is HMGB1 or HMGN1. [E8] The antigen-binding molecule of [E1], wherein the first antigen is selected from the group consisting of F-actin, GRP78, phosphatidylserine, HSP90, and SAP130. [E9] The antigen-binding molecule of any one of [E1] to [E8], wherein the first portion is an Ig-type binding portion selected from the group consisting of an IgG-type antibody, VHH, VH, VL, sdAb, scFv, single-chain antibody, Fab, and F(ab')2. [E10] The antigen-binding molecule of any one of [E1] to [E8], wherein the first portion is a non-Ig type-binding portion. [E11] The antigen-binding molecule of [E1], wherein the first antigen is F-actin and the first moiety is a Clec9A polypeptide. [E12] The antigen-binding molecule of [E1], wherein the first antigen is selected from the group consisting of GRP78, phosphatidylserine, and HSP90, and the first moiety is an Ig-type binding moiety. [E13] The antigen-binding molecule of [E1], wherein the first antigen is SAP130 and the first moiety is an mClec4e polypeptide. [E14] An antigen-binding molecule of any one of [E1] to [E13], wherein the second antigen is selected from the group consisting of a T cell receptor, CD3, CD137, CD40, and CTLA4. [E15] The antigen-binding molecule of any one of [E1] to [E14], wherein the second portion is an Ig-type binding portion. [E16] The antigen-binding molecule of any one of [E1] to [E15], wherein the molecule is an IgG type antibody containing both the first portion and the second portion. [E17] The antigen-binding molecule of [E16], wherein the IgG antibody is capable of binding to a membrane protein of an immune cell and comprises at least one binding polypeptide. [E18] The antigen-binding molecule of [E17], wherein the binding polypeptide is linked to the Fc region of an IgG antibody. [E19] The antigen-binding molecule of [E17] or [E18], wherein the binding polypeptide comprises a Clec9A polypeptide or an mClec4e polypeptide. [E20] The IgG antibody, an anti-CD3 antibody comprising a Clec9A polypeptide; an anti-CD137 antibody comprising a Clec9A polypeptide; an anti-CD3 antibody comprising a mClec4e polypeptide, and Anti-CD137 Antibodies Containing mClec4e Polypeptides The antigen-binding molecule of [E17], selected from [E21] The antigen-binding molecule of [E16], wherein the IgG antibody is a bispecific antibody. [E22] The antigen-binding molecule of [E21], wherein the bispecific antibody is directed against a membrane protein of an immune cell and against a DAMP. [E23] The bispecific antibody, bispecific anti-HSP90 anti-CD3 antibody, bispecific anti-HSP90 anti-CD3 antibody, bispecific anti-GRP78 anti-CD3 antibody, bispecific anti-GRP78 anti-CD137 antibody, bispecific anti-phosphatidylserine anti-CD3 antibodies, and Bispecific anti-phosphatidylserine anti-CD137 antibody The antigen-binding molecule of [E21], selected from [E24] A pharmaceutical composition comprising any one of the antigen-binding molecules of [E1] to [E23]. [E25] An isolated polynucleotide encoding any one of the antigen-binding molecules [E1] to [E23]. A vector comprising the polynucleotide of [E26][E25]. A host cell comprising the polynucleotide of [E27][E25]. [E28] A method for treating a medical condition accompanied by cell damage or cell death in a subject suffering from such a condition, the method comprising the step of administering to the subject an antigen-binding molecule of any one of [E1] to [E23]. [E29] A method for treating cancer in a subject in need thereof, comprising the step of administering to the subject an antigen-binding molecule of any one of [E1] to [E23]. [E30] A method for treating an immune disease in a subject in need thereof, comprising the step of administering to the subject an antigen-binding molecule of any one of [E1] to [E23]. [E31] The method of [E30], wherein the immune disease is an autoimmune disease. [E32] A method comprising culturing the host cell of [E27] under conditions suitable for expression of the antigen-binding molecule, and optionally recovering the antigen-binding molecule. [Brief description of the drawings]

[0020] [Figure 1] A drawing which illustrates, in a schematic manner, the technical concept encompassed and achieved by the present invention, where the molecule of the present invention is an antibody-like molecule comprising a portion which binds to F-actin, which is exposed to the extracellular environment upon cell death, and which also illustrates that the molecule forms a complex with F-actin, and that the complex binds via the molecule to one or more of the membrane proteins in the cell, thereby conferring a signal or blockage in the cell. [Diagram 2] FIG. 1 is a diagram illustrating the technical features of one embodiment of the molecule of the present invention, which is an antibody-like molecule comprising a moiety that binds to a cellular component or part thereof that is exposed to the extracellular environment upon cell death. [Diagram 3]Figures illustrating the technical features of various embodiments of the molecules of the present invention, which are antibody-like molecules that contain a moiety that binds to a component or part of a cell exposed to the extracellular environment upon cell death. For example, a circle (open) refers to a "first moiety" that binds to a "first antigen". In a further example, when two first moieties (open circles) are conjugated, at least one moiety binds to the "first antigen" and the other can bind to any antigen different from the "first antigen" and the "second antigen". In another example, at least one Fab arm binds to the "second antigen" and the other Fab arm can bind to any antigen different from the "first antigen" and the "second antigen". In a further example, at least one Fab arm binds to the "second antigen" and the other Fab arm can bind to the "first antigen", and the conjugated first moiety (open circle) can bind to any antigen different from the "first antigen" and the "second antigen". [Figure 4] Figure 1 shows the results of measuring the binding of Clec9A-conjugated antibodies to live cells and necrotic cells or components thereof, respectively. [Diagram 5] FIG. 1 shows the results of measuring T cell activation through clustering of Clec9A-conjugated anti-CD3 antibodies on T cells in the presence of dead cells. [Figure 6] Figure 1 shows the results of measuring the binding of anti-phosphatidylserine or anti-HSP90 to live or dead cells. [Figure 7] FIG. 1 shows the results of measuring CD3+ T cell activation through clustering of anti-phosphatidylserine / / CD3 bispecific antibody or anti-HSP90 / / CD3 bispecific antibody on T cells in the presence of live or dead cells. [Figure 8] FIG. 1 shows the results of measuring CD137+ T cell activation through clustering of Clec9A-conjugated anti-CD137 antibodies on T cells in the presence of live or dead cells. [Figure 9]FIG. 1 shows the results of measuring CD137+ T cell activation through clustering of anti-phosphatidylserine / / CD137 antibodies on T cells in the presence of live or dead cells. [Figure 10] A diagram showing the results of CD3 activation by Clec9A-conjugated antibodies in the presence or absence of F-actin. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Description of the Aspects The following definitions and detailed descriptions are provided to facilitate understanding of the invention exemplified herein. In particular, these definitions and detailed descriptions are useful for interpreting the embodiments of Groups A, B, C, and D above.

[0022] cell death There are several types of cell death that have been reported, which are classified into two types: programmed cell death (PCD) and non-PCD (Cell Research volume 29, pages 347-364, 2019; Cell Biol. Int., 2019 Jun; 43(6): 582-592; Nature Reviews Cancer, Vol.12, p.860-875, 2012). PCD is tightly controlled by signaling cascades and molecularly defined mechanisms. Apoptosis is the best known PCD, and recently, many non-apoptotic types of PCD, such as pyroptosis, NETosis, and necroptosis, have been reported. On the other hand, non-PCD is a biologically uncontrollable process, and necrosis is classified as non-PCD.

[0023] In the present invention, "cell death" refers to, but is not limited to, one of the aspects of programmed cell death (PCD) of any kind or type of cell, including but not limited to somatic cells (e.g., epithelial cells, fibroblasts, stromal cells, bone cells, muscle cells, nerve cells, blood cells, etc.), germ cells, or diseased or abnormal cells (e.g., tumor cells, autoreactive cells), including but not limited to apoptosis; necrosis; accidental cell death; or controlled cell death (e.g., apoptosis, controlled necrosis, autophagic cell death, necroptosis, ferroptosis, or pyroptosis). Such cells can be located in any tissue, including but not limited to epithelial tissue, muscle tissue, nerve tissue, connective tissue. Here, necrosis, which is a form of non-programmed cell death caused by injury or inflammation, also results in increased cell death. Specifically, in the present invention, "cell death" can be, for example, cell death observed in diseased tissues due to cancer or autoimmune disease.

[0024] Cell Damage Similarly, in the present invention, "cell damage" is not particularly limited and includes any cell damage resulting in exposure of DAMPs, including in particular all of the above-mentioned forms of cell death.

[0025] First antigen / damage-associated molecular pattern (DAMP) In the present invention, the term "first antigen", which is also referred to herein as "damage-associated molecular pattern" or "DAMP" according to one general aspect of the present invention, and is used interchangeably with "substance exposed to extracellular environment due to cell damage", refers to a biomolecule associated with cell damage, which in the present invention can also be referred to as "damaged cell-derived molecule", or "substance derived from damaged cell", "damaged cell-associated molecule", "damaged cell-associated substance", and "danger-associated molecular pattern", which are used interchangeably. These biomolecules and substances associated with cell damage can be upregulated in expression, expressed on the cell surface, modified, passively diffused, or actively secreted outside the cell upon cell damage. These changes are positively correlated with the presence or increase of damaged cells, or with the decrease of intact cells. In other words, in cells facing cell damage, components or parts of the cytoskeleton, cell membrane, organelle, cytoplasmic protein, or metabolite in the cell are exposed to the extracellular environment, and any of these are engulfed by substances or biomolecules associated with cell damage.

[0026] In any case, "damage-associated molecular patterns" ("DAMPs") are well known to those skilled in the art as "substances that are exposed to the extracellular environment due to cellular damage."

[0027] In the present invention, the terms "damage-associated molecular pattern", "DAMP", and "substance exposed to the extracellular environment due to cell damage" can be interchangeably referred to as "a component or part of the cytoskeleton, cell membrane, organelle, cytoplasmic protein, or metabolic product exposed to the extracellular environment due to cell damage", and also as "first antigen". That is, in the present invention, unless otherwise defined to mean differently, the term "first antigen" means to include but is not limited to "a component or part of the cytoskeleton, cell membrane, organelle, cytoplasmic protein, or metabolic product exposed to the extracellular environment due to cell damage".

[0028] In certain embodiments, the DAMP is selected from the group consisting of: (A) actin, particularly F-actin; (B) heat shock proteins, especially HSP90, selected from HSP90 and GRP78; (C) phosphatidylserine (PS), especially phosphatidylserine that is part of the cell membrane; (D) histones or components thereof; (E) histone deacetylase complex subunits, especially SAP130; (F) HMGN proteins, especially selected from HMGB1 and HMGN1; (G) hepatoma-derived growth factor; (H) BCL-2; (I) calreticulin; and (J) cyclophilin A.

[0029] In the present invention, cell damage preferably comprises cell death. In a preferred embodiment of the present invention, the cell damage is cell death. Thus, in a further general aspect of the present invention, the following applies:

[0030] The first antigen and substances exposed to the extracellular environment upon cell death In the present invention, the term "first antigen", also referred to as "substance exposed to extracellular environment by cell death", refers to a biomolecule associated with cell death, and may be referred to interchangeably as "dead cell-derived molecule", or "dead cell-derived substance", "dead cell-associated molecule", "dead cell-associated substance", and "danger-associated molecular pattern" in the present invention. These biomolecules and substances associated with cell death can be upregulated in expression, expressed on the cell surface, modified, passively diffused, or actively secreted outside the cell upon cell death. These changes are positively correlated with the presence or increase of dead cells, or with the decrease of live cells. In other words, in cells heading for cell death, components or parts of the cytoskeleton, cell membrane, organelle, cytoplasmic protein, or metabolite in the cell are exposed to the extracellular environment, and any of these are engulfed by the substance or biomolecule associated with cell death.

[0031] That is, the term "substance exposed to the extracellular environment due to cell death" (or "substance exposed to the extracellular environment due to cell damage", "damage-associated molecular pattern", or "DAMP") can be referred to as "a component or a part of the cytoskeleton, cell membrane, organelle, cytoplasmic protein, or metabolite exposed to the extracellular environment due to cell death", and also interchangeably as "first antigen" (or "a component or a part of the cytoskeleton, cell membrane, organelle, cytoplasmic protein, or metabolite exposed to the extracellular environment due to cell damage", respectively). That is, in the present invention, unless otherwise defined to mean differently, the term "first antigen" means, but is not limited to, "a component or a part of the cytoskeleton, cell membrane, organelle, cytoplasmic protein, or metabolite exposed to the extracellular environment due to cell death", or "a component or a part of the cytoskeleton, cell membrane, organelle, cytoplasmic protein, or metabolite exposed to the extracellular environment due to cell damage", respectively.

[0032] In any case, in the present invention, all examples and embodiments of "dead cell-derived molecules" etc., respectively, are specifically included within DAMPs in the context of the present invention.

[0033] In the present invention, the term "cytoskeleton" refers to a filament structure in the cytoplasm that generates the physical force required for intracellular and extracellular movement of cells and also to maintain the shape of the cell. In the present invention, "filament" includes, but is not limited to, intermediate filaments, actin filaments, myosin filaments, and keratin filaments. Eukaryotic cells have actin filaments, intermediate filaments, myosin filaments, keratin filaments, and microtubules as cytoskeleton. Actin filaments are composed of actin; intermediate filaments are composed of vimentin, glial fibrillary proteins, neurofilament proteins, nuclear lamins, etc.; and microtubules are composed of α-tubulin and β-tubulin. In the present invention, the "cytoskeleton" exposed to the extracellular environment due to cell death is not limited to the protein components that form the above cytoskeleton, and they may be conjugated with one or more any other proteins.

[0034] Biological membranes such as cell membranes are composed of lipid bilayers in which proteins are embedded. In the present invention, components of biological membranes such as cell membranes include, but are not limited to, lipids and proteins. Lipids include, but are not limited to, phospholipids, glycolipids, and sterols. Proteins include, but are not limited to, transmembrane proteins such as ion channels, G protein-coupled receptors (GPCRs), proton pumps, lipid-anchored proteins such as G proteins, and peripheral membrane proteins such as enzymes, hormones, and the like.

[0035] Generally, in the present invention, "membrane protein" refers to membrane proteins, which are generally well known to those skilled in the art as general proteins that are part of or interact with biological membranes and include several broad categories depending on their location, including in particular integral membrane proteins, which are permanent parts of the cell membrane and can either span the membrane (transmembrane) or associate with one or the other side of the membrane (integral monotopic), and peripheral membrane proteins, which are transiently associated with the cell membrane (e.g., via so-called anchors or by a certain type or combination of non-covalent interactions). This term in particular includes any of the membrane proteins described herein.

[0036] In the present invention, "cell membranes or organelles exposed to the extracellular environment due to cell death" includes, but is not limited to, the above-mentioned phospholipids or proteins; any biological membranes that constitute organelles such as nucleoli, nuclei, ribosomes, endoplasmic reticulum, Golgi apparatus, mitochondria, etc.; and also proteins embedded in biological membranes.

[0037] In the present invention, the "substance / molecule exposed to the extracellular environment due to cell death" ("dead cell-derived molecule") and DAMP include, but are not limited to, the above-mentioned components or parts thereof, and any substance or molecule exposed to the extracellular environment due to cell death may be utilized in the present invention. Furthermore, the dead cell-derived molecule may be a complex of one or more of the above-mentioned components or parts thereof. The dead cell-derived molecule may be a whole or truncated form of a protein. In preferred embodiments, the dead cell-derived molecule includes, but is not limited to, cytoskeleton, organelle, and cytoplasmic proteins in cells. Cell membranes and any fragments of cell membranes are also included in the dead cell-derived molecule as preferred embodiments. In the present invention, more preferred embodiments of the dead cell-derived molecule include, but are not limited to, filaments constituting the cytoskeleton or nucleoskeletons, and nucleosomes or molecules forming nucleosomes.

[0038] In the present invention, the dead cell-derived molecule includes, as a non-limiting embodiment, a molecule released from a cell into the extracellular environment by immunogenic cell death, i.e., a damage-associated molecular pattern (DAMP) released by immunogenic cell death. DAMPs include molecules reported by Dmitri et al., Nature Reviews Cancer, Vol. 12, p. 860-875, 2012. In other words, all DAMPs described in Dmitri et al. are specifically included as embodiments of DAMPs according to the present invention.

[0039] Further and somewhat reiterating, in the present invention, specific embodiments of "substances exposed to the extracellular environment due to cell death" or "components or parts of cytoskeleton, cell membrane, organelle, cytoplasmic protein, or metabolites exposed to the extracellular environment due to cell death" and "DAMPs" include, but are not limited to, filaments or histones (e.g., intermediate filaments such as F-actin) forming the cytoskeleton or nucleoskeleton, phosphatidylserine, heat shock proteins, and further proteins, RNA, DNA, or metabolites, or combinations thereof resulting in modified molecules or complexes. Here, the proteins can be full-length proteins or fragments thereof.

[0040] In addition, molecules such as glypican and syndecan are expressed on the surface of living cells, but can be modified by the microenvironment rich in dead cells, and these molecules are also included in the dead cell-derived molecules. Furthermore, biglycan, decorin, and lumican can also be sequestered in the extracellular matrix under normal physiological conditions, but are proteolytically released with an increase in dead cells. These molecules are also included in the present invention as dead cell-associated molecules in the present invention.

[0041] Dead cell-derived molecules can also be molecules that are released when cell death is induced or that are exposed or abundantly expressed on the cell surface. All cells, such as somatic and germ cells, are capable of producing dead cell-derived molecules. A common feature of solid tumors is the presence of hypoxia and low nutrition conditions resulting from reduced blood flow. These hypoxic and low nutrition conditions stress tumor cells, resulting in abundant cell death in solid tumors. Since cell death is involved in the pathogenesis of autoimmune diseases, cell death is usually found in many types of autoimmune diseases, such as vitiligo, type I diabetes, in addition to tumors. For example, vitiligo is an autoimmune disease induced by the destruction of melanocytes. In vitiligo, the presence of many types of autoantibodies that recognize lamin A, tyrosinase-related protein 1, HSP90, HSP70, etc. has been reported (Hamid et al., Pigmentary Disorders 2015, 2:6). These molecules are usually localized in the cytosol or nucleus under steady-state conditions. Because antibodies are raised against molecules that are located on the outside of the cell, these molecules must be released or exposed to the plasma membrane at the time of cell death.

[0042] In the present invention, the source of the dead cell-derived molecule is not limited to the above-mentioned sources. Any molecule or substance of or in a cell exposed to the extracellular environment in which cell death is observed can be the source of the dead cell-derived molecule. Whether or not a certain molecule or substance is a dead cell-derived molecule can be confirmed by examining whether the molecule or substance is a molecule or substance exposed to the extracellular environment in which cell death is induced.

[0043] To examine and confirm whether cell death is induced, the following methods can be employed, including but not limited to:

[0044] Methods for detecting dead cells or molecules or substances derived from dead cells may include detecting an increase in the rate of one or more of the characteristics, processes associated with dead cells, or a decrease in the rate of viable cells. Such methods include, but are not limited to, detecting cells that stain positive for live cell-impermeable DNA-binding dyes, live cell-impermeable amine-reactive dyes, measuring enzyme activity such as lactate dehydrogenase in dead cell supernatants, or a decrease in the level of intracellular ATP associated with metabolically active live cells. In addition, methods for detecting dead cells, such as those reported by Riss, may also be used (Riss, Cytotoxicity Assays: In Vitro Methods to Measure Dead Cells; May 1, 2019).

[0045] In the present invention, whether a certain molecule is a dead cell-derived molecule can be determined or confirmed by comparing the supernatant of a cell culture to which a cytotoxic drug such as mitoxantrone or mitomycin C has been added with the supernatant of a cell culture without drug treatment. By performing proteome analysis and / or metabolome analysis of proteins and / or metabolites contained in each of the supernatant of a cell culture cultured in the presence of a cytotoxic drug and the supernatant of a cell culture without drug addition, a molecule detected in a relatively large amount in the supernatant of a cell culture cultured with the addition of a cytotoxic drug can be determined to be a dead cell-derived molecule (i.e., a substance exposed to the extracellular environment due to cell death). Here, the drug added to the cell culture is not limited to mitoxantrone or mitomycin C, and any drug may be used as long as it has cytotoxicity.

[0046] In the present invention, the method of inducing cell death is not limited to the above-mentioned method using a cytotoxic drug. For example, cell death can be physically induced by freezing and thawing the cells.

[0047] Furthermore, in the present invention, whether a certain molecule or substance is a dead cell-derived molecule or a dead cell-derived substance can be determined or confirmed by, for example, preparing cells treated with the above-mentioned drug that induces cell death or untreated cells, and using flow cytometry to measure and compare the expression level of one or more membrane proteins in each group of cells.The membrane protein that is found to have increased expression in drug-treated cells compared to the expression in untreated cells is determined as a molecule that can be used as a dead cell-derived molecule.

[0048] In the present invention, the above dead cell-derived molecules, including but not limited to RNA, DNA, filamentous action, histone, phosphatidylserine, or heat shock protein, can be quantified using any means and / or methods for quantification well known by those skilled in the art, such as column chromatography, mass spectrography (this term is used interchangeably with mass spectrometry herein), ELISA, etc. Any other means and / or methods for quantification may be used as long as the dead cell-derived molecules in the present invention can be quantified.

[0049] Can be combined In the context of the present invention, the terms "capable of binding" and "binding" are used interchangeably and refer to the ability of a moiety to bind to an antigen, especially under physiological conditions, i.e., conditions found in an animal, especially the human body. Certain exemplary methods for determining said ability are described herein below. As used herein, terms such as "ability to bind to an antigen" may also be designated as terms such as "binding affinity for an antigen". Furthermore, in the case of an antibody, said ability may also be described by using the term "antibody against" or by using the term "antigen X antibody".

[0050] A first moiety that binds to a first antigen In the present invention, the "first portion" of the "molecule comprising a first portion that binds to a first antigen and a second portion that binds to a second antigen" binds to a "first antigen," which is also referred to interchangeably as a "substance exposed to the extracellular environment upon cell death" and a "component or a portion thereof constituting the cytoskeleton, cell membrane, organelle, cytoplasmic protein, or metabolic product that is exposed to the extracellular environment upon cell death," as described above.

[0051] The "first part" of the molecule in the present invention may have any structure as long as it binds to the "first antigen" as described above. The structure of the "first part" may include, but is not limited to, a polypeptide or a part thereof, or a small or medium-sized chemical compound or a part thereof, or a polynucleotide or a part thereof. The polypeptide or a part thereof includes, but is not limited to, a cell membrane protein or a part thereof (e.g., an extracellular domain, any unique domain thereof) expressed on a cell (e.g., an immune cell such as a dendritic cell); an antibody (including, but not limited to, a human antibody, a chimeric antibody, a humanized antibody, and a VHH antibody) or an antigen-binding domain (also referred to as a part, part, or fragment of an antibody). Antigen-binding domains of antibodies include, but are not limited to, antibody heavy chain variable (VH) regions, antibody light chain variable (VL) regions (preferably a combination of an antibody heavy chain variable (VH) region and an antibody light chain variable (VL) region), single domain antibodies (sdAbs), single chain Fvs (scFvs), single chain antibodies, Fvs, single chain Fv2 (scFv2), Fab, and F(ab')2.

[0052] The polypeptide or a portion thereof may also be used in a variety of applications, including a module called the A domain of Avimer, which has approximately 35 amino acids contained in an in vivo cell membrane protein (WO2004 / 044011 and WO2005 / 040229); an adnectin having a 10Fn3 domain acting as a protein-binding domain derived from the glycoprotein fibronectin expressed on the cell membrane (WO2002 / 032925); an affibody having an IgG-binding domain scaffold consisting of a 58 amino acid three-helix bundle of protein A (WO1995 / 001937); and DARPins (designed ankyrin repeat proteins), which are molecular surface exposed regions of ankyrin repeats (AR), each of which has a structure of 33 amino acid residues folded into a subunit of a turn, two antiparallel helices, and a loop. The antigen-binding polypeptide may be an antigen-binding polypeptide such as a lipocalin molecule having four loop regions connecting eight antiparallel strands bent towards a central axis at one end of a highly conserved barrel structure such as neutrophil gelatinase-associated lipocalin (NGAL) (WO2003 / 029462), and a concave region in a horseshoe-shaped internal parallel sheet structure consisting of repeated leucine-rich repeat (LRR) modules of variable lymphocyte receptors (VLR) that do not have an immunoglobulin structure, as found in the adaptive immune system of jawless vertebrates such as lampreys or hagfish (WO2008 / 016854).

[0053] An embodiment of the polypeptide or a portion thereof belonging to the above-mentioned "first portion that binds to the first antigen" in the present invention includes, but is not limited to, a cell membrane protein (e.g., a receptor) expressed on a cell or a portion thereof (e.g., an extracellular domain, any unique domain thereof). A representative of a receptor or a portion thereof belonging to the "first portion that binds to the first antigen" in the present invention includes, but is not limited to, a scavenger receptor, a toll-like receptor (TLR), a C-type lectin (CLEC) such as Clec9A expressed on dendritic cells, NOD-like receptor (NLR) (J Biol Chem. 2014 Dec 19;289(51):35237-45), or a portion thereof. One of the preferred embodiments of the "first portion that binds to the first antigen" in the present invention is Clec9A, which is a C-type lectin expressed on dendritic cells (DC) and binds to a complex of F-actin, a cytoskeleton-forming filament, when the F-actin complex is exposed to the extracellular environment due to cell death. In the present invention, the "first portion that binds to a first antigen" includes, but is not limited to, the entire polypeptide of Clec9A (GenBank: NP_001192292.1), an extracellular domain (ECD) of any amino acid length, a C-type lectin domain (CTLD), or any portion thereof.

[0054] A second moiety that binds to a second antigen In the present invention, the "second portion" of the "molecule comprising a first portion that binds to a first antigen and a second portion that binds to a second antigen" binds to a "second antigen" that is different from the "first antigen" as described above. The "second antigen" may be any antigen as long as it is different from the first antigen. In the present invention, the "second antigen" is also referred to as a "target molecule" that is targeted by the "second portion" of the "molecule comprising a first portion that binds to a first antigen and a second portion that binds to a second antigen". Upon targeting of the "second antigen" by the "second portion" of the molecule in the present invention, the target molecule, i.e., the "second antigen", results in a desired outcome for the indicated disease, for example, immune activation to fight infection or cancer, immune suppression for autoimmune disease and / or cytokine release syndrome (CRS), direct inhibition of cell proliferation in cancer, or promotion of cell regeneration after tissue damage.

[0055] In the present invention, one common embodiment of immune activation target includes, but is not limited to, CD3 ("CDε"), CD4, CD8, CD28, OX40, 4-1BB, and T cell receptor (TCR) expressed on T cells, which results in activation of cytotoxic or T helper functions for the elimination of pathogens, infected cells, or transformed cells. Target molecules (i.e., "second antigens") may also be expressed on antigen-presenting cells (APCs), including XCR1, Clec9A, DEC-205, DCIR2, TLR3, TLR5, Flt3 on dendritic cells (DCs). Activation of DCs can indirectly result in the elimination of pathogens and cancer cells through the priming and activation of adaptive immune cell types, including cytotoxic T cells and helper T cells. A case in point would be the targeting of CD40 with CD40 agonists for antitumor effects through enhanced antigen cross-presentation and costimulatory capacity for more effective activation of cytotoxic T cells (ESMO Open. 2019; 4(Suppl 3): e000510). Furthermore, synergistic effects resulting in stronger antigen-processing immune cell priming capacity can be achieved with parallel activation of TLR3, a polyinosinic:polycytidylic acid (polyIC)-sensing receptor, also expressed by DCs (J Clin Invest. 2018;128(10): 4387-4396). In other cases, inhibition of targeted molecules may be desirable, for example antagonizing CTLA-4, PD-1, LAG-3, TIM-3, VISTA on T cells to prevent suppression of immune responses against tumor cells. The targeted molecule (i.e., the "second antigen") may also be any molecule expressed on or in other immune cell types, such as natural killer cells, macrophages, neutrophils, and their corresponding activating or inhibitory receptors. Oncogenic receptors on cancer cells, or immune evasion molecules on transformed or infected cells may also be targeted (i.e., the "second antigen").

[0056] In the present invention, the "second antigen" briefly described above includes, but is not limited to, membrane proteins expressed on the cell membrane and / or endosomal membrane in cells. One embodiment of the membrane protein (i.e., second antigen) to which the "second part" binds includes, but is not limited to, membrane proteins involved in signal transduction in cells, expressed on the cell membrane and / or endosome of immune cells such as T cells, killer cells, helper T cells, regulatory T cells, B cells, memory B cells, NK cells, NKT cells, dendritic cells, macrophages, eosinophils, neutrophil cells, and basophils. Another embodiment of the membrane protein (i.e., second antigen) to which the "second part" binds includes, but is not limited to, membrane proteins expressed on tumor cells or autoreactive cells.

[0057] In the present invention, the membrane proteins belonging to the above-mentioned "second antigen" include costimulatory molecules such as CD28 (TP44), CD278 (ICOS), CD27 (TNFRSF-7), CD30, CD134 (OX40), CD357 (GITR), Tim-2, CD28H (TMIGD2), CD137 (4-1BB, TNFRSF-9), CD226 (DNAM-1), and CD244 (2B4); or costimulatory molecules such as CD279 (PD-1), CD152 (CTLA-4), TIGIT, BTLA, CD366 (Tim-3), CD96, CD112R, and CD200R; or costimulatory molecules such as CD274 (PD-L1), CD273 (PD-L2), CD80, CD86, CD276 (ICOSL), CD276 (VIC N1), VISTA, HHLA2, members of the butyrophilin family, costimulatory or co-inhibitory molecules such as CD270 (HVEM), CD137L (TNFSF-9), CD252 (OX40L), CD70 (TNFSF-7), CD40 (TNFRSF-7), GIRL (TNFSF-18), CD155 (PVR, NECL5), CD112 (NECTIN-2), CD200 (MOX1), CD48 (BCM-1), or Gal-9.

[0058] Further embodiments of membrane proteins belonging to the "second antigen" as described above include CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3 (e.g., CD3ε), CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD1 9. CD20, CD21, CD22, CD23, CD25, CD27L, CD29, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147 , CD148, CD152, CD164, CEACAM5, CFTR, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6; fibroblast activation protein (FAP), Fas, cytokine receptors, such as IL-1R, IL-2R, IL-4R, IL-5R, IL-6R, IL-10R, IL-12R, IL-15R, IL-17R, IL-18R, IL-20R, or IL-3 1R; Integrin family, such as α1β1 (VLA-1), α2β1 (VLA-2), α3β1 (VLA-3), α4β1 (VLA-4), α5β1 (VLA-5), α6β1 (VLA-6), α7β1 , α8β1, α9β1, α11bβ3(GPIIb / IIIa), αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, αLβ2(LFA-1), αMβ2(Mac-1), αXβ2, αDβ2, α4β7;TNF-RI、TNF-RII、TNFRSF10A(TRAIL R1 Apo-2、DR4)、TNFRSF10B(TRAIL R2 DR5、KILLER、TRICK-2A、TRICK-B)、TNFRSF10C(TRAIL R3). DcR1、LIT、TRID)、TNFRSF10D(TRAIL R4DcR2、TRUNDD)、TNFRSF11A(RANK ODF R、TRANCE R)、TNFRSF11B(OPG OCIF、TR1)、TNFRSF12(TWEAK R). FN14)、TNFRSF13B(TACI)、TNFRSF13C(BAFF R)、TNFRSF14(HVEM ATAR、HveA、LIGHT R、TR2)、TNFRSF16(NGFR p75NTR)、TNFRSF17(BCMA)、TNFRSF18(GITR). AITR)、TNFRSF19(TROY CROWN、TRADE)、TNFRSF19L(RELT)、TNFRSF1A(TNF RI CD120a、p55-60)、TNFRSF1B(TNF RII). CD120b, p75-80, TNFRSF26(TNFRH3), TNFRSF3(LTbR TNF RIII, TNFC R), TNFRSF4(OX40 ACT35, TXGP1 R), TNFRSF6(Fas). Apo-1, APT1, CD95, TNFRSF6B(DcR3 M68, TR6), TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-1BB CD137, ILA), TNFRSF21(DR6), TNFRSF22(DcTRAIL R2). TNFRH2)、TNFRST23(DcTRAIL R1 TNFRH1)、TNFRSF25(DR3). Apo-3, LARD, TR-3, TRAMP, WSL-1, TLR(Toll Liquids)1, TLR2, TLR3, T LR4, TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10 are all free;

[0059] In the present invention, the "second antigen" also includes, but is not limited to, soluble proteins that can bind to membrane proteins as described above. In the present invention, the soluble proteins that belong to the "second antigen" include, but are not limited to, cytokines such as IL-1, IL-2, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IFNβ, IFNγ, IL-18, IL21, IL-23, and IL-27.

[0060] In the present invention, soluble proteins belonging to the "second antigen" further include, but are not limited to, the following molecules or any soluble form of said molecules (e.g., truncated forms of membrane proteins): 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA. ALK-2, activin RIBALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAM TS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, α-1-antitrypsin, α-V / β-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, artemin, anti-Id, ASPARTIC, atrial natriuretic factor, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulatory factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-associated antigen, cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V,Cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, Botulinum toxin, Clostridium perfringens toxin, Ckb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, PD-1, PD-L1, LAG3, TIM3, Galectin-9, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, Cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, Decay accelerating factor, des(1-3)-IGF-I (Brain IGF-1), Dhh, Digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOS, Eot, Eotaxin 1, EpCAM, ephrin B2 / EphB4, ePO, ERCC, E-selectin, ET-1, factor IIa, factor VII, factor VIIIc, factor IX, fibroblast activation protein (FAP), Fas, FcR1, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-α1, GFR-α2, GFR-α3, gITR, glucagon, Glut4, glycoprotein IIb / IIIa (GPIIb / IIIa), GM-CSF, gp130, gp72, GRO, growth hormone releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, I-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg,Ig, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-21, IL-23, IL-27, interferon (INF)-α, INF-β, INF-γ, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, integrin α2, integrin α3, integrin α4, integrin Integrin α4 / β1, integrin α4 / β7, integrin α5 (αV), integrin α5 / β1, integrin α5 / β3, integrin α6, integrin β1, integrin β2, interferon gamma, IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-1 bp1, LBP, LDGF, LECT2, lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, lung surface surface), luteinizing hormone, lymphotoxin β receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloprotease, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-α, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Muc1), MUC18, Müllerian inhibitory substance, Mug, MuSK, NAIP,NAP, NCAD, N-cadherin, NCA 90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, nerve growth factor (NGF), NGFR, NGF-β, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDGF, PDK-1, PE CAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PlGF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV)F, RSV Fgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (e.g., T cell receptor α / β), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-α, TGF-β, TGF-βPan Specific, TGF-βRI(ALK-5), TGF-βRII, TGF-βRIIb, TGF-βRIII, TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, thrombin, thymus Ck-1, thyroid Stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-α, TNF-α / β, TNF-β2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4),TNFRSF10B(TRAIL R2 DR5、KILLER、TRICK-2A、TRICK-B)、TNFRSF10C(TRAIL R3 DcR1、LIT、TRID)、TNFRSF10D(TRAIL R4 DcR2、TRUNDD)、TNFRSF11A(RANK ODF R、TRANCE R)、TNFRSF11B(OPG OCIF、TR1)、TNFRSF12(TWEAK RFN14)、TNFRSF13B(TACI)、TNFRSF13C(BAFF R)、TNFRSF14(WHO ATAR、HveA、LIGHT R、TR2)、TNFRSF16(NGFR p75NTR)、TNFRSF17(BCMA)、TNFRSF18(GITRAITR)、TNFRSF19(TROY TAJ、TRADE)、TNFRSF19L(RELT) TNFRSF1A(TNF RICD120a, p55-60, TNFRSF1B(TNF RII CD120b, p75-80), TNFRSF26(TNFRH3), TNFRSF3(LTbR TNF RIII, TNFC R), TNFRSF4(OX40 ACT35, TXGP1). R), TNFRSF5(CD40 p50), TNFRSF6(Fas Apo-1, APT1, CD95), TNFRSF6B(DcR3 M68, TR6), TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-1BB). CD137, ILA, TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRST23 (DcTRAIL R1 TNFRH1), TNFRSF25 (DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL). Apo-2 polymer TL2) TNFSF11(TRANCE / RANK dimer ODF) TNFSF12(TWEAK). Apo-3リンド, DR3リンド, TNFSF13(APRIL TALL2), TNFSF13B(BAFF LIGHT, TALL1, THANK, TNFSF20), TNFSF14(LIGHT HVEM LTg, TNFSF15 (TL1A / VEGI), TNFSF18 (GITR). AITR TL6, TNFSF1A (TNF-a Connectin) and DIF.TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand, Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand, CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TLR (toll-like receptor) 1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigen expressed Lewis-Y-related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, Urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, viral antigen, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT 5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, HMGB1, IgA, CD81, CD97, CD98, DDR1, DKK1, EREG, Hsp90, IL-17 / IL-17R, IL-20 / IL- 20R, oxidized LDL, PCSK9, prekallikrein, RON, TMEM16F, SOD1, chromogranin A, chromogranin B, tau, VAP1, high molecular weight kininogen, IL-31, IL-31R, Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, Nav1.9, EPCR, C1, C1q, C1r, C1s, C2, C2a, C2b,C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, C9, factor B, factor D, factor H, properdin, sclerostin, fibrinogen, fibrin, prothrombin, thrombin, tissue factor, factor V, factor Va, factor VII, factor VIIa, factor VIiI, factor VIIIa, factor IX, factor IXa, factor X, factor Xa factor XI, factor XIa, factor XII, factor XIIa, factor XIII, factor XIIIa, TFPI, antithrombin III, EPCR, thrombomodulin, TAPI, tPA, plasminogen, plasmin, PAI-1, PAI-2, GPC3, syndecan-1, syndecan-2, syndecan-3, syndecan-4, LPA, S1P, and receptors for hormones or growth factors.

[0061] The above-listed examples of molecules also include receptors (membrane proteins), which may exist in a soluble form (e.g., truncated form) in body fluids and may be used as a "second antigen" (target molecule) to which the "second portion" of the present invention binds. One non-limiting example of a soluble form of such a receptor (membrane protein) may include the protein represented by soluble IL-6R, as described by Mullberg et al. (J. Immunol. (1994) 152 (10), 4958-4968).

[0062] In the present invention, when the "second antigen" is a soluble protein, for example, IL-6, the molecule comprising the first and second parts of the present invention binds to a dead cell-derived molecule (i.e., the first antigen), for example, an F-actin complex, via the first part of the molecule, and binds to IL-6 (i.e., the second antigen) via the second part of the molecule, more specifically neutralizing IL-6 in diseased tissues, where dead cell-derived molecules are more prevalent due to cell death than in non-diseased tissues. When IL-6 is neutralized, the immune response triggered by immune cells is indirectly controlled.

[0063] In the present invention, the "second portion" that binds to the "second antigen" as described above may have any structure as long as it binds to the "second antigen" as described above. The structure of the "second portion" may include, but is not limited to, a polypeptide or a portion thereof, or a small or medium-sized chemical compound or a portion thereof, or a polynucleotide or a portion thereof. The polypeptide or a portion thereof includes, but is not limited to, an antibody (including, but not limited to, a human antibody, a chimeric antibody, a humanized antibody, and a VHH antibody) or an antigen-binding domain (also referred to as a portion, part, or fragment of an antibody). The antigen-binding domain of an antibody includes, but is not limited to, an antibody heavy chain variable (VH) region, an antibody light chain variable (VL) region (preferably a combination of an antibody heavy chain variable (VH) region and an antibody light chain variable (VL) region), a single domain antibody (sdAb), a single chain Fv (scFv), a single chain antibody, Fv, a single chain Fv2 (scFv2), Fab, and F(ab')2.

[0064] A molecule comprising a first portion that binds to a first antigen and a second portion that binds to a second antigen In the present invention, one embodiment of the "molecule comprising a first portion that binds to a first antigen and a second portion that binds to a second antigen" includes, but is not limited to, a molecule having flow properties.

[0065] i. An antibody in which a "first molecule that binds to a first antigen" is conjugated to the Fc region and at least one of the two Fabs binds to a "second antigen". A schematic example of this embodiment is as shown in FIG. 2. Alternatively, the "first moiety that binds to a first antigen" may be conjugated to one of the Fab arms instead of or in addition to conjugation to the Fc region. Furthermore, more than two "first moieties that bind to a first antigen" may be conjugated to the Fc region. Further various examples of the molecular embodiment of the present invention are illustrated diagrammatically in FIG. 3. The two Fab arms can be conjugated to the Fc region in various formats as illustrated in FIG. 3. An example of the "first moiety that binds to a first antigen" in this embodiment is the whole or part of Clec9A or VHH.

[0066] ii. An antibody in which one Fab arm binds to a "first antigen" and another Fab arm binds to a "second antigen". An example of this embodiment can be a bispecific antibody or bispecific antigen-binding domain in which one Fab arm binds to a dead cell-derived molecule (e.g., F-actin complex) as described above, and the other Fab arm binds to a membrane protein expressed on the cell membrane of a cell (e.g., immune cell, cancer cell, autoreactive cell). The antibody or antibody-like molecule in this example can have various formats, as illustrated in FIG. 3.

[0067] A polypeptide comprising a "first portion that binds a first antigen" and a "second portion that binds a second antigen", wherein the first portion and the second portion are fused together with or without a linker. An example of this embodiment is a fusion protein comprising the extracellular domain of Clec9A and a ligand for any membrane protein expressed on the cell membrane of a cell (e.g., an immune cell, a cancer cell, an autoreactive cell).

[0068] iv. A chemical compound comprising a "first part that binds to a first antigen" and a "second part that binds to a second antigen". An example of this embodiment may be a chemical compound comprising at least two parts, one part that binds to a dead cell-derived molecule as described above (e.g., F-actin complex, nucleosome, which is a complex of histone and nucleic acid), and the other part arm that binds to a membrane protein expressed on the cell membrane of a cell (e.g., immune cell, cancer cell, autoreactive cell). Such a chemical compound may be implemented by using bioconjugate techniques (Bioconjugate Techniques, 3rd Edition, 2013, by Greg T. Hermanson). For example, if the "first antigen" is a nucleosome, which is a complex of histone and nucleic acid, and the "second antigen" is mTOR (mechanistic Target Of Rapamycin), the chemical compound may be a compound that includes distamycin as a "first moiety" that binds to nucleic acid, and rapamycin as a "second moiety" that binds to mTOR, and further includes a photoreactive group (e.g., aryl azide, diazirine, psoralen). Upon receipt of photo (light) energy by the compound, the first antigen and the second antigen are crosslinked by the compound, and the residence of the second moiety on the cell membrane to the second antigen provides signal transduction or signal blocking in the cell.

[0069] Provides signal transduction or signal blocking In the present invention, "imparting signaling or signal blocking" in a cell by a "molecule comprising a first portion that binds to a first antigen and a second portion that binds to a second antigen" can be achieved by providing modulation in the cell, such as agonist activity, antagonist activity, allosteric modulation, allosteric modulation, conformational change, internalization, stabilization, or whatever the target molecule (i.e., the second antigen) is affected by the molecule of the present invention.

[0070] Affected tissue In the present invention, the term "disease tissue" refers to tissue that is different from that of normal tissue and has any condition or characteristic that is specific to a disease. Examples of disease tissue include, but are not limited to, tumor tissue, inflammatory tissue, tissue related to autoimmune disease, etc. In such disease tissue, the above-mentioned cell death occurs more frequently than in normal tissue, producing the above-mentioned dead cell-derived molecules.

[0071] Tumor tissue In the present invention, the term "tumor tissue" refers to tissue that contains at least one tumor cell. Usually, tumor tissue is made up of a group of tumor cells (parenchyma) that constitutes the tumor body, and the connective tissue and blood vessels (stroma) that exist between tumor cells and support the tumor. In some cases, these are clearly distinguishable, but they may also be mixed up. In some cases, cells such as immune cells that infiltrate into tumor tissue exist. In contrast, "non-tumor tissue" refers to tissue in a living body other than tumor tissue. Healthy tissue / normal tissue that does not have disease is representative of such non-tumor tissue.

[0072] inflammatory tissue In the present invention, "inflammatory tissue" includes, but is not limited to, the following: i. Joint tissue associated with rheumatoid arthritis or osteoarthritis. ii. Lung tissue associated with bronchial asthma. iii. Gastrointestinal tissue associated with inflammatory bowel disease, Crohn's disease, or ulcerative colitis. iv. Fibrous tissue associated with fibrosis in the liver, kidney, or lung. v. Tissues involved in immune rejection following organ transplantation. vi. Vascular or cardiac tissue associated with arteriosclerosis or heart failure. vii. Visceral adipose tissue associated with metabolic syndrome. viii. Skin tissue associated with atopic dermatitis or any other dermatitis. ix. Spinal nerve tissue associated with herniated discs or chronic back pain. x. Bone tissue associated with a fracture. xi. Burn-related tissues. xii. Tissue of organ tissues damaged by external mechanical, physical or chemical forces.

[0073] Antigens formed by the multimerization of multiple molecules In the present invention, one embodiment of the "first antigen" as described includes, but is not limited to, an "antigen formed by the multimerization of a plurality of molecules." In the present invention, the "antigen formed by the multimerization of a plurality of molecules" includes, but is not limited to, an antigen formed by the multimerization of a plurality of proteins or parts thereof and / or lipids that constitute a cytoskeleton, a biological membrane such as a cell membrane, a nucleosome, a chaperone molecule, etc.

[0074] Proteins or portions thereof include, but are not limited to, proteins or portions thereof constituting the cytoskeleton, which are composed of actin filaments, intermediate filaments, myosin filaments, keratin filaments, microtubules, and the like. Biological membranes, such as cell membranes, are composed of different proteins, different lipids, and / or various complexes formed by one or more proteins and one or more lipids. Such proteins include, but are not limited to, transmembrane proteins, such as ion channels, G protein-coupled receptors (GPCRs), proton pumps, lipid-anchored proteins, such as G proteins, and peripheral membrane proteins, such as enzymes, hormones, and the like. Lipids include, but are not limited to, phospholipids, glycolipids, sterols, and the like. As described above, in diseased tissues, such as tissues associated with cancer, inflammation, autoimmune diseases, and the like, such complexes of proteins and / or lipids in biological membranes, such as cell membranes, or portions thereof, are exposed to the extracellular environment due to cell death. Such complexes or portions thereof are also included as "first antigens" (also called dead cell-derived molecules) as described above.

[0075] Nucleosomes are complexes of histones and nucleic acids, and chaperone molecules, mainly consisting of heat shock proteins (HSPs), are also included as "first antigens" (also called dead cell-derived molecules).

[0076] Cells expressing membrane proteins as "second antigens" In the present invention, as described above, the "second antigen" includes, but is not limited to, membrane proteins expressed on cell membranes and / or endosomal membranes that are involved in signal transduction in cells. In the present invention, such cells in which membrane proteins as "second antigens" are expressed may be any kind or type of cells, including, but not limited to, immune cells (T cells, killer cells, helper T cells, regulatory T cells, B cells, memory B cells, NK cells, NKT cells, dendritic cells, macrophages, eosinophils, neutrophils, and basophils, etc.), somatic cells (e.g., epithelial cells, fibroblasts, stromal cells, bone cells, muscle cells, nerve cells, blood cells, etc.), germ cells, or diseased or abnormal cells (e.g., tumor cells, autoreactive cells). Such cells may be located in any tissue, including, but not limited to, epithelial tissue, muscle tissue, nerve tissue, connective tissue, or diseased tissue associated with cancer, inflammation, and autoimmune diseases, etc. A preferred embodiment of the cell expressing the "second antigen" (also referred to as the target molecule) may be an immune cell such as a T cell, a killer cell, a helper T cell, a regulatory T cell, a B cell, a memory B cell, a NK cell, a NKT cell, a dendritic cell, a macrophage, an eosinophil, a neutrophil cell, and a basophil. Another preferred embodiment of the cell expressing the "second antigen" (also referred to as the target molecule) may be a cell in a diseased tissue associated with cancer, inflammation, autoimmune disease, and the like.

[0077] In the present invention, the binding of the above-mentioned "molecule comprising a first portion that binds to a first antigen and a second portion that binds to a second antigen" of the present invention to the above-mentioned "substance (molecule) exposed to the extracellular environment due to cell death" ("dead cell-derived molecule") can be examined by preparing cells treated with a cytotoxic drug such as mitoxantrone or mitomycin or untreated cells (treatment with the drug induces cell death), and measuring and comparing the degree of binding of the molecule to each of the drug-treated and untreated cells using flow cytometry. An increase in the degree of binding of the above-mentioned molecule of the present invention to the drug-treated cells indicates that the molecule of the present invention specifically binds to the above-mentioned dead cell-derived molecule.

[0078] The present invention provides molecules that bind to a component or a portion of a cell exposed to the extracellular environment due to cell death as described above ("first antigen", also called dead cell-derived molecule), and at the same time bind to a target molecule ("second antigen"; e.g., a membrane protein as described above) on or in a cell (e.g., immune cells, diseased cells, such as tumor cells, autoreactive cells, and virus-infected cells). The molecules enable cross-linking between a component or a portion of a cell exposed to the extracellular environment due to cell death and a target molecule on or in a cell, such as an immune cell or diseased cell (e.g., cancer cells, autoreactive cells, and virus-infected cells), and provide a signal transduction or blockage in the cell in the tissue.

[0079] In the present invention, the above-mentioned signal transmission or blocking in cells by the molecules of the present invention can be examined, for example, as follows. i. Providing cells that are untreated or treated with a cytotoxic drug, where treatment with the drug induces cell death. ii. For example, provide a molecule (e.g., an antibody) that binds to CD3 or a costimulatory or co-inhibitory molecule ("second antigen") expressed on an immune cell [control molecule], as well as a molecule having a portion that binds to the same ("second antigen") and a portion that binds to, for example, F-actin ("first antigen"; an embodiment of a dead cell-derived molecule) [molecule of the invention]. iii. Proving reporter cells that have been engineered to provide a detectable signal upon signal transduction via binding of the molecule in (ii). iv. Reporter cells are cultured with each of the molecules of (ii) in the presence of drug-treated cells or supernatants of drug-treated cell cultures or untreated cells or supernatants of untreated cells of (i). v. Measuring the reporter signal in each of the cell cultures of (iv) and comparing the reporter signals so measured for each of the cell cultures. vi. A higher reporter signal detected in a cell culture of the molecule of the present invention (ii) in the presence of drug-treated cells or supernatant of the drug-treated cell culture than in a cell culture of the molecule of the present invention (ii) in untreated cells or supernatant of the untreated cell culture indicates that the molecule of the present invention (ii) has conferred signaling in the cells by binding to an antigen expressed in immune cells, e.g., CD3 or a costimulatory or co-inhibitory molecule ("second antigen"). vii. A higher reporter signal in the cell culture of the molecule of the present invention (ii) in the presence of drug-treated cells or the supernatant of the drug-treated cell culture than that in the control cell culture of (ii) indicates that the molecule of the present invention (ii) conferred stronger signaling in the cells than the control molecule that is unable to bind to a dead cell-derived molecule, e.g., F-actin (the "first antigen").

[0080] In the present invention, any kind and / or type of reporter and any kind and / or type of reporter cell may be used depending on the "second antigen", as long as it is possible to detect signal transduction into cells induced by binding of the molecule of the present invention (above (ii)) to a "second antigen", for example, CD3 or a costimulatory molecule or a costimulatory molecule expressed in immune cells.

[0081] In the present invention, a "stronger" signal in (vii) above means a difference in maximum activity of at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 50-fold, 100-fold, 200-fold, or 1,000-fold. A "stronger" signal in (vii) above can also be a difference in EC50 or IC50 value of at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 50-fold, 100-fold, 200-fold, or 1,000-fold.

[0082] When the terms described or referenced below are used in the present invention, they have meanings that include, but are not limited to, the meanings as described or referenced below.

[0083] Moreover, the techniques and procedures described or referenced herein are generally well understood and may be found in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (FM Ausubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (RI Freshney, ed. (1987)); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1998) Academic Press; Animal Cell Culture (RI Freshney), ed., 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (DMWeir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (JE Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993), which are commonly used using conventional methodologies by those of skill in the art.

[0084] amino acid As used herein, amino acids are described by the one-letter or three-letter code, or both, e.g., Ala / A, Leu / L, Arg / R, Lys / K, Asn / N, Met / M, Asp / D, Phe / F, Cys / C, Pro / P, Gln / Q, Ser / S, Glu / E, Thr / T, Gly / G, Trp / W, His / H, Tyr / Y, Ile / I, or Val / V.

[0085] Amino acid modification For amino acid modification (also referred to as "amino acid substitution" or "amino acid mutation" in this description) in the amino acid sequence of an antigen-binding molecule, known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR may be used as appropriate. In addition, some known methods may also be used as amino acid modification methods for substitution with non-natural amino acids (Annu Rev. Biophys. Biomol. Struct. (2006) 35, 225-249; and Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, it is preferable to use a cell-free translation system (Clover Direct (Protein Express)) that contains a tRNA in which a non-natural amino acid is bound to the complementary amber suppressor tRNA of the UAG codon (amber codon), which is one of the termination codons.

[0086] In the present specification, the meaning of the term "and / or" when describing the site of amino acid modification includes any suitable combination of "and" and "or". Specifically, for example, "amino acids at positions 33, 55, and / or 96 are substituted" includes the following variations of amino acid modification: (a) amino acids at positions 33, (b) 55, (c) 96, (d) 33 and 55, (e) 33 and 96, (f) 55 and 96, and (g) 33, 55, and 96.

[0087] Furthermore, in the present specification, as an expression indicating an amino acid modification, an expression indicating the one-letter code or three-letter code of the amino acid before and after the modification, respectively, before and after the number indicating a specific position may be appropriately used. For example, the modification N100bL or Asn100bLeu used when replacing an amino acid contained in an antibody variable region indicates the replacement of Asn at position 100b (according to Kabat numbering) with Leu. That is, the number indicates the amino acid position according to Kabat numbering, the one-letter or three-letter amino acid code written before the number indicates the amino acid before the replacement, and the one-letter or three-letter amino acid code written after the number indicates the amino acid after the replacement. Similarly, the modification P238D or Pro238Asp used when replacing an amino acid in the Fc region contained in an antibody constant region indicates the replacement of Pro at position 238 (according to EU numbering) with Asp. That is, the numbers indicate the amino acid positions according to EU numbering, the one-letter or three-letter amino acid code written before the number indicates the amino acid before substitution, and the one-letter or three-letter amino acid code written after the number indicates the amino acid after substitution.

[0088] Polypeptides As used herein, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain of two or more amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other term used to refer to a chain of two or more amino acids are included within the definition of "polypeptide," and the term "polypeptide" may be used in place of or interchangeably with any of these terms.

[0089] The term "polypeptide" is also intended to refer to the product of post-expression modification of a polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-natural amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a specified nucleic acid sequence. A polypeptide may be generated in any manner, including chemical synthesis. A polypeptide as described herein may be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. A polypeptide may have a defined three-dimensional structure, but does not necessarily have such a structure. A polypeptide that has a defined three-dimensional structure is referred to as folded, and a polypeptide that does not have a defined three-dimensional structure, but rather can adopt a number of different conformations, is referred to as unfolded.

[0090] Percent (%) Amino Acid Sequence Identity "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps if necessary to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes herein, % amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is the copyright of Genentech, Inc., and its source code has been filed with user documentation in the US Copyright Office, Washington DC, 20559, where it is registered under US Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.In the context of using ALIGN-2 for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (alternatively, one may say a given amino acid sequence A has or contains a % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in said program's alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B is not equal to the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0091] Recombinant Methods and Compositions Antibodies, antigen-binding molecules, antigen-binding domains, molecules, including those belonging to the present invention, can be produced using recombinant methods and compositions, for example, as described in U.S. Pat. No. 4,816,567. When the molecule of the present invention is an antibody, in one embodiment, an isolated nucleic acid is provided that encodes an antibody as described herein. Such a nucleic acid may encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of the antibody (e.g., the light chain and / or the heavy chain of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such a nucleic acid are provided. In a further embodiment, a host cell comprising such a nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., has been transformed with) (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, such as a Chinese Hamster Ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp2 / 0 cell). In one embodiment, a method of making a molecule of the invention is provided, the method comprising culturing a host cell comprising a nucleic acid encoding such a molecule (e.g., an antibody) under conditions suitable for expression of such a molecule (e.g., an antibody), and, optionally, recovering such a molecule (e.g., an antibody) from the host cell (or host cell culture medium).

[0092] For recombinant production of such molecules (e.g., antibodies), nucleic acids encoding such molecules (e.g., antibodies) are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., in the case of an antibody, by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0093] Suitable host cells for cloning or expressing the vector encoding the molecule of the present invention include prokaryotic or eukaryotic cells as described herein. For example, the molecule of the present invention may be produced in bacteria, especially when glycosylation and Fc effector function are not required. For the expression of the molecule of the present invention in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523 (also see Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp.245-254, which describes the expression of antibody fragments in E. coli). After expression, the molecule of the present invention may be isolated in a soluble fraction from bacterial cell paste and can be further purified.

[0094] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast, including fungal and yeast strains in which the glycosylation pathway has been "humanized," resulting in the production of the molecules of the invention with partial or fully human glycosylation patterns, are suitable cloning or expression hosts for vectors encoding the molecules of the invention. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0095] Suitable host cells for the expression of glycosylated molecules of the present invention are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified that can be used with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0096] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing the PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0097] Vertebrate cells may also be used as hosts, for example mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (e.g., 293 or 293 cells described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary carcinoma (MMT 060562); TRI cells (e.g., Mather et al., Annals NY Acad. Sci. 383:44-68 (1982); MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR- CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0098] Recombinant production of the molecules of the invention can be carried out in a manner similar to that described above, by using host cells that contain (e.g., have been transformed with) one or more vectors that contain nucleic acid encoding an amino acid sequence comprising the entire molecule or a portion thereof.

[0099] In the present invention, as described above, one embodiment of the molecule of the present invention, i.e., the molecule comprising a first portion that binds to a first antigen and a second portion that binds to a second antigen, as described above, is an antibody or its antigen-binding domain / molecule, which may comprise an additional portion conjugated to the Fc region, for example. In the present invention, such a molecule may be called an antigen-binding molecule. When the molecule is an antigen-binding molecule having multiple specificities, for example, for two or more different antigens, it may be called a multispecific antigen-binding molecule.

[0100] Antigen-binding molecules and multispecific antigen-binding molecules The term "antigen-binding molecule" as used herein refers to any molecule that contains an antigen-binding site or has binding activity to an antigen, and may further refer to molecules such as peptides or proteins having a length of about 5 amino acids or more. Peptides and proteins are not limited to those derived from living organisms, and may be, for example, polypeptides produced from artificially designed sequences. They may also be natural polypeptides, synthetic polypeptides, recombinant polypeptides, etc. Scaffold molecules that contain a known stable conformational structure such as an α / β barrel as a scaffold, and a part of the molecule is an antigen-binding site, are also an embodiment of the antigen-binding molecules described herein.

[0101] "Multispecific antigen-binding molecule" refers to an antigen-binding molecule that specifically binds to more than one antigen. The term "bispecific" means that an antigen-binding molecule can specifically bind to at least two distinct antigenic determinants. The term "trispecific" means that an antigen-binding molecule can specifically bind to at least three distinct antigenic determinants.

[0102] Antigen-binding domain As described above, when the molecule of the invention, i.e., the "molecule comprising a first portion that binds a first antigen and a second portion that binds a second antigen", is an antigen-binding molecule such as, for example, an antibody, also referred to as an antigen-binding molecule as described above, in some embodiments the "second portion that binds a second antigen" and, where appropriate, further the "first portion that binds the first antigen" may be an antigen-binding domain of, or derived from, an antibody.

[0103] The term "antigen-binding domain" refers to a portion of an antibody that comprises an area that specifically binds to and is complementary to a portion or all of an antigen. An antigen-binding domain may be provided, for example, by one or more antibody variable domains (also called antibody variable regions). Preferably, an antigen-binding domain contains both an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). Such preferred antigen-binding domains include, for example, "single-chain Fv (scFv)", "single-chain antibody", "Fv", "single-chain Fv2 (scFv2)", "Fab", and "F(ab')2".

[0104] Ig type binding part The terms "Ig-type binding moiety", "Ig-type binding moiety" and the like as used herein are well understood by those skilled in the art and generally refer to an Ig-type (immunoglobulin-type) antigen-binding moiety that is familiar to those skilled in the art. In certain embodiments herein, it includes all "antigen-binding domains" described herein (provided that they include at least one immunoglobulin structure), and in particular includes all examples of the latter term described herein. Preferably, as used herein, said Ig-type binding moiety is selected from the group consisting of single domain antibodies (VHH), antibody heavy chain variable (VH) regions, antibody light chain variable (VL) regions, single domain antibodies (sdAb), single chain Fvs (scFvs), single chain antibodies, Fvs, single chain Fv2s (scFv2s), Fabs, and F(ab')2. More preferably, said Ig-type binding moiety is selected from the group consisting of VHHs, scFvs, scFv2s, Fabs, and F(ab')2. It particularly includes any of the group consisting of "single-chain Fv (scFv)", "single-chain antibody", "Fv", "single-chain Fv2 (scFv2)", "Fab", and "F(ab')2", and in particular specific embodiments of any of those described elsewhere herein.

[0105] Variable region The term "variable region" or "variable domain" refers to the domain of the heavy or light chain of an antibody that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of a natural antibody (VH and VL, respectively) usually have a similar structure, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, for example, Kindt et al. Kuby Immunology, 6 thed., WH Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Moreover, antibodies that bind to a specific antigen may be isolated by screening a library of complementary VL or VH domains, respectively, with a VH or VL domain from an antibody that binds that antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0106] HVR or CDR The term "hypervariable region" or "HVR" as used herein refers to each region of an antibody variable domain that is hypervariable in sequence ("complementarity determining region" or "CDR") and / or forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contacts"). Hypervariable regions (HVRs) are also called "complementarity determining regions" (CDRs), and these terms are used interchangeably herein for the portions of the variable regions that form the antigen binding region. Typically, antibodies contain six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3).

[0107] Exemplary HVRs herein include the following: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigenic contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and (d) a combination of (a), (b), and / or (c), comprising HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3). Unless otherwise indicated, HVR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al., supra. HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 are also referred to as "H-CDR1," "H-CDR2," "H-CDR3," "L-CDR1," "L-CDR2," and "L-CDR3," respectively.

[0108] Capable of binding to antigens Whether an antibody variable region is "capable of binding to an antigen" can be determined by methods known in the art, for example, by electrochemiluminescence (ECL) (BMC Research Notes 2011, 4: 281). Specifically, for example, a region capable of binding to the antigen of the biotin-labeled antigen-binding molecule to be tested, such as a low molecular weight antibody composed of a Fab region, or a monovalent antibody thereof (an antibody lacking one of the two Fab regions possessed by a normal antibody) is mixed with an antigen labeled with a sulfo-tag (Ru complex), and the mixture is added onto a streptavidin-immobilized plate. In this operation, the biotin-labeled antigen-binding molecule to be tested binds to the streptavidin on the plate. Light is generated from the sulfo-tag, and the luminescence signal is detected using a Sector Imager 600 or 2400 (MSD KK), etc., thereby allowing the binding of the antigen-binding molecule to the antigen of the above-mentioned region to be tested to be confirmed. Alternatively, the assay may be performed by ELISA, FACS (fluorescence activated cell sorting), ALPHAScreen (amplified luminescent proximity homogeneous assay screen), BIACORE method based on the surface plasmon resonance (SPR) phenomenon, etc. (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

[0109] Specifically, the assay can be carried out using, for example, an interaction analyzer Biacore (GE Healthcare Japan Corp.) based on the surface plasmon resonance (SPR) phenomenon. Biacore analyzers include any model, such as Biacore T100, T200, X100, A100, 4000, 3000, 2000, 1000, or C. Any sensor chip for Biacore, such as CM7, CM5, CM4, CM3, C1, SA, NTA, L1, HPA, or Au chip, can be used as the sensor chip. Proteins for capturing the antigen-binding molecules of the present invention, such as protein A, protein G, protein L, anti-human IgG antibody, anti-human IgG-Fab, anti-human L chain antibody, anti-human Fc antibody, antigen protein, or antigen peptide, are immobilized on the sensor chip by a coupling method such as amine coupling, disulfide coupling, or aldehyde coupling. An antigen is injected thereon as an analyte, and the interaction is measured to obtain a sensorgram. In this procedure, the concentration of the antigen can be selected within the range of several μM to several pM according to the strength of the interaction (eg, KD) of the assay sample.

[0110] Alternatively, an antigen may be immobilized on a sensor chip instead of the antigen-binding molecule, and the antibody sample to be evaluated may then be allowed to interact with it. Whether or not the antibody variable region of the antigen-binding molecule of the present invention has binding activity to an antigen can be confirmed based on the dissociation constant (KD) value calculated from the sensorgram of the interaction, or based on the degree of increase in the sensorgram after the action of the antigen-binding molecule sample above the level before the action.

[0111] In some embodiments, the binding activity or affinity of the antibody variable region of the present invention to an antigen of interest (i.e., antigen) is evaluated, for example, at 37°C or 25°C (for antigen) using a Biacore T200 instrument (GE Healthcare) or a Biacore 8K instrument (GE Healthcare). Anti-human Fc (e.g., GE Healthcare) is immobilized on all flow cells of a CM4 sensor chip using an amine coupling kit (e.g., GE Healthcare). The antigen-binding molecule or antibody variable region is captured on the anti-Fc sensor surface, and then the antigen is injected over the flow cell. The capture level of the antigen-binding molecule or antibody variable region can be targeted at 200 resonance units (RU). Recombinant human antigen may be injected at 400 to 25 nM prepared by two-fold serial dilution, followed by dissociation. All antigen-binding molecules or antibody variable regions and analytes are prepared in ACES pH 7.4, containing 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, 0.005% NaN3. The sensor surface is regenerated with 3M MgCl2 every cycle. The binding affinity is determined by processing data and fitting to a 1:1 binding model, for example, using Biacore T200 Evaluation software, version 2.0 (GE Healthcare) or Biacore 8K Evaluation software (GE Healthcare). The KD value is calculated to evaluate the specific binding activity or affinity of the antigen-binding domain.

[0112] ALPHAScreen is implemented by ALPHA technology using two types of beads (donor and acceptor) based on the following principle: luminescence signals are detected only when the two beads are located in close proximity through biological interactions between the molecules bound to the donor beads and the molecules bound to the acceptor beads. The photosensitizer in the donor beads excited by a laser converts the surrounding oxygen into singlet oxygen with an excited state. The singlet oxygen diffuses around the donor beads and reaches the nearby acceptor beads, thereby triggering a chemiluminescence reaction in the beads, which ultimately emits light. In the absence of interactions between the molecules bound to the donor beads and the molecules bound to the acceptor beads, the singlet oxygen produced by the donor beads does not reach the acceptor beads. Therefore, no chemiluminescence reaction occurs.

[0113] One of the substances (ligands) whose interaction is to be observed is immobilized on the gold film of the sensor chip. Light is applied from the back of the sensor chip so that total reflection occurs at the interface between the gold film and the glass. As a result, a region of reduced reflection intensity (SPR signal) is formed in a portion of the reflected light. The other substance (analyte) whose interaction is to be observed is injected onto the surface of the sensor chip. When the analyte binds to the ligand, the mass of the immobilized ligand molecule increases, changing the refractive index of the solvent on the sensor chip surface. This change in refractive index shifts the position of the SPR signal (conversely, dissociation of the bound molecule returns the signal to its original position). The Biacore system plots the amount of shift, i.e., the change in mass on the sensor chip surface, on the vertical axis, and displays the change in mass over time as assay data (sensorgram). The amount of analyte bound to the ligand captured on the sensor chip surface (the amount of change in response on the sensorgram between before and after the interaction of the analyte) can be determined from the sensorgram. However, the amount of binding also depends on the amount of ligand, so comparison must be performed under conditions where substantially the same amount of ligand is used. Kinetics, i.e., the association rate constant (ka) and the dissociation rate constant (kd) can be determined from the curve of the sensorgram, and affinity (KD) can be determined from the ratio between these constants. Inhibition assays are also preferably used in the BIACORE method. Examples of inhibition assays are described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010.

[0114] For example, a biotin-labeled antigen-binding molecule to be tested is bound to streptavidin on donor beads, while an antigen tagged with glutathione S-transferase (GST) is bound to acceptor beads. The antigen-binding molecule to be tested interacts with the antigen in the absence of a competing second antigen to generate a signal at 520 to 620 nm. The untagged second antigen competes with the antigen for interaction with the antigen-binding molecule to be tested. The decrease in fluorescence caused as a result of the competition is quantified, thereby determining the relative binding activity. Polypeptide biotinylation using sulfo-NHS-biotin or the like is known in the art. For example, an antigen can be tagged with GST by an appropriately adopted method, including a step of fusing a polynucleotide encoding an antigen with a polynucleotide encoding GST in frame; and expressing the resulting fusion gene by a cell carrying a vector that enables its expression, followed by purification using a glutathione column. The resulting signals are preferably analyzed by fitting to a one-site competition model based on non-linear regression analysis, for example using the software GRAPHPAD PRISM (GraphPad Software, Inc., San Diego).

[0115] Tagging is not limited to GST tagging, and may be performed with any tag, such as but not limited to histidine tag, MBP, CBP, Flag tag, HA tag, V5 tag, or c-myc tag. The binding of the antigen-binding molecule to be tested to the donor bead is not limited to binding using biotin-streptavidin reaction. In particular, when the antigen-binding molecule to be tested contains Fc, a possible method includes binding the antigen-binding molecule to be tested on the donor bead via Fc recognition protein such as protein A or protein G.

[0116] Fab molecule / Fab region / Fab arm In the present invention, the terms "Fab molecule", "Fab region", or "Fab arm", which may be used interchangeably, refer to a protein consisting of the VH and CH1 domains of an immunoglobulin heavy chain ("Fab heavy chain"), and the VL and CL domains of the light chain ("Fab light chain").

[0117] Fab, F(ab') 2 , and Fab' A "Fab" consists of a single light chain and the CH1 domain and variable region from a single heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0118] "F(ab')2" or "Fab" refers to an antibody fragment produced by treating an immunoglobulin (monoclonal antibody) with a protease such as pepsin and papain, and digesting the immunoglobulin (monoclonal antibody) near the disulfide bond present between the hinge regions in each of the two H chains. For example, papain cleaves IgG upstream of the disulfide bond present between the hinge regions in each of the two H chains to produce two homologous antibody fragments, in which an L chain containing a VL (L chain variable region) and a CL (L chain constant region) is linked to an H chain fragment containing a VH (H chain variable region) and a CHγ1 (γ1 region in the H chain constant region) via a disulfide bond at their C-terminal regions. Each of these two homologous antibody fragments is called Fab'.

[0119] "F(ab')2" consists of two light chains and two heavy chains containing a constant region of a part of the CH1 domain and the CH2 domain, so that a disulfide bond is formed between the two heavy chains. The F(ab')2 disclosed herein can be preferably produced as follows: A whole monoclonal antibody containing a desired antigen-binding site is partially digested with a protease such as pepsin; and the Fc fragment is removed by adsorption onto a protein A column. The protease is not particularly limited as long as it can selectively cleave the whole antibody to produce F(ab')2 under appropriately set enzyme reaction conditions such as pH. Such proteases include, for example, pepsin and ficin.

[0120] Fused / Fused The terms "fused" and "fusion" mean that two or more different polypeptides (e.g., a first polypeptide that binds a first antigen and a second polypeptide that binds a second antigen; a Fab arm and an Fc domain) are linked by a peptide bond, either directly or via one or more peptide linkers.

[0121] Thus, as used herein, the term "fusion protein" (which may be used interchangeably with the term "fusion polypeptide" herein) preferably means that two or more different polypeptides are covalently linked, either directly or via one or more peptide linkers. A specific example of a fusion protein herein is an antigen-binding molecule of the present invention in which an antibody (or at least an Ig-type binding portion) is linked to a binding polypeptide that is a non-Ig-type binding portion.

[0122] Crossover Fab A "crossover" Fab molecule (also referred to as "Crossfab") refers to a Fab molecule in which either the variable or constant regions of the heavy and light chains of the Fab are exchanged, i.e., the crossover Fab molecule comprises a peptide chain composed of a light chain variable region and a heavy chain constant region, and a peptide chain composed of a heavy chain variable region and a light chain constant region. For clarity, in a crossover Fab molecule in which the variable regions of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain constant region is referred to herein as the "heavy chain" of the crossover Fab molecule. Conversely, in a crossover Fab molecule in which the constant regions of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain variable region is referred to herein as the "heavy chain" of the crossover Fab molecule.

[0123] Conventional Fab In contrast, a "conventional" Fab molecule refers to a Fab molecule in its natural form, i.e., containing a heavy chain composed of a heavy chain variable and constant region (VH-CH1), and a light chain composed of a light chain variable and constant region (VL-CL). The term "immunoglobulin molecule" refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of about 150,000 daltons composed of two light chains and two heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called the variable heavy domain or the heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also called the heavy chain constant region. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called the variable light domain or the light chain variable domain, followed by a constant light chain (CL) domain, also called the light chain constant region. The heavy chains of an immunoglobulin can be assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further divided into subtypes, e.g., γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chains of an immunoglobulin can be assigned to one of two types, called κ and λ, based on the amino acid sequence of their constant domains. Immunoglobulins essentially consist of two Fab molecules and an Fc domain linked via an immunoglobulin hinge region.

[0124] Affinity "Affinity" refers to the strength of the total non-covalent interactions between a single binding site of a molecule (e.g., a first or second moiety; an antibody or an antigen-binding domain) and its binding partner (e.g., a first or second antigen; or an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to the inherent binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., a first moiety and a first antigen; a second moiety and a second antigen; an antigen-binding molecule and an antigen, or an antibody and an antigen). The affinity of a molecule X for its partner Y can usually be expressed by a dissociation constant (KD), which is the ratio of the dissociation rate constant and the association rate constant (koff and k, respectively). Thus, equivalent affinities may include different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by well-established methods known in the art, including those described herein. A particular method for measuring affinity is surface plasmon resonance (SPR).

[0125] How to determine affinity In certain embodiments, each of the first or second portions of the "molecule comprising a first portion that binds to a first antigen and a second portion that binds to a second antigen" of the present invention, and the antigen-binding molecule or antibody as described above, is at a concentration of 1 μM or less, 120 nM or less, 100 nM or less, 80 nM or less, 70 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 2 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, 10 -8 M~10 -13 M, 10 -9 M~10 -13The molecule of the present invention has a dissociation constant (KD) for its antigen (i.e., first antigen, second antigen) of 1-40, 1-50, 1-70, 1-80, 30-50, 30-70, 30-80, 40-70, 40-80, or 60-80 nM. In certain embodiments, the KD values ​​of the molecules of the present invention for the first antigen and second antigen fall within the range of 1-40, 1-50, 1-70, 1-80, 30-50, 30-70, 30-80, 40-70, 40-80, or 60-80 nM, respectively.

[0126] In one embodiment, KD is measured by a radiolabeled antigen binding assay (RIA). For example, the binding affinity of the molecules of the invention to an antigen (i.e., a first antigen, a second antigen) in solution is determined by measuring the binding affinity of the molecule to an antigen (i.e., a first antigen, a second antigen) at a minimum concentration of ( 125 I) Equilibration of the molecule with labeled antigen and then capture of the bound antigen with a plate coated with an antibody that binds the molecule (see, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999)). To establish the conditions for the assay, MICROTITER® multi-well plates (Thermo Scientific) are coated overnight with 5 μg / ml of an antibody that binds the capture molecule (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and then blocked with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23° C.). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM of [ 125I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., as in the evaluation of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The molecule of interest is then incubated overnight, although this incubation may be continued for longer (e.g., about 65 hours) to ensure that equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed and the plate is washed 8 times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate has dried, 150 μL / well of scintillant (MICROSCINT-20®; Packard) is added and the plate is counted for 10 minutes in a TOPCOUNT® gamma counter (Packard). The concentration of each Fab that gives 20% or less of maximum binding is selected for use in competitive binding assays.

[0127] According to another embodiment, Kd is measured using a BIACORE® surface plasmon resonance assay. For example, an assay using a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) is performed at 25°C using a CM5 chip with antigen immobilized at about 10 response units (RU). In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted to 5 μg / ml (about 0.2 μM) in 10 mM sodium acetate, pH 4.8, prior to injection at a flow rate of 5 μL / min to achieve approximately 10 response units (RU) of protein binding. After injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS containing 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate of approximately 25 μL / min. The binding rate (k on ) and dissociation rate (k off ) is calculated by simultaneously fitting the association and dissociation sensorgrams with a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2). The equilibrium dissociation constant (Kd) is k off / k on The on-rate is calculated as a ratio of 10 to 10 by the surface plasmon resonance assay described above. 6 M -1 s -1If the on-rate exceeds 100%, it can be determined by using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, band pass 16 nm) at 25°C of antibody binding to 20 nM of the molecule in PBS, pH 7.2 in the presence of increasing concentrations of antigen, as measured in a spectrometer, for example a stopped-flow spectrophotometer (Aviv Instruments) or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) using a stirred cuvette.

[0128] Following the methods for measuring the affinity of the molecules of the invention to antigens described above, one skilled in the art can carry out affinity measurements for any other kind or type of molecules of the invention to various kinds of antigens.

[0129] antibody As described above, when the molecule of the invention, i.e., the "molecule comprising a first portion that binds a first antigen and a second portion that binds a second antigen", is an antigen-binding molecule such as, for example, an antibody, also referred to as an antigen-binding molecule as described above, in some embodiments the "second portion that binds a second antigen" and, where appropriate, further the "first portion that binds the first antigen" may be an antigen-binding domain of, or derived from, an antibody.

[0130] The term "antibody" as used herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0131] The terms "full length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain that includes an Fc region as defined herein.

[0132] antibody fragmentAn "antibody fragment" (also referred to as a portion of an antibody) refers to a molecule other than a complete antibody that contains a portion of the complete antibody that binds to the antigen to which the complete antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and single domain antibodies. For a review of certain antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments that contain salvage receptor binding epitope residues and have increased in vivo half-lives, see U.S. Pat. No. 5,869,046. Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Single-domain antibodies are antibody fragments that contain all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, single-domain antibodies are human single-domain antibodies (Domantis, Inc., Waltham, MA; see, for example, U.S. Patent No. 6,248,516 B1).Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of whole antibodies and production by recombinant host cells (e.g., E. coli or phages), as described herein.

[0133] In certain embodiments herein, the antibody fragment is an Ig-type binding moiety.

[0134] Fragment variable (Fv) As used herein, the term "variable fragment (Fv)" refers to the minimum unit of an antigen-binding site derived from an antibody, which is composed of a pair of an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). In 1988, Skerra and Pluckthun found that a homogeneous and active antibody can be prepared from the periplasmic fraction of E. coli by inserting an antibody gene downstream of a bacterial signal sequence and inducing expression of the gene in E. coli (Science (1988) 240(4855), 1038-1041). In the Fv prepared from the periplasmic fraction, VH associates with VL in such a manner that it binds to an antigen.

[0135] scFv, single chain antibodies, and sc(Fv) 2 As used herein, the terms "scFv", "single-chain antibody" and "sc(Fv)2" all refer to a single polypeptide chain antibody fragment that contains variable regions derived from heavy and light chains but does not contain constant regions. Single-chain antibodies usually also contain a polypeptide linker between the VH and VL domains, which allows the formation of a desired structure that would allow antigen binding. Single-chain antibodies are discussed in detail by Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, 269-315 (1994). See also International Patent Publication WO 1988 / 001649; U.S. Patent Nos. 4,946,778 and 5,260,203. In certain embodiments, single-chain antibodies can be bispecific and / or humanized.

[0136] scFv is a single-chain low molecular weight antibody in which the VH and VL forming the Fv are linked together by a peptide linker (Proc. Natl. Acad. Sci. USA (1988) 85(16), 5879-5883). The VH and VL can be held in close proximity by the peptide linker.

[0137] sc(Fv)2 is a single-chain antibody in which four variable regions, two VL and two VH, are linked by a linker such as a peptide linker to form a single chain (J Immunol. Methods (1999) 231(1-2), 177-189). The two VH and two VL may be derived from different monoclonal antibodies. Such sc(Fv)2 preferably includes bispecific sc(Fv)2 that recognizes two epitopes present in a single antigen, as disclosed in, for example, Journal of Immunology (1994) 152(11), 5368-5374. sc(Fv)2 can be produced by methods known to those skilled in the art. For example, sc(Fv)2 can be produced by linking scFvs by a linker such as a peptide linker.

[0138] As used herein, sc(Fv)2 comprises two VH units and two VL units arranged in the order of VH, VL, VH, and VL starting from the N-terminus of a single-chain polypeptide ([VH]-linker-[VL]-linker-[VH]-linker-[VL]). The order of the two VH units and the two VL units is not limited to the above form and may be arranged in any order. Examples of forms are listed below. [VL]-linker-[VH]-linker-[VH]-linker-[VL] [VH]-linker-[VL]-linker-[VL]-linker-[VH] [VH]-linker-[VH]-linker-[VL]-linker-[VL] [VL]-linker-[VL]-linker-[VH]-linker-[VH] [VL]-linker-[VH]-linker-[VL]-linker-[VH]

[0139] The molecular form of sc(Fv)2 is also described in detail in WO 2006 / 132352. Following these descriptions, those skilled in the art can appropriately prepare the desired sc(Fv)2 to produce the polypeptide complexes disclosed herein.

[0140] Antibody classes The "class" of an antibody refers to the type of constant domain or constant region present in the antibody's heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0141] Unless otherwise indicated, amino acid residues in the light chain constant region are numbered herein according to Kabat et al., and numbering of amino acid residues in the heavy chain constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0142] Framework "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences typically appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0143] Human Consensus Framework A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Usually, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Usually, the subgroup of sequences is a subgroup in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup kappa I according to Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III according to Kabat et al., supra.

[0144] Chimeric antibodies The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species. Similarly, the term "chimeric antibody variable domain" refers to an antibody variable region in which a portion of the heavy and / or light chain variable region is derived from a particular source or species, while the remainder of the heavy and / or light chain variable region is derived from a different source or species.

[0145] Humanized antibodies A "humanized" antibody refers to a chimeric antibody that contains amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody contains substantially all of at least one, and typically two, variable domains, in which all or substantially all HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization. A "humanized antibody variable region" refers to the variable region of a humanized antibody.

[0146] Human antibodies A "human antibody" is an antibody with an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell, or from a non-human source that utilizes a human antibody repertoire or other human antibody coding sequence. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues. "Human antibody variable region" refers to the variable region of a human antibody.

[0147] Polynucleotides (nucleic acids) "Polynucleotide" or "nucleic acid", as used interchangeably herein, refers to a polymer of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substance that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides can include modified nucleotides, such as methylated nucleotides and their analogs. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can include modifications made after synthesis, such as conjugation to a label. Other types of modifications include, for example, "caps", substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications, such as those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) and charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalating agents (e.g., acridine, psoralen, etc.), those containing chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.), those containing alkylating agents, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of polynucleotides. In addition, any hydroxyl groups normally present in the sugar can be replaced by, for example, phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare for further linkage to additional nucleotides, or conjugated to solid or semi-solid supports. The 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of 1 to 20 carbon atoms. The other hydroxyls can also be derivatized to standard protecting groups.Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl-, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose or xylose or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and basic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages can be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which phosphate is replaced by: P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where each R or R' is independently H or substituted or unsubstituted alkyl (1-20 C), optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all linkages in a polynucleotide need be identical. The above description applies to all polynucleotides referred to herein, including RNA and DNA.

[0148] Isolated (nucleic acid) An "isolated" nucleic acid molecule is one which is separated from a component of its natural environment. Isolated nucleic acid molecules further include nucleic acid molecules contained in a cell which ordinarily contains the nucleic acid molecule, but which are present extrachromosomally or at a chromosomal location which is different from its natural chromosomal location.

[0149] vector The term "vector" as used herein refers to a nucleic acid molecule that can propagate another nucleic acid to which it is linked. This term includes vectors as self-replicating nucleic acid structures and vectors that are integrated into the genome of a host cell to which it is introduced. Certain vectors can direct the expression of a nucleic acid to which it is operably linked. Such vectors are referred to herein as "expression vectors." Vectors can be introduced into host cells using viruses or electroporation. However, the introduction of vectors is not limited to in vitro methods. For example, vectors can also be directly introduced into a subject using in vivo methods.

[0150] subject In the present invention, the subject is not particularly limited. According to a preferred embodiment herein, the subject is an animal, preferably a mammal, more preferably a human.

[0151] In a generally preferred embodiment herein, the subject matter (including, but not limited to, any medical use) relates to an animal subject, preferably a mammalian subject, more preferably a human subject.

[0152] Therefore, in a preferred embodiment herein, the first antigen is an antigen derived from an animal, preferably a mammal, more preferably a human.Therefore, in a preferred embodiment herein, the second antigen is an antigen derived from an animal, preferably a mammal, more preferably a human.Therefore, in a preferred embodiment herein, the antibody herein is an antibody derived from an animal, preferably a mammal, more preferably a human.In an alternative embodiment, the antibody herein is a humanized antibody.

[0153] host cell The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the originally transformed cell and progeny derived from that cell regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell, and may contain mutations. Mutant progeny that have the same function or biological activity as the originally transformed cell screened or selected for are included herein.

[0154] specificity "Specific" means that a molecule that specifically binds to one or more binding partners does not show any significant binding to molecules other than the partners. Furthermore, "specific" is also used when an antigen-binding site is specific to a particular epitope among multiple epitopes contained in an antigen. When an antigen-binding molecule specifically binds to an antigen, it is also described as "the antigen-binding molecule has / shows specificity to / for an antigen". When the epitope that an antigen-binding site binds to is contained in multiple different antigens, the antigen-binding molecule that contains the antigen-binding site can bind to various antigens that have that epitope.

[0155] Furthermore, the molecules of the present invention (i.e., "molecules comprising a first portion that binds to a first antigen and a second portion that binds to a second antigen") may be conjugated to a carrier polymer such as PEG or an organic compound such as an anticancer drug. Alternatively, a glycosylation sequence is preferably inserted into the molecule so that the glycosylation produces the desired effect.

[0156] Linker Linkers used to link two or more different components (e.g., polypeptides, peptides) include any peptide linker that can be introduced by genetic engineering, synthetic linkers, and linkers disclosed in, for example, Protein Engineering, 9(3), 299-305, 1996. However, in the present disclosure, peptide linkers are preferred. The length of the peptide linker is not particularly limited and can be suitably selected by those skilled in the art depending on the purpose. The length is preferably 5 amino acids or more (although not particularly limited, the upper limit is usually 30 amino acids or less, preferably 20 amino acids or less), and is particularly preferably 15 amino acids. For example, when sc(Fv)2 contains three peptide linkers, their lengths may all be the same or different.

[0157] For example, such peptide linkers include: Ser, Gly-Ser, Gly-Gly-Ser, Ser-Gly-Gly, Gly-Gly-Gly-Ser (SEQ ID NO: 11), Ser-Gly-Gly-Gly (SEQ ID NO: 12), Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 13), Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 14), Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 15), Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 16), Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 17), Ser-Gly-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 18), (Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 13)), and (Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 14)) where n is an integer equal to or greater than 1. The length or sequence of the peptide linker can be appropriately selected by those skilled in the art depending on the purpose.

[0158] Synthetic linkers (chemical cross-linkers) are routinely used to cross-link peptides, examples include: N-hydroxysuccinimide (NHS), Disuccinimidyl suberate (DSS), Bis(sulfosuccinimidyl) suberate (BS3), Dithiobis(succinimidyl propionate) (DSP), Dithiobis(sulfosuccinimidyl propionate) (DTSSP), Ethylene glycol bis(succinimidyl succinate) (EGS), Ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), Disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), Bis[2-(succinimidoxycarbonyloxy)ethyl]sulfone (BSOCOES), and Bis[2-(sulfosuccinimidoxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES). These crosslinkers are commercially available.

[0159] For example, three linkers are usually required to link together four antibody variable regions. The linkers used may be of the same type or of different types.

[0160] Fc area The term "Fc region" or "Fc domain" refers to a region including a hinge or a part thereof and a fragment consisting of a CH2 domain and a CH3 domain in an antibody molecule. The Fc region of the IgG class means, for example, but is not limited to, a region from cysteine ​​226 (EU numbering (also referred to herein as EU index)) to the C-terminus, or from proline 230 (EU numbering) to the C-terminus. The Fc region can be preferably obtained, for example, by partially digesting an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody with a protease such as pepsin, followed by re-elution of the fraction adsorbed on a protein A column or a protein G column. Such a protease is not particularly limited as long as the enzyme can digest the entire antibody under appropriately set reaction conditions (e.g., pH) of the enzyme to form Fab or F(ab')2 only. Examples thereof may include pepsin and papain.

[0161] For example, an Fc region derived from a naturally occurring IgG can be used as the "Fc region" of the present invention. In this context, naturally occurring IgG refers to a polypeptide that contains the same amino acid sequence as that of an IgG found in nature and belongs to a class of antibodies substantially encoded by an immunoglobulin gamma gene. Naturally occurring human IgG refers to, for example, naturally occurring human IgG1, naturally occurring human IgG2, naturally occurring human IgG3, or naturally occurring human IgG4. Naturally occurring IgG also includes naturally derived variants thereof. Multiple allotype sequences based on genetic polymorphisms are described in Sequences of proteins of immunological interest, NIH Publication No. 91-3242 as constant regions of human IgG1, human IgG2, human IgG3, and human IgG4 antibodies, any of which can be used in the present invention. In particular, the sequence of human IgG1 can have DEL or EEM as the amino acid sequence at positions 356 to 358 of the EU numbering system.

[0162] In some embodiments, the Fc domain of the antibody belonging to the molecule of the present invention is composed of a pair of polypeptide chains that contain the heavy chain domain of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which contains the CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain can stably associate with each other. In one embodiment, the molecule of the present invention may contain no more than one Fc domain.

[0163] In one embodiment, the Fc domain that the molecule may comprise is an IgG Fc domain. In a particular embodiment, the Fc domain is an IgG1 Fc domain. In another embodiment, the Fc domain is an IgG1 Fc domain. In a further particular embodiment, the Fc domain is a human IgG1 Fc region.

[0164] Production and purification of multispecific antibodies As described above, when the molecule of the present invention, i.e., the molecule comprising a "first portion that binds to a first antigen and a second portion that binds to a second antigen", is an antigen-binding molecule, such as an antibody, also referred to as an antigen-binding molecule as described above, in some embodiments, the "second portion that binds to a second antigen" and, where appropriate, further the "first portion that binds to a first antigen" may be an antigen-binding domain of or derived from an antibody. In one embodiment, the molecule, i.e., the antigen-binding molecule, such as an antibody, may be a multispecific antigen-binding molecule, such as a bispecific, trispecific, and tetraspecific antibody or antigen-binding molecule.

[0165] In one example, the multispecific antigen-binding molecules described herein contain two different antigen-binding moieties (e.g., a "first antigen-binding moiety" (i.e., the first moiety) and a "second antigen-binding moiety" (i.e., the second moiety) fused to one or the other of the two subunits of the Fc domain, and thus the two subunits of the Fc domain are typically contained in two non-identical polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization results in several possible combinations of the two polypeptides. To improve the yield and purity of the multispecific antigen-binding molecules in recombinant production, it would be advantageous to introduce modifications to the Fc domain of the multispecific antigen-binding molecule that promote the association of the desired polypeptides.

[0166] Thus, in certain embodiments, the Fc domain of the multispecific antigen-binding molecule described herein comprises a modification that promotes the association of the first and second subunits of the Fc domain.The most extensive protein-protein interaction site between the two subunits of the human IgG Fc domain is in the CH3 domain of the Fc domain.Thus, in one embodiment, said modification is in the CH3 domain of the Fc domain. In a specific embodiment, said modification is a so-called "knob-into-hole" modification, comprising a "knob" modification in one of the two subunits of the Fc domain and a "hole" modification in the other of the two subunits of the Fc domain.

[0167] Knob-into-hole technology is described, for example, in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Typically, the method involves introducing a protrusion ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, such that the protrusion can be positioned into the cavity to promote heterodimer formation and prevent homodimer formation. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A compensatory cavity of the same or similar size as the protrusion is created at the interface of the second polypeptide by replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

[0168] Thus, in a particular embodiment, in the CH3 domain of a first subunit of the Fc domain of a multispecific antigen-binding molecule, amino acid residues are replaced with amino acid residues having a larger side chain volume, thereby generating a protrusion in the CH3 domain of the first subunit that can be positioned in a cavity in the CH3 domain of the second subunit, and in the CH3 domain of a second subunit of the Fc domain, amino acid residues are replaced with amino acid residues having a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second subunit into which the protrusion in the CH3 domain of the first subunit can be positioned.

[0169] The protrusions and cavities can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis.

[0170] In a specific embodiment, in the CH3 domain of the first subunit of the Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domain of the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one embodiment, in the second subunit of the Fc domain, additionally, the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A).

[0171] In yet a further embodiment, in the first subunit of the Fc domain, the serine residue at position 354 is additionally replaced by a cysteine ​​residue (S354C), and in the second subunit of the Fc domain, the tyrosine residue at position 349 is additionally replaced by a cysteine ​​residue (Y349C). The introduction of these two cysteine ​​residues results in the formation of a disulfide bridge between the two subunits of the Fc domain, which further stabilizes the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0172] In other embodiments, other techniques for promoting association between H chains and between L chains and H chains with desired combinations can be applied to the multispecific antigen-binding molecules of the present invention.

[0173] For example, techniques for suppressing undesired heavy chain association by introducing electrostatic repulsion at the interface of the second or third constant region (CH2 or CH3) of the antibody heavy chain can be applied to multispecific antibody association (WO2006 / 106905).

[0174] In a technique for suppressing unintended H-chain association by introducing electrostatic repulsion into the CH2 or CH3 interface, examples of amino acid residues that contact the interface of other constant regions of the H-chain include the regions corresponding to residues at EU numbering positions 356, 439, 357, 370, 399, and 409 in the CH3 region.

[0175] More specifically, examples include antibodies comprising two types of H chain CH3 regions, in which one to three pairs of amino acid residues in a first H chain CH3 region selected from the pairs of amino acid residues shown in (1) to (3) below carry the same type of charge: (1) amino acid residues contained in the H chain CH3 region at positions 356 and 439 (EU numbering); (2) amino acid residues contained in the H chain CH3 region at positions 357 and 370 (EU numbering); and (3) amino acid residues contained in the H chain CH3 region at positions 399 and 409 (EU numbering).

[0176] Furthermore, the antibody may be an antibody in which pairs of amino acid residues in a second H chain CH3 region different from the above-mentioned first H chain CH3 region are selected from the above-mentioned pairs of amino acid residues (1) to (3), and 1 to 3 pairs of amino acid residues corresponding to the above-mentioned pairs of amino acid residues (1) to (3) having the same type of charge in the above-mentioned first H chain CH3 region have an opposite charge to the corresponding amino acid residues in the above-mentioned first H chain CH3 region.

[0177] The amino acid residues shown in (1) to (3) above approach each other during association. Those skilled in the art can find positions corresponding to the above-mentioned amino acid residues in (1) to (3) in the desired H chain CH3 region or H chain constant region by homology modeling using commercially available software, and can appropriately modify the amino acid residues at these positions.

[0178] In the above-mentioned antibodies, the "charged amino acid residues" are preferably selected, for example, from amino acid residues included in any one of the following groups: (a) glutamic acid (E) and aspartic acid (D); and (b) Lysine (K), arginine (R), and histidine (H).

[0179] In the above-mentioned antibodies, the phrase "bearing the same charge" means, for example, that all of the two or more amino acid residues are selected from the amino acid residues included in any one of the above groups (a) and (b). The phrase "bearing the opposite charge" means, for example, that if at least one of the amino acid residues between two or more amino acid residues is selected from the amino acid residues included in any one of the above groups (a) and (b), the remaining amino acid residue is selected from the amino acid residues included in the other group.

[0180] In a preferred embodiment, the above-mentioned antibody can have its first H chain CH3 region and second H chain CH3 region cross-linked by a disulfide bond.

[0181] In the present invention, the amino acid residues that are modified are not limited to the above-mentioned amino acid residues in antibody variable region or antibody constant region.Those skilled in the art can identify the amino acid residues that form the interface in mutant polypeptide or heteromultimer by using commercially available software, such as homology modeling; then, the amino acid residues at these positions can be modified to control the association.

[0182] In addition, other known techniques can also be used to form multispecific antibodies. Association of polypeptides with different sequences can be efficiently introduced by complementary association of CH3 using chain exchange engineered domain CH3, which is produced by changing a part of one of the H chain CH3s of an antibody to a corresponding IgA-derived sequence, and introducing the corresponding IgA-derived sequence into the complementary part of another H chain CH3 (Protein Engineering Design & Selection, 23; 195-202, 2010). This known technique can also be used to efficiently form the multispecific antibody of interest.

[0183] In addition, techniques for producing antibodies using the association of antibody CH1 and CL and VH and VL, as described in WO2011 / 028952, WO2014 / 018572, and Nat Biotechnol. 2014 Feb; 32(2):191-8; techniques for producing bispecific antibodies using a combination of separately prepared monoclonal antibodies (Fab Arm Exchange), as described in WO2008 / 119353 and WO2011 / 131746; techniques for controlling the association between antibody heavy chain CH3, as described in WO2012 / 058768 and WO2013 / 063702; techniques for producing multispecific antibodies composed of two types of light chains and one type of heavy chain, as described in WO2012 / 023053; Christoph et al. (Nature Biotechnology Vol. 31, p 753-758 Techniques for producing multispecific antibodies using two bacterial cell lines each expressing one of the chains of an antibody comprising a single heavy chain and a single light chain, such as that described by Hoffmann et al. (2013), may be used to form multispecific antibodies.

[0184] Alternatively, even if the multispecific antibody of interest cannot be efficiently formed, it can be obtained by separating and purifying the multispecific antibody of interest from the produced antibody.For example, a method has been reported for purifying two types of homomeric forms and the heteromeric antibody of interest by ion exchange chromatography by introducing amino acid substitutions into the variable regions of two types of H chains to impart a difference in isoelectric point (WO2007114325).To date, as a method for purifying heteromeric antibodies, a method has been reported for purifying a heterodimeric antibody comprising mouse IgG2a H chain that binds to protein A and rat IgG2b H chain that does not bind to protein A using protein A (WO98050431 and WO95033844). Furthermore, heterodimeric antibodies can be efficiently purified independently by using H chains that contain substitutions of amino acid residues at positions 435 and 436 (EU numbering), which are the IgG-Protein A binding site, with amino groups such as Tyr and His that result in different Protein A affinities, or by using H chains with different Protein A affinities in order to change the interaction of each of the H chains with Protein A, followed by a Protein A column.

[0185] Furthermore, an Fc region with improved C-terminal heterogeneity can be appropriately used as the Fc region of the present invention. More specifically, the present invention provides an Fc region produced by deleting glycine at position 446 and lysine at position 447, as specified by EU numbering, from the amino acid sequences of two polypeptides that constitute an Fc region derived from IgG1, IgG2, IgG3, or IgG4.

[0186] The multispecific antigen-binding molecules prepared as described herein can be purified by techniques known in the art, such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions used to purify a particular protein will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to those skilled in the art. For affinity chromatography purification, the antibody, ligand, receptor, or antigen to which the multispecific antigen-binding molecule binds can be used. For example, a matrix containing protein A or protein G can be used for affinity chromatography purification of the multispecific antigen-binding molecule. Sequential protein A or G affinity chromatography and size exclusion chromatography can be used to isolate the multispecific antigen-binding molecule. The purity of the multispecific antigen-binding molecule can be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high pressure liquid chromatography, etc.

[0187] Antibody-dependent cell-mediated cytotoxicity "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcR) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages), thereby enabling these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. NK cells, the primary cells mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. Expression of FcRs on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To evaluate the ADCC activity of the molecule of interest, in vitro ADCC assays can be carried out, such as those described in US Patent No. 5,500,362 or 5,821,337 or US Patent No. 6,737,056 (Presta).Effector cells useful for such assays include PBMC and NK cells.Alternatively or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo, for example, in animal models, such as those disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0188] Complement-dependent cytotoxicity "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate subclass) that is bound to its corresponding antigen. To assess complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996). Polypeptide variants with altered Fc region amino acid sequences (polypeptides with variant Fc regions) and increased or decreased C1q binding ability are described, for example, in U.S. Patent No. 6,194,551 B1 and WO 1999 / 51642. See also, for example, Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0189] Pharmaceutical Compositions In one aspect, the disclosure provides a pharmaceutical composition comprising a molecule of the invention as described above, e.g., a molecule comprising a first portion that binds to a first antigen and a second portion that binds to a second antigen.

[0190] In certain embodiments, the pharmaceutical composition of the present invention is for providing signal transduction or signal blocking in cells (e.g., immune cells, cells in diseased tissues, as described above), in other words, for directly or indirectly activating, regulating, suppressing, or inhibiting immune cells, or for suppressing or inhibiting cells (e.g., tumor cells) in diseased tissues.In one embodiment, the pharmaceutical composition of the present invention is a therapeutic agent for inducing cellular cytotoxicity.In certain embodiments, the pharmaceutical composition of the present disclosure is a pharmaceutical composition used for treating and / or preventing cancer, autoimmune disease, inflammation, viral infection, etc.

[0191] In the present invention, the pharmaceutical composition for treatment may also be referred to as a therapeutic agent (including, but not limited to, a cytostatic agent, an anticancer agent, etc.).

[0192] In the present invention, the terms "cancer", "carcinoma" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by uncontrolled cell growth / proliferation. In the present invention, the term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "carcinoma", "cancer", "cancerous", "cell proliferative disorder", "proliferative disorder", and "tumor" are not mutually exclusive as used herein. The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

[0193] In the present invention, examples of cancer include, but are not limited to, gastric cancer, head and neck cancer, esophageal cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, ovarian cancer, breast cancer, colon cancer, kidney cancer, skin cancer, muscle tumor, pancreatic cancer, prostate cancer, testicular cancer, uterine cancer, bile duct cancer, Merkel cell carcinoma, bladder cancer, thyroid cancer, schwannoma, adrenal cancer, anal cancer, central nervous system tumor, neuroendocrine tissue tumor, penile cancer, pleural tumor, salivary gland tumor, vulvar cancer, thymoma, lymphoma, myeloid leukemia, and childhood cancer (Wilms' tumor, neuroblastoma, sarcoma, hepatoblastoma, and germ cell tumor). More preferred cancer types include, but are not limited to, gastric cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, breast cancer, skin cancer, lymphoma, and myeloid leukemia (Tumori. (2012) 98, 478-484;Tumor Biol. (2015) 36, 4671-4679;Am J Clin Pathol (2008) 130, 224-230;Adv Anat Pathol (2014) 21, 450-460;Med Oncol (2012) 29, 663-669;Clinical Cancer Research (2004) 10, 6612-6621;Appl Immunohistochem Mol Morphol (2009) 17, 40-46;Eur J Pediatr Surg (2015) 25, 138-144;J Clin Pathol (2011) 64, 587-591;Am J Surg Pathol (2006) 30, 1570-1575;Oncology (2007) 73, 389-394;Diagnostic Pathology (2010) 64, 1-6;Diagnostic Pathology (2015) 34, 1-6;Am J Clin Pathol (2008) 129, 899-906;Virchows Arch (2015) 466, 67-76). More favorable cancer types include gastric cancer, lung cancer, mesothelioma, liver cancer, breast cancer, skin cancer, lymphoma, and myeloid leukemia.

[0194] In the present invention, "autoimmune diseases" include, but are not limited to, vitiligo, type I diabetes, and the like.

[0195] In the present invention, "inflammation" includes, but is not limited to, inflammation in the following tissues: i. Joint tissue associated with rheumatoid arthritis or osteoarthritis. ii. Lung tissue associated with bronchial asthma. iii. Gastrointestinal tissue associated with inflammatory bowel disease, Crohn's disease, or ulcerative colitis. iv. Fibrous tissue associated with fibrosis in the liver, kidney, or lung. v. Tissues involved in immune rejection following organ transplantation. vi. Vascular or cardiac tissue associated with arteriosclerosis or heart failure. vii. Visceral adipose tissue associated with metabolic syndrome. viii. Skin tissue associated with atopic dermatitis or any other dermatitis. ix. Spinal nerve tissue associated with herniated discs or chronic back pain. x. Bone tissue associated with a fracture. xi. Burn-related tissues. xii. Tissue of organ tissues damaged by external mechanical, physical or chemical forces.

[0196] The pharmaceutical compositions of the present invention can be formulated with one or more of the molecules of the present invention, if necessary. For example, the activation, regulation, suppression, or inhibition of immune cell immune responses, or the cytotoxic effect against cells expressing an antigen, can be enhanced by a cocktail of two or more of the molecules of the present invention.

[0197] Pharmaceutical compositions comprising the molecules of the invention are prepared in the form of lyophilized formulations or aqueous solutions by mixing the molecules of the invention as described above having the desired purity with one or more pharma- ceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are typically non-toxic to recipients at the dosages and concentrations used, and include, but are not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight ( Polypeptides (less than about 10 residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersion agents, such as soluble neutral activated hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968.In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases, such as a chondroitinase.

[0198] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter formulations including a histidine-acetate buffer.

[0199] The formulations herein may also contain more than one active ingredient, preferably those with complementary activities that do not adversely affect each other, as necessary for the particular indication being treated. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.

[0200] If necessary, the molecules of the present invention as described above may be encapsulated in microcapsules (microcapsules made of hydroxymethylcellulose, gelatin, poly[methyl methacrylate], etc.) or may be components of colloidal drug delivery systems (liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) (see, for example, "Remington's Pharmaceutical Science 16th edition", Oslo Ed. (1980)). In addition, methods for preparing agents as sustained release agents are known and can be applied to the antigen-binding molecules of the present disclosure (J. Biomed. Mater. Res. (1981) 15, 267-277; Chemtech. (1982) 12, 98-105; U.S. Patent No. 3,773,719; European Patent Application (EP) Nos. EP58481 and EP133988; Biopolymers (1983) 22, 547-556).

[0201] The pharmaceutical composition comprising the molecule of the present invention as described above may be administered to a patient either orally or parenterally. Parenteral administration is preferred. Specifically, such administration methods include injection, nasal administration, pulmonary administration, and transdermal administration. Injection includes, for example, intravenous injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection. For example, the pharmaceutical composition comprising the molecule of the present invention as described above can be administered locally or systemically by injection. Furthermore, an appropriate administration method can be selected according to the age and symptoms of the patient. The dose to be administered can be selected, for example, from the range of 0.0001 mg to 1,000 mg per kg of body weight for each administration. Alternatively, the dose can be selected, for example, from the range of 0.001 mg / body to 100,000 mg / body per patient. However, the dose of the pharmaceutical composition of the present invention is not limited to these doses.

[0202] The present invention provides a method for providing signaling or signal blocking in cells (e.g., immune cells, cells in diseased tissues, as described above), in other words, directly or indirectly activating, regulating, suppressing, or inhibiting immune cells, or suppressing or inhibiting cells in diseased tissues (e.g., tumor cells), comprising administering to a subject in need thereof a molecule of the present invention as described above. In one embodiment, the method of the present invention is for inducing cell-mediated cytotoxicity in diseased tissues as described above. The present invention further provides a method for treating and / or preventing cancer, autoimmune diseases, inflammation, viral infections, and the like, comprising administering to a subject in need thereof a molecule of the present invention as described above.

[0203] The present disclosure also provides kits for use in the methods of the present invention, containing the molecules of the present invention as described above. The kits may be packaged with additional pharma- ceutically acceptable carriers or vehicles, or instructions for use in the method of using the kit, or the like.

[0204] In another aspect of the present invention, an article of manufacture is provided that contains materials useful for treating, preventing, and / or diagnosing the above disorders. The article of manufacture includes a container and a label on the container or a package insert associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container holds a composition alone or in combination with another composition effective for treating, preventing, and / or diagnosing a condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic injection needle). At least one active ingredient in the composition is an antibody of the present invention.

[0205] The label or package insert indicates that the composition is used for treating a selected condition. Additionally, the article of manufacture may include (a) a first container having contained therein a composition comprising a molecule of the invention as described above; and (b) a second container having contained therein a composition comprising an additional cytotoxic or non-therapeutic agent.

[0206] The article of manufacture in this aspect of the invention may further include a package insert indicating that the composition can be used to treat a particular condition.Alternatively or additionally, the article of manufacture may further include a second (or third) container containing a pharma-ceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution.It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0207] Attached document The term "package insert" is used to refer to instructions typically included in commercial packaging of therapeutic products that contain information about the indications, usage, dosage, administration, concomitant therapy, contraindications, and / or warnings regarding the use of such therapeutic product.

[0208] Pharmaceutical preparations The terms "pharmaceutical formulation" or "pharmaceutical composition" are used interchangeably to mean a preparation that is in a form that allows the biological activity of the active ingredients contained therein to be effective, and that does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered.

[0209] Pharmaceutically acceptable carriers "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0210] treatment As used herein, "treatment" (and its grammatical derivatives, such as "treat" or "treating", used interchangeably herein) refers to a clinical intervention that seeks to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of a clinical condition. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, relief of symptoms, attenuation of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing the rate of disease progression, amelioration or alleviation of the disease state, and remission or improvement of prognosis. In some embodiments, the antigen-binding molecules or antibodies of the present disclosure are used to delay the onset of disease or to slow the progression of disease.

[0211] Other Agents and Treatments The molecules of the present invention as described above may be administered in combination with one or more other agents in a therapeutic regimen. For example, the molecules of the present invention as described above may be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" encompasses any drug administered to treat a condition or disease in an individual in need of such treatment. Such additional therapeutic agents may include any active ingredient suitable for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. In certain embodiments, the additional therapeutic agent is an immunomodulatory agent, a cytostatic agent, an inhibitor of cell adhesion, a cytotoxic agent, an activator of cell apoptosis, or an agent that increases the sensitivity of cells to apoptosis inducers. In certain embodiments, the additional therapeutic agent is an anti-cancer agent, such as a microtubule disrupting agent, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, a hormone therapy, a kinase inhibitor, a receptor antagonist, an activator of tumor cell apoptosis, or an anti-angiogenic agent.

[0212] Such other agents are suitably present in the combination in an amount that is effective for the intended purpose. The effective amount of such other agents depends on the amount of the molecule of the present invention used, the type of disorder or treatment, and other factors discussed above. The molecule of the present invention as described above is typically used in the same dosages and by the administration route as described herein, or about 1-99% of the dosages described herein, or any dosage and by any route that is empirically / clinically determined to be appropriate.

[0213] Such combination therapy as described above encompasses combined administration (wherein the two or more therapeutic agents are contained in the same composition or in separate compositions) and separate administration, where administration of the antibodies of the invention as described above can occur prior to, simultaneously with, and / or after administration of the additional therapeutic agent and / or adjuvant. The molecules of the invention as described above can also be used in combination with radiation therapy.

[0214] In the following, certain aspects of the invention are presented in more detail, which in part relate to, but are not limited to, the aspects outlined above as D1 to D37. All definitions and further details given above relate inter alia also to the following aspects.

[0215] That is, the present invention particularly relates to an antigen-binding molecule comprising (1) at least one first portion that binds to a first antigen, and (2) at least one second portion that binds to a second antigen, wherein the first antigen is a damage-associated molecular pattern (DAMP), and the second antigen is different from the first antigen.

[0216] Furthermore, the present invention relates in particular to (1) at least one first moiety that binds to a first antigen; and (2) at least one second moiety that binds to a second antigen; An antigen-binding molecule comprising: the first antigen is a tissue-derived antigen characteristic of a tissue condition, in particular, the antigen is exposed to the extracellular environment, e.g., a damage-associated molecular pattern (DAMP); and the second antigen is different from the first antigen, Concerning antigen-binding molecules.

[0217] According to the present invention, the term "tissue-derived antigens characteristic of tissue conditions" relates to antigens exposed to the extracellular environment by the various different cell death programs characterized in organs and tissues as a result of microbial infection, cell stress, injury, and exposure to chemotherapy. The various cell death programs are well known as apoptosis, necroptosis, and pyroptosis. Dying and dead cells release various self-proteins and bioactive chemicals originating from the cytosol, nucleus, endoplasmic reticulum, and mitochondria. These endogenous factors have been named pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), cell death-associated molecular patterns (CDAMPs), and alarmins (Beatriz Sangiuliano et al, Mediators Inflammation (2014) 2014:Article ID 821043, pages 1-14).

[0218] Generally, as used herein, a damage-associated molecular pattern (DAMP) is preferably exposed to the extracellular environment upon cell damage, in particular upon cell death. Preferably, the molecule is capable of providing cell contact and / or a signal upon said cell damage, said contact and / or signal involving said second antigen.

[0219] Generally, as used herein, said cell damage preferably (i) comprises, in particular is, cell death, and / or (ii) occurs in particular in diseased tissue selected from tissues affected by a condition or disease, in particular by cancer or an autoimmune disease, especially in the tumor microenvironment (TME), and / or (iii) is due to any one selected from the group consisting of necrosis, apoptosis, autophagic cell death and accidental cell death, and / or (iv) is cell death selected from the group consisting of necrosis, apoptosis, autophagic cell death and accidental cell death.

[0220] Therefore, in certain embodiments herein, the cell damage preferably includes cell death, and is particularly cell death.Therefore, in certain embodiments herein, the cell damage preferably occurs in diseased tissue, particularly selected from tissues affected by condition or disease, particularly cancer or autoimmune disease, and especially in tumor microenvironment (TME).Therefore, in certain embodiments herein, the cell damage is preferably caused by any one selected from the group consisting of necrosis, apoptosis, autophagy cell death, and accidental cell death.Therefore, in certain embodiments herein, the cell damage is preferably cell death selected from the group consisting of necrosis, apoptosis, autophagy cell death, and accidental cell death.

[0221] The first antigen as defined herein is not particularly limited, and certain preferred embodiments correspond to those herein above.

[0222] In fact, the first antigen may be any DAMP, which are generally well known and include all DAMPs described elsewhere herein, including in particular the molecules described elsewhere herein that are exposed to the extracellular environment upon cell death.

[0223] In certain preferred embodiments, the first antigen is a cytoskeletal component or a portion thereof, a cell membrane component or a portion thereof, an organelle component or a portion thereof, a cytoplasmic protein or a portion thereof, and a metabolite.

[0224] In certain preferred embodiments, the first antigen is located on any one selected from the group consisting of filaments (particularly intermediate filaments), nucleosomes, the cytoskeleton, and / or the nucleoskeletal.

[0225] In certain preferred embodiments, the first antigen is capable of multimerization of multiple molecules. In certain additional / alternative preferred embodiments, the first antigen is present in a multimeric molecule.

[0226] In a particular embodiment, the first antigen is selected from the group consisting of a cytoskeletal component or a portion thereof, a cell membrane component or a portion thereof, an organelle component or a portion thereof, a cytoplasmic protein or a portion thereof, and a metabolite, and is located on any one selected from the group consisting of a filament (particularly an intermediate filament), a nucleosome, a cytoskeleton, and a nucleoskeletal. In certain preferred embodiments, the first antigen is selected from the group consisting of: (A) actin, particularly F-actin; (B) heat shock proteins, especially HSP90, selected from HSP90 and GRP78; (C) phosphatidylserine (PS), especially phosphatidylserine that is part of the cell membrane; (D) histones or components thereof; (E) histone deacetylase complex subunits, especially SAP130; (F) HMGN proteins, especially selected from HMGB1 and HMGN1; (G) hepatoma-derived growth factor; (H) BCL-2; (I) calreticulin; and (J) cyclophilin A. In certain preferred embodiments, the first antigen is selected from the group consisting of F-actin, GRP78, phosphatidylserine, HSP90, and SAP130; more preferably, selected from the group consisting of F-actin, phosphatidylserine, and HSP90; even more preferably, selected from the group consisting of F-actin and HSP90, especially where the first antigen is F-actin.

[0227] The first moiety as defined herein is not particularly limited, and certain preferred embodiments correspond to those herein above.

[0228] This aspect relating to the first antigen may additionally or alternatively be further defined by reference to the first moiety.

[0229] In certain preferred embodiments, the first moiety is an Ig-type binding moiety.

[0230] In another preferred embodiment, the first moiety is a binding polypeptide. Preferably, said binding polypeptide is a non-Ig type binding moiety.

[0231] According to certain preferred embodiments, the first portion is selected from the group consisting of: (A) a portion capable of binding to actin, in particular F-actin; (B) a portion capable of binding to heat shock proteins, in particular selected from HSP90 and GRP78; (C) a portion capable of binding to phosphatidylserine (PS), in particular to phosphatidylserine that is part of the cell membrane; (D) a portion capable of binding to histones or components thereof; (E) a portion capable of binding to a histone deacetylase complex subunit, in particular SAP130; (F) a portion capable of binding to an HMGN protein, in particular selected from HMGB1 and HMGN1; (G) a portion capable of binding to hepatoma-derived growth factor; (H) a portion capable of binding to BCL-2; (I) a portion capable of binding to calreticulin; and (J) a portion capable of binding to cyclophilin A.

[0232] In certain preferred embodiments, the first moiety is selected from the group consisting of a moiety capable of binding to F-actin, a moiety capable of binding to GRP78, a moiety capable of binding to phosphatidylserine, a moiety capable of binding to HSP90, and a moiety capable of binding to SAP130. More preferably, the first moiety is selected from the group consisting of a moiety capable of binding to F-actin, a moiety capable of binding to phosphatidylserine, and a moiety capable of binding to HSP90. In particular, the first moiety is selected from the group consisting of a moiety capable of binding to F-actin and a moiety capable of binding to HSP90, and the first moiety is, among others, a moiety capable of binding to actin, preferably F-actin.

[0233] In certain embodiments where the first antigen is actin, preferably F-actin, the first moiety is an Ig-type binding moiety. In other preferred embodiments where the first antigen is actin, preferably F-actin, the first moiety is a binding polypeptide, preferably a Clec9A polypeptide.

[0234] In certain embodiments in which the first antigen is GRP78, the first moiety is an Ig-type binding moiety, preferably an Ig-type binding moiety derived from any respective antibody set forth herein, more preferably an Ig-type binding moiety derived from antibody GA20 or Mab159.

[0235] In certain embodiments in which the first antigen is phosphatidylserine, the first moiety is an Ig-type binding moiety, preferably an Ig-type binding moiety from any respective antibody set forth herein, more preferably an Ig-type binding moiety from antibody 3G4.

[0236] In certain embodiments in which the first antigen is HSP90, the first moiety is an Ig-type binding moiety, preferably an Ig-type binding moiety derived from any respective antibody set forth herein, more preferably an Ig-type binding moiety derived from antibody 1.5.1 or 6H8.

[0237] In certain embodiments in which the first antigen is SAP130, the first moiety is an Ig-type binding moiety. In other preferred embodiments in which the first antigen is SAP130, the first moiety is a binding polypeptide, preferably a mClec4e polypeptide.

[0238] Preferably, and in the above embodiment, the Ig-type binding moiety is selected from the group consisting of a single domain antibody (VHH), an antibody heavy chain variable (VH) region, an antibody light chain variable (VL) region, a single domain antibody (sdAb), a single chain Fv (scFv), a single chain antibody, an Fv, a single chain Fv2 (scFv2), Fab, and F(ab')2. More preferably, said Ig-type binding moiety is selected from the group consisting of VHH, scFv, scFv2, Fab, and F(ab')2.

[0239] In a preferred general embodiment, the molecule comprises said first portion at the Fc terminus of the antigen-binding molecule.

[0240] The second antigen as defined herein is not particularly limited, and certain preferred embodiments correspond to those herein above.

[0241] In a particularly preferred embodiment, the second antigen is a membrane protein of an immune cell.Preferably, the second antigen is a membrane protein in the cell membrane of a T cell selected from the group consisting of a T cell receptor, CD3, CD137, CD40, CTLA4, a co-stimulatory molecule, and a co-inhibitory molecule.

[0242] In a preferred embodiment, the second antigen herein is a membrane protein involved in signal transduction in a cell, and preferably, the cell is (i) an immune cell (among which, the immune cell is selected from the group consisting of T cells, killer cells, helper T cells, regulatory T cells, B cells, memory B cells, NK cells, NKT cells, dendritic cells, macrophages, eosinophils, neutrophil cells, and basophils), (ii) a tumor cell, or (iii) an autoreactive cell.

[0243] In certain preferred embodiments, the second antigen is a signal transduction receptor.In certain preferred embodiments, the second antigen is an immune cell receptor antigen.In certain preferred embodiments, the second antigen is a signal transduction receptor and an immune cell receptor antigen.

[0244] In certain embodiments, the second antigen is a membrane protein of endosome.In certain embodiments, the second antigen is a membrane protein in the cell membrane of T cell, in particular, the membrane protein in the cell membrane of T cell selected from the group consisting of T cell receptor, CD3, CD137, CD40, CTLA4, costimulatory molecule, or co-inhibitory molecule.In certain embodiments, the second antigen is a membrane protein in the cell membrane of cells other than T cell, which can be specifically bound to costimulatory molecule or co-inhibitory molecule in the cell membrane of T cell. In certain preferred embodiments, the costimulatory molecule is selected from the group consisting of CD28 (TP44), CD278 (ICOS), CD27 (TNFRSF-7), CD30, CD134 (OX40), CD357 (GITR). Tim-2, CD28H (TMIGD2), CD137 (4-1BB, TNFRSF-9), CD226 (DNAM-1), and CD244 (2B4).

[0245] In certain preferred embodiments, the co-inhibitory molecule is selected from the group consisting of CD279 (PD-1), CD152 (CTLA-4), TIGIT, BTLA, CD366 (Tim-3), CD96, CD112R, or CD200R.

[0246] In certain preferred embodiments, the membrane protein capable of binding a costimulatory or co-inhibitory molecule is selected from CD274 (PD-L1), CD273 (PD-L2), CD80, CD86, CD276 (ICOSL), CD276 (VICN1), VISTA, HHLA2, a member of the butyrophilin family, CD270 (HVEM), CD137L (TNFSF-9), CD252 (OX40L), CD70 (TNFSF-7), CD40 (TNFRSF-7), GIRL (TNFSF-18), CD155 (PVR, NECL5), CD112 (NECTIN-2), CD200 (MOX1), CD48 (BCM-1), and Gal-9.

[0247] In particularly preferred embodiments, the second antigen is selected from the group consisting of CD3, CD137, CD40, and CTLA4, preferably selected from the group consisting of CD3 and CD137, more preferably the second antigen is CD3.

[0248] The second moiety as defined herein is not particularly limited, and certain preferred embodiments correspond to those herein above.

[0249] This embodiment relating to the second antigen may additionally or alternatively be further defined by reference to the second moiety.

[0250] In a preferred embodiment, the second moiety is an Ig-type binding moiety. Thus, in a particular preferred embodiment, the present invention provides a method for the preparation of a medicament for the treatment of a pulmonary edema. (1) at least one first moiety that binds to a first antigen; and (2) at least one second moiety that binds to a second antigen; An antigen-binding molecule comprising: the first antigen is a damage-associated molecular pattern (DAMP); the second antigen is different from the first antigen, and the second antigen is a membrane protein of an immune cell; and the second portion is an Ig-type binding portion; Concerning antigen-binding molecules.

[0251] In a particularly preferred embodiment, the present invention provides (1) at least one first moiety that binds to a first antigen; and (2) at least one second moiety that binds to a second antigen; An antigen-binding molecule comprising: the first antigen is a damage-associated molecular pattern (DAMP); the first portion is an Ig-type binding portion, the second antigen is different from the first antigen; the second antigen is a membrane protein of an immune cell; and the second portion is an Ig-type binding portion; Concerning antigen-binding molecules.

[0252] In a further particularly preferred aspect, the present invention provides a method for producing a method for treating a cancer cell comprising the steps of: (1) at least one first moiety that binds to a first antigen; and (2) at least one second moiety that binds to a second antigen; An antigen-binding molecule comprising: the first antigen is a damage-associated molecular pattern (DAMP); the first portion is Clec9a or mClec4e, the second antigen is different from the first antigen; the second antigen is a membrane protein of an immune cell; and the second portion is an Ig-type binding portion; Concerning antigen-binding molecules.

[0253] In certain embodiments, the second moiety is a moiety capable of binding to an endosomal membrane protein.

[0254] In certain embodiments, the second moiety is a moiety capable of binding to a membrane protein in the cell membrane of a T cell, preferably a membrane protein in the cell membrane of a T cell selected from the group consisting of a T cell receptor, CD3, CD137, CD40, CTLA4, a costimulatory molecule, or a co-inhibitory molecule.

[0255] In certain preferred embodiments, the membrane protein capable of binding a costimulatory or co-inhibitory molecule is selected from CD274 (PD-L1), CD273 (PD-L2), CD80, CD86, CD276 (ICOSL), CD276 (VICN1), VISTA, HHLA2, a member of the butyrophilin family, CD270 (HVEM), CD137L (TNFSF-9), CD252 (OX40L), CD70 (TNFSF-7), CD40 (TNFRSF-7), GIRL (TNFSF-18), CD155 (PVR, NECL5), CD112 (NECTIN-2), CD200 (MOX1), CD48 (BCM-1), and Gal-9.

[0256] In certain embodiments, the second portion is a portion capable of binding to a membrane protein in the cell membrane of a cell other than a T cell, particularly capable of binding to a costimulatory or co-inhibitory molecule in the cell membrane of a T cell.

[0257] In certain preferred embodiments, the costimulatory molecule is selected from the group consisting of CD28 (TP44), CD278 (ICOS), CD27 (TNFRSF-7), CD30, CD134 (OX40), CD357 (GITR). Tim-2, CD28H (TMIGD2), CD137 (4-1BB, TNFRSF-9), CD226 (DNAM-1), and CD244 (2B4).

[0258] In certain preferred embodiments, the co-inhibitory molecule is selected from the group consisting of CD279 (PD-1), CD152 (CTLA-4), TIGIT, BTLA, CD366 (Tim-3), CD96, CD112R, or CD200R.

[0259] In particularly preferred embodiments, the second moiety is selected from the group consisting of a moiety capable of binding to CD3, a moiety capable of binding to CD137, a moiety capable of binding to CD40, and a moiety capable of binding to CTLA4, more preferably selected from the group consisting of a moiety capable of binding to CD3 and a moiety capable of binding to CD137, and even more preferably the second moiety is a moiety capable of binding to CD3.

[0260] In the above embodiments, preferably the moiety capable of binding to CD3 is an Ig-type binding moiety. In the above embodiments, preferably the moiety capable of binding to CD137 is an Ig-type binding moiety. In the above embodiments, preferably the moiety capable of binding to CD40 is an Ig-type binding moiety. In the above embodiments, preferably the moiety capable of binding to CTLA4 is an Ig-type binding moiety.

[0261] Preferably, the Ig-type binding portion is selected from the group consisting of a single domain antibody (VHH), a combination of an antibody heavy chain variable (VH) region and an antibody light chain variable (VL) region, a single domain antibody (sdAb), a single chain Fv (scFv), a single chain antibody, Fv, a single chain Fv2 (scFv2), Fab, and F(ab')2. More preferably, said Ig-type binding moiety is selected from the group consisting of VHH, scFv, scFv2, Fab, and F(ab')2.

[0262] In a preferred general embodiment, the molecule comprises said second portion in the antigen-binding region, preferably the F(ab')2 region, of the antigen-binding molecule.

[0263] In a preferred general embodiment, the molecule comprises at least one first portion and at least one second portion, in particular the antigen-binding molecule comprises one first portion and one second portion, more particularly the antigen-binding molecule comprises at least two, preferably two first portions, and at least two, preferably two second portions. In certain embodiments herein, the antigen-binding molecule is capable of binding to a first antigen and a second antigen upon irradiation with light.

[0264] In a preferred general embodiment, the antigen-binding molecule is characterized in that the complex formed by the binding of one or more of the molecules to the first antigen can bind to more than one membrane protein via the second part.In a specific embodiment, the complex formed by the binding of one or more of the molecules to the first antigen can bind to one or more of the membrane proteins via the second part to provide signal transduction through the one or more membrane proteins in cells.In another specific embodiment, the complex formed by the binding of one or more of the molecules to the first antigen can bind to one or more of the membrane proteins via the second part to provide signal transduction through the one or more membrane proteins in cells.

[0265] Typically, the antigen-binding molecule herein may be selected from the group consisting of: (A) a polypeptide complex, optionally a fusion protein; (B) a polynucleotide (preferably composed of two entities, preferably connected by a linker); (C) a medium-sized chemical compound (preferably composed of two entities, preferably connected by a linker); and (D) a small-sized chemical compound (preferably composed of two entities, preferably connected by a linker).

[0266] In a preferred general embodiment herein, the antigen-binding molecule is a polypeptide complex.

[0267] In a particularly preferred embodiment herein, the antigen-binding molecule is an antibody or an antibody fragment, especially an antibody. In a particular embodiment, said antibody is further characterized as described elsewhere herein.

[0268] In certain preferred embodiments, the antibody, antigen-binding molecule is an IgG type antibody. In certain preferred embodiments, the antibody, antigen-binding molecule is a bispecific IgG type antibody.

[0269] In another preferred general embodiment herein, the antigen-binding molecule is a polypeptide complex that is a fusion protein. Preferably, said molecule is an antibody that is a fusion protein, or a fragment thereof, especially an antibody that is a fusion protein.

[0270] Preferably, said antibody is an IgG type antibody having at least one binding polypeptide, preferably two binding polypeptides, fused to its Fc domain, in particular an antibody having at least one binding polypeptide, preferably two binding polypeptides, fused to its Fc domain.

[0271] The binding polypeptide may be fused to the antigen-binding molecule using one or more linkers, preferably any of the linkers described elsewhere herein.

[0272] Typically, in certain preferred embodiments where the antigen-binding molecule is an antibody, (i-1) the first moiety is an Ig-type binding moiety, or (i-2) the first moiety is a binding polypeptide, and / or (ii) the second moiety is an Ig-type binding moiety. In certain preferred embodiments where the antigen-binding molecule is an antibody (especially a fusion protein), the antibody preferably comprises at least one binding polypeptide capable of binding to a membrane protein of an immune cell, and further preferably linked to an Fc region.

[0273] Preferably, the binding polypeptide is selected from a Clec9A polypeptide and an mClec4e polypeptide, and more preferably, the antibody is selected from the group consisting of an anti-CD3 antibody comprising a Clec9A polypeptide, an anti-CD137 antibody comprising a Clec9A polypeptide, an anti-CD3 antibody comprising a mClec4e polypeptide, and an anti-CD137 antibody comprising a mClec4e polypeptide.

[0274] In certain embodiments, the antigen binding molecule comprises at least one, preferably two, Clec9A polypeptides, among which the antigen binding molecule further comprises (i) at least two, preferably two, moieties that are involved in signal transduction in a cell, or (ii) at least two, preferably two, moieties that are immune cell receptor antigens.

[0275] Similarly, in certain embodiments, the antigen binding molecule comprises at least one, preferably two, mClec4e polypeptides, among which the antigen binding molecule further comprises (i) at least two, preferably two, moieties that are involved in signal transduction in a cell, or (ii) at least two, preferably two, moieties that are immune cell receptor antigens.

[0276] In a generally preferred embodiment where the antigen-binding molecule is an antibody, the antibody is a bispecific antibody against a membrane protein of an immune cell and against a DAMP, and more preferably the antibody is selected from the group consisting of a bispecific anti-HSP90 anti-CD3 antibody, a bispecific anti-HSP90 anti-CD3 antibody, a bispecific anti-GRP78 anti-CD3 antibody, a bispecific anti-GRP78 anti-CD137 antibody, a bispecific anti-phosphatidylserine anti-CD3 antibody, and a bispecific anti-phosphatidylserine anti-CD137 antibody.

[0277] Typically, in certain preferred embodiments where the antigen-binding molecule is an antibody, (i-1) the first moiety is an Ig-type binding moiety, or (i-2) the first moiety is a binding polypeptide and (ii) the second moiety is an Ig-type binding moiety.

[0278] In certain embodiments herein, (a) the first moiety is an Ig-type binding moiety selected from the group consisting of a single domain antibody (VHH), a combination of an antibody heavy chain variable (VH) region and an antibody light chain variable (VL) region, a single domain antibody (sdAb), a single chain Fv (scFv), a single chain antibody, Fv, a single chain Fv2 (scFv2), Fab, and F(ab')2; and / or (b) the second moiety is an Ig-type binding moiety selected from the group consisting of a single domain antibody (VHH), a combination of an antibody heavy chain variable (VH) region and an antibody light chain variable (VL) region, a single domain antibody (sdAb), a single chain Fv (scFv), a single chain antibody, Fv, a single chain Fv2 (scFv2), Fab, and F(ab')2; and / or (c) said binding polypeptide is a non-Ig-type binding moiety.

[0279] In further preferred specific embodiments herein, (a*) the first portion is a binding domain selected from the group consisting of VHH, scFv, scFv2, Fab, and F(ab')2, and / or (b*) the second portion is a binding domain selected from the group consisting of VHH, scFv, scFv2, Fab, and F(ab')2, and / or (c*) the binding polypeptide is a non-Ig type binding moiety, in particular a non-Ig type binding moiety attached to the Fc domain of the antigen-binding molecule.

[0280] In a preferred specific embodiment herein, the antigen-binding molecule is an antibody selected from the group consisting of: (A) an antibody capable of binding to F-actin and an antigen involved in signal transduction in cells; (B) an antibody capable of binding to F-actin and an immune cell receptor; (C) an antibody capable of binding to HSP90 and an antigen involved in signal transduction in cells; (D) an antibody capable of binding to HSP90 and an immune cell receptor; (E) an antibody capable of binding to GRP78 and an antigen involved in signal transduction in cells; (F) an antibody capable of binding to GRP78 and an immune cell receptor; (G) an antibody capable of binding to phosphatidylserine and an antigen involved in signal transduction in cells; (H) an antibody capable of binding to phosphatidylserine and an immune cell receptor; (I) an antibody capable of binding to SAP130 and an antigen involved in signal transduction in cells; and (J) an antibody capable of binding to SAP130 and an immune cell receptor.

[0281] More preferably, the antibody is selected from the group consisting of: (A-1) an antibody capable of binding to F-actin and CD3; (A-2) an antibody capable of binding to F-actin and CD137; (C-1) an antibody capable of binding to HSP90 and CD3; (C-2) an antibody capable of binding to HSP90 and CD137; (E-1) an antibody capable of binding to GRP78 and CD3; (E-2) an antibody capable of binding to GRP78 and CD137; (G-1) an antibody capable of binding to phosphatidylserine and CD3; (G-2) an antibody capable of binding to phosphatidylserine and CD137; (I-1) an antibody capable of binding to SAP130 and CD3; (I-2) an antibody capable of binding to SAP130 and CD137.

[0282] In certain preferred embodiments herein, the antibody comprises (A) an antibody comprising two first moieties capable of binding to F-actin, among others being a binding polypeptide, preferably a Clec9A polypeptide, attached to the Fc domain of the antibody, and two second moieties involved in signal transduction in a cell, among others being Ig-type binding moieties; (B) an antibody comprising two first moieties capable of binding to F-actin, among others, which are binding polypeptides, preferably Clec9A polypeptides, attached to the Fc domain of the antibody, and two second moieties which are immune cell receptor antigens, among others, Ig type binding moieties; (C) an antibody comprising two first moieties capable of binding to HSP90, among them Ig-type binding moieties, and two second moieties involved in signal transduction in a cell, among them Ig-type binding moieties; (D) an antibody comprising two first moieties capable of binding to HSP90, among which Ig-type binding moieties, and two second moieties which are immune cell receptor antigens, among which Ig-type binding moieties; (E) an antibody comprising two first moieties capable of binding to GRP78, among them Ig-type binding moieties, and two second moieties involved in signal transduction in a cell, among them Ig-type binding moieties; (F) an antibody comprising two first moieties capable of binding to GRP78, among which are Ig-type binding moieties, and two second moieties which are immune cell receptor antigens, among which are Ig-type binding moieties; (G) An antibody comprising two first moieties capable of binding to phosphatidylserine, among them Ig-type binding moieties, and two second moieties involved in signal transduction in cells, among them Ig-type binding moieties; (H) An antibody comprising two first moieties capable of binding to phosphatidylserine, among which Ig-type binding moieties, and two second moieties which are immune cell receptor antigens, among which Ig-type binding moieties; (I) an antibody comprising two first moieties capable of binding to SAP130, preferably a binding polypeptide, preferably an mClec4e polypeptide, attached to the Fc domain of the antibody, among others, and two second moieties involved in signal transduction in cells, among others Ig-type binding moieties; (J) An antibody comprising two first moieties capable of binding to SAP130, preferably a binding polypeptide attached to the Fc domain of the antibody, preferably an mClec4e polypeptide, among others, and two second moieties which are immune cell receptor antigens, among others Ig type binding moieties. is selected from the group consisting of:

[0283] More preferably, the antibody (A1) An antibody comprising two first moieties capable of binding to F-actin, preferably a binding polypeptide attached to the Fc domain of the antibody, preferably a Clec9A polypeptide, among others, and two second moieties capable of binding to CD3, among others Ig-type binding moieties; (A2) An antibody comprising two first moieties capable of binding to F-actin, preferably a binding polypeptide attached to the Fc domain of the antibody, preferably a Clec9A polypeptide, among others, and two second moieties capable of binding to CD137, among others Ig-type binding moieties; (C1) An antibody comprising two first moieties capable of binding to HSP90, among which Ig-type binding moieties, and two second moieties capable of binding to CD3, among which Ig-type binding moieties; (C2) An antibody comprising two first moieties capable of binding to HSP90, among which Ig-type binding moieties, and two second moieties capable of binding to CD137, among which Ig-type binding moieties; (E1) An antibody comprising two first moieties capable of binding to GRP78, among which Ig-type binding moieties, and two second moieties capable of binding to CD3, among which Ig-type binding moieties; (E2) an antibody comprising two first moieties capable of binding to GRP78, among which Ig-type binding moieties, and two second moieties capable of binding to CD137, among which Ig-type binding moieties; (G1) An antibody comprising two first moieties capable of binding to phosphatidylserine, among others Ig-type binding moieties, and two second moieties capable of binding to CD3, among others Ig-type binding moieties; (G2) An antibody comprising two first moieties capable of binding to phosphatidylserine, among them Ig-type binding moieties, and two second moieties capable of binding to CD137, among them Ig-type binding moieties; (I1) An antibody comprising two first moieties capable of binding to SAP130, preferably a binding polypeptide attached to the Fc domain of the antibody, preferably an mClec4e polypeptide, among others, and two second moieties capable of binding to CD3, among others Ig-type binding moieties; (I2) An antibody comprising two first moieties capable of binding to SAP130, preferably a binding polypeptide attached to the Fc domain of the antibody, preferably a mClec4e polypeptide, and two second moieties capable of binding to CD137, preferably Ig-type binding moieties. is selected from the group consisting of:

[0284] A further general aspect of the invention relates to a pharmaceutical composition comprising an antigen-binding molecule as described herein.

[0285] Further general aspects of the present invention relate to an antigen-binding molecule of the present invention for use in medicine, and / or (ii) in a method for treating a medical condition, particularly a medical condition involving cell death, and / or (iii) in a method for treating a medical condition in a subject, comprising a step of contacting the antigen-binding molecule with a damaged cell, and / or (iv) in a method for treating a medical condition in a subject, comprising a step of contacting the antigen-binding molecule with a damage-associated molecular pattern (DAMP) in the patient, in particular further comprising a step of contacting the antigen-binding molecule with the second antigen, and / or (v) in a method for treating a medical condition in a subject, comprising a step of administering the antigen-binding molecule to a patient suffering from a condition involving cell damage, particularly cell death.

[0286] Thus, in certain preferred embodiments, the antigen-binding molecule of the present invention is provided for use in medicine. Thus, in certain preferred embodiments, the antigen-binding molecule of the present invention is provided for use in a method for treating a medical condition, particularly a medical condition involving cell damage, particularly involving cell death. Thus, in certain preferred embodiments, the antigen-binding molecule of the present invention is provided for use in a method for treating a medical condition in a subject, comprising a step of contacting the antigen-binding molecule with a damaged cell. Thus, in certain preferred embodiments, the antigen-binding molecule of the present invention is provided for use in a method for treating a medical condition in a subject, comprising a step of contacting the antigen-binding molecule with a damage-associated molecular pattern (DAMP) in the patient, particularly further comprising a step of contacting the antigen-binding molecule with the second antigen. Thus, in certain preferred embodiments, the antigen-binding molecule of the present invention is provided for use in a method for treating a medical condition in a subject, comprising a step of administering the antigen-binding molecule to a patient suffering from a condition involving cell damage, particularly cell death.

[0287] In a further related general aspect, the invention relates to a pharmaceutical composition for any of the above-mentioned uses of the molecules of the invention.

[0288] In certain preferred embodiments, methods of treatment in the context of the present invention comprise contacting a first and a second antigen with multiple antigen-binding molecules in close proximity.

[0289] In certain preferred embodiments, the method of treatment in the context of the present invention is a method for activating or inhibiting an immune response. Preferably, the method of treatment in the context of the present invention is a method for activating an immune response.

[0290] In certain preferred embodiments, the method of treatment in the context of the present invention is for conferring or blocking signaling in a cell, preferably for conferring signaling in a cell.

[0291] In certain preferred embodiments herein, the method of treatment comprises administering an antigen-binding molecule according to the invention to a patient, particularly a human patient, preferably characterized as described elsewhere herein.

[0292] In certain preferred embodiments herein, the method of treatment is a method for treating or preventing cancer or an immune (preferably autoimmune) disease, among which the method is a method for treating or preventing cancer, especially the method is a method for treating cancer.

[0293] Thus, in certain preferred embodiments, the present invention provides a method for treating cancer comprising: (1) at least one first moiety that binds to a first antigen; and (2) at least one second moiety that binds to a second antigen; An antigen-binding molecule comprising: the first antigen is a damage-associated molecular pattern (DAMP); the second antigen is different from the first antigen, and the second antigen is a membrane protein of an immune cell; and the second portion is an Ig-type binding portion; Concerning antigen-binding molecules.

[0294] In a preferred embodiment, the present invention provides a method for treating cancer comprising: (1) at least one first moiety that binds to a first antigen; and (2) at least one second moiety that binds to a second antigen; An antigen-binding molecule comprising: the first antigen is a damage-associated molecular pattern (DAMP); the first portion is an Ig-type binding portion, the second antigen is different from the first antigen; the second antigen is a membrane protein of an immune cell; and the second portion is an Ig-type binding portion; Concerning antigen-binding molecules.

[0295] In a further preferred embodiment, the present invention provides a method for treating cancer comprising: (1) at least one first moiety that binds to a first antigen; and (2) at least one second moiety that binds to a second antigen; An antigen-binding molecule comprising: the first antigen is a damage-associated molecular pattern (DAMP); the first portion is Clec9a or mClec4e, the second antigen is different from the first antigen; the second antigen is a membrane protein of an immune cell; and the second portion is an Ig-type binding portion; Concerning antigen-binding molecules.

[0296] In a particular embodiment, the method for treatment in the context of the present invention comprises contacting an antigen-binding molecule with a damage-associated molecular pattern (DAMP) exposed to the extracellular environment due to cell damage, among others, the DAMP being selected from the group consisting of F-actin, GRP78, phosphatidylserine, HSP90, and SAP130.

[0297] In a particular embodiment, the method for treatment in the context of the present invention comprises contacting the antigen-binding molecule with, inter alia, a cell selected from (ai) an immune cell (inter alia, the immune cell is selected from the group consisting of a T cell, a killer cell, a helper T cell, a regulatory T cell, a B cell, a memory B cell, a NK cell, a NKT cell, a dendritic cell, a macrophage, an eosinophil, a neutrophil cell, and a basophil), (a-ii) a tumor cell, and (a-iii) an autoreactive cell.

[0298] In certain preferred embodiments, there is provided an antigen binding molecule or a pharmaceutical composition of the invention for use in medicine. In certain preferred embodiments, there is provided an antigen binding molecule or pharmaceutical composition of the invention for use in a method of treating or preventing a medical condition.

[0299] In a preferred general embodiment herein, the medical condition is selected from the group consisting of cancer and immune disease, preferably an autoimmune disease.Accordingly, in a particular preferred embodiment herein, the medical condition is cancer, particularly as described elsewhere herein.Accordingly, in a particular preferred embodiment herein, the medical condition is an immune disease, particularly as described elsewhere herein.Preferably, said immune disease is an autoimmune disease, particularly as described elsewhere herein.

[0300] A further general aspect of the invention pertains to polynucleotides encoding the antigen-binding molecules of the invention.

[0301] A further general aspect of the invention relates to a vector comprising a polynucleotide of the invention.

[0302] A further general aspect of the invention relates to a cell comprising the vector of the invention.

[0303] In addition, the present invention also relates to a use of at least a first portion and at least a second portion for the manufacture of an antigen-binding molecule comprising at least a first portion and at least a second portion, comprising: (1) at least one first portion binds to a first antigen; and (2) at least one second moiety binds to a second antigen; the first antigen is a damage-associated molecular pattern (DAMP); and The second antigen is different from the first antigen, preferably the second antigen is a membrane protein of an immune cell. Regarding use.

[0304] The present invention further relates to the use of an antigen-binding molecule for targeting cells in a tissue in which cell death is to be observed or detected, comprising: (1) at least one first portion binds to a first antigen; and (2) at least one second moiety binds to a second antigen; the first antigen is a damage-associated molecular pattern (DAMP); and The second antigen is different from the first antigen, preferably the second antigen is a membrane protein of an immune cell. Regarding use.

[0305] In addition, the present invention also relates to the use of an antigen-binding molecule for inducing or blocking signal transduction in cells in tissues in which cell death is observed, comprising: (1) at least one first portion binds to a first antigen; and (2) at least one second moiety binds to a second antigen; the first antigen is a damage-associated molecular pattern (DAMP); and The second antigen is different from the first antigen, preferably the second antigen is a membrane protein of an immune cell. Regarding use.

[0306] Furthermore, the present invention relates to a kit for producing a molecule that specifically acts in a tissue in which cell death is observed, the molecule comprising: (1) at least one first moiety that binds to a first antigen; and (2) at least one second moiety that binds to a second antigen; Includes; the first antigen is a damage-associated molecular pattern (DAMP); and the second antigen is different from the first antigen, preferably the second antigen is a membrane protein of an immune cell, The kit comprises: at least a first container containing at least a first portion; and At least a second container containing at least a second portion. The present invention relates to a kit comprising:

[0307] For the above uses and kits of the present invention, all the aspects and definitions discussed above also apply.

[0308] All documents cited herein are hereby incorporated by reference.

[0309] The following are examples of the methods and compositions of the present disclosure. Given the general description provided above, it is understood that various other embodiments may be implemented. The examples are provided to illustrate, but not to limit, the present invention. EXAMPLES

[0310] Example 1 Recombinant antibody expression and purification Recombinant antibodies were transiently expressed using the Expi293 cell line (Thermo Fisher, Carlsbad, CA, USA). Antibody purification was performed using protein G or A affinity chromatography and gel filtration. The combinations of genes encoding the heavy and light chains of each antibody used for co-transfection are summarized in Table 1. Each expression vector encoding the antibody sequence was designed for a mammalian expression system. Preparation of bispecific antibodies using Fab arm exchange (FAE) was performed according to the method described (Proc Natl Acad Sci US A. 2013 Mar 26; 110(13): 5145-5150). The concentration of the purified antibodies was measured by measuring their absorbance at 280 nm using a spectrophotometer. The antibody concentration was calculated from the obtained value using the extinction coefficient calculated by a method such as PACE (Protein Science 1995; 4: 2411-2423).

[0311] [Table 1]

[0312] [Table 1-1] TIFF2024504903000004.tif216170TIFF2024504903000005.tif171170

[0313] Example 2 Binding of Clec9A-conjugated antibodies to live and dead cells Clec9A-mediated binding of live and dead cells was examined by FACS analysis. Cell death of Ba / F3 cells was induced by treatment with 20 μM mitomycin C (MMC) for 24 h at 37°C, followed by immunization with 2.5 × 10 5Cells were used for staining of each sample. Briefly, cells were incubated with 1000-fold diluted zombie violet (ZV) dye (BioLegend, #423114) for 20 min at RT, washed, and blocked with anti-mouse CD16 / 32 antibody (BioLegend #101320) at a final concentration of 2 μg / mL for 10 min on ice. Surface staining with AF488-labeled anti-keyhole limpet hemocyanin (KLH) antibody (αKLH) either unconjugated or conjugated to Clec9A ECD (SEQ ID NO:9) or Celc-9A CTLD (SEQ ID NO:10) was performed at a final concentration of 10 μg / mL for 30 min on ice. Annexin V (AnnV) staining was performed using a commercial kit (BioLegend #640930) according to the manufacturer's instructions. Data was acquired on a BD LSRFortessa™ X-20 flow cytometer and analyzed using Flowjo v10.4.2 software.

[0314] Binding of Clec9A-conjugated antibodies was observed primarily on ZV+AnnV+ necrotic cells, whereas minimal to no binding was observed on ZV-AnnV+ apoptotic cells (Figure 4). ZV-AnnV- live cell populations did not show any binding. Taken together, the data suggest that necrotic cells or their components specifically bind Clec9A and mediate the clustering of its conjugated antibodies.

[0315] Example 3 CD3 activation by Clec9A-mediated clustering In mouse B cell line Ba / F3, 1 × 10 6Cell death was induced by treatment with 5 μM methotrexate (MTX) (Sigma, #M2305000) at a concentration of cells / mL for 24 hours at 37° C., 5% CO2. Untreated or MTX-treated Ba / F3 cells were then harvested, washed with fresh culture medium, and co-cultured at a ratio of 1 Ba / F3 cells:5 Jurkat luciferase reporter T cells (Promega, #J1601). To assess the effect of Clec9A-mediated T cell activation, the extracellular (ECD) domain of Clec9A (SEQ ID NO:9) or the C-type lectin domain (CTLD) of Clec9A (SEQ ID NO:10) was conjugated to the C-terminus of the Fc region on anti-human CD3 antibody (αCD3). Clec9A-conjugated anti-CD3 antibody or non-conjugated control anti-CD3 antibody was added to co-cultures of BaF3 and Jurkat T cell cultures for 24 hours, and supernatants were assessed for luciferase activity using the Bio-Glo™ Luciferase Assay System (Promega, #G7941). Data and graphs were analyzed using GraphPad Prism software v8.4.2. Fold induction was calculated relative to the negative control for each condition.

[0316] In the presence of "live" Ba / F cells not treated with MTX, unconjugated anti-CD3 antibodies induced a maximum of 22-fold relative luciferase signal, whereas only a 5-fold induction was observed with CD3-targeting antibodies conjugated to Clec9A (Figure 5). However, in the presence of "dead" Ba / F3 cells treated with MTX, a maximum of 54-fold and 46-fold relative luciferase activity was induced by Clec9A-ECD and Clec9A-CTLD conjugated antibodies, respectively. This is significantly greater than the 20-fold relative fold induction observed with unconjugated anti-CD3 antibodies. These data suggest that the presence of dead cells mediates the clustering of Clec9A-conjugated anti-CD3 antibodies, resulting in immune T cell stimulation.

[0317] Example 4 Binding of anti-phosphatidylserine and anti-HSP90 to the surfaces of live and dead cells Phosphatidylserine (PS) and heat shock protein 90 (HSP90) are sequestered in normal cells but are exposed to the extracellular environment during cellular stress. To confirm this, anti-PS antibody (Immunotargets Ther. 2018 Jan 23;7:1-14) and anti-HSP90 antibody clones 1.5.1 (US9068987B2) and 6H8 (WO2011 / 129379) were used to detect their respective targets on live and dead cells by flow cytometry. For PS detection, Ba / F3 mouse pro-B cell line was first treated with 20 μM mitomycin C (MMC) for 48 hours to induce cell death. For HSP90 detection, FreeStyle™ 293-F (Thermofisher Scientific #R790-07) human embryonic kidney cell line was pretreated with 100 μM MMC for 72 hours. For subsequent staining, cells were incubated with 1:1000 diluted zombie violet (ZV) dye (BioLegend, #423114) for 20 min at room temperature (RT), washed, and blocked with anti-mouse CD16 / 32 antibody (BioLegend #101320) at a final concentration of 2 μg / mL or human FcR blocking reagent (#130-059-901) at a final dilution of 20 μg / mL for 10 min at RT. Surface staining with anti-PS (anti-PS(3G4)) or anti-HSP90 (anti-HSP90(1.5.1) and anti-HSP90(6H8)) was performed at a final antibody concentration of 10 μg / mL for 30 min at RT. Data were acquired on a BD LSRFortessa™ X-20 flow cytometer and analyzed using Flowjo v10.4.2 software.

[0318] Binding of anti-PS and anti-HSP90 antibodies was observed on ZV+ gated dead cells treated with MMC, but no binding was detected on ZV- live cells (Figure 6). Thus, surface expression of PS or HSP90 was specific for dead cells only.

[0319] Example 5 CD3 activation by anti-PS and anti-HSP90 antibodies To investigate whether dead cell-specific surface expression of PS or HSP90 translates into dead cell-specific immune cell activation, live or dead cells were co-cultured with CD3-expressing Jurkat reporter T cells (Promega, #J1600) in the presence of anti-PS / / CD3 (anti-PS(3G4) / / CD3) or anti-HSP90 / / CD3 (anti-HSP90(1.5.1) / / CD3 or anti-HSP90(6H8) / / CD3) bispecific antibodies. For evaluation of anti-PS / / CD3 activity, Ba / F3 cells were pre-treated with 5 μM mitoxantrone (MTX) (Sigma, #M2305000) for 48 h to induce cell death. Untreated or MTX-treated Ba / F3 cells were then co-cultured with five times the number of Jurkat reporter T cells. For evaluation of anti-PS / HSP90 activity, untreated or MMC-treated FreeStyle™ 293-F cells were co-cultured with an equal number of Jurkat reporter T cells. 24 hours after antibody stimulation, luciferase activity was detected using Bio-Glo™ Luciferase Assay System (Promega, #G7941) and GloMax® Explorer (#GM3500). Data and graphs were analyzed using GraphPad Prism software v9.0.2. Fold induction was calculated relative to the no antibody treatment control for each co-culture condition.

[0320] In the presence of dead cells, anti-PS / CD3 antibodies induced 16-fold and 70-fold luciferase signals at antibody concentrations of 5 and 25 nM, respectively (Figure 7). This activity induction was significantly higher compared to co-culture with live cells at the same antibody concentrations. Similarly, anti-HSP90 / CD3 antibodies showed induction of luciferase activity in co-culture with dead cells at antibody concentrations of 5 and 25 nM, but did not show any relative fold induction when co-cultured with live cells (Figure 7). This phenomenon was not dependent on antibody clonality, since both anti-HSP90 clones induced similar luciferase reporter activity only in the presence of dead cells. Taken together, the results are consistent with CD3 activation in a dead cell-dependent manner, suggesting that various dead cell-associated molecules can be targeted for immune cell modulation.

[0321] Example 6 CD137 activation by Clec9A-mediated clustering To examine whether the concept of dead cell immunomodulation could be extended to effector targets other than CD3, the extracellular domain of Clec9A was conjugated to the C-terminus of the Fc region of an anti-human CD137 agonist antibody (CAS: 1417318-27-4) (anti-CD137-Clec9A). Dead cell-dependent activation of CD137 was assessed in live or MTX-treated dead Ba / F3 cells co-cultured with CD137 Jurkat reporter T cells (Promega #J2332) at an effector:target ratio of 1:5. Cells were treated with Clec9A-conjugated anti-CD137-Clec9A or control unconjugated anti-human CD137 antibody (anti-CD137) for 24 hours and then analyzed by detecting luciferase activity.

[0322] In the presence of MTX-treated dead Ba / F3 cells, induction of CD137 was observed in anti-CD137-Clec9A antibody-treated conditions (Figure 8). This induction was Clec9A-dependent, as unconjugated anti-CD137 antibodies did not result in a relative fold induction of luciferase activity. Remarkably, fold induction was also not observed by anti-CD137-Clec9A antibodies in the presence of live Ba / F3. These results suggest that conjugation of dead cell-binding moieties such as Clec9A to CD137 agonist antibodies results in dead cell-dependent immune activation, similar to that observed with dead cell-binding CD3 agonist antibodies. Thus, dead cell specificity can be conferred to distinct effector molecules through combination with dead cell-binding moieties.

[0323] Example 7 CD137 activation by anti-PS To investigate whether dead cell-specific surface expression of PS or HSP90 translates into dead cell-specific immune cell activation via CD137, anti-PS / / CD137 bispecific antibodies were used in a similar assay setup as described for anti-CD137-Clec9A and luciferase reporter activity was assessed. Anti-PS / / CD137 induced enhanced reporter cell activity in the presence of dead cells when compared to co-culture with live cells. In contrast, anti-KLH / / CD137 antibodies, which do not bind PS, did not show any difference in reporter activity between live and dead cell co-cultures (Figure 9). Taken together, the data indicate that the concept of dead cell-specific regulation can be applied through the binding of various dead cell-associated molecules to distinct effector targets.

[0324] Example 8 Clec9A fusion antibodies activate CD3 in an F-actin-dependent manner In this invention, we described dead cell-dependent regulation by Clec9A-conjugated antibodies. To confirm that this phenomenon occurs through binding of filamentous actin (F-actin) on dead cells (Immunity. 2012 Apr 20;36(4):646-57), CD3 activation was examined in the presence or absence of F-actin. Briefly, recombinant F-actin (Cytoskeleton, #AKF99) reconstituted to 0.4 mg / mL was coated onto the wells of Nunc Maxisorp™ plates (Thermofisher Scientific #442404) overnight at 4°C. Uncoated wells were filled with 50 uL of germinal actin buffer (Cytoskeleton, #BSA01). The next day, wells were washed and blocked with Dulbecco's phosphate-buffered saline (DPBS) supplemented with 5% bovine serum albumin (BSA, Sigma-Aldrich #A7888) for 1 h at room temperature (RT). After the blocking step, titrated anti-CD3 antibodies, either unconjugated (anti-CD3) or conjugated to the Clec9A C-type lectin domain (anti-CD3-Clec9A), were added to F-actin-coated or -uncoated wells for 15 min at 37°C, followed by addition of 5 × 10 per well. 4 Jurkat luciferase reporter T cells (Promega, #J1601) were added. After 24 hours of incubation at 37° C., 5% CO2, luciferase activity was assessed using the Bio-Glo™ Luciferase Assay System (Promega, #G7940) and the GloMax® Explorer System (Promega #GM3500). Results were analyzed using GraphPad Prism software v9.0.2.

[0325] In the presence of F-actin, Clec9A-conjugated CD3 antibodies induced significantly higher T cell reporter activation than in wells not coated with F-actin (Figure 10). This enhanced activation was dependent on Clec9A, as unconjugated CD3 antibodies did not show any difference in reporter activity between wells coated with F-actin and those not coated with F-actin. Therefore, the modulation of CD3 activity by anti-CD3-Clec9A antibodies appears to be dependent on the interaction between Clec9A and F-actin. This result confirms the mechanism of dead cell-dependent activation by Clec9A-conjugated antibodies. [Industrial Applicability]

[0326] The present invention provides molecules that bind to cellular components or parts thereof (e.g., filaments or histones forming the cytoskeleton or nucleoskeleton) that are exposed to the extracellular environment upon cell death, which are specifically observed in diseased tissues (e.g., the TME), and at the same time bind to target molecules (e.g., membrane proteins) on or in cells (e.g., immune cells, diseased cells, such as tumor cells, autoreactive cells, and virus-infected cells), and act specifically in tissues where cell death is observed, such as diseased tissues, abnormal tissues, etc.

Claims

1. (1) at least one first moiety that binds to a first antigen; and (2) at least one second moiety that binds to a second antigen; An antigen-binding molecule comprising: the first antigen is a damage-associated molecular pattern (DAMP); and The second antigen is different from the first antigen, preferably the second antigen is a membrane protein of an immune cell. Antigen-binding molecules.

2. The first antigen is (i) selected from the group consisting of cytoskeletal components or parts thereof, cell membrane components or parts thereof, organelle components or parts thereof, cytoplasmic proteins or parts thereof, and metabolites; and / or (ii) located on any one selected from the group consisting of filaments (particularly intermediate filaments), nucleosomes, the cytoskeleton, and / or the nucleoskeletal system; The antigen-binding molecule of claim 1.

3. The first antigen is (A) Phosphatidylserine (PS), specifically phosphatidylserine that is part of the cell membrane; (B) Actin, especially F-actin, (C) heat shock proteins, particularly selected from HSP90 and GRP78, among which HSP90; (D) histones or components thereof; (E) Histone deacetylase complex subunits, especially SAP130; (F) HMGN proteins, in particular selected from HMGB1 and HMGN1; (G) hepatoma-derived growth factor, (H) BCL-2, (I) Calreticulin, and (J) Cyclophilin A is selected from the group consisting of In particular, the first antigen is selected from the group consisting of phosphatidylserine, F-actin, GRP78, HSP90, and SAP130; Among them, the first antigen is selected from the group consisting of phosphatidylserine, F-actin, and HSP90; An antigen-binding molecule according to any one of claims 1 to 2.

4. The first part is (A) Ig-type binding moiety, or (B) A binding polypeptide, particularly a non-Ig type binding moiety. The antigen-binding molecule according to any one of claims 1 to 3, selected from the group consisting of:

5. (A) when the first antigen is actin, preferably F-actin, the first moiety is a Clec9A polypeptide; or (B) if the first antigen is GRP78, phosphatidylserine, or HSP90, the first moiety is an Ig-type binding moiety; or (C) when the first antigen is SAP130, the first moiety is a mClec4e polypeptide; The antigen-binding molecule of claim 4.

6. The antigen-binding molecule of claim 4 or 5, wherein when the first antigen is actin or SAP130, the first binding moiety is an Ig-type binding moiety.

7. the second antigen is a membrane protein of an immune cell, in particular a membrane protein in the cell membrane of a T cell selected from the group consisting of a T cell receptor, CD3, CD137, CD40, CTLA4, a costimulatory molecule, or a costimulatory molecule, Among them, the second antigen is a membrane protein involved in signal transduction in cells, preferably a signal transduction receptor. An antigen-binding molecule according to any one of claims 1 to 6.

8. The antigen-binding molecule of any one of claims 1 to 7, wherein the second portion is an Ig-type binding portion.

9. The antigen-binding molecule according to any one of claims 1 to 8, wherein the molecule is an antibody, particularly an IgG type antibody.

10. the antibody further comprises at least one binding polypeptide capable of binding to a membrane protein of an immune cell and preferably linked to an Fc region, In particular, the binding polypeptide is selected from a Clec9A polypeptide and a mClec4e polypeptide, Among others, the antibody is an anti-CD3 antibody comprising a Clec9A polypeptide; an anti-CD137 antibody comprising a Clec9A polypeptide; an anti-CD3 antibody comprising a mClec4e polypeptide, and Anti-CD137 Antibodies Containing mClec4e Polypeptides Selected from the group consisting of: The antigen-binding molecule of claim 9.

11. the antibody is a bispecific antibody, In particular, said antigen-binding molecule is a bispecific antibody against a membrane protein of an immune cell and against a DAMP, Among others, the antibody is bispecific anti-HSP90 anti-CD3 antibody, bispecific anti-HSP90 anti-CD3 antibody, bispecific anti-GRP78 anti-CD3 antibody, bispecific anti-GRP78 anti-CD137 antibody, bispecific anti-phosphatidylserine anti-CD3 antibodies, and Bispecific anti-phosphatidylserine anti-CD137 antibody Selected from the group consisting of: The antigen-binding molecule of claim 9.

12. A pharmaceutical composition comprising an antigen-binding molecule according to any one of claims 1 to 11.

13. 13. An antigen-binding molecule or a pharmaceutical composition according to any one of claims 1 to 12 for use in medicine.

14. 14. An antigen-binding molecule or pharmaceutical composition according to any one of claims 1 to 13 for use in a method for treating or preventing a medical condition.

15. The antigen-binding molecule or pharmaceutical composition for use according to claim 14, wherein the medical condition is selected from the group consisting of cancer and an immune disease, preferably an autoimmune disease.

16. A polynucleotide encoding the antigen-binding molecule described in any one of claims 1 to 11.

17. A vector comprising the polynucleotide described in claim 16.

18. A host cell comprising the vector described in claim 17.

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