Molecules that act specifically in tissues where cell death is observed

Abstract

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, and simultaneously bind to target molecules on or in cells that act specifically in tissues where cell death is observed, such as diseased tissues, abnormal tissues, etc. The molecules of the present invention are useful as drugs that provide significant therapeutic or preventive effects while reducing side effects.

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 signaling or blocking in cells in tissues such as immune cells or diseased cells; molecules for use as drugs; use of the molecules in the preparation of drugs; pharmaceutical compositions comprising the molecules; methods for conferring signaling or blocking in cells in tissues; polynucleotides encoding the molecules; vectors comprising the polynucleotides; cells harboring the vectors, etc.

[0002] In one aspect herein, the present invention relates to an antigen-binding molecule comprising a first portion that binds to a first antigen that is a damage-associated molecular pattern (DAMP) and a second portion that binds to an antigen different from the first antigen, and its pharmaceutical use. In one aspect herein, the first antigen is selected from the group consisting of (a) splicing factor 3B subunit 3 (SF3B3); and (b) proliferating cell nuclear antigen (PCNA). In one aspect herein, an antigen-binding molecule comprising a first portion that binds to a first antigen that is a damage-associated molecular pattern (DAMP) and a second portion that binds to an antigen different from the first antigen, wherein the molecule comprises at least one third portion that binds to a third antigen; the third antigen is different from the first antigen; and the second and / or third antigen is a membrane protein of an immune cell or tumor cell, and its pharmaceutical use are provided. In one aspect, the first antigen is selected from the group consisting of F-actin; phosphatidylserine (PS); splicing factor 3B subunit 3 (SF3B3); and proliferating cell nuclear antigen (PCNA). [Background technology]

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

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

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

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

[0007] As one of the possible strategies to overcome systemic toxicity, bispecific antibodies targeting tumor antigens and antigens that regulate immune cell function have been evaluated. Anti-CD137 and tumor antigen-binding bispecific antibodies have shown 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 the 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 [Patent Document 3] WO2011 / 146902 A1 [Patent Document 4] WO2017 / 059231 A1 [Patent Document 5] US7714109 B2 [Patent Document 6] WO2018 / 187227 A1 [Patent Document 7] WO2000 / 002584 A2 [Patent Document 8] WO2009 / 071892 A2 [Patent Document 9] WO2018 / 031419 A1 [Patent Document 10] WO2010 / 028306 A2 [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 [Non-Patent Document 3] Huang X et al; Cancer Res 15 April 2010; 70 (8_Supplement): 1919 [Non-Patent Document 4] Chang W et al. Theranostics. 2020 Jul 23;10(20):9214-9229. [Non-Patent Document 5] Silvestris F et al. Blood. 1996 Jun 15;87(12):5185-95 [Non-Patent Document 6] Ran S and Thorpe PE. Int J Radiat Oncol Biol Phys. 2002 Dec 1;54(5):1479-84. [Non-Patent Document 7] Yamasaki S. et al. Nat Immunol. 2008 Oct;9(10):1179-88 [Non-Patent Document 8] Brown GD. Nat Immunol. 2008 Oct;9(10):1099-100 [Summary of the Invention] [Problems to be Solved by the Invention]

[0010] In one aspect, it is an object of the present invention to overcome the systemic toxicity caused by therapeutic agents that target one or more antigens expressed in diseased tissues such as cancer, and to provide a strategy for conditionally regulating cellular function in the unique environment of diseased tissues, such as the TME. In a further aspect, it is an object of the present invention to provide molecules that act specifically in the tissue sought to be treated or targeted, such as diseased tissue, abnormal tissue, etc., but do not act or act poorly in healthy or normal tissues, thereby making it possible to provide molecules useful in pharmaceuticals that provide significant therapeutic or preventive effects while having reduced side effects.

[0011] In one aspect, 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 that elicit no or poor immune response in healthy or normal tissue. [Means for solving the problem]

[0012] In one aspect, the present invention provides molecules that act specifically in tissues where cell death is observed, such as diseased tissues and abnormal tissues, by binding to cellular components or portions thereof (e.g., filaments or histones forming the cytoskeleton or nucleoskeleton) that are specifically observed in diseased tissues (e.g., TME) that are exposed to the extracellular environment due to cell death, and simultaneously binding to target molecules (e.g., membrane proteins) on or in cells (e.g., immune cells, diseased cells, e.g., tumor cells, autoreactive cells, and virus-infected cells). In a further aspect, molecules are provided that enable bridging between cellular components or portions thereof that are exposed to the extracellular environment due to cell death and target molecules on or in cells such as immune cells or diseased cells (e.g., cancer cells, autoreactive cells, and virus-infected cells), and imparting signal transduction or signal blocking to cells in tissues. In a further aspect, increased cross-linking by multiple molecules between cellular components or portions thereof that are exposed to the extracellular environment due to cell death and target molecules on or in cells such as immune cells or diseased cells is achieved, thereby achieving increased signal transduction or increased signal blocking via target molecules in cells.

[0013] As explained and outlined in more detail below and in the Examples, the present invention provides antigen-binding molecules that bind to antigens found extracellularly, 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 extracellularly due to the cellular 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] It will be understood by those skilled in the art that the following aspects and embodiments are part of the present invention, and that the embodiments included below are intended independently as embodiments with respect to each and every embodiment and aspect described below. [AA1] (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 or a tumor cell, and The first antigen is (a) splicing factor 3B subunit 3 (SF3B3); and (b) Proliferating cell nuclear antigen (PCNA) selected from the group consisting of Antigen-binding molecules. [AA2] An antigen-binding molecule of [AA1] comprising at least one third moiety that binds to a third antigen; (i) the third antigen is different from the first and second antigens, or (ii) the third antigen is different from the first antigen but the same as the second antigen; preferably, the third antigen is a membrane protein of an immune cell or a tumor cell. [AA3] The antigen-binding molecule of [AA1] or [AA2], wherein the first antigen is exposed to the extracellular environment due to cell damage, particularly cell death. [AA4] The first part is (A) an Ig-type binding moiety; or (B) a binding polypeptide, particularly a non-Ig type binding moiety An antigen-binding molecule selected from the group consisting of [AA1] to [AA3]. [AA5] (A) if the first antigen is splicing factor 3B subunit 3 (SF3B3), the first moiety is an Ig-type binding moiety; or (B) when the first antigen is splicing factor 3B subunit 3 (SF3B3), the first moiety is a Clec4e polypeptide; or (C) when the first antigen is proliferating cell nuclear antigen (PCNA), the first moiety is an Ig-type binding moiety; Antigen-binding molecule of [AA4]. [AA6] the second and / or third 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 these, the second and / or third antigen is a membrane protein involved in signal transduction in cells, preferably a signal transduction receptor. Any one of the antigen-binding molecules [AA1] to [AA5]. [AA7] The antigen-binding molecule of any one of [AA1] to [AA6], wherein the second and / or third antigen is a membrane protein of a tumor cell. [AA8] The antigen-binding molecule of any one of [AA1] to [AA7], wherein the second and / or third moiety is an Ig-type binding moiety. [AA9] An antigen-binding molecule of any one of [AA1] to [AA8], which is an antibody, particularly an IgG antibody. [AA10] the antibody further comprises at least one binding polypeptide capable of binding to a membrane protein of an immune cell or a tumor cell, and preferably linked to an Fc region; In particular, the binding polypeptide is selected from a Clec9A polypeptide and a Clec4e polypeptide; Among others, the antibody an anti-CD3 antibody comprising a Clec9A polypeptide; an anti-CD137 antibody comprising a Clec9A polypeptide; an anti-CD3 antibody comprising a Clec4e polypeptide, and Anti-CD137 Antibodies Containing Clec4e Polypeptides selected from the group consisting of Antigen-binding molecule of [AA9]. [AA11] the antigen-binding molecule is an antibody, particularly an IgG antibody, and the first portion is linked to an Fc region and is an Ig-type binding portion; In particular, the antibody (A) Anti-CD3 antibody; (B) anti-CD137 antibody; and (C) Bispecific anti-CD3 anti-CD137 antibody selected from the group consisting of An antigen-binding molecule selected from [AA1] to [AA8]. [AA12] the antibody is a bispecific antibody, In particular, the antigen-binding molecule is a bispecific antibody against a membrane protein of an immune cell or a tumor cell and against a first antigen selected from the group consisting of: (a) splicing factor 3B subunit 3 (SF3B3); and (b) proliferating cell nuclear antigen (PCNA); Among others, the antibody bispecific anti-SF3B3 anti-CD3 antibody, bispecific anti-SF3B3 anti-CD137 antibody, bispecific anti-PCNA anti-CD3 antibodies, and Bispecific anti-PCNA anti-CD137 antibody selected from the group consisting of Antigen-binding molecule of [AA9]. [AA13] the antibody is a trispecific antibody; In particular, the antigen-binding molecule is (i) against membrane proteins of immune cells and against membrane proteins of tumor cells; and (a) splicing factor 3B subunit 3 (SF3B3); and (b) proliferating cell nuclear antigen (PCNA), or (ii) against a first membrane protein of an immune cell and a second membrane protein of an immune cell that is different from the first membrane protein; and (a) splicing factor 3B subunit 3 (SF3B3); and (b) proliferating cell nuclear antigen (PCNA), or (iii) to a first membrane protein of a tumor cell and a second membrane protein of the tumor cell that is different from the first membrane protein; and (a) splicing factor 3B subunit 3 (SF3B3); and (b) proliferating cell nuclear antigen (PCNA). It is a trispecific antibody, Among others, the antibody a trispecific anti-SF3B3 anti-CD3 anti-CD137 antibody, and Trispecific anti-PCNA anti-CD3 anti-CD137 antibody selected from the group consisting of Antigen-binding molecule of [AA9]. [AA14] A pharmaceutical composition comprising any one of the antigen-binding molecules [AA1] to [AA13]. [AA15] A pharmaceutical composition of any one of the antigen-binding molecules [AA1] to [AA13] or [AA14] for use in medicine. [AA16] An antigen-binding molecule of any one of [AA1] to [AA13] or a pharmaceutical composition of [AA14] for use in a method for treating or preventing a medical condition, wherein the medical condition is preferably selected from the group consisting of cancer and immune diseases, preferably autoimmune diseases. [AA17] A method for screening an antigen-binding molecule, comprising the steps of: (a) selecting one or more nucleic acids encoding at least one first portion, wherein the first portion binds to a first antigen, and the first antigen is a damage-associated molecular pattern (DAMP), the first antigen being selected from the group consisting of: a) splicing factor 3B subunit 3 (SF3B3); and b) proliferating cell nuclear antigen (PCNA); (b) selecting one or more nucleic acids encoding at least one second portion, wherein the second portion binds to a second antigen and the second antigen is different from the first antigen, preferably the second antigen is a membrane protein of an immune cell or a tumor cell; (b1) optionally, providing one or more nucleic acids encoding at least one third portion, wherein the third portion binds to a third antigen, the third antigen being different from the first antigen, and preferably the third antigen being a membrane protein of an immune cell or a tumor cell; (c) obtaining one or more nucleic acids encoding the antigen-binding molecule, wherein at least one first portion provided in (a), at least one second portion provided in (b), and optionally at least one third portion provided in (b1) are linked; and (d) producing an antigen-binding molecule using one or more nucleic acids prepared in (c), thereby obtaining the antigen-binding molecule. [AA18] A method for producing an antigen-binding molecule, comprising the steps of: (a) providing one or more nucleic acids encoding at least one first portion, wherein the first portion binds to a first antigen, and the first antigen is a damage-associated molecular pattern (DAMP), the first antigen being selected from the group consisting of: a) splicing factor 3B subunit 3 (SF3B3); and b) proliferating cell nuclear antigen (PCNA); (b) providing one or more nucleic acids encoding at least one second portion, wherein the second portion binds to a second antigen and the second antigen is different from the first antigen, preferably the second antigen is a membrane protein of an immune cell or a tumor cell; (b1) optionally, providing one or more nucleic acids encoding at least one third portion, wherein the third portion binds to a third antigen, the third antigen being different from the first antigen, and preferably the third antigen being a membrane protein of an immune cell or a tumor cell; (c) obtaining one or more nucleic acids encoding the antigen-binding molecule, wherein at least one first portion provided in (a), at least one second portion provided in (b), and optionally at least one third portion provided in (b1) are linked; and (d) producing an antigen-binding molecule using one or more nucleic acids prepared in (c), thereby obtaining the antigen-binding molecule. [AA19] A method for conferring signal transduction or blocking in a cell, comprising the step of contacting a cell with an antigen-binding molecule or administering an antigen-binding molecule to a subject, wherein the antigen-binding molecule: (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; Including, 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 or a tumor cell, and The first antigen is (a) splicing factor 3B subunit 3 (SF3B3); and (b) Proliferating cell nuclear antigen (PCNA) selected from the group consisting of method. [AA20] A method for activating or inhibiting an immune response, comprising the step of contacting an antigen-binding molecule with a cell or administering an antigen-binding molecule to a subject, wherein the antigen-binding molecule: (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; Including, 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 or a tumor cell, and The first antigen is (a) splicing factor 3B subunit 3 (SF3B3); and (b) Proliferating cell nuclear antigen (PCNA) selected from the group consisting of method. [AA21] A bispecific anti-phosphatidylserine (PS) anti-CD3 antibody comprising a heavy chain of the IgG1 isotype and a light chain of either the κ or λ isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers. [AA22] A bispecific antibody or pharmaceutical composition of [AA21] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA23] A bispecific anti-phosphatidylserine (PS) anti-CD3 antibody comprising a heavy chain of the IgG2 isotype and a light chain of either the κ or λ isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers. [AA24] A bispecific antibody or pharmaceutical composition of [AA23] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA25] A bispecific anti-phosphatidylserine (PS) anti-CD3 antibody comprising a heavy chain of the IgG3 isotype and a light chain of either the κ or λ isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers. [AA26] A bispecific antibody or pharmaceutical composition of [AA25] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA27] 1. A bispecific anti-phosphatidylserine (PS) anti-CD3 antibody comprising a heavy chain of the IgG4 isotype and a light chain of either the κ or λ isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers. [AA28] A bispecific antibody or pharmaceutical composition of [AA27] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA29] A bispecific anti-phosphatidylserine (PS) anti-CD137 antibody comprising a heavy chain of the IgG1 isotype and a light chain of either the κ or λ isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers. [AA30] A bispecific antibody or pharmaceutical composition of [AA29] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA31] A bispecific anti-phosphatidylserine (PS) anti-CD137 antibody comprising a heavy chain of the IgG2 isotype and a light chain of either the κ or λ isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers. [AA32] A bispecific antibody or pharmaceutical composition of [AA31] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA33] A bispecific anti-phosphatidylserine (PS) anti-CD137 antibody comprising a heavy chain of the IgG3 isotype and a light chain of either the kappa or lambda isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers. [AA34] A bispecific antibody or pharmaceutical composition of [AA33] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA35] 1. A bispecific anti-phosphatidylserine (PS) anti-CD137 antibody comprising a heavy chain of the IgG4 isotype and a light chain of either the kappa or lambda isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers. [AA36] A bispecific antibody or pharmaceutical composition of [AA35] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA37] A bispecific anti-F-actin anti-CD3 antibody comprising a heavy chain of the IgG1 isotype and a light chain of the κ or λ isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers. [AA38] A bispecific antibody or pharmaceutical composition of [AA37] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA39] A bispecific anti-F-actin anti-CD3 antibody comprising a heavy chain of the IgG2 isotype and a light chain of the κ or λ isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers. [AA40] A bispecific antibody or pharmaceutical composition of [AA39] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA41] 1. A bispecific anti-F-actin, anti-CD3 antibody comprising a heavy chain of the IgG3 isotype and a light chain of the κ or λ isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers. [AA42] A bispecific antibody or pharmaceutical composition of [AA41] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA43] 1. A bispecific anti-F-actin, anti-CD3 antibody comprising a heavy chain of the IgG4 isotype and a light chain of the κ or λ isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers. [AA44] A bispecific antibody or pharmaceutical composition of [AA43] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA45] A bispecific anti-F-actin anti-CD137 antibody comprising a heavy chain of the IgG1 isotype and a light chain of the κ or λ isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers. [AA46] A bispecific antibody or pharmaceutical composition of [AA45] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA47] A bispecific anti-F-actin anti-CD137 antibody comprising a heavy chain of the IgG2 isotype and a light chain of the κ or λ isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers. [AA48] A bispecific antibody or pharmaceutical composition of [AA47] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA49] A bispecific anti-F-actin anti-CD137 antibody comprising a heavy chain of the IgG3 isotype and a light chain of either the κ or λ isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers. [AA50] A bispecific antibody or pharmaceutical composition of [AA49] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA51] 1. A bispecific anti-F-actin anti-CD137 antibody comprising a heavy chain of the IgG4 isotype and a light chain of the κ or λ isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers. [AA52] A bispecific antibody or pharmaceutical composition of [AA51] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA53] A bispecific antigen-binding molecule that is an anti-CD3 antibody comprising a Clec9A polypeptide, wherein the bispecific antigen-binding molecule is an IgG-type antibody comprising a heavy chain of the IgG1 isotype and a light chain of the κ or λ isotype, or a pharmaceutical composition comprising the bispecific antigen-binding molecule and one or more pharmaceutically acceptable carriers. [AA54] A bispecific molecule or pharmaceutical composition of [AA53] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA55] A bispecific antigen-binding molecule that is an anti-CD3 antibody comprising a Clec9A polypeptide, wherein the bispecific antigen-binding molecule is an IgG type antibody comprising a heavy chain of an IgG2 isotype and a light chain of a κ or λ isotype, or a pharmaceutical composition comprising the bispecific antigen-binding molecule and one or more pharmaceutically acceptable carriers. [AA56] A bispecific molecule or pharmaceutical composition of [AA55] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA57] A bispecific antigen-binding molecule that is an anti-CD3 antibody comprising a Clec9A polypeptide, wherein the bispecific antigen-binding molecule is an IgG-type antibody comprising a heavy chain of the IgG3 isotype and a light chain of either the κ isotype or the λ isotype, or a pharmaceutical composition comprising the bispecific antigen-binding molecule and one or more pharmaceutically acceptable carriers. [AA58] A bispecific molecule or pharmaceutical composition of [AA57] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA59] A bispecific antigen-binding molecule that is an anti-CD3 antibody comprising a Clec9A polypeptide, wherein the bispecific antigen-binding molecule is an IgG-type antibody comprising a heavy chain of an IgG4 isotype and a light chain of a κ or λ isotype, or a pharmaceutical composition comprising the bispecific antigen-binding molecule and one or more pharmaceutically acceptable carriers. [AA60] A bispecific molecule or pharmaceutical composition of [AA59] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA61] A bispecific antigen-binding molecule that is an anti-CD137 antibody comprising a Clec9A polypeptide, wherein the bispecific antigen-binding molecule is an IgG type antibody comprising a heavy chain of IgG1 isotype and a light chain of κ or λ isotype, or a pharmaceutical composition comprising the bispecific antigen-binding molecule and one or more pharmaceutically acceptable carriers. [AA62] A bispecific molecule or pharmaceutical composition of [AA61] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA63] A bispecific antigen-binding molecule that is an anti-CD137 antibody comprising a Clec9A polypeptide, wherein the bispecific antigen-binding molecule is an IgG type antibody comprising a heavy chain of an IgG2 isotype and a light chain of a κ or λ isotype, or a pharmaceutical composition comprising the bispecific antigen-binding molecule and one or more pharmaceutically acceptable carriers. [AA64] A bispecific molecule or pharmaceutical composition of [AA63] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA65] A bispecific antigen-binding molecule that is an anti-CD137 antibody comprising a Clec9A polypeptide, wherein the bispecific antigen-binding molecule is an IgG-type antibody comprising a heavy chain of an IgG3 isotype and a light chain of a κ or λ isotype, or a pharmaceutical composition comprising the bispecific antigen-binding molecule and one or more pharmaceutically acceptable carriers. [AA66] A bispecific molecule or pharmaceutical composition of [AA65] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA67] A bispecific antigen-binding molecule that is an anti-CD137 antibody comprising a Clec9A polypeptide, wherein the bispecific antigen-binding molecule is an IgG-type antibody comprising a heavy chain of an IgG4 isotype and a light chain of a κ or λ isotype, or a pharmaceutical composition comprising the bispecific antigen-binding molecule and one or more pharmaceutically acceptable carriers. [AA68] A bispecific molecule or pharmaceutical composition of [AA67] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA69] A bispecific anti-splice factor 3B subunit 3 (SF3B3) anti-CD3 antibody comprising a heavy chain of the IgG1 isotype and a light chain of the kappa or lambda isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers. [AA70] A bispecific antibody or pharmaceutical composition of [AA69] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA71] A bispecific anti-splice factor 3B subunit 3 (SF3B3) anti-CD3 antibody comprising a heavy chain of the IgG2 isotype and a light chain of the kappa or lambda isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers. [AA72] A bispecific antibody or pharmaceutical composition of [AA71] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA73] A bispecific anti-splice factor 3B subunit 3 (SF3B3) anti-CD3 antibody comprising a heavy chain of the IgG3 isotype and a light chain of the kappa or lambda isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers. [AA74] A bispecific antibody or pharmaceutical composition of [AA73] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA75] 1. A bispecific anti-splice factor 3B subunit 3 (SF3B3) anti-CD3 antibody comprising a heavy chain of the IgG4 isotype and a light chain of the kappa or lambda isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers. [AA76] A bispecific antibody or pharmaceutical composition of [AA75] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA77] 1. A bispecific anti-splice factor 3B subunit 3 (SF3B3) anti-CD137 antibody comprising a heavy chain of the IgG1 isotype and a light chain of the kappa or lambda isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers. [AA78] A bispecific antibody or pharmaceutical composition of [AA77] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA79] 1. A bispecific anti-splice factor 3B subunit 3 (SF3B3) anti-CD137 antibody comprising a heavy chain of the IgG2 isotype and a light chain of the kappa or lambda isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers. [AA80] A bispecific antibody or pharmaceutical composition of [AA79] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA81] 1. A bispecific anti-splice factor 3B subunit 3 (SF3B3) anti-CD137 antibody comprising a heavy chain of the IgG3 isotype and a light chain of the kappa or lambda isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers. [AA82] A bispecific antibody or pharmaceutical composition of [AA81] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA83] 1. A bispecific anti-splice factor 3B subunit 3 (SF3B3) anti-CD137 antibody comprising a heavy chain of the IgG4 isotype and a light chain of the kappa or lambda isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers. [AA84] A bispecific antibody or pharmaceutical composition of [AA83] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA85] A bispecific antigen-binding molecule that is an anti-CD3 antibody comprising a Clec4e polypeptide, wherein the bispecific antigen-binding molecule is an IgG-type antibody comprising a heavy chain of the IgG1 isotype and a light chain of the κ or λ isotype, or a pharmaceutical composition comprising the bispecific antigen-binding molecule and one or more pharmaceutically acceptable carriers. [AA86] A bispecific molecule or pharmaceutical composition of [AA85] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA87] A bispecific antigen-binding molecule that is an anti-CD3 antibody comprising a Clec4e polypeptide, wherein the bispecific antigen-binding molecule is an IgG-type antibody comprising a heavy chain of the IgG2 isotype and a light chain of the κ or λ isotype, or a pharmaceutical composition comprising the bispecific antigen-binding molecule and one or more pharmaceutically acceptable carriers. [AA88] A bispecific molecule or pharmaceutical composition of [AA87] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA89] A bispecific antigen-binding molecule that is an anti-CD3 antibody comprising a Clec4e polypeptide, wherein the bispecific antigen-binding molecule is an IgG-type antibody comprising a heavy chain of the IgG3 isotype and a light chain of the κ or λ isotype, or a pharmaceutical composition comprising the bispecific antigen-binding molecule and one or more pharmaceutically acceptable carriers. [AA90] A bispecific molecule or pharmaceutical composition of [AA89] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA91] A bispecific antigen-binding molecule that is an anti-CD3 antibody comprising a Clec4e polypeptide, wherein the bispecific antigen-binding molecule is an IgG-type antibody comprising a heavy chain of the IgG4 isotype and a light chain of the κ or λ isotype, or a pharmaceutical composition comprising the bispecific antigen-binding molecule and one or more pharmaceutically acceptable carriers. [AA92] A bispecific molecule or pharmaceutical composition of [AA91] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA93] A bispecific antigen-binding molecule that is an anti-CD137 antibody comprising a Clec4e polypeptide, wherein the bispecific antigen-binding molecule is an IgG type antibody comprising a heavy chain of IgG1 isotype and a light chain of κ or λ isotype, or a pharmaceutical composition comprising the bispecific antigen-binding molecule and one or more pharmaceutically acceptable carriers. [AA94] A bispecific molecule or pharmaceutical composition of [AA93] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA95] A bispecific antigen-binding molecule that is an anti-CD137 antibody comprising a Clec4e polypeptide, wherein the bispecific antigen-binding molecule is an IgG type antibody comprising a heavy chain of an IgG2 isotype and a light chain of a κ or λ isotype, or a pharmaceutical composition comprising the bispecific antigen-binding molecule and one or more pharmaceutically acceptable carriers. [AA96] A bispecific molecule or pharmaceutical composition of [AA95] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA97] A bispecific antigen-binding molecule that is an anti-CD137 antibody comprising a Clec4e polypeptide, wherein the bispecific antigen-binding molecule is an IgG type antibody comprising a heavy chain of IgG3 isotype and a light chain of κ or λ isotype, or a pharmaceutical composition comprising the bispecific antigen-binding molecule and one or more pharmaceutically acceptable carriers. [AA98] A bispecific molecule or pharmaceutical composition of [AA97] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA99] A bispecific antigen-binding molecule that is an anti-CD137 antibody comprising a Clec4e polypeptide, wherein the bispecific antigen-binding molecule is an IgG-type antibody comprising a heavy chain of the IgG4 isotype and a light chain of the κ or λ isotype, or a pharmaceutical composition comprising the bispecific antigen-binding molecule and one or more pharmaceutically acceptable carriers. [AA100] A bispecific molecule or pharmaceutical composition of [AA99] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA101] 1. A bispecific anti-proliferating cell nuclear antigen (PCNA) anti-CD3 antibody comprising a heavy chain of the IgG1 isotype and a light chain of either the κ or λ isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers. [AA102] A bispecific antibody or pharmaceutical composition of [AA101] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA103] 1. A bispecific anti-proliferating cell nuclear antigen (PCNA) anti-CD3 antibody comprising a heavy chain of the IgG2 isotype and a light chain of either the kappa or lambda isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers. [AA104] A bispecific antibody or pharmaceutical composition of [AA103] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA105] 1. A bispecific anti-proliferating cell nuclear antigen (PCNA) anti-CD3 antibody comprising a heavy chain of the IgG3 isotype and a light chain of either the kappa or lambda isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers. [AA106] A bispecific antibody or pharmaceutical composition of [AA105] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA107] 1. A bispecific anti-proliferating cell nuclear antigen (PCNA) anti-CD3 antibody comprising a heavy chain of the IgG4 isotype and a light chain of either the kappa or lambda isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers. [AA108] A bispecific antibody or pharmaceutical composition of [AA107] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA109] 1. A bispecific anti-proliferating cell nuclear antigen (PCNA) anti-CD137 antibody comprising a heavy chain of the IgG1 isotype and a light chain of either the kappa or lambda isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers. [AA110] A bispecific molecule or pharmaceutical composition of [AA109] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA111] 1. A bispecific anti-proliferating cell nuclear antigen (PCNA) anti-CD137 antibody comprising a heavy chain of the IgG2 isotype and a light chain of either the kappa or lambda isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers. [AA112] A bispecific antibody or pharmaceutical composition of [AA111] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA113] 1. A bispecific anti-proliferating cell nuclear antigen (PCNA) anti-CD137 antibody comprising a heavy chain of the IgG3 isotype and a light chain of either the kappa or lambda isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers. [AA114] A bispecific antibody or pharmaceutical composition of [AA113] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA115] 1. A bispecific anti-proliferating cell nuclear antigen (PCNA) anti-CD137 antibody comprising a heavy chain of the IgG4 isotype and a light chain of either the kappa or lambda isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers. [AA116] A bispecific antibody or pharmaceutical composition of [AA115] for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor. [AA117] (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 or a tumor cell, and the molecule comprises at least one third moiety that binds to a third antigen; the third antigen is different from the first antigen; preferably, the third antigen is a membrane protein of an immune cell or a tumor cell; and The first antigen is (a) F-actin; (b) phosphatidylserine (PS); (c) splicing factor 3B subunit 3 (SF3B3); and (d) Proliferating cell nuclear antigen (PCNA) selected from the group consisting of Antigen-binding molecules. [AA118] The antigen-binding molecule of [AA117], wherein (i) the third antigen is different from the first and second antigens, or (ii) the third antigen is different from the first antigen but the same as the second antigen. [AA119] The first part is (A) an Ig-type binding moiety; or (B) a binding polypeptide, particularly a non-Ig type binding moiety An antigen-binding molecule selected from the group consisting of [AA117] to [AA118]. [AA120] (A) if the first antigen is splicing factor 3B subunit 3 (SF3B3), the first moiety is an Ig-type binding moiety; or (B) when the first antigen is splicing factor 3B subunit 3 (SF3B3), the first moiety is a Clec4e polypeptide; or (C) if the first antigen is proliferating cell nuclear antigen (PCNA), the first moiety is an Ig-type binding moiety; or (D) if the first antigen is F-actin, the first moiety is an Ig-type binding moiety; or (E) when the first antigen is F-actin, the first moiety is a Clec9A polypeptide; or (F) if the first antigen is PS, the first moiety is an Ig-type binding moiety; Antigen-binding molecule of [AA119]. [AA121] the second and / or third 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 these, the second and / or third antigen is a membrane protein involved in signal transduction in cells, preferably a signal transduction receptor. An antigen-binding molecule selected from [AA117] to [AA120]. [AA122] The antigen-binding molecule of any one of [AA117] to [AA121], wherein the second and / or third antigen is a membrane protein of a tumor cell. [AA123] The antigen-binding molecule of any one of [AA117] to [AA122], wherein the second and / or third moiety is an Ig-type binding moiety. [AA124] An antigen-binding molecule of any one of [AA117] to [AA123], which is an antibody, particularly an IgG antibody. [AA125] the antibody further comprises at least one binding polypeptide capable of binding to a membrane protein of an immune cell or a tumor cell, and preferably linked to an Fc region; In particular, the binding polypeptide is selected from a Clec9A polypeptide and a Clec4e polypeptide; Among others, the antibody an anti-CD3 antibody comprising a Clec9A polypeptide; an anti-CD137 antibody comprising a Clec9A polypeptide; an anti-CD3 antibody comprising a Clec4e polypeptide, and Anti-CD137 Antibodies Containing Clec4e Polypeptides selected from the group consisting of Antigen-binding molecule of [AA124]. [AA126] the antibody is a trispecific antibody; In particular, the antigen-binding molecule is (i) against membrane proteins of immune cells and against membrane proteins of tumor cells; and (a) F-actin; (b) phosphatidylserine (PS); (c) splicing factor 3B subunit 3 (SF3B3); and (d) proliferating cell nuclear antigen (PCNA), or (ii) against a first membrane protein of an immune cell and a second membrane protein of an immune cell that is different from the first membrane protein; and (a) F-actin; (b) phosphatidylserine (PS); (c) splicing factor 3B subunit 3 (SF3B3); and (d) proliferating cell nuclear antigen (PCNA), or (iii) to a first membrane protein of a tumor cell and a second membrane protein of the tumor cell that is different from the first membrane protein; and (a) F-actin; (b) phosphatidylserine (PS); (c) splicing factor 3B subunit 3 (SF3B3); and (d) proliferating cell nuclear antigen (PCNA). It is a trispecific antibody, Among others, the antibody trispecific anti-SF3B3 anti-CD3 anti-CD137 antibody, trispecific anti-PCNA anti-CD3 anti-CD137 antibody, trispecific anti-PS anti-CD3 anti-CD137 antibody, Trispecific anti-F-actin, anti-CD3, and anti-CD137 antibodies a bispecific anti-CD3 anti-CD137 antibody comprising a Clec9A polypeptide, and Bispecific anti-CD3 anti-CD137 antibodies containing Clec4e polypeptides selected from the group consisting of Antigen-binding molecule of [AA125]. [AA127] A method for screening an antigen-binding molecule, comprising the steps of: (a) selecting one or more nucleic acids encoding at least one first portion, wherein the first portion binds to a first antigen, and the first antigen is a damage-associated molecular pattern (DAMP), the first antigen being selected from the group consisting of: a) F-actin; b) phosphatidylserine (PS); c) splicing factor 3B subunit 3 (SF3B3); and d) proliferating cell nuclear antigen (PCNA); (b) selecting one or more nucleic acids encoding at least one second portion, wherein the second portion binds to a second antigen and the second antigen is different from the first antigen, preferably the second antigen is a membrane protein of an immune cell or a tumor cell; (b1) providing one or more nucleic acids encoding at least one third portion, wherein the third portion binds to a third antigen, the third antigen being different from the first antigen, and preferably the third antigen being a membrane protein of an immune cell or a tumor cell; (c) obtaining one or more nucleic acids encoding the antigen-binding molecule, wherein at least one first portion provided in (a), at least one second portion provided in (b), and at least one third portion provided in (b1) are linked; and (d) producing an antigen-binding molecule using one or more nucleic acids prepared in (c), thereby obtaining the antigen-binding molecule. [AA128] A method for producing an antigen-binding molecule, comprising the steps of: (a) providing one or more nucleic acids encoding at least one first portion, wherein the first portion binds to a first antigen, and the first antigen is a damage-associated molecular pattern (DAMP); the first antigen is selected from the group consisting of: a) F-actin; b) phosphatidylserine (PS); c) splicing factor 3B subunit 3 (SF3B3); and d) proliferating cell nuclear antigen (PCNA); (b) providing one or more nucleic acids encoding at least one second portion, wherein the second portion binds to a second antigen and the second antigen is different from the first antigen, preferably the second antigen is a membrane protein of an immune cell or a tumor cell; (b1) providing one or more nucleic acids encoding at least one third portion, wherein the third portion binds to a third antigen, the third antigen being different from the first antigen, and preferably the third antigen being a membrane protein of an immune cell or a tumor cell; (c) obtaining one or more nucleic acids encoding the antigen-binding molecule, wherein at least one first portion provided in (a), at least one second portion provided in (b), and at least one third portion provided in (b1) are linked; and (d) producing an antigen-binding molecule using one or more nucleic acids prepared in (c), thereby obtaining the antigen-binding molecule. [AA129] A method for conferring signal transduction or blocking in a cell, comprising the step of contacting a cell with an antigen-binding molecule or administering an antigen-binding molecule to a subject, wherein the antigen-binding molecule: (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; Including, 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 or a tumor cell, and the molecule comprises at least one third moiety that binds to a third antigen; the third antigen is different from the first antigen; preferably, the third antigen is a membrane protein of an immune cell or a tumor cell; and The first antigen is (a) F-actin; (b) phosphatidylserine (PS); (c) splicing factor 3B subunit 3 (SF3B3); and (d) Proliferating cell nuclear antigen (PCNA) selected from the group consisting of method. [AA130] A method for activating or inhibiting an immune response, comprising the step of contacting an antigen-binding molecule with a cell or administering an antigen-binding molecule to a subject, wherein the antigen-binding molecule: (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; Including, 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 or a tumor cell, and the molecule comprises at least one third moiety that binds to a third antigen; the third antigen is different from the first antigen; preferably, the third antigen is a membrane protein of an immune cell or a tumor cell; and The first antigen is (a) F-actin; (b) phosphatidylserine (PS); (c) splicing factor 3B subunit 3 (SF3B3); and (d) Proliferating cell nuclear antigen (PCNA) selected from the group consisting of method.

[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 metabolite 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, histone, or histone-containing nucleosome that forms the cytoskeleton or 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] Any one of the molecules [A1] to [A8], wherein the second antigen is a membrane protein involved in signal transduction in cells. [A10] The molecule according to [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, 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 binding of one or more of the molecules to a first antigen binds to one or more 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], wherein the molecule confers signal transduction via a membrane protein in a cell. [A15] The molecule according to [A13], wherein the molecule provides signal blocking via a membrane protein in a cell. [A16] Any one of the molecules [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 [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 molecule or a 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 of [A1] to [A20], wherein the first portion is an antibody variable region or a single domain antibody (VHH). [A22] The molecule of any one of [A1] to [A20], wherein the first antigen is F-actin and the first moiety 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] The molecule of any one 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 comprising two Fab regions that binds to a second antigen; and The first moiety is a molecule that binds to F-actin or a portion thereof, and 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 according to [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, 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 binding of one or more of the molecules to F-actin binds to one or more membrane proteins via a second portion, thereby providing signal transduction or blocking via the membrane protein in a cell. [A30] The molecule according to [A29], wherein the molecule confers signal transduction via a membrane protein in a cell. [A31] The molecule according to [A29], wherein the molecule 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 molecule or a 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 of [A25] to [A36], wherein the first portion is an antibody variable region or a single domain antibody (VHH). [A38] The molecule of any one of [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] The molecule of any one 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 of any one of [A1] to [A20], which includes a compound comprising a first portion and a second portion, or which is a compound comprising a first portion and a second portion. [A41] The molecule of [A40], wherein the compound is a compound that binds to a first antigen and a second antigen upon receiving light irradiation.

[0016] A pharmaceutical composition comprising any one of the molecules [B1][A1] to [A41]. [B2] The pharmaceutical composition of [B1] for providing signal transduction or blocking in cells. [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] A pharmaceutical composition according to any one 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] Molecules of [B6] to provide signaling or blocking in cells. [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 immunity. [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 providing or blocking signaling in a cell. [B13] The use of [B11] or [B12], wherein the agent is an agent for providing signaling or blocking in immune cells. [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 signal transduction in cells, 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 any one of the molecules of [B17][A1] to [A41] to a subject. [B18] The method according to any one of [B15] to [B17], wherein the cells are immune cells, cancer cells, or autoreactive cells. A method for treating or preventing cancer or an autoimmune disease, comprising the step of administering to a subject a molecule selected from any one of [B19][A1] to [A41]. [B20] A method for producing any one of the molecules [A1] to [A39]. 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]. A vector comprising the polynucleotide of [B23][B22]. A vector comprising a DNA sequence encoding an antigen-binding molecule as defined in any one of [B24], [A1] to [A39], particularly for recombinant protein expression. Host cells carrying the [B25][B23] or [B24] vector.

[0017] In addition, the present disclosure also particularly 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 metabolite 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 according to [C1], wherein the cell death is cell death in an affected tissue. [C3] The molecule of [C1] or [C2], wherein the first antigen is a filament, histone, or 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] Any one of the molecules [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 cells. [C9] The molecule according to [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 moiety 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 comprising two Fab regions that binds to a second antigen; and The first moiety is a molecule that binds to F-actin or a portion thereof, and 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 also 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, particularly an antigen 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] The antigen-binding molecule of any one of [D1] and [D2], wherein the damage-associated molecular pattern (DAMP) is exposed to the extracellular environment due to cell damage, particularly cell death. [D4] The molecule is (A) the cell damage can provide cell contact and / or signaling, the contact and / or signaling 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 the antigen-binding region, preferably the 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, 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; 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, cell death; and / or (ii) occurs in diseased tissues, particularly selected from tissues affected by a condition or disease, particularly cancer or an autoimmune disease, especially in the tumor microenvironment (TME); and / or (iii) caused by any one selected from the group consisting of 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 (especially intermediate filaments), nucleosomes, the cytoskeleton, and / or the nucleoskeleton; 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 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, cytoskeleton, and / or nucleoskeletons; Any one of the antigen-binding molecules [D1] to [D5]. [D7] The first antigen is (A) Actin, especially F-actin, or (B) heat shock proteins, particularly HSP90, selected from HSP90 and GRP78; (C) phosphatidylserine (PS), specifically phosphatidylserine that is part of the cell membrane; (D) histones or their components; (E) Histone deacetylase complex subunits, particularly 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 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 others, 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, particularly selected from HSP90 and GRP78; (C) a moiety capable of binding to phosphatidylserine (PS), particularly 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, particularly 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 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 antibody 1.5.1 or 6H8; and / or (E) the moiety capable of binding to SAP130 is a binding polypeptide, preferably a Clec4e 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, Fv, a single chain Fv2 (scFv2), Fab, and 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 Clec4e polypeptide; An antigen-binding molecule selected from [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 these, the second antigen is a membrane protein involved in signal transduction in cells, preferably a signal transduction receptor. An antigen-binding molecule selected from [D1] to [D12]. [D14] The antigen-binding molecule of any one of [D1] to [D13], wherein the second moiety is an Ig-type binding moiety. [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 signaling receptor, and / or (A-3) the cell is (i) an immune cell (particularly, 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), (ii) a tumor cell, or (iii) an autoreactive cell; and / or (B) immune cell receptor antigen, An antigen-binding molecule selected from any one of [D1] to [D14]. [D16] The second antigen is (A) Endosomal membrane proteins, (B) Membrane proteins in the plasma membrane of T cells. membrane proteins in the plasma membrane of T cells selected from the group consisting of T cell receptors, CD3, CD137, CD40, CTLA4, costimulatory molecules, or costimulatory molecules, (Among the membrane proteins capable of binding to costimulatory or costinhibitory molecules are selected from 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. An antigen-binding molecule selected from any one of [D1] to [D15]. [D17] The second part is (A) A moiety capable of binding to an endosomal membrane protein; (B) a moiety capable of binding to a membrane protein in the plasma membrane of a T cell, particularly 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; (Among the membrane proteins capable of binding to costimulatory or costimulatory molecules are selected from 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.) selected from the group consisting of Among others, 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; 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. An antigen-binding molecule selected from any one of [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, Fv, a single chain Fv2 (scFv2), Fab, and 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 through said one or more membrane proteins in a cell; 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 confers signal blocking via said one or more membrane proteins in a cell; An antigen-binding molecule selected from any one of [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) selected from the group consisting of especially, (A-1) the molecule is a polypeptide complex; Among others, 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, An antigen-binding molecule selected from any one of [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 Clec4e polypeptide; Among others, the antibody an anti-CD3 antibody comprising a Clec9A polypeptide; an anti-CD137 antibody comprising a Clec9A polypeptide; an anti-CD3 antibody comprising a Clec4e polypeptide, and Anti-CD137 Antibodies Containing Clec4e Polypeptides selected from the group consisting of An antigen-binding molecule selected from [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 bispecific anti-HSP90 anti-CD3 antibody, bispecific anti-HSP90 anti-CD3 antibody, bispecific anti-GRP78 anti-CD3 antibody, bispecific anti-GRP78 anti-CD137 antibody, a bispecific anti-phosphatidylserine anti-CD3 antibody, and Bispecific anti-phosphatidylserine anti-CD137 antibody selected from the group consisting of An antigen-binding molecule selected from [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 moiety is an Ig-type binding moiety; 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, Fv, a single chain Fv2 (scFv2), Fab, and 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, particularly a non-Ig binding moiety attached to the Fc domain of the antigen-binding molecule. An antigen-binding molecule selected from any one of [D1] to [D24]. [D26] The molecule is (A) Antibodies capable of binding to F-actin and antigens 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 cells; (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 cells; (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 cells; and (J) Antibodies capable of binding to SAP130 and immune cell receptors 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) Antibodies 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, among others, two first moieties capable of binding to F-actin, which are binding polypeptides, preferably Clec9A polypeptides, attached to the Fc domain of the antibody, and two second moieties involved in signal transduction in cells, among others, Ig-type binding moieties; (B) an antibody comprising, among others, two first moieties capable of binding to F-actin, 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 cells, among them Ig-type binding moieties; (D) an antibody comprising two first moieties capable of binding to HSP90, among them Ig-type binding moieties, and two second moieties that are immune cell receptor antigens, among them Ig-type binding moieties; (E) An antibody comprising two first moieties capable of binding to GRP78, among which Ig-type binding moieties, and two second moieties involved in signal transduction in a cell, among which Ig-type binding moieties; (F) An antibody comprising two first moieties capable of binding to GRP78, among which Ig-type binding moieties, and two second moieties that are immune cell receptor antigens, among which 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 them Ig-type binding moieties, and two second moieties which are immune cell receptor antigens, among them Ig-type binding moieties; (I) an antibody comprising, among others, two first moieties capable of binding to SAP130, which are binding polypeptides, preferably Clec4e polypeptides, attached to the Fc domain of the antibody, 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, which are binding polypeptides, preferably Clec4e polypeptides, among others, attached to the Fc domain of the antibody, and two second moieties which are immune cell receptor antigens, among others, Ig-type binding moieties. selected from the group consisting of Preferably, the molecule is (A1) An antibody comprising, among others, two first moieties capable of binding to F-actin, which are binding polypeptides, preferably Clec9A polypeptides, attached to the Fc domain of the antibody, and two second moieties capable of binding to CD3, among others, Ig-type binding moieties; (A2) An antibody comprising, among others, two first moieties capable of binding to F-actin, which are binding polypeptides, preferably Clec9A polypeptides, attached to the Fc domain of the antibody, 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 them Ig-type binding moieties, and two second moieties capable of binding to CD3, among them Ig-type binding moieties; (C2) An antibody comprising two first moieties capable of binding to HSP90, among them Ig-type binding moieties, and two second moieties capable of binding to CD137, among them 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 them Ig-type binding moieties, and two second moieties capable of binding to CD137, among them Ig-type binding moieties; (G1) 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 CD3, among them Ig-type binding moieties; (G2) An antibody comprising two first moieties capable of binding to phosphatidylserine, particularly Ig-type binding moieties, and two second moieties capable of binding to CD137, particularly Ig-type binding moieties; (I1) An antibody comprising two first moieties capable of binding to SAP130, among others, which are binding polypeptides, preferably Clec4e polypeptides, attached to the Fc domain of the antibody, and two second moieties capable of binding to CD3, among others, which are Ig-type binding moieties; (I2) An antibody comprising two first moieties capable of binding to SAP130, preferably Clec4e polypeptides, among others, attached to the Fc domain of the antibody, and two second moieties capable of binding to CD137, among others, Ig-type binding moieties. selected from the group consisting of An antigen-binding molecule selected from any one of [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 for 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, the method comprising contacting the antigen-binding molecule with a damage-associated molecular pattern (DAMP) in the patient, and in particular further comprising contacting the antigen-binding molecule with the second antigen; (v) a method for 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, particularly cell death; (Among other things, 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 [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 according to [D31], wherein the medical condition is selected from the group consisting of cancer and immune diseases, 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 for activating or inhibiting an immune response, and / or (iii) the method is for conferring signal transduction or blocking in a cell; especially, (a) the method includes a step of contacting the antigen-binding molecule with a damage-associated molecular pattern (DAMP) exposed to the extracellular environment due to cellular 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 (in particular, 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 others, the antigen-binding molecule further comprises (c-1) at least two, preferably two moieties involved in signal transduction in cells, 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 a method 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]. A polynucleotide, particularly DNA, encoding any one of the molecules [D36][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 containing 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 to target cells in a tissue where 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 tissues where 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 acts specifically 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; Includes a kit.

[0019] In addition, the present disclosure also particularly 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 different 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 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-binding moiety 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 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 a Clec4e polypeptide. [E14] The 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 moiety is an Ig-type binding moiety. [E16] The antigen-binding molecule of any one of [E1] to [E15], wherein the molecule is an IgG 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 a Clec4e 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 Clec4e polypeptide, and Anti-CD137 Antibodies Containing Clec4e 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, a bispecific anti-phosphatidylserine anti-CD3 antibody, 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 containing 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, the method 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 explanation of the drawings]

[0020] [Figure 1] A drawing that schematically illustrates the technical concept encompassed and achieved by the present invention when the molecule of the present invention is an antibody-like molecule that comprises a portion that binds to F-actin, which is exposed to the extracellular environment upon cell death, and also illustrates that the molecule forms a complex with F-actin, and the complex binds to one or more membrane proteins in the cell via the molecule, thereby imparting a signal or blockage in the cell. [Figure 2] FIG. 1 is a drawing 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. [Figure 3]

[0023] Figures schematically illustrate the technical features of various embodiments of the molecules of the present invention, which are antibody- or Ig-like molecules comprising a moiety that binds to a cellular component or portion thereof exposed to the extracellular environment upon cell death. More specifically, the figures schematically illustrate various embodiments of the antibody- or Ig-like molecules of the present invention comprising at least one "first moiety," at least one "second moiety," and an Fc region, as well as embodiments comprising at least one "first moiety," at least one "second moiety," at least one "third moiety," and an Fc region. For example, a circle (with a white horizontal brick pattern) represents a "first moiety" that binds to a "first antigen." In a further example, when two first moieties (circles with a white horizontal brick pattern) 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 another Fab arm can bind to any antigen different from the "first antigen" and the "second antigen," and in particular, the third Fab arm can bind to the "third antigen." In a further example, at least one Fab arm binds to the "second antigen," and another Fab arm can bind to the "first antigen," and the conjugated first moiety (white horizontal brick circle) can bind to any antigen different from the "first antigen" and the "second antigen," and in particular, the third Fab arm can bind to the "third antigen." In one example, at least one Fab arm binds to the "first antigen," and the second Fab arm can bind to the "second antigen," and the third Fab arm can bind to any antigen different from the "first antigen" and the "second antigen," and in particular, the third Fab arm can bind to the "third antigen."In another example, at least one first moiety binds to a "first antigen," and the second Fab arm is capable of binding to a "second antigen," and the third Fab arm is capable of binding to any antigen different from the "first antigen" and the "second antigen," and in particular, the third Fab arm is capable of binding to a "third antigen." Further embodiments, schematically illustrated in Figure 3, are described in more detail below. [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. [Figure 5] Figure 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] Figure 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] Figure 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] Figure 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] Figure 1 shows the results of CD3 activation by Clec9A-conjugated antibodies in the presence or absence of F-actin. [Figure 11A] A drawing showing the results of measuring the binding of anti-SF3B3 antibody and anti-PCNA antibody to dead cells. [Figure 11B] A diagram showing the results of measuring the binding of anti-SF3B3 antibody and anti-PCNA antibody to live cells. [Figure 12] Figure 1 shows the results of measuring CD3+ T cell activation through clustering of anti-SF3B3 / / CD3 bispecific antibodies on T cells in the presence of live or dead cells. [Figure 13] Figure 1 shows the results of measuring CD3+ T cell activation through clustering of anti-PCNA / / CD3 bispecific antibodies on T cells in the presence of live or dead cells. [Figure 14A] Figure showing the results of measuring the binding of anti-PS(1N11) antibody to live or dead cells. [Figure 14B] A figure showing the results of measuring the binding of anti-actin (8E5D12A9) antibody to live or dead cells. [Figure 15A] Figure 1 shows the results of measuring CD3+ T cell activation through clustering of anti-PS(1N11) / / CD3+ bispecific antibodies on T cells in the presence of live or dead cells. [Figure 15B] Figure showing the results of measuring CD137+ T cell activation through clustering of anti-PS(1N11) / / CD137+ bispecific antibody on T cells in the presence of live or dead cells. [Figure 16] Figure showing the results of in vivo distribution of dead cell-binding molecules in a muscle injury model. DETAILED DESCRIPTION OF THE INVENTION

[0021] Description of 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 above aspects and embodiments of Group [AA] and embodiments of Groups [A], [B], [C], [D], and [E].

[0022] cell death Several types of cell death have been reported, 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, pp. 860-875, 2012). PCD is tightly controlled by signaling cascades and molecularly defined mechanisms. Apoptosis is the most well-known form of PCD, but 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 any 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, which is one of the aspects of programmed cell death (PCD); necrosis; accidental cell death; or regulated cell death (e.g., apoptosis, regulated necrosis, autophagic cell death, necroptosis, ferroptosis, or pyroptosis). Such cells may be located in any tissue, including but not limited to epithelial tissue, muscle tissue, nervous tissue, and connective tissue. Here, necrosis, a form of unprogrammed cell death caused by injury or inflammation, also results in increased cell death. Specifically, in the present invention, "cell death" may refer to cell death observed in tissues affected by, for example, cancer or autoimmune disease.

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

[0025] First antigen / damage-associated molecular pattern (DAMP) In the present invention, the term "first antigen," also referred to herein as "damage-associated molecular pattern" or "DAMP" and interchangeably used herein with "substances exposed to the extracellular environment due to cell damage" in accordance with one general aspect of the present invention, refers to a biological molecule associated with cell damage, which may also be referred to interchangeably as "damaged cell-derived molecule," "damaged cell-derived substance," "damaged cell-associated molecule," "damaged cell-associated substance," and "danger-associated molecular pattern." These biological molecules and substances associated with cell damage can be upregulated, expressed on the cell surface, modified, passively diffused, or actively secreted extracellularly upon cell damage. These changes are positively correlated with the presence or increase of damaged cells or the decrease of intact cells. In other words, in cells undergoing cell damage, components or portions thereof constituting the cytoskeleton, cell membrane, organelles, cytoplasmic proteins, or metabolites in the cell are exposed to the extracellular environment, and any of these are encapsulated by substances or biological molecules 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 metabolite, respectively, 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 metabolite, respectively, exposed to the extracellular environment due to cell damage."

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

[0029] In one aspect herein, the first antigen is selected from the group consisting of: (a) splicing factor 3B subunit 3 (SF3B3); and (b) proliferating cell nuclear antigen (PCNA). The terms "SF3B3 splicing factor 3b subunit 3" and "SF3B3" are used interchangeably herein to refer to subunit 3 of the splicing factor 3b protein complex. The sequence of human SF3B3, as a translated protein sequence, is disclosed in the May 22, 2022 version of NCBI Reference Sequence: NP_036558.3. The terms "proliferating cell nuclear antigen" and "PCNA" are used interchangeably herein to refer to the protein known in the art as proliferating cell nuclear antigen. Proliferating cell nuclear antigen, or PCNA, has been described to act as a processivity factor for DNA polymerase δ in eukaryotic cells and to function as a DNA clamp essential for replication. In vivo, PCNA has been described to be a homotrimer. PCNA has been described to act as a scaffold that recruits proteins involved in DNA replication, DNA repair, chromatin remodeling, and epigenetics. The sequence of human PCNA, as a translated protein sequence, is disclosed in the NCBI Reference Sequence: NP_872590.1, version of June 19, 2022. 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 "substances exposed to the extracellular environment upon cell death," refers to biomolecules associated with cell death, and may also be interchangeably referred to as "dead cell-derived molecules," "dead cell-derived substances," "dead cell-associated molecules," "dead cell-associated substances," and "danger-associated molecular patterns." These biomolecules and substances associated with cell death can be upregulated, expressed on the cell surface, modified, passively diffused, or actively secreted extracellularly upon cell death. These changes are positively correlated with the presence or increase of dead cells or the decrease in viable cells. In other words, in cells undergoing cell death, components or parts of the cytoskeleton, cell membrane, organelles, cytoplasmic proteins, or metabolic products in the cell are exposed to the extracellular environment, and any of these are engulfed by substances or biomolecules 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 also be referred to interchangeably as "a component or a portion of the cytoskeleton, cell membrane, organelle, cytoplasmic protein, or metabolite that is exposed to the extracellular environment due to cell death," and also as "first antigen" (or, respectively, "a component or a portion of the cytoskeleton, cell membrane, organelle, cytoplasmic protein, or metabolite that is exposed to the extracellular environment due to cell damage"). 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 portion of the cytoskeleton, cell membrane, organelle, cytoplasmic protein, or metabolite that is exposed to the extracellular environment due to cell death," or "a component or a portion of the cytoskeleton, cell membrane, organelle, cytoplasmic protein, or metabolite that is 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 forces required for intracellular and extracellular movement of cells and for maintaining the cell's shape. In the present invention, "filaments" include, but are 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 their 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 upon cell death is not limited to the protein components that form the cytoskeleton described above, and may be conjugated with one or more or 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, etc., lipid-anchored proteins such as G proteins, etc., and peripheral membrane proteins such as enzymes, hormones, etc.

[0035] Generally, in the present invention, the term "membrane protein" refers to membrane proteins, which are generally 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 be associated 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 certain types or combinations of non-covalent interactions). This term particularly 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 proteins embedded in biological membranes.

[0037] In the present invention, "substances / molecules exposed to the extracellular environment upon cell death" ("dead cell-derived molecules") and DAMPs include, but are not limited to, the above-mentioned components or portions thereof. Any substance or molecule exposed to the extracellular environment upon cell death may be utilized in the present invention. Furthermore, dead cell-derived molecules may be complexes of one or more of the above-mentioned components or portions thereof. Dead cell-derived molecules may be whole or truncated proteins. Preferred embodiments of dead cell-derived molecules include, but are not limited to, cytoskeleton, organelles, nuclear proteins, and cytoplasmic proteins in cells. Cell membranes and any fragments of cell membranes are also preferred embodiments of dead cell-derived molecules. In the present invention, more preferred embodiments of dead cell-derived molecules include, but are not limited to, filaments constituting the cytoskeleton or nucleoskeleton, and nucleosomes or molecules forming nucleosomes. In the present invention, further preferred embodiments of dead cell-derived molecules include, but are not limited to, components of the spliceosome.

[0038] In the present invention, molecules derived from dead cells include, but are not limited to, molecules released from cells into the extracellular environment due to immunogenic cell death, i.e., damage-associated molecular patterns (DAMPs) released due to immunogenic cell death. DAMPs include those reported by Dmitri et al., Nature Reviews Cancer, Vol. 12, pp. 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 upon cell death" or "components or parts of the cytoskeleton, cell membrane, organelle, cytoplasmic protein, or metabolites exposed to the extracellular environment upon 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. Furthermore, in some preferred embodiments, the organelle may be a mitochondrion, a nucleus, a lysosome, a Golgi apparatus, or an endoplasmic reticulum. By way of further example, and not limitation, such dead cell-derived molecules of the present invention include molecules normally present in the cytosol or nucleus, including amyloid beta, adenosine triphosphate (ATP), uric acid crystals, myosin light chain, α1-microglobulin, α2-microglobulin, cyclophilin A, F-actin, HSP70, HSP90, gp96, GRP94, HSP110, HSP170, calgranulin A, calgranulin B, calreticulin, Gc-globulin, SF3B3, HMBG1, HMGN1, IL1a, IL1b, IL33, serum amyloid protein, and the like. As a further example, a "DAMP" may be mitochondrial DNA (mtDNA), mitochondrial ROS (mROS) or mitochondrial TFA (TFAM), or a formyl peptide.

[0040] In addition, molecules such as glypicans and syndecans are expressed on the surface of live cells but can be modified by the microenvironment rich in dead cells, and these molecules are also included in dead cell-derived molecules. Furthermore, biglycan, decorin, lumican, fibrinogen, fibronectin, the EDA domain of fibronectin, heparan sulfate, perlecan, low-molecular-weight hyaluronan, versican, and tenascin-C can also be sequestered in the extracellular matrix under normal physiological conditions but are proteolytically released as the number of dead cells increases. Furthermore, granules originally present intracellularly in live cells, such as cathelicidin (LL37), defensins, eosinophil-derived neurotoxin (EDN), and granulysin, can be released and localized in the microenvironment rich in dead cells, and are also included in dead cell-derived molecules. These molecules are also included in the present invention as dead cell-associated molecules.

[0041] Dead cell-derived molecules can also be molecules released upon induction of cell death or molecules exposed or abundantly expressed on the cell surface. All cells, including somatic and germ cells, are capable of producing dead cell-derived molecules. A common feature of solid tumors is the presence of hypoxia and nutrient deficiency due to reduced blood flow. These hypoxic and nutrient deficiency conditions stress tumor cells, resulting in extensive cell death in solid tumors. Because cell death is involved in the pathogenesis of autoimmune diseases, it is commonly observed in many types of autoimmune diseases, such as vitiligo and type 1 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 recognizing 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 at the plasma membrane at the time of cell death.

[0042] In the present invention, the source of dead cell-derived molecules is not limited to the above sources. Any molecule or substance present in or in cells exposed to the extracellular environment in which cell death is observed can be a source of dead cell-derived molecules. Whether a particular 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 characteristics or 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 a viable cell-impermeable DNA-binding dye, a viable cell-impermeable amine-reactive dye, measuring enzyme activity such as lactate dehydrogenase in the supernatant of dead cells, or a decrease in the level of intracellular ATP associated with metabolically active viable cells. Additionally, 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 particular 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 proteomic and / or metabolomic analysis of proteins and / or metabolites contained in each of the supernatants of cell cultures cultured in the presence of a cytotoxic drug and the supernatant of a cell culture without drug addition, molecules detected in relatively large amounts in the supernatant of a cell culture cultured with the addition of a cytotoxic drug can be determined to be dead cell-derived molecules (i.e., substances 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; any drug may be used as long as it has cytotoxicity.

[0046] In the present invention, the method for 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 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 measuring and comparing the expression level of one or more membrane proteins in each group of cells using flow cytometry.Membrane proteins whose expression is increased in drug-treated cells compared to that in untreated cells are determined to be molecules that can be used as dead cell-derived molecules.

[0048] In the present invention, the dead cell-derived molecules, including but not limited to RNA, DNA, filamentous action, histone, phosphatidylserine, or heat shock proteins, can be quantified using any means and / or method 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 method for quantification may also be used as long as the dead cell-derived molecules of the present invention can be quantified.

[0049] In one aspect of the invention, the first antigen is selected from the group consisting of: (a) splicing factor 3B subunit 3 (SF3B3); and (b) proliferating cell nuclear antigen (PCNA). The terms "SF3B3 splicing factor 3b subunit 3" and "SF3B3" are used interchangeably herein to refer to subunit 3 of the splicing factor 3b protein complex. The sequence of human SF3B3, as a translated protein sequence, is disclosed in the May 22, 2022 version of NCBI Reference Sequence: NP_036558.3. In one embodiment, the SF3B3 is human SF3B3. In one embodiment, SF3B3 is human SF3B3, as disclosed as the translated protein sequence in the May 22, 2022 version of NCBI Reference Sequence: NP_036558.3. Splicing factor 3b, together with splicing factor 3a and the 12S RNA subunit, forms the U2 small nuclear ribonucleoprotein complex (U2 snRNP). The splicing factor 3b / 3a complex binds to pre-mRNA upstream of the intron branch site in a sequence-independent manner and can anchor the U2 snRNP to the pre-mRNA. Splicing factor 3b is also a component of the minor U12 spliceosome. Subunit 3 has also been identified as a component of the STAGA (SPT3-TAF(II)31-GCN5L acetylase) transcriptional coactivator-HAT (histone acetyltransferase) complex and the TFTC (TATA-binding protein-free TAF(II)-containing complex). These complexes may function in chromatin modification, transcription, splicing, and DNA repair.

[0050] The terms "proliferating cell nuclear antigen" and "PCNA" are used interchangeably herein to refer to the protein known in the art as proliferating cell nuclear antigen. Proliferating cell nuclear antigen, or PCNA, has been described to act as a processivity factor for DNA polymerase δ in eukaryotic cells and to function as a DNA clamp essential for replication. In vivo, PCNA has been described to be a homotrimer. PCNA has been described to act as a scaffold that recruits proteins involved in DNA replication, DNA repair, chromatin remodeling, and epigenetics. The sequence of human PCNA is disclosed as a translated protein sequence in NCBI Reference Sequence: NP_872590.1, version of June 19, 2022. Additionally, the sequence of human PCNA is disclosed as a translated protein sequence in NCBI Reference Sequence: NM_182649.2, version of June 19, 2022. In one embodiment, the PCNA is human PCNA. In one embodiment, PCNA is human PCNA, as disclosed as the translated protein sequence in the June 19, 2022 version of NCBI Reference Sequence: NP_872590.1.

[0051] Can be combined In the context of the present invention, the terms "capable of binding" and "bind" 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. Specific exemplary methods for determining said ability are described herein below. As used herein, "ability to bind to an antigen" and the like may also be designated as terms such as "binding affinity for an antigen." Furthermore, in the case of antibodies, said ability may also be described by using the term "antibody against" or by using the term "anti-antigen X antibody."

[0052] 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 part thereof that constitutes the cytoskeleton, cell membrane, organelle, cytoplasmic protein, or metabolic product that is exposed to the extracellular environment upon cell death," as described above.

[0053] The "first portion" of a molecule of the present invention may have any structure as long as it binds to the "first antigen" described above. The structure of the "first portion" may include, but is not limited to, a polypeptide or a portion thereof, a small or medium-sized chemical compound or a portion thereof, or a polynucleotide or a portion thereof. Polypeptides or portions thereof include, but are not limited to, cell membrane proteins expressed on cells (e.g., immune cells such as dendritic cells) or portions thereof (e.g., extracellular domains, any unique domains thereof); antibodies (including, but not limited to, human antibodies, chimeric antibodies, humanized antibodies, and VHH antibodies) or antigen-binding domains (also referred to as portions, parts, or fragments of antibodies). Antibody antigen-binding domains 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 Fv2s (scFv2s), Fabs, and F(ab')2s.

[0054] The polypeptide or a portion thereof may also be a module called an A domain of an Avimer (WO2004 / 044011 and WO2005 / 040229), which has approximately 35 amino acids contained in an in vivo cell membrane protein; an adnectin (WO2002 / 032925), which has an Fn3 domain that acts as a protein-binding domain derived from the glycoprotein fibronectin expressed on the cell membrane; an affibody (WO1995 / 001937), which has an IgG-binding domain scaffold that constitutes a three-helix bundle consisting of 58 amino acids of protein A; 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 anticalin having four loop regions connecting eight antiparallel strands bent toward a central axis at one end of a highly conserved barrel structure in lipocalin molecules 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 (VLRs) without immunoglobulin structure, as found in the adaptive immune systems of jawless vertebrates such as lampreys or hagfish (WO2008 / 016854).

[0055] In one embodiment of the polypeptide or a portion thereof that corresponds to the "first portion that binds to a first antigen" of the present invention, the polypeptide or a portion thereof 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). Representative receptors or portions thereof that correspond to the "first portion that binds to a first antigen" of the present invention include, but are not limited to, scavenger receptors, toll-like receptors (TLRs), C-type lectins (CLECs) such as Clec9A expressed on dendritic cells, NOD-like receptors (NLRs) (J Biol Chem. 2014 Dec 19;289(51):35237-45), and portions thereof. For example, polypeptides or portions thereof that belong to the "first portion that binds to a first antigen" as described above in the present invention include, but are not limited to, CD36, RAGE, NLRP1, NLRP3, CD147, CD91, TLR2, TLR3, TLR4, TLR7, TLR8, TLR9, AIM2, TIM3, SREC1, FEEL1, LOX-1, LRP1, CD14, IL-1R, ST2, RIG-I, MDA5, NKP44, and the like.

[0056] One preferred embodiment of the "first moiety that binds to a first antigen" of the present invention is Clec9A, a C-type lectin expressed on dendritic cells (DCs) that binds to the cytoskeleton-forming filament F-actin complex when the F-actin complex is exposed to the extracellular environment due to cell death. In the present invention, the "first moiety that binds to a first antigen" includes, but is not limited to, the entire polypeptide of Clec9A (GenBank: NP_001192292.1), the extracellular domain (ECD) of any amino acid length, the C-type lectin domain (CTLD), or any portion thereof. One preferred embodiment of the "first moiety that binds to a first antigen" of the present invention is Clec4e, also known as "C-type lectin domain family 4 member E," "macrophage-inducible Ca2+-dependent lectin receptor," or "Mincle." Splicing factor 3B subunit 3 (SF3B3) is reportedly released during cell death and signals through Mincle. In the present invention, a "first moiety that binds to a first antigen" includes, but is not limited to, the entire polypeptide or antigen-binding portion of Clec4e as set forth in UniProt Accession No. Q9ULY5 (version effective July 28, 2022). In a preferred embodiment herein, the Clec4e is human Clec4e. In one preferred embodiment, the "first portion that binds to a first antigen" of the present invention is the entire polypeptide or an antigen-binding portion of human Clec4e, for example, as described in UniProt Accession No. Q9ULY5 (version effective July 28, 2022).

[0057] A second moiety that binds to a second antigen In the present invention, the "second portion" of a "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" 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" targeted by the "second portion" of a "molecule comprising a first portion that binds to a first antigen and a second portion that binds to a second antigen." When the "second portion" of a molecule in the present invention targets the "second antigen," the target molecule, i.e., the "second antigen," brings about a desired outcome for the indicated disease, such as immune activation to combat infection or cancer, immune suppression for autoimmune disease and / or cytokine release syndrome (CRS), direct inhibition of cancer cell proliferation, or promotion of cell regeneration after tissue damage.

[0058] In the present invention, one common embodiment of immune activation targets includes, but is not limited to, CD3 ("CDε"), CD4, CD8, CD28, OX40, 4-1BB, and T cell receptors (TCRs) expressed on T cells, which result in the 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, and 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 targeting 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 acid:polycytidylic acid (PolyIC)-sensing receptor also expressed by DCs (J Clin Invest. 2018;128(10): 4387-4396). In other cases, inhibition of target molecules, such as antagonizing CTLA-4, PD-1, LAG-3, TIM-3, or VISTA on T cells, may be desirable to prevent suppression of the immune response against tumor cells. The targeted molecule (i.e., "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., "second antigens").

[0059] 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 of a cell. One embodiment of the membrane protein (i.e., second antigen) to which the "second moiety" binds includes, but is not limited to, membrane proteins involved in cell signal transduction, which are 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, neutrophils, and basophils. Another embodiment of the membrane protein (i.e., second antigen) to which the "second moiety" binds includes, but is not limited to, membrane proteins expressed on tumor cells or autoreactive cells.

[0060] In the present invention, membrane proteins belonging to the "second antigen" as described above 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), and 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.

[0061] 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, CD19, CD20 , CD21, CD22, CD23, CD25, CD27L, CD29, CD30L, CD32, CD33 (p67 protein), CD34, CD36, CD38, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD89, CD91, 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-31R; IL1 RL1 (ST2), integrin family, such as α1β1 (VLA-1), α2β1 (VLA-2), α3β1 (VLA-3), α4β1 (VLA-4), α5β1 (VLA-5), α6β1 (VLA-6), α 7β1, α8β1, α9β1, α11bβ3(GPIIb / IIIa), αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, αLβ2(LFA-1), αMβ2(Mac-1), αXβ2, αDβ2, α4β7;LOX-1, MDA5(MDA5, 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(WHO ATAR、HveA、LIGHT R、TR2)、TNFRSF16(NGFR p75NTR)、TNFRSF17(BCMA)、TNFRSF18(GITR AITR)、TNFRSF19(TROY TAJ、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, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, and TLR 10. RIG-1;RAGE, P2Y2, P2X7 )、、おインフェロ。。。。。。(A IM2) is also smooth;

[0062] In the present invention, the term "second antigen" also includes, but is not limited to, soluble proteins that can bind to membrane proteins such as those described above. In the present invention, soluble proteins that fall under the category of "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.

[0063] In the present invention, soluble proteins belonging to the category of "second antigens" further include, but are not limited to, the following molecules or any soluble form of said molecules (e.g., truncated 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, CD36, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD91, 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-related antigen, DAN, DCC, DcR3, DC-SIGN, Decay accelerating factor, des(1-3)-IGF-I (Brain IGF-1), Dhh, Digoxin, DNAM-1, Dnase, DppDPPIV / CD26, Dtk, 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 α4 / β1, integrin α4 / β7, integrin α5 (αV), integrin α5 / β1, integrin α5 / β3, integrin α6, integrin β1, integrin β2, interferon γ, 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, metalloproteinase, 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, 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, PDGF, PDK-1, PECAM, P EM, 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, 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, X CR1, XCR1, XEDAR, XIAP, XPD, HMGB1, IgA, CD81, CD97, CD98, DDR1, DKK1, EREG, Hsp90, IL-17 / IL-17R, IL-20 / IL-2 0R, oxidized LDL, PCSK9, prekallikrein, RON, TMEM16F, SOD1, chromogranin A, chromogranin B, tau, VAP1, high molecular weight kininogen, IL-31, IL-31R, IL1RL1(ST2), 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.

[0064] The examples of molecules listed above also include receptors (membrane proteins), which, even if present in a soluble form (e.g., truncated form) in body fluids, can be used as a "second antigen" (target molecule) to which the "second moiety" 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).

[0065] In the present invention, when the "second antigen" is a soluble protein, e.g., IL-6, a molecule comprising a first portion and a second portion of the present invention binds to a dead cell-derived molecule (i.e., the first antigen), e.g., an F-actin complex, via the first portion of the molecule, and to IL-6 (i.e., the second antigen) via the second portion of the molecule, thereby neutralizing IL-6, more specifically, in diseased tissues where dead cell-derived molecules are more abundant than in non-diseased tissues due to cell death. Neutralization of IL-6 indirectly controls the immune response triggered by immune cells.

[0066] In the present invention, the "second moiety" that binds to the "second antigen" described above may have any structure as long as it binds to the "second antigen" described above. The structure of the "second moiety" may include, but is not limited to, a polypeptide or a portion thereof, a small or medium-sized chemical compound or a portion thereof, or a polynucleotide or a portion thereof. Polypeptides or portions thereof include, but are not limited to, antibodies (including, but not limited to, human antibodies, chimeric antibodies, humanized antibodies, and VHH antibodies) or antigen-binding domains (also referred to as antibody portions, parts, or fragments). Antibody antigen-binding domains 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 Fv2s (scFv2s), Fabs, and F(ab')2s.

[0067] 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 a "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, molecules having the following properties:

[0068] 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 shown in Figure 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 molecular embodiments of the present invention are schematically illustrated in Figure 3. The two Fab arms can be conjugated to the Fc region in various formats, as illustrated in Figure 3. In this embodiment, an example of a "first moiety that binds to a first antigen" is all or a portion of Clec9A or a VHH.

[0069] ii. An antibody in which one Fab arm binds to a "first antigen" and the other Fab arm binds to a "second antigen." An example of this embodiment would be a bispecific antibody or bispecific antigen-binding domain in which one Fab arm binds to a dead cell-derived molecule (e.g., an 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., an immune cell, a cancer cell, an autoreactive cell). The antibody or antibody-like molecule in this example can have a variety of formats, as illustrated in Figure 3.

[0070] iii. A polypeptide comprising a "first portion that binds to a first antigen" and a "second portion that binds to 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).

[0071] iv. A chemical compound comprising a "first portion that binds to a first antigen" and a "second portion that binds to a second antigen." An example of this embodiment is a chemical compound comprising at least two portions, one of which binds to a dead cell-derived molecule (e.g., an F-actin complex or a nucleosome, which is a complex of histone and nucleic acid) as described above, and the other portion of which binds to a membrane protein expressed on the cell membrane of a cell (e.g., an immune cell, a cancer cell, an autoreactive cell). Such a chemical compound can be produced using bioconjugate techniques (Greg T. Hermanson, Bioconjugate Techniques, 3rd Edition, 2013). 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 contains distamycin as a "first moiety" that binds to nucleic acid, and rapamycin as a "second moiety" that binds to mTOR, and further contains a photoreactive group (e.g., aryl azide, diazirine, psoralen). Upon receiving photo (light) energy by the compound, the first antigen and the second antigen are crosslinked by the compound, and the binding of the second moiety to the second antigen on the cell membrane causes signal transduction or signal blocking in the cell.

[0072] Conferring 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, conformational change, internalization, stabilization, or whatever the target molecule (i.e., the second antigen) is affected by the molecule of the present invention.

[0073] Affected tissue In the present invention, the term "diseased tissue" refers to tissue in which any condition or characteristic that is different from that of normal tissue and is specific to a disease is found. Examples of diseased tissue include, but are not limited to, tumor tissue, inflammatory tissue, tissue associated with autoimmune disease, etc. In such diseased tissue, the above-mentioned cell death occurs more frequently than in normal tissue, resulting in the production of the above-mentioned dead cell-derived molecules.

[0074] Tumor tissue In the present invention, the term "tumor tissue" refers to tissue containing at least one tumor cell. Usually, tumor tissue is composed of a group of tumor cells (parenchyma) that constitutes the tumor body, and connective tissue and blood vessels (stroma) that exist between the tumor cells and support the tumor. In some cases, these are clearly distinguishable, but they may also be confused. In some cases, cells such as immune cells infiltrate into tumor tissue. In contrast, "non-tumor tissue" refers to tissue in a living body other than tumor tissue. Healthy / normal tissue that does not have a disease is a representative of such non-tumor tissue.

[0075] inflammatory tissue In the present invention, "inflammatory tissue" includes, but is not limited to: 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 from organ transplants. 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 fracture. xi. Burn-related tissues. xii. Tissue of organ tissues damaged by external mechanical, physical, or chemical forces.

[0076] 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 multiple molecules." In the present invention, "antigens formed by the multimerization of multiple molecules" include, but are not limited to, antigens formed by the multimerization of multiple proteins or portions thereof and / or lipids that constitute the cytoskeleton, biological membranes such as cell membranes, nucleosomes, chaperone molecules, etc.

[0077] 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, etc. Biological membranes, such as cell membranes, are composed of various complexes formed by different proteins, different lipids, and / or 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), and proton pumps, lipid-anchored proteins such as G proteins, and peripheral membrane proteins such as enzymes and hormones. Lipids include, but are not limited to, phospholipids, glycolipids, sterols, etc. As described above, in diseased tissues, such as those associated with cancer, inflammation, autoimmune diseases, etc., such protein and / or lipid complexes, or portions thereof, in biological membranes, such as cell membranes, are exposed to the extracellular environment upon cell death. Such complexes or portions thereof are also included as "first antigens" (also referred to as dead cell-derived molecules) as described above.

[0078] Nucleosomes are complexes of histones and nucleic acids, and chaperone molecules, primarily heat shock proteins (HSPs), are also included as "first antigens" (also called dead cell-derived molecules).

[0079] Cells expressing membrane proteins as "second antigens" In the present invention, as described above, the term "second antigen" includes, but is not limited to, membrane proteins expressed on the cell membrane and / or endosomal membrane that are involved in signal transduction in cells. In the present invention, such cells expressing membrane proteins as "second antigens" 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, 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, autoimmune disease, etc. Preferred embodiments of cells expressing the "second antigen" (also referred to as a target molecule) may be 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, neutrophils, and basophils. Other preferred embodiments of cells expressing the "second antigen" (also referred to as a target molecule) may be cells in diseased tissues associated with cancer, inflammation, autoimmune diseases, and the like.

[0080] In the present invention, the binding of the above-described "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, or to the above-described "substance (molecule) exposed to the extracellular environment upon cell death" ("dead cell-derived molecule"), can be examined by preparing cells treated with a cytotoxic drug such as mitoxantrone or mitomycin (treatment with the drug induces cell death), or by preparing untreated cells, 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-described molecule of the present invention to drug-treated cells indicates that the molecule specifically binds to the above-described dead cell-derived molecule.

[0081] The present invention provides molecules that bind to a cellular component or a portion thereof (a "first antigen," also called a dead cell-derived molecule) exposed to the extracellular environment upon cell death as described above, and simultaneously bind to a target molecule (a "second antigen"; e.g., a membrane protein as described above) on or in a cell (e.g., an immune cell, a diseased cell, e.g., a tumor cell, an autoreactive cell, or a virus-infected cell). The molecules enable cross-linking between a cellular component or a portion thereof exposed to the extracellular environment upon cell death and a target molecule on or in a cell, such as an immune cell or a diseased cell (e.g., a cancer cell, an autoreactive cell, or a virus-infected cell), thereby imparting signal transduction or blocking to cells in a tissue.

[0082] 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 have been treated with or are untreated 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"; one embodiment of a dead cell-derived molecule) [molecule of the invention]. iii. Providing a reporter cell that has been modified to produce a detectable signal when signal transduction occurs due to binding of the molecule of (ii). iv. Reporter cells are cultured with each of the molecules in (ii) in the presence of the drug-treated cells or the supernatant of a drug-treated cell culture in (i), or the untreated cells or the supernatant of untreated cells. 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 the supernatant of a drug-treated cell culture than in a cell culture of the molecule of the present invention (ii) in the presence of untreated cells or the supernatant of an untreated cell culture indicates that the molecule of the present invention (ii) conferred signaling in the cells by binding to an antigen expressed in immune cells, e.g., CD3 or a costimulatory or co-inhibitory molecule (a "second antigen"). vii. A higher reporter signal detected 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 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 cannot bind to a dead cell-derived molecule, e.g., F-actin (the "first antigen").

[0083] 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 the "second antigen," for example, CD3 or a costimulatory molecule or co-inhibitory molecule expressed in immune cells.

[0084] 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.

[0085] 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.

[0086] Furthermore, the techniques and procedures described or referenced herein are generally well understood and can be found in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd 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 J.D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V.T. DeVita et al., eds., J.B. Lippincott Company, 1993).

[0087] a third moiety that binds to a third antigen In the present invention, the "third portion" of an "antigen-binding molecule comprising at least one third portion that binds to a third antigen" binds to a "third antigen" that is different from the "first antigen" described above. The third antigen is a membrane protein of an immune cell or tumor cell, as described above. The "third antigen" may be any membrane protein of an immune cell or tumor cell, as long as it is different from the first antigen. In the present invention, the "third antigen" is also referred to as a "target molecule" targeted by the "third portion" of an "antigen-binding molecule comprising at least one third portion that binds to a third antigen." When the "third antigen" is targeted by the "third portion" of a molecule of the present invention that comprises a third portion, the target molecule, i.e., the "third antigen," brings about a desired outcome for the indicated disease, such as immune activation to combat infection or cancer, immune suppression for autoimmune disease and / or cytokine release syndrome (CRS), direct inhibition of cancer cell proliferation, or promotion of cell regeneration after tissue damage.

[0088] In some aspects of the present invention, the third antigen is different from the first and second antigens. In some aspects of the present invention, the third antigen is different from the first antigen but the same as the second antigen. In the present invention, one common embodiment of immune activation targets includes, but is not limited to, CD3 ("CDε"), CD4, CD8, CD28, OX40, 4-1BB, and T cell receptors (TCRs) expressed on T cells, which result in the 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, and 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 targeting 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 acid:polycytidylic acid (PolyIC)-sensing receptor also expressed by DCs (J Clin Invest. 2018;128(10): 4387-4396). In other cases, inhibition of target molecules, such as antagonizing CTLA-4, PD-1, LAG-3, TIM-3, or VISTA on T cells, may be desirable to prevent suppression of the immune response against tumor cells. The targeted molecule (i.e., "third 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., "third antigens").

[0089] In the present invention, the "third antigen" briefly described above includes, but is not limited to, membrane proteins expressed on the cell membrane and / or endosomal membrane of a cell. One embodiment of the membrane protein (i.e., third antigen) to which the "third moiety" binds includes, but is not limited to, membrane proteins involved in cell signal transduction, which are 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, neutrophils, and basophils. Another embodiment of the membrane protein (i.e., third antigen) to which the "third moiety" binds includes, but is not limited to, membrane proteins expressed on tumor cells or autoreactive cells.

[0090] In the present invention, membrane proteins belonging to the "third antigen" as described above 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), and 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.

[0091] Further embodiments of membrane proteins belonging to the "third 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, CD19, CD20 , CD21, CD22, CD23, CD25, CD27L, CD29, CD30L, CD32, CD33 (p67 protein), CD34, CD36, CD38, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD89, CD91, 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-31R; IL1 RL1 (ST2), integrin family, such as α1β1 (VLA-1), α2β1 (VLA-2), α3β1 (VLA-3), α4β1 (VLA-4), α5β1 (VLA-5), α6β1 (VLA-6), α 7β1, α8β1, α9β1, α11bβ3(GPIIb / IIIa), αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, αLβ2(LFA-1), αMβ2(Mac-1), αXβ2, αDβ2, α4β7;LOX-1, MDA5(MDA5, 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(WHO ATAR、HveA、LIGHT R、TR2)、TNFRSF16(NGFR p75NTR)、TNFRSF17(BCMA)、TNFRSF18(GITR AITR)、TNFRSF19(TROY TAJ、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, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, and TLR 10. RIG-I;RAGE, P2Y2, P2X7, and RIG-I; )、、おインフェロ。。。。。。(A IM2) is also smooth;

[0092] In the present invention, the term "third antigen" also includes, but is not limited to, soluble proteins that can bind to membrane proteins such as those described above. In the present invention, soluble proteins that fall under the category of "third 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.

[0093] In the present invention, soluble proteins belonging to the "third antigen" category further include, but are not limited to, the following molecules or any soluble form of said molecules (e.g., truncated 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, CD36, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD91, 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-related antigen, DAN, DCC, DcR3, DC-SIGN, Decay accelerating factor, des(1-3)-IGF-I (Brain IGF-1), Dhh, Digoxin, DNAM-1, Dnase, DppDPPIV / CD26, Dtk, 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 α4 / β1, integrin α4 / β7, integrin α5 (αV), integrin α5 / β1, integrin α5 / β3, integrin α6, integrin β1, integrin β2, interferon γ, 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, metalloproteinase, 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, 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, PDGF, PDK-1, PECAM, P EM, 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, 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, LOX-1, MDA5 (melanoma differentiation-related protein 5), 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(HVEM 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 LINK, LTg, TNFSF15(TL1A / VEGI), TNFSF18 (GITR LINK, AITR LINK, TL6)TNFSF1A (TNF-α connectin, DIF, TNFSF2), TNFSF1B (TNF-β 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, RIG-I; RAGE, P2Y2, P2X7, secreted frizzled-related protein 1 (FRP1), and interferon-inducible protein 2 (AIM2); 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 antiviral Hara, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, 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, IL1RL1(ST2), 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. The examples of molecules listed above also include receptors (membrane proteins), which, even if present in a soluble form (e.g., truncated form) in body fluids, can be used as a "third antigen" (target molecule) to which the "third moiety" 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).

[0094] In the present invention, when the "third antigen" is a soluble protein, such as IL-6, the molecule comprising the first and third portions of the present invention binds to a dead cell-derived molecule (i.e., the first antigen), such as an F-actin complex, via the first portion of the molecule and to IL-6 (i.e., the third antigen) via the third portion of the molecule, thereby neutralizing IL-6, specifically in diseased tissues where dead cell-derived molecules are more abundant than in non-diseased tissues due to cell death. Upon neutralization of IL-6, the immune response triggered by immune cells is indirectly controlled. The second portion may bind to a second target, which may be the same target as the third target (in this case, IL-6) or a different target, such as CD3 or CD137.

[0095] In the present invention, the "third moiety" that binds to the "third antigen" described above may have any structure as long as it binds to the "third antigen" described above. The structure of the "third moiety" may include, but is not limited to, a polypeptide or a portion thereof, a small or medium-sized chemical compound or a portion thereof, or a polynucleotide or a portion thereof. Polypeptides or portions thereof include, but are not limited to, antibodies (including, but not limited to, human antibodies, chimeric antibodies, humanized antibodies, and VHH antibodies) or antigen-binding domains (also referred to as antibody portions, parts, or fragments). Antibody antigen-binding domains 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 Fv2s (scFv2s), Fabs, and F(ab')2s.

[0096] Cells expressing membrane proteins as "third antigens" In the present invention, as described above, the "third antigen" refers to a membrane protein of an immune cell or tumor cell, including, but not limited to, a membrane protein expressed on the cell membrane and / or endosomal membrane, which is involved in signal transduction in the cell. In the present invention, the cell expressing the membrane protein as the "third antigen" may be any kind or type of immune cell or tumor cell, including, but not limited to, 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, neutrophils, and basophils, 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, autoimmune disease, etc. A preferred embodiment of the cell expressing the "third antigen" (also referred to as the target molecule) may be an immune cell selected from 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. Another preferred embodiment of the cell expressing the "third antigen" (also referred to as the target molecule) may be a cell in an affected tissue associated with cancer, inflammation, autoimmune disease, and the like. The aspects disclosed above for the "second antigen" also apply to the "third antigen."

[0097] A molecule comprising a first portion that binds to a first antigen and a second portion that binds to a second antigen, and at least a third portion that binds to a third 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, and comprising at least one third portion that binds to a third antigen" includes, but is not limited to, molecules having the following properties:

[0098] i. An antibody in which one of the two Fabs binds to a "second antigen" and one of the two Fabs binds to a "third antigen." The two Fab arms can form an F(ab')2 conjugated to Fc. In embodiments, the "first moiety that binds to the first antigen" can be conjugated to both chains or a single chain at the C-terminus of the Fc region. A further example is an antibody in which the "first moiety that binds to the first antigen" is conjugated to the C-terminus of one or both light chain regions. Alternatively, the "first moiety that binds to the first antigen" can be conjugated to one or both of the two Fab arms. The "first moiety that binds to the first antigen" can be conjugated to one or both of the two Fab arms in various ways. In an exemplary embodiment, the "first moiety that binds to the first antigen" can be conjugated to one or both of the two Fab arms by conjugation of one Fab arm to the N-terminus of the light chain. In exemplary embodiments, the "first moiety that binds to the first antigen" can be conjugated to one or both of the two Fab arms by conjugation to the N-terminus of the heavy chain of one or both of the Fab arms. In certain embodiments, the "first moiety that binds to the first antigen" is an additional Ig-like moiety such as an scFv or VHH, or the entire or antigen-binding portion of Clec9A or Clec4e. In exemplary embodiments, the "first moiety that binds to the first antigen" can be conjugated to one or both of the two Fab arms by conjugation to the VL and CL domains of one or both of the Fab arms. In exemplary embodiments, the "first moiety that binds to the first antigen" can be conjugated to one or both of the two Fab arms by conjugation to the VH and CH1 domains of one or both of the Fab arms. In exemplary embodiments, the "first moiety that binds to the first antigen" can be conjugated to one or both of the two Fab arms by conjugation to the VL domains of one or both of the Fab arms.In exemplary embodiments, the "first moiety that binds to the first antigen" may be conjugated to one or both of the two Fab arms by conjugation to the VH domain of one or both of the Fab arms. In exemplary embodiments, the "first moiety that binds to the first antigen" may be conjugated to one or both of the two Fab arms by conjugation to the VH and CH1 domain of one or both of the Fab arms. In exemplary embodiments, the "first moiety that binds to the first antigen" may be conjugated to one or both of the two Fab arms by conjugation to the VH and CH1 domain of one or both of the Fab arms. Further alternatively, the "first moiety that binds to the first antigen" may be conjugated to one of the Fab arms instead of or in addition to conjugation to the Fc region, and one of the two Fabs binds to a "third antigen," and the "third moiety" is conjugated to the Fc region. An example of this embodiment may be a trispecific antibody or trispecific antigen-binding domain in which one Fab arm binds to a dead cell-derived molecule (e.g., an F-actin complex) as described above, another Fab arm binds to a membrane protein expressed on the cell membrane of a cell (e.g., an immune cell, a cancer cell, an autoreactive cell), and an additional Ig-like moiety, such as an scFv or VHH, binds to a membrane protein expressed on the cell membrane of a cell (e.g., an immune cell, a cancer cell, an autoreactive cell), which may be the same or a different membrane protein. Furthermore, more than two "first moieties that bind to a first antigen" may be conjugated to the Fc region. The two Fab arms may be conjugated to the Fc region in various formats. An example of a "first moiety that binds to a first antigen" in a specific embodiment is the entire or antigen-binding portion of Clec9A or Clec4e, or an Ig-like moiety, such as an scFv or VHH. Another example of a "first moiety that binds to a first antigen" in certain embodiments is an Ig-like moiety, such as an scFv or VHH, that binds to PCNA. Another example of a "first moiety that binds to a first antigen" in certain embodiments is an Ig-like moiety, such as an scFv or VHH, that binds to SF3B3.Another example of a "first moiety that binds to a first antigen" in certain embodiments is an Ig-like moiety, such as an scFv or VHH, that binds to PS. Another example of a "first moiety that binds to a first antigen" in certain embodiments is an Ig-like moiety, such as an scFv or VHH, that binds to F-actin. In any of the above embodiments, the two Fab arms can be linked to each other via one or more bonds, such as a disulfide bond. In embodiments, the amino acid residues that form the bond between the antigen-binding domains are located in the variable or constant region. An exemplary embodiment is shown schematically in Figure 3. In one preferred embodiment, the antibody can have its first H chain CH3 region and second H chain CH3 region cross-linked by a disulfide bond.

[0099] ii. An antibody in which one of the two Fabs binds to the "first antigen" and one of the two Fabs binds to the "second antigen" (or "third antigen"). The two Fab arms can form an F(ab')2 conjugated to Fc. The "third moiety that binds to the third antigen" (or "second moiety that binds to the second antigen") can, in embodiments, be conjugated to both chains or a single chain at the C-terminus of the Fc region. A further example is an antibody in which the "third moiety that binds to the third antigen" (or "second moiety that binds to the second antigen") is conjugated to the C-terminus of one or both light chain regions. Alternatively, the "third moiety that binds to the third antigen" (or "second moiety that binds to the second antigen") can be conjugated to one or both of the two Fab arms. The "third moiety that binds to the third antigen" (or "second moiety that binds to the second antigen") can be conjugated to one or both of the two Fab arms in various ways. In exemplary embodiments, the "third moiety that binds to the third antigen" (or "second moiety that binds to the second antigen") can be conjugated to one or both of the two Fab arms by conjugation to the N-terminus of the light chain of one Fab arm. In exemplary embodiments, the "third moiety that binds to the third antigen" (or "second moiety that binds to the second antigen") can be conjugated to one or both of the two Fab arms by conjugation to the N-terminus of the heavy chain of one or both Fab arms. In certain embodiments, the "third moiety that binds to the third antigen" (or "second moiety that binds to the second antigen") is an additional Ig-like moiety, such as an scFv or VHH, or is the entire or antigen-binding portion of, for example, a receptor ligand or cytokine. In exemplary embodiments, the "third moiety that binds to the third antigen" (or "second moiety that binds to the second antigen") can be conjugated to one or both of the two Fab arms by conjugation to the VL and CL domains of one or both Fab arms.In exemplary embodiments, the "third portion that binds to the third antigen" (or "second portion that binds to the second antigen") may be conjugated to one or both of the two Fab arms by conjugation to the VH and CH1 domains of one or both of the Fab arms. In exemplary embodiments, the "third portion that binds to the third antigen" (or "second portion that binds to the second antigen") may be conjugated to one or both of the two Fab arms by conjugation to the VL domains of one or both of the Fab arms. In exemplary embodiments, the "third portion that binds to the third antigen" (or "second portion that binds to the second antigen") may be conjugated to one or both of the two Fab arms by conjugation to the VH domains of one or both of the Fab arms. In exemplary embodiments, the "third portion that binds to the third antigen" (or "second portion that binds to the second antigen") may be conjugated to one or both of the two Fab arms by conjugation to the VH and CH1 domains of one or both of the Fab arms. In exemplary embodiments, the "third moiety that binds a third antigen" (or "second moiety that binds a second antigen") can be conjugated to one or both of the two Fab arms by conjugation to the VH and CH1 domains of one or both Fab arms. Further alternatively, the "third moiety that binds a third antigen" (or "second moiety that binds a second antigen") can be conjugated to one of the Fab arms instead of, or in addition to, conjugation to the Fc region, and one of the two Fabs binds the "first antigen" and the "first moiety" is conjugated to the Fc region.An example of this embodiment may be a trispecific antibody or trispecific antigen-binding domain in which one Fab arm binds to a dead cell-derived molecule (e.g., an F-actin complex) as described above, another Fab arm binds to a membrane protein expressed on the cell membrane of a cell (e.g., an immune cell, a cancer cell, an autoreactive cell), and an additional Ig-like moiety such as an scFv or VHH binds to a membrane protein expressed on the cell membrane of a cell (e.g., an immune cell, a cancer cell, an autoreactive cell), which may be the same or a different membrane protein. Furthermore, more than two "third moieties that bind to a third antigen" (or "second moieties that bind to a second antigen") may be conjugated to the Fc region. The two Fab arms may be conjugated to the Fc region in various formats. In certain embodiments, an example of a "first portion that binds to a first antigen" is the entirety or an antigen-binding portion of Clec9A or Clec4e, or an Ig-like portion such as an scFv or VHH. Another example of a "first portion that binds to a first antigen" in certain embodiments is an Ig-like portion such as an scFv or VHH that binds to PCNA. Another example of a "first portion that binds to a first antigen" in certain embodiments is an Ig-like portion such as an scFv or VHH that binds to SF3B3. Another example of a "first portion that binds to a first antigen" in certain embodiments is an Ig-like portion such as an scFv or VHH that binds to PS. Another example of a "first portion that binds to a first antigen" in certain embodiments is an Ig-like portion such as an scFv or VHH that binds to F-actin.

[0100] In any of the above embodiments, the two Fab arms can be linked to each other via one or more bonds, such as a disulfide bond. In embodiments, the amino acid residues responsible for the bond between the antigen-binding domains are present in the variable region or the constant region. An exemplary embodiment is shown schematically in Figure 3. In one preferred embodiment, the antibody can have its first H chain CH3 region and second H chain CH3 region cross-linked by a disulfide bond.

[0101] iii. A polypeptide comprising a "first portion that binds to a first antigen," a "second portion that binds to a second antigen," and a "third portion that binds to a third antigen," wherein the first portion, second portion, and third portion are fused with or without a linker. An example of this embodiment is a fusion protein comprising the extracellular domain of Clec9A or Clec4e, a first ligand for any membrane protein expressed on the cell membrane of a cell (e.g., an immune cell, cancer cell, or autoreactive cell), and a second ligand for any membrane protein expressed on the cell membrane of a cell (e.g., an immune cell, cancer cell, or autoreactive cell), wherein the first and second ligands can bind to the same or different membrane proteins. For example, the second portion can be an Ig-like portion, such as an scFv or VHH, that binds to CD3, and the third portion can be an Ig-like portion, such as an scFv or VHH, that binds to CD137. Another example of this embodiment is a fusion protein comprising an Ig-like moiety, such as an scFv or VHH, that binds to PCNA, and a first ligand for any membrane protein expressed on the cell membrane of a cell (e.g., an immune cell, a cancer cell, an autoreactive cell), and a second ligand for any membrane protein expressed on the cell membrane of a cell (e.g., an immune cell, a cancer cell, an autoreactive cell), where the first and second ligands can bind to the same or different membrane proteins. For example, the second moiety can be an Ig-like moiety, such as an scFv or VHH, that binds to CD3, and the third moiety can be an Ig-like moiety, such as an scFv or VHH, that binds to CD137. Another example of this embodiment is a fusion protein comprising an Ig-like moiety, such as an scFv or VHH, that binds to SF3B3, and a first ligand for any membrane protein expressed on the cell membrane of a cell (e.g., an immune cell, a cancer cell, an autoreactive cell), and a second ligand for any membrane protein expressed on the cell membrane of a cell (e.g., an immune cell, a cancer cell, an autoreactive cell), wherein the first and second ligands can bind to the same or different membrane proteins.Another example of this embodiment is a fusion protein comprising an Ig-like moiety, such as an scFv or VHH, that binds to PS, a first ligand for any membrane protein expressed on the cell membrane of a cell (e.g., an immune cell, a cancer cell, an autoreactive cell), and a second ligand for any membrane protein expressed on the cell membrane of a cell (e.g., an immune cell, a cancer cell, an autoreactive cell), where the first and second ligands can bind to the same or different membrane proteins. For example, the second moiety can be an Ig-like moiety, such as an scFv or VHH, that binds to CD3, and the third moiety can be an Ig-like moiety, such as an scFv or VHH, that binds to CD137. An exemplary embodiment comprising an Fc region is schematically illustrated in Figure 3. For example, the third moiety, which can be an Ig-like moiety, such as an scFv or VHH, and the second moiety, which can be an Ig-like moiety, such as an scFv or VHH, can be fused (conjugated) and then conjugated to the Fc region via one of the two chains. The first moiety may then be conjugated thereto in various ways. For example, a "first moiety that binds to a first antigen" that is not an Ig-like moiety, such as the entirety or an antigen-binding portion of Clec9A or Clec4e, is conjugated to the second chain of the Fc region, such as the N- or C-terminus of the Fc region, or to an internal side chain of the Fc region. Such an embodiment is shown in Figure 3. In another exemplary embodiment shown in Figure 3, the "first moiety that binds to a first antigen" is conjugated to the VL and / or CL region of the second or third moiety. In a further alternative embodiment, as shown schematically in Figure 3, the "second moiety" (or "third moiety") corresponding to Fab is conjugated to the Fc region via one of the two chains of the Fc region, and the "third moiety" (or "second moiety") may be conjugated to the CH2 or CH3 region of one chain of the Fc region. A "first moiety that binds to a first antigen," which is not an Ig-like moiety, such as the entirety or an antigen-binding portion of Clec9A or Clec4e, is conjugated to the second chain of the Fc region, such as the N-terminus of the Fc region (or the C-terminus of the Fc region, or an internal side chain of the Fc region).In yet further alternative embodiments, also shown schematically in Figure 3, a "first moiety that binds to a first antigen" that is not an Ig-like moiety, such as the entirety or an antigen-binding portion of Clec9A or Clec4e, is conjugated to one chain of the Fc region, such as the N-terminus or C-terminus of the Fc region, or to an internal side chain of the Fc region. In some embodiments, a second "first moiety that binds to a first antigen" that is not an Ig-like moiety, such as the entirety or an antigen-binding portion of Clec9A or Clec4e, is conjugated to a second chain of the Fc region, such as the N-terminus or C-terminus of the Fc region, or to an internal side chain of the Fc region. For example, the third moiety, which may be an Ig-like moiety such as an scFv or VHH, and the second moiety, which may be an Ig-like moiety such as an scFv or VHH, may be fused (conjugated) and then conjugated to the CH2 or CH3 domain of the Fc region via one of the two chains. Alternatively, the third moiety, which may be an Ig-like moiety such as an scFv or VHH, may then be conjugated to the CH2 or CH3 domain of the Fc region via one of the two chains, and the second moiety, which may be an Ig-like moiety such as an scFv or VHH, may then be conjugated to the CH2 or CH3 domain of the Fc region via the second of the two chains.

[0102] In any of the above embodiments, the two Fab arms can be linked to each other via one or more bonds, such as a disulfide bond. In embodiments, the amino acid residues responsible for the bond between the antigen-binding domains are present in the variable region or the constant region. An exemplary embodiment is shown schematically in Figure 3. In one preferred embodiment, the antibody can have its first H chain CH3 region and second H chain CH3 region cross-linked by a disulfide bond.

[0103] iv. A chemical compound comprising a "first portion that binds to a first antigen" and a "second portion that binds to a second antigen." An example of this embodiment is a chemical compound comprising at least two portions, one of which binds to a dead cell-derived molecule (e.g., an F-actin complex or a nucleosome, which is a complex of histone and nucleic acid) as described above, and the other portion of which binds to a membrane protein expressed on the cell membrane of a cell (e.g., an immune cell, a cancer cell, an autoreactive cell). Such a chemical compound can be produced using bioconjugate techniques (Greg T. Hermanson, Bioconjugate Techniques, 3rd Edition, 2013). 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 receiving photo (light) energy by the compound, the first antigen and the second antigen are crosslinked by the compound, and the binding of the second moiety to the second antigen on the cell membrane causes signal transduction or signal blocking in the cell.

[0104] In one aspect, (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 or a tumor cell, and the first antigen is (a) splicing factor 3B subunit 3 (SF3B3); and (b) Proliferating cell nuclear antigen (PCNA) selected from the group consisting of Antigen-binding molecules are provided.

[0105] In one embodiment, the antigen-binding molecule comprises at least one third moiety that binds to a third antigen; (i) the third antigen is different from the first and second antigens, or (ii) the third antigen is different from the first antigen but the same as the second antigen; preferably, the third antigen is a membrane protein of an immune cell or a tumor cell. In one embodiment, the third antigen is different from the first and second antigens. For example, the first antigen is PCNA or SF3B3, the second antigen is CD3, and the third antigen is CD137. For example, the second antigen is CD137, and the third antigen is CD3. In one embodiment, the third antigen is different from the first antigen but the same as the second antigen. For example, the first antigen is PCNA or SF3B3, the second antigen is CD3, and the third antigen is CD3. For example, the first antigen is PCNA or SF3B3, the second antigen is CD137, and the third antigen is CD137.

[0106] In embodiments, the first portion is (A) an Ig-type binding moiety; or (B) a binding polypeptide, particularly a non-Ig type binding moiety is selected from the group consisting of: In embodiments, when the first antigen is splicing factor 3B subunit 3 (SF3B3), the first portion is an Ig-type binding portion. In embodiments, when the first antigen is splicing factor 3B subunit 3 (SF3B3), the first portion is a Clec4e polypeptide. In embodiments, when the first antigen is proliferating cell nuclear antigen (PCNA), the first portion is an Ig-type binding portion. In embodiments, the second and / or third 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, and among them, the second and / or third antigen is a membrane protein involved in signal transduction in cells, preferably a signaling receptor. In one embodiment, the second and third antigens are membrane proteins of immune cells, particularly membrane proteins in the cell membrane of T cells selected from the group consisting of T cell receptors, CD3, CD137, CD40, CTLA4, costimulatory molecules, or co-inhibitory molecules; among them, the second and third antigens are membrane proteins involved in signal transduction in cells, preferably signal transduction receptors. In one embodiment, the second or third antigen is a membrane protein of immune cells, particularly membrane proteins in the cell membrane of T cells selected from the group consisting of T cell receptors, CD3, CD137, CD40, CTLA4, costimulatory molecules, or co-inhibitory molecules; among them, the second or third antigen is a membrane protein involved in signal transduction in cells, preferably signal transduction receptors.

[0107] In one embodiment, the second and / or third antigen is a membrane protein of a tumor cell. In one embodiment, the second and third antigens are membrane proteins of a tumor cell. In one embodiment, the second or third antigen is a membrane protein of a tumor cell. In one embodiment, 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, costimulatory molecule, or co-inhibitory molecule; among others, the second antigen is a membrane protein involved in signal transduction in cells, preferably a signal transduction receptor; and the third antigen is a membrane protein of a tumor cell. In one embodiment, the second antigen is a membrane protein of a tumor cell, and the third 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, costimulatory molecule, or co-inhibitory molecule; among others, the second antigen is a membrane protein involved in signal transduction in cells, preferably a signal transduction receptor.

[0108] In one embodiment, the second and / or third moiety is an Ig-type binding moiety. In one embodiment, the antigen-binding molecule is an antibody, particularly an IgG antibody. In one embodiment, the antibody further comprises at least one binding polypeptide capable of binding to a membrane protein of an immune cell or tumor cell, and preferably linked to an Fc region, in particular, the binding polypeptide is selected from a Clec9A polypeptide and a Clec4e polypeptide; among others, 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 Clec4e polypeptide, and an anti-CD137 antibody comprising a Clec4e polypeptide.

[0109] In one embodiment, the antigen-binding molecule is an antibody, particularly an IgG type antibody, and said first portion is linked to the Fc region and is an Ig type binding portion, particularly the antibody (A) Anti-CD3 antibody; (B) anti-CD137 antibody; and (C) Bispecific anti-CD3 anti-CD137 antibody is selected from the group consisting of:

[0110] In one embodiment, the antigen-binding molecule is an antibody, particularly an IgG antibody, and the antigen-binding molecule is (1) one first moiety linked to an Fc region, in particular an Ig-type binding moiety; (2) one second moiety linked to the Fc region and which is an Ig-type binding moiety; and (3) one third portion linked to the Fc region and which is an Ig-type binding portion; Includes. In one embodiment, one second portion and one third portion together form F(ab')2. In one embodiment, the antigen-binding molecule is an antibody, particularly an IgG-type antibody, wherein the first portion is linked to an Fc region and is an Ig-type binding portion, and the antibody is a bispecific antibody, particularly, the antigen-binding molecule is a bispecific antibody against a membrane protein of an immune cell or a tumor cell, and against a first antigen selected from the group consisting of: (a) splicing factor 3B subunit 3 (SF3B3); and (b) proliferating cell nuclear antigen (PCNA), Among others, the antibody bispecific anti-SF3B3 anti-CD3 antibody, bispecific anti-SF3B3 anti-CD137 antibody, bispecific anti-PCNA anti-CD3 antibodies, and Bispecific anti-PCNA anti-CD137 antibody is selected from the group consisting of:

[0111] In one embodiment, the antigen-binding molecule is an antibody, particularly an IgG type antibody, wherein the first portion is linked to an Fc region and is an Ig type binding portion, and the antibody is a trispecific antibody, particularly wherein the antigen-binding molecule is (i) against membrane proteins of immune cells and against membrane proteins of tumor cells; and (a) splicing factor 3B subunit 3 (SF3B3); and (b) proliferating cell nuclear antigen (PCNA), or (ii) to a first membrane protein of an immune cell and a second membrane protein of the immune cell that is different from the first membrane protein; and (a) splicing factor 3B subunit 3 (SF3B3); and (b) proliferating cell nuclear antigen (PCNA), or (iii) to a first membrane protein of a tumor cell and a second membrane protein of the tumor cell that is different from the first membrane protein; and (a) splicing factor 3B subunit 3 (SF3B3); and (b) proliferating cell nuclear antigen (PCNA). It is a trispecific antibody, Among others, the antibody a trispecific anti-SF3B3 anti-CD3 anti-CD137 antibody, and Trispecific anti-PCNA anti-CD3 anti-CD137 antibody is selected from the group consisting of: In another aspect, (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; the second antigen is a membrane protein of an immune cell or a tumor cell, and the molecule comprises at least one third moiety that binds to a third antigen; the third antigen is different from the first antigen; and the third antigen is a membrane protein of an immune cell or a tumor cell; The first antigen is (a) F-actin; (b) phosphatidylserine (PS); (c) splicing factor 3B subunit 3 (SF3B3); and (d) proliferating cell nuclear antigen (PCNA). selected from the group consisting of Antigen-binding molecules are provided.

[0112] In one embodiment, the third antigen is different from the first and second antigens. In one embodiment, the third antigen is different from the first antigen but the same as the second antigen. In one embodiment, the third antigen is different from the first and second antigens. For example, the first antigen is F-actin, the second antigen is CD3, and the third antigen is CD137. For example, the second antigen is CD137, and the third antigen is CD3. In one embodiment, the third antigen is different from the first antigen but the same as the second antigen. For example, the first antigen is F-actin, the second antigen is CD3, and the third antigen is CD3. For example, the first antigen is F-actin, the second antigen is CD137, and the third antigen is CD137. For example, the first antigen is phosphatidylserine (PS), the second antigen is CD3, and the third antigen is CD3. For example, the first antigen is phosphatidylserine (PS), the second antigen is CD137, and the third antigen is CD137. For example, the first antigen is phosphatidylserine (PS), the second antigen is CD3, and the third antigen is CD137. For example, the second antigen is CD137, and the third antigen is CD3. For example, the first antigen is proliferating cell nuclear antigen (PCNA), the second antigen is CD3, and the third antigen is CD3. For example, the first antigen is proliferating cell nuclear antigen (PCNA), the second antigen is CD137, and the third antigen is CD137. For example, the first antigen is proliferating cell nuclear antigen (PCNA), the second antigen is CD3, and the third antigen is CD137. For example, the second antigen is CD137, and the third antigen is CD3. For example, the first antigen is splicing factor 3B subunit 3 (SF3B3), the second antigen is CD3, and the third antigen is CD3. For example, the first antigen is splicing factor 3B subunit 3 (SF3B3), the second antigen is CD137, and the third antigen is CD137. For example, the first antigen is splicing factor 3B subunit 3 (SF3B3), the second antigen is CD3, and the third antigen is CD137. For example, the second antigen is CD137, and the third antigen is CD3.

[0113] In embodiments, the first moiety is selected from the group consisting of: (A) an Ig-type binding moiety; or (B) a binding polypeptide, particularly a non-Ig-type binding moiety. (A) if the first antigen is splicing factor 3B subunit 3 (SF3B3), the first moiety is an Ig-type binding moiety; or (B) if the first antigen is splicing factor 3B subunit 3 (SF3B3), the first moiety is a Clec4e polypeptide; or (C) if the first antigen is proliferating cell nuclear antigen (PCNA), the first moiety is an Ig-type binding moiety; or (D) if the first antigen is F-actin, the first moiety is an Ig-type binding moiety; or (E) if the first antigen is F-actin, the first moiety is a Clec9A polypeptide; or (F) When the first antigen is PS, the first moiety is an Ig-type binding moiety.

[0114] In one embodiment, the second and / or third 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 co-inhibitory molecule; among others, the second and / or third antigen is a membrane protein involved in signal transduction in cells, preferably a signal transduction receptor. In one embodiment, the second and third antigen are 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 co-inhibitory molecule; among others, the second and third antigen is a membrane protein involved in signal transduction in cells, preferably a signal transduction receptor. In one embodiment, the second or third 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 others, the second or third antigen is a membrane protein involved in signal transduction in the cell, preferably a signal transduction receptor.

[0115] In one embodiment, the second and / or third antigen is a membrane protein of a tumor cell. In one embodiment, the second and third antigens are membrane proteins of a tumor cell. In one embodiment, the second or third antigen is a membrane protein of a tumor cell. In one embodiment, 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, costimulatory molecule, or co-inhibitory molecule; among others, the second antigen is a membrane protein involved in signal transduction in cells, preferably a signal transduction receptor; and the third antigen is a membrane protein of a tumor cell. In one embodiment, the second antigen is a membrane protein of a tumor cell, and the third 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, costimulatory molecule, or co-inhibitory molecule; among others, the second antigen is a membrane protein involved in signal transduction in cells, preferably a signal transduction receptor. In one embodiment, the second and / or third moiety is an Ig-type binding moiety.

[0116] In one embodiment, the antigen-binding molecule is an antibody, particularly an IgG-type antibody. For example, an antigen-binding molecule can be provided in which an anti-PS portion and an anti-CD3 portion linked to Fc together form F(ab')2. An additional Ig-like portion, such as an scFv, VHH, or variable domain, that binds to a membrane protein of an immune cell or tumor cell can be linked to the Fc. In one embodiment, the antibody further comprises at least one binding polypeptide that can bind to a membrane protein of an immune cell or tumor cell and is preferably linked to the Fc region, and in particular, the binding polypeptide is selected from a Clec9A polypeptide and a Clec4e polypeptide; Among them, antibodies an anti-CD3 antibody comprising a Clec9A polypeptide; an anti-CD137 antibody comprising a Clec9A polypeptide; an anti-CD3 antibody comprising a Clec4e polypeptide, and Anti-CD137 Antibodies Containing Clec4e Polypeptides is selected from the group consisting of:

[0117] In embodiments, the antibody is a trispecific antibody, In particular, the antigen-binding molecule (i) against membrane proteins of immune cells and against membrane proteins of tumor cells; and (a) F-actin; (b) phosphatidylserine (PS); (c) splicing factor 3B subunit 3 (SF3B3); and (d) proliferating cell nuclear antigen (PCNA), or (ii) against a first membrane protein of an immune cell and a second membrane protein of an immune cell that is different from the first membrane protein; and (a) F-actin; (b) phosphatidylserine (PS); (c) splicing factor 3B subunit 3 (SF3B3); and (d) proliferating cell nuclear antigen (PCNA), or (iii) to a first membrane protein of a tumor cell and a second membrane protein of the tumor cell that is different from the first membrane protein; and (a) F-actin; (b) phosphatidylserine (PS); (c) splicing factor 3B subunit 3 (SF3B3); and (d) proliferating cell nuclear antigen (PCNA). It is a trispecific antibody, Among them, antibodies trispecific anti-SF3B3 anti-CD3 anti-CD137 antibody, trispecific anti-PCNA anti-CD3 anti-CD137 antibody, trispecific anti-PS anti-CD3 anti-CD137 antibody, Trispecific anti-F-actin, anti-CD3, and anti-CD137 antibodies a bispecific anti-CD3 anti-CD137 antibody comprising a Clec9A polypeptide, and Bispecific anti-CD3 anti-CD137 antibodies containing Clec4e polypeptides is selected from the group consisting of:

[0118] In one embodiment, the antigen-binding molecule is an antibody, particularly an IgG antibody, and the antigen-binding molecule is (1) one first moiety linked to an Fc region, in particular an Ig-type binding moiety; (2) one second moiety linked to the Fc region and which is an Ig-type binding moiety; and (3) one third portion linked to the Fc region and which is an Ig-type binding portion; Includes.

[0119] In one embodiment herein, one second portion and one third portion together form F(ab')2. For example, an antigen-binding molecule can be provided in which an F(ab')2 that binds to CD3 and CD137 is linked to Fc and further linked to an Ig-like portion, such as an scFv, VHH, or variable domain, that binds to SF3B3, PCNA, PS, or F-actin. In one aspect, there is provided a pharmaceutical composition comprising an antigen-binding molecule according to any one of the embodiments herein. In one embodiment, the antigen-binding molecule is as defined in any one of the embodiments of invention group [AA].

[0120] Further provided herein is an antigen-binding molecule of any of the embodiments or aspects herein, or a pharmaceutical composition comprising an antigen-binding molecule of any of the embodiments or aspects herein, for use in medicine. In one embodiment, the antigen-binding molecule is as specified in any one of the embodiments of invention group [AA].

[0121] In one embodiment, an antigen-binding molecule of an embodiment or aspect herein, or a pharmaceutical composition comprising an antigen-binding molecule of an embodiment or aspect herein, is provided for use in a method for treating or preventing a medical condition, preferably the medical condition is selected from the group consisting of cancer and immune disease, preferably autoimmune disease. In one embodiment, the cancer disease is as provided herein. In one embodiment, the antigen-binding molecule is as specified in any one of the embodiments of the invention group [AA].

[0122] In an embodiment, in any of the antigen binding molecules of any of the aspects and embodiments herein, the antigen binding molecule comprises: (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) the Ig-type binding portion of the antigen-binding molecule, preferably the F(ab')2 region, comprises the second portion; and / or (D) comprises at least one first portion and at least one second portion, wherein (i) the antigen-binding molecule comprises one first portion and one second portion, or (ii) the antigen-binding molecule comprises at least two, preferably two, first portions and at least two, preferably two, second portions; and / or (E) the Ig-type binding portion of the antigen-binding molecule, preferably the F(ab')2 region, comprises the third portion; and / or (F) comprises at least one first portion, at least one second portion, and at least one third portion, particularly wherein (i) the antigen-binding molecule comprises one first portion, one second portion, and one third portion, or (ii) the antigen-binding molecule comprises at least two, preferably two, first portions, and at least two, preferably two second portions, and at least two, preferably two third portions; and / or (G) The antigen-binding molecule is capable of binding to the first antigen and the second antigen upon light irradiation. In any of the aspects herein, the cell damage is (i) including, in particular, cell death; and / or (ii) occurs in diseased tissues, particularly selected from tissues affected by a condition or disease, particularly cancer or an autoimmune disease, especially in the tumor microenvironment (TME); and / or (iii) caused by any one selected from the group consisting of 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. In any of the aspects or embodiments herein, the antigen-binding molecules are characterized in that a complex formed by binding of one or more of the antigen-binding molecules to a first antigen can bind to more than one membrane protein via at least one second moiety and / or at least one third moiety; especially, (i) a complex formed by the binding of one or more of the antigen-binding molecules to a first antigen can bind to one or more membrane proteins via at least one second moiety and confer signal transduction through the one or more membrane proteins in a cell; or (ii) a complex formed by the binding of one or more of the antigen-binding molecules to a first antigen can bind to one or more membrane proteins via at least one second moiety to provide signal blocking via the one or more membrane proteins in a cell; or (iii) a complex formed by the binding of one or more of the antigen-binding molecules to a first antigen can bind to one or more membrane proteins via at least one third moiety and confer signal transduction through the one or more membrane proteins in a cell; or (iv) A complex formed by the binding of one or more of the antigen-binding molecules to a first antigen can bind to one or more membrane proteins via at least one third moiety to provide signal blocking in a cell via the one or more membrane proteins. In any of the aspects or embodiments herein, the antigen-binding molecule comprises: (A) a polypeptide complex, optionally a fusion protein; (B) a polynucleotide (preferably composed of two or more entities, preferably connected by a linker); (C) a medium-sized chemical compound (preferably composed of two or more entities, preferably connected by a linker); (D) a small chemical compound (preferably composed of two or more entities, preferably connected by a linker) selected from the group consisting of especially, (A-1) The antigen-binding molecule is a polypeptide complex, Among these, the antigen-binding molecule is an antibody or an antibody fragment thereof, In particular, the antigen-binding molecule is an antibody, Preferably, the antibody is an IgG type antibody and / or a bispecific antibody; or (A-2) The antigen-binding molecule is a polypeptide complex that is a fusion protein, Among these, the antigen-binding molecule is an antibody that is a fusion protein, or an antibody fragment thereof, In particular, the antigen-binding 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, and / or (c) a trispecific antibody having at least one binding polypeptide, preferably two binding polypeptides or three binding polypeptides fused to its Fc domain; or (A-3) The antigen-binding molecule is a polypeptide complex that is a fusion protein, Among these, the antigen-binding molecule is an antibody that is a fusion protein, or an antibody fragment thereof, In particular, the antigen-binding molecule is an antibody that is a fusion protein, Preferably, the antibody is (a) an IgG-type antibody having at least one Ig-type binding moiety, preferably two IgG-type binding moieties, fused to its Fc domain, or three Ig-type binding moieties fused to its Fc domain, and / or (b) a bispecific antibody having at least one Ig-type binding moiety, preferably two Ig-type binding moieties, fused to its Fc domain, and / or (c) a trispecific antibody having at least one Ig-type binding moiety, preferably two Ig-type binding moieties, fused to its Fc domain, or three Ig-type binding moieties fused to its Fc domain.

[0123] In some embodiments, the Ig-type binding moiety is selected from the group consisting of an IgG-type antibody, 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 in particular, the Ig-type binding moiety is selected from the group consisting of VHH, scFv, scFv2, Fab, and F(ab')2.

[0124] In any of the aspects or embodiments herein, the antigen-binding molecule comprises: (i-1) the first portion is an Ig-type binding portion, or (i-2) the first portion is a binding polypeptide; and / or (ii) the second moiety is an Ig-type binding moiety; and / or (iii) the third portion is an Ig-type binding portion; It is characterized by the fact that 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, Fv, a single chain Fv2 (scFv2), Fab, and 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; and / or (d) the third 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; 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, particularly a non-Ig binding moiety attached to the Fc domain of the antigen-binding molecule; and / or (d*) The third portion is a binding domain selected from the group consisting of VHH, scFv, scFv2, Fab, and F(ab')2.

[0125] In any of the aspects herein, the antigen-binding molecule is characterized in that it is a bispecific antibody, in which the two antigen-binding domains of the bispecific antibody of any of the aspects herein are linked to each other via one or more bonds. Methods for producing such molecules are disclosed, for example, in EP3831854. In an embodiment, at least one of the amino acid residues responsible for the bond between the antigen-binding domains is present in the variable region. In an embodiment, at least one of the amino acid residues responsible for the bond between the antigen-binding domains is present in the hinge region.

[0126] In any of the aspects herein, the antigen-binding molecule comprises: the first portion is an Ig-type binding moiety or binding polypeptide; and the second portion is an Ig-type binding moiety, and / or the third portion is an Ig-type binding portion, and the second and / or third portion is capable of binding to CD3 and CD137, but does not simultaneously bind to CD3 and CD137. Such molecules and methods for producing them are disclosed, for example, in EP 3720963.

[0127] In any of the aspects herein, the antigen-binding molecule is an antigen-binding molecule in which the second and / or third antigen is (A) Involved in signal transduction in cells; especially, (A-1) the second and / or third antigen is a membrane protein, and / or (A-2) the second and / or third antigen is a signal transduction receptor, and / or (A-3) The cell is (i) an immune cell (among other things, 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), (ii) a tumor cell, or (iii) an autoreactive cell; and / or (B) Immune cell receptor antigen It is characterized by: In any of the aspects herein, the antigen-binding molecule is an antigen-binding molecule in which the second and / or third antigen is (A) Endosomal membrane proteins, (B) Membrane proteins in the plasma membrane of T cells. membrane proteins in the plasma membrane of T cells selected from the group consisting of T cell receptors, CD3, CD137, CD40, CTLA4, costimulatory molecules, or costimulatory molecules, (Among the membrane proteins capable of binding to costimulatory or costinhibitory molecules are selected from 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 and / or third antigen is selected from the group consisting of CD3, CD137, CD40, and CTLA4; In particular, the second and / or third antigens are independently selected from the group consisting of CD3 and CD137; Preferably, the second and / or third antigen is CD3. It is characterized by: In embodiments, the second and / or third moiety is (A) A moiety capable of binding to an endosomal membrane protein; (B) a moiety capable of binding to a membrane protein in the plasma membrane of a T cell, particularly 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 others, 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.) are independently selected from the group consisting of: Among others, the second and / or third 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; In particular, the second and / or third moieties are independently selected from the group consisting of a moiety capable of binding to CD3 and / or a moiety capable of binding to CD137; Preferably, the second and / or third moiety is a moiety capable of binding to CD3.

[0128] In any of the aspects herein, the antigen-binding molecule comprises: (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, Fv, a single chain Fv2 (scFv2), Fab, and F(ab')2; Among these, the Ig-type binding moiety is selected from the group consisting of VHH, scFv, scFv2, Fab, and F(ab')2. It is characterized by:

[0129] amino acid As used herein, amino acids are described by one-letter or three-letter codes, or both, such as 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.

[0130] Amino acid modification For amino acid modifications (also referred to as "amino acid substitutions" or "amino acid mutations" 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. Furthermore, several known methods for amino acid modification for substitution with unnatural amino acids may also be used (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)) containing a tRNA in which an unnatural amino acid is bound to an amber suppressor tRNA complementary to the UAG codon (amber codon), which is one of the termination codons.

[0131] As used herein, the term "and / or" when describing the site of amino acid modification includes any suitable combination of "and" and "or." Specifically, for example, "the amino acids at positions 33, 55, and / or 96 are substituted" includes the following amino acid modification variations: (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.

[0132] Furthermore, herein, expressions indicating amino acid modifications may be appropriately expressed by using one-letter or three-letter codes for the amino acids before and after the modification, respectively, before and after a number indicating a specific position. For example, the modification N100bL or Asn100bLeu, used when substituting an amino acid contained in an antibody variable region, indicates a substitution 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 substitution, and the one-letter or three-letter amino acid code written after the number indicates the amino acid after substitution. Similarly, the modification P238D or Pro238Asp, used when substituting an amino acid in the Fc region contained in an antibody constant region, indicates a substitution of Pro at position 238 (according to EU numbering) with Asp. That is, the numbers indicate 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.

[0133] 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, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," 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" can be used in place of or interchangeably with any of these terms.

[0134] The term "polypeptide" is also intended to refer to the products of post-expression modifications of polypeptides, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-natural amino acids. Polypeptides can be derived from natural biological sources or produced by recombinant technology, but are not necessarily translated from a designated nucleic acid sequence. Polypeptides may be generated by any method, including chemical synthesis. Polypeptides as described herein may be 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 in size. Polypeptides can have a defined three-dimensional structure, but do not necessarily have such a structure. Polypeptides with a defined three-dimensional structure are referred to as folded, while polypeptides that do not have a defined three-dimensional structure but rather can adopt a number of different conformations are referred to as unfolded.

[0135] 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 those 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 purposes 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 appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment across 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, together with user documentation, has been filed with the U.S. Copyright Office, Washington, DC, 20559, where it is registered under U.S. 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 situations where ALIGN-2 is used 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 can say that a given amino acid sequence A has or contains a certain % 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 its 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 will not equal 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.

[0136] Recombinant methods and compositions Antibodies, antigen-binding molecules, antigen-binding domains, and molecules, including those of the present invention, can be produced using recombinant methods and compositions, such as those described in U.S. Patent No. 4,816,567. When the molecule of the present invention is an antibody, in one embodiment, an isolated nucleic acid encoding the antibody as described herein is provided. 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 heavy chain of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In a further embodiment, a host cell comprising such nucleic acids 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 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, e.g., a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., a Y0, NS0, Sp2 / 0 cell). In one embodiment, a method of producing a molecule of the invention is provided, the method comprising culturing a host cell comprising 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).

[0137] 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., if the molecule is an antibody, by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0138] Suitable host cells for cloning or expressing vectors encoding the molecules of the present invention include prokaryotic or eukaryotic cells as described herein. For example, the molecules of the present invention may be produced in bacteria, particularly if glycosylation and Fc effector function are not required. For expression of the molecules 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 molecules of the present invention may be isolated in a soluble fraction from the bacterial cell paste and further purified.

[0139] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast, including fungal and yeast strains in which the glycosylation pathways have 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).

[0140] Suitable host cells for expressing glycosylated molecules of the 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.

[0141] 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 PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0142] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 (COS-7); human embryonic kidney (e.g., 293 or 293 cells described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney (BHK) cells; mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney (CV1); African green monkey kidney (VERO-76); human cervical carcinoma (HELA); canine kidney (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung (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).

[0143] Recombinant production of the molecules of the invention can be carried out in a similar manner to that described above by using host cells that contain (e.g., have been transformed with) one or more vectors that contain nucleic acids encoding amino acid sequences comprising the entire molecule or a portion thereof.

[0144] 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, for example, an antibody or its antigen-binding domain / molecule, which may comprise an additional portion conjugated to the Fc region. In the present invention, such a molecule may be referred to as an antigen-binding molecule. When the molecule is an antigen-binding molecule having multiple specificities for, for example, two or more different antigens, it may be referred to as a multispecific antigen-binding molecule.

[0145] In one aspect, one polynucleotide or two or more polynucleotides are provided that encode the antigen-binding molecule described in any one of the embodiments or aspects herein.For example, when the antigen-binding molecule comprises or consists of more than one polypeptide chain, the antigen-binding molecule can be encoded by one polynucleotide, or the antigen-binding molecule can be encoded by two or more polynucleotides.Such two or more polynucleotides can be, for example, on the same single vector, or separately on two or more vectors.For example, two or more vectors can be expressed in a single host cell, or can be expressed separately.

[0146] In one aspect, there is provided a polynucleotide, particularly a DNA, encoding the antigen-binding molecule of any one of the embodiments or aspects herein. In one aspect, there is provided a vector comprising the polynucleotide of any one of the embodiments or aspects herein.

[0147] In one aspect, there is provided a vector comprising a DNA sequence encoding an antigen-binding molecule as defined in any one of the embodiments or aspects herein, the vector being particularly for recombinant protein expression. In one aspect, there is provided a host cell comprising the vector of any one of the embodiments or aspects herein.

[0148] In one aspect, a host cell is provided comprising a vector of any embodiment or aspect herein, the host cell being particularly for recombinant protein expression of an antigen binding molecule encoded in the vector.

[0149] In one aspect, a method for producing an antigen-binding molecule is provided, comprising the steps of: (a) providing one or more nucleic acids encoding at least one first portion, wherein the first portion binds to a first antigen, and the first antigen is a damage-associated molecular pattern (DAMP), the first antigen being selected from the group consisting of: a) splicing factor 3B subunit 3 (SF3B3); and b) proliferating cell nuclear antigen (PCNA); (b) providing one or more nucleic acids encoding at least one second portion, wherein the second portion binds to a second antigen and the second antigen is different from the first antigen, preferably the second antigen is a membrane protein of an immune cell or a tumor cell; (b1) optionally, providing one or more nucleic acids encoding at least one third portion, wherein the third portion binds to a third antigen, the third antigen being different from the first antigen, and preferably the third antigen being a membrane protein of an immune cell or a tumor cell; (c) obtaining one or more nucleic acids encoding the antigen-binding molecule, wherein at least one first portion provided in (a), at least one second portion provided in (b), and optionally at least one third portion provided in (b1) are linked; and (d) producing an antigen-binding molecule using one or more nucleic acids prepared in (c), thereby obtaining the antigen-binding molecule.

[0150] In an embodiment, the first portion in step (a) is as defined in any one of embodiments [AA1] to [AA13]. In an embodiment, the second portion in step (b) is as defined in any one of embodiments [AA1] to [AA13]. In an embodiment, the third portion in step (b1) is as defined in any one of embodiments [AA2] to [AA13]. In an embodiment, the one or more nucleic acids are RNA or DNA. In an embodiment, the one or more nucleic acids obtained in step (c) are one nucleic acid or two nucleic acids, optionally, the two nucleic acids are on a single expression vector or on separate expression vectors.

[0151] In embodiments, step (d) comprises recombinant expression of the antigen binding molecule in a host cell. In another aspect, there is provided a method for producing an antigen-binding molecule, comprising the steps of: (a) providing one or more nucleic acids encoding at least one first portion, wherein the first portion binds to a first antigen, and the first antigen is a damage-associated molecular pattern (DAMP); the first antigen is selected from the group consisting of: a) F-actin; b) phosphatidylserine (PS); c) splicing factor 3B subunit 3 (SF3B3); and d) proliferating cell nuclear antigen (PCNA); (b) providing one or more nucleic acids encoding at least one second portion, wherein the second portion binds to a second antigen and the second antigen is different from the first antigen; preferably, the second antigen is a membrane protein of an immune cell or a tumor cell; (b1) providing one or more nucleic acids encoding at least one third portion, wherein the third portion binds to a third antigen, the third antigen being different from the first antigen, and preferably the third antigen being a membrane protein of an immune cell or a tumor cell; (c) obtaining one or more nucleic acids encoding the antigen-binding molecule, wherein at least one first portion provided in (a), at least one second portion provided in (b), and at least one first portion provided in (b1) are linked; and (d) producing an antigen-binding molecule using one or more nucleic acids prepared in (c), thereby obtaining the antigen-binding molecule.

[0152] In an embodiment, the third antigen is different from the first and second antigens. In an embodiment, the third antigen is different from the first antigen but the same as the second antigen. In an embodiment, the antigen-binding molecule obtained in step (b) is as defined in any one of embodiments [AA117] to [AA126]. In an embodiment, the first portion in step (a) is as defined in any one of embodiments [AA117] to [AA126]. In an embodiment, the second portion in step (b) is as defined in any one of embodiments [AA117] to [AA126]. In an embodiment, the third portion in step (b') is as defined in any one of embodiments [AA117] to [AA126]. In an embodiment, the one or more nucleic acids are RNA or DNA. In an embodiment, the one or more nucleic acids obtained in step (c) are one nucleic acid or two nucleic acids, and optionally, the two nucleic acids are on a single expression vector or separate expression vectors. In embodiments, step (d) comprises recombinant expression of the antigen binding molecule in a host cell.

[0153] In one aspect, a use of at least one first portion and at least one second portion for the manufacture 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 or a tumor cell, and the first antigen is (a) splicing factor 3B subunit 3 (SF3B3); and (b) Proliferating cell nuclear antigen (PCNA) selected from the group consisting of Use is provided.

[0154] In one embodiment, the antigen-binding molecule comprises at least one third moiety that binds to a third antigen; (i) the third antigen is different from the first and second antigens, or (ii) the third antigen is different from the first antigen but the same as the second antigen; preferably, the third antigen is a membrane protein of an immune cell or a tumor cell. In one embodiment, the use is an in vitro use. In one embodiment, the use is an in vivo use. In one embodiment, the use is an ex vivo use. In one embodiment, the first moiety is as defined in any one of embodiments [AA1] to [AA13]. In one embodiment, the second moiety is as defined in any one of embodiments [AA1] to [AA13]. In one embodiment, the third moiety is as defined in any one of embodiments [AA2] to [AA13].

[0155] In one aspect, (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; Use of at least one first portion, at least one second portion, and at least one third portion for the manufacture of 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; the second antigen is a membrane protein of an immune cell or a tumor cell, and the molecule comprises at least one third moiety that binds to a third antigen; the third antigen is different from the first antigen; preferably, the third antigen is a membrane protein of an immune cell or a tumor cell; The first antigen is (a) F-actin; (b) phosphatidylserine (PS); (c) splicing factor 3B subunit 3 (SF3B3); and (d) Proliferating cell nuclear antigen (PCNA) selected from the group consisting of Use is provided.

[0156] In embodiments, (i) said third antigen is different from the first and second antigens, or (ii) said third antigen is different from the first antigen but the same as the second antigen. In embodiments, the first portion is as defined in any one of embodiments [AA117]-[AA126]. In embodiments, the second portion is as defined in any one of embodiments [AA117]-[AA126]. In embodiments, the third portion is as defined in any one of embodiments [AA117]-[AA126].

[0157] Antigen-binding molecules and multispecific antigen-binding molecules As used herein, the term "antigen-binding molecule" refers to any molecule containing an antigen-binding site or any molecule having 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, for example, be 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 portion of the molecule serves as the antigen-binding site, are also one embodiment of the antigen-binding molecules described herein.

[0158] A "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.

[0159] antigen-binding domain As described above, when the molecule of the present invention, i.e., "a 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, for example, an antibody, also referred to as an antigen-binding molecule as described above, in some embodiments, the "second portion that binds to the second antigen," and, if appropriate, further the "first portion that binds to the first antigen," may be an antigen-binding domain of or derived from an antibody.

[0160] The term "antigen-binding domain" refers to a portion of an antibody that comprises an area that specifically binds to and is complementary to part or all of an antigen. An antigen-binding domain can be provided, for example, by one or more antibody variable domains (also called antibody variable regions). Preferably, the 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."

[0161] Ig type binding part The terms "Ig-type binding moiety," "Ig-type binding moiety," "Ig-like moiety," "Ig-like binding moiety," and the like, which are used interchangeably 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 comprise at least one immunoglobulin structure), and in particular includes all examples of the latter term described herein. Preferably, the Ig-type binding moiety used herein 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, 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, including in particular 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 any particular embodiment of those described elsewhere herein.

[0162] Variable region The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains (VH and VL, respectively) of a natural antibody 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., W.H. 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 using a VH or VL domain from an antibody that binds that antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0163] HVR or CDR As used herein, the term "hypervariable region" or "HVR" 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-contacting residues ("antigen contacts"). Hypervariable regions (HVRs) are also called "complementarity-determining regions" (CDRs), and these terms are used interchangeably herein in reference to the portions of the variable region that form the antigen-binding region. Typically, antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3).

[0164] 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 (e.g., 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.

[0165] Able to bind to antigens Whether an antibody variable region is "capable of binding to an antigen" can be determined by methods known in the art, such as 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 small antibody composed of the 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 to a streptavidin-immobilized plate. In this procedure, the biotin-labeled antigen-binding molecule to be tested binds to the streptavidin on the plate. Light is emitted from the sulfo-tag, and the luminescence signal is detected using a Sector Imager 600 or 2400 (MSD KK), etc., thereby confirming the binding of the above-mentioned region of the antigen-binding molecule to the antigen to be tested. 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).

[0166] Specifically, the assay can be performed using, for example, the Biacore (GE Healthcare Japan Corp.) interaction analyzer, which is based on the surface plasmon resonance (SPR) phenomenon. Biacore analyzers include any model, such as the Biacore T100, T200, X100, A100, 4000, 3000, 2000, 1000, or C. Any Biacore sensor chip, such as the 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 onto the sensor chip as an analyte, and the interaction is measured to obtain a sensorgram. In this procedure, the antigen concentration can be selected within the range of several μM to several pM depending on the strength of the interaction (for example, KD) of the assay sample.

[0167] 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 the antibody variable region of the antigen-binding molecule of the present invention has antigen-binding activity 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.

[0168] In some embodiments, the binding activity or affinity of the antibody variable regions 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, and 0.005% NaN3. The sensor surface is regenerated with 3 M MgCl2 at each cycle. 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). KD values are calculated to evaluate the specific binding activity or affinity of antigen-binding domains.

[0169] ALPHAScreen is implemented using ALPHA technology, which uses two types of beads (donor and acceptor), based on the following principle: a luminescent signal is detected only when the two beads are positioned in close proximity through biological interactions between molecules bound to the donor beads and molecules bound to the acceptor beads. A photosensitizer in the donor beads, excited by a laser, converts ambient oxygen into singlet oxygen, which has an excited state. The singlet oxygen diffuses around the donor beads and reaches the nearby acceptor beads, thereby triggering a chemiluminescent reaction in the beads, which ultimately emits light. In the absence of interactions between molecules bound to the donor beads and molecules bound to the acceptor beads, the singlet oxygen produced by the donor beads does not reach the acceptor beads. Therefore, the chemiluminescent reaction does not occur.

[0170] One of the substances (ligands) whose interaction will be monitored is immobilized on a thin gold film on a sensor chip. Light is shone on the back of the sensor chip to cause total internal reflection 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 will be monitored is then 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 (a 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 analyte interaction) can be determined from the sensorgram. However, since the amount of binding also depends on the amount of ligand, comparison should be made 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.An example of an inhibition assay is described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010.

[0171] 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, generating a signal at 520-620 nm. The untagged second antigen competes with the antigen for interaction with the antigen-binding molecule to be tested. The resulting decrease in fluorescence is quantified, thereby determining relative binding activity. Polypeptide biotinylation using sulfo-NHS-biotin or similar is known in the art. For example, an antigen can be tagged with GST by any suitable method, including fusing a polynucleotide encoding the antigen with a polynucleotide encoding GST in frame; expressing the resulting fusion gene in cells carrying a vector enabling its expression; and then purifying it using a glutathione column. The resulting signals are preferably analyzed by fitting to a one-site competition model based on nonlinear regression analysis, for example, using the software GRAPHPAD PRISM (GraphPad Software, Inc., San Diego).

[0172] 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. Binding of the antigen-binding molecule to be tested to the donor beads 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 to the donor beads via an Fc-recognizing protein such as protein A or protein G.

[0173] 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").

[0174] 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.

[0175] "F(ab')2" or "Fab" refers to an antibody fragment produced by treating an immunoglobulin (monoclonal antibody) with a protease such as pepsin or papain, digesting the immunoglobulin (monoclonal antibody) near the disulfide bond between the hinge regions of each of the two H chains. For example, papain cleaves IgG upstream of the disulfide bond between the hinge regions of each of the two H chains to produce two homologous antibody fragments, in which an L chain containing a VL (light chain variable region) and a CL (light chain constant region) is linked to an H chain fragment containing a VH (heavy chain variable region) and a CHγ1 (γ1 region in the heavy chain constant region) via a disulfide bond at their C-terminal regions. Each of these two homologous antibody fragments is called Fab'.

[0176] "F(ab')2" consists of two light chains and two heavy chains containing constant regions, namely, the CH1 domain and a portion of the CH2 domain, resulting in the formation of a disulfide bond between the two heavy chains. The F(ab')2 disclosed herein can be preferably produced as follows: a whole monoclonal antibody containing the 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 appropriate enzyme reaction conditions, such as pH. Examples of such proteases include pepsin and ficin.

[0177] fused / fused The terms "fused" and "fusion" mean that two or more different polypeptides (e.g., a first polypeptide that binds to a first antigen and a second polypeptide that binds to a second antigen; a Fab arm and an Fc domain) are linked by peptide bonds, either directly or via one or more peptide linkers.

[0178] Thus, as used herein, the term "fusion protein" (which may be used interchangeably with the term "fusion polypeptide") 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.

[0179] 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 have been 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 have been 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 have been exchanged, the peptide chain comprising the heavy chain variable region is referred to herein as the "heavy chain" of the crossover Fab molecule.

[0180] Traditional 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 approximately 150,000 daltons composed of two disulfide-bonded light chains and two heavy chains. From the N-terminus to the C-terminus, each heavy chain contains a variable region (VH), also called a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also called heavy chain constant regions. Similarly, from the N-terminus to the C-terminus, each light chain contains a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light (CL) domain, also called a light chain constant region. Immunoglobulin heavy chains 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). Immunoglobulin light chains 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 one Fc domain linked via an immunoglobulin hinge region.

[0181] Affinity "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a first or second moiety; an antibody or 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 intrinsic 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 the dissociation constant (KD), which is the ratio of the dissociation rate constant to the association rate constant (koff and kon, respectively). Thus, equivalent affinities may involve 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).

[0182] How affinity is determined In certain embodiments, each of the first or second moieties of the "molecule comprising a first moiety that binds to a first antigen and a second moiety that binds to a second antigen" of the present invention, and the antigen-binding molecule or antibody as described above, has 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 molecules of the invention have a dissociation constant (KD) for their antigens (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, respectively.

[0183] In one embodiment, KD is measured by a radiolabeled antigen binding assay (RIA). For example, the solution binding affinity of a molecule of the invention to an antigen (i.e., a first antigen, a second antigen) is measured at a minimum concentration of ( 125 I) Measurement is performed by equilibrating the molecule with labeled antigen, then capturing the bound antigen on a plate coated with an antibody that binds to the molecule (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish assay conditions, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 μg / ml of antibody (Cappel Labs) that binds to the capture molecule in 50 mM sodium carbonate (pH 9.6), and then blocked with 2...

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 or a tumor cell, and The first antigen is (a) splicing factor 3B subunit 3 (SF3B3); and (b) Proliferating cell nuclear antigen (PCNA) selected from the group consisting of Antigen-binding molecules.

2. The antigen-binding molecule of claim 1, comprising at least one third moiety that binds to a third antigen; (i) the third antigen is different from the first and second antigens, or (ii) the third antigen is different from the first antigen but the same as the second antigen; preferably, the third antigen is a membrane protein of an immune cell or a tumor cell.

3. The antigen-binding molecule of claim 1 or 2, wherein the first antigen is exposed to the extracellular environment due to cell damage, particularly cell death.

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

5. (A) if the first antigen is splicing factor 3B subunit 3 (SF3B3), the first moiety is an Ig-type binding moiety; or (B) when the first antigen is splicing factor 3B subunit 3 (SF3B3), the first moiety is a Clec4e polypeptide; or (C) if the first antigen is proliferating cell nuclear antigen (PCNA), the first moiety is an Ig-type binding moiety; The antigen-binding molecule of claim 4.

6. the second and / or third 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 these, the second and / or third antigen is a membrane protein involved in signal transduction in cells, preferably a signal transduction receptor. The antigen-binding molecule of any one of claims 1 to 5.

7. The antigen-binding molecule of any one of claims 1 to 6, wherein the second and / or third antigen is a membrane protein of a tumor cell.

8. The antigen-binding molecule of any one of claims 1 to 7, wherein the second and / or third moiety is an Ig-type binding moiety.

9. The antigen-binding molecule of any one of claims 1 to 8, which 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 or a tumor cell, and preferably linked to an Fc region; In particular, the binding polypeptide is selected from a Clec9A polypeptide and a Clec4e polypeptide; Among others, the antibody an anti-CD3 antibody comprising a Clec9A polypeptide; an anti-CD137 antibody comprising a Clec9A polypeptide; an anti-CD3 antibody comprising a Clec4e polypeptide, and Anti-CD137 Antibodies Containing Clec4e Polypeptides selected from the group consisting of The antigen-binding molecule of claim 9.

11. the antigen-binding molecule is an antibody, particularly an IgG antibody, and the first portion is linked to an Fc region and is an Ig-type binding portion; In particular, the antibody (A) Anti-CD3 antibody; (B) an anti-CD137 antibody; and (C) Bispecific anti-CD3 anti-CD137 antibody selected from the group consisting of The antigen-binding molecule of any one of claims 1 to 8.

12. the antibody is a bispecific antibody, In particular, the antigen-binding molecule is a bispecific antibody against a membrane protein of an immune cell or a tumor cell and against a first antigen selected from the group consisting of: (a) splicing factor 3B subunit 3 (SF3B3); and (b) proliferating cell nuclear antigen (PCNA); Among others, the antibody bispecific anti-SF3B3 anti-CD3 antibody, bispecific anti-SF3B3 anti-CD137 antibody, bispecific anti-PCNA anti-CD3 antibodies, and Bispecific anti-PCNA anti-CD137 antibody selected from the group consisting of The antigen-binding molecule of claim 9.

13. the antibody is a trispecific antibody; In particular, the antigen-binding molecule is (i) against membrane proteins of immune cells and against membrane proteins of tumor cells; and (a) splicing factor 3B subunit 3 (SF3B3); and (b) proliferating cell nuclear antigen (PCNA), or (ii) to a first membrane protein of an immune cell and a second membrane protein of an immune cell that is different from the first membrane protein; and (a) splicing factor 3B subunit 3 (SF3B3); and (b) proliferating cell nuclear antigen (PCNA), or (iii) to a first membrane protein of a tumor cell and a second membrane protein of a tumor cell that is different from the first membrane protein; and (a) splicing factor 3B subunit 3 (SF3B3); and (b) proliferating cell nuclear antigen (PCNA). It is a trispecific antibody, Among others, the antibody a trispecific anti-SF3B3 anti-CD3 anti-CD137 antibody, and Trispecific anti-PCNA anti-CD3 anti-CD137 antibody selected from the group consisting of The antigen-binding molecule of claim 9.

14. A pharmaceutical composition comprising the antigen-binding molecule of any one of claims 1 to 13.

15. An antigen-binding molecule according to any one of claims 1 to 13 or a pharmaceutical composition according to claim 14 for use in medicine.

16. The antigen-binding molecule of any one of claims 1 to 13 or the pharmaceutical composition of claim 14, for use in a method for treating or preventing a medical condition, preferably wherein the medical condition is selected from the group consisting of cancer and an immune disease, preferably an autoimmune disease.

17. A method for screening an antigen-binding molecule, comprising the steps of: (a) selecting one or more nucleic acids encoding at least one first portion, wherein the first portion binds to a first antigen, and the first antigen is a damage-associated molecular pattern (DAMP), the first antigen being selected from the group consisting of: a) splicing factor 3B subunit 3 (SF3B3); and b) proliferating cell nuclear antigen (PCNA); (b) selecting one or more nucleic acids encoding at least one second portion, wherein the second portion binds to a second antigen and the second antigen is different from the first antigen, preferably the second antigen is a membrane protein of an immune cell or a tumor cell; (b1) optionally, providing one or more nucleic acids encoding at least one third portion, wherein the third portion binds to a third antigen, the third antigen being different from the first antigen, and preferably the third antigen being a membrane protein of an immune cell or a tumor cell; (c) obtaining one or more nucleic acids encoding the antigen-binding molecule, wherein at least one first portion provided in (a), at least one second portion provided in (b), and optionally at least one third portion provided in (b1) are linked; and (d) producing an antigen-binding molecule using one or more nucleic acids prepared in (c), thereby obtaining the antigen-binding molecule.

18. A method for producing an antigen-binding molecule, comprising the steps of: (a) providing one or more nucleic acids encoding at least one first portion, wherein the first portion binds to a first antigen, and the first antigen is a damage-associated molecular pattern (DAMP), the first antigen being selected from the group consisting of: a) splicing factor 3B subunit 3 (SF3B3); and b) proliferating cell nuclear antigen (PCNA); (b) providing one or more nucleic acids encoding at least one second portion, wherein the second portion binds to a second antigen and the second antigen is different from the first antigen, preferably the second antigen is a membrane protein of an immune cell or a tumor cell; (b1) optionally, providing one or more nucleic acids encoding at least one third portion, wherein the third portion binds to a third antigen, the third antigen being different from the first antigen, and preferably the third antigen being a membrane protein of an immune cell or a tumor cell; (c) obtaining one or more nucleic acids encoding the antigen-binding molecule, wherein at least one first portion provided in (a), at least one second portion provided in (b), and optionally at least one third portion provided in (b1) are linked; and (d) producing an antigen-binding molecule using one or more nucleic acids prepared in (c), thereby obtaining the antigen-binding molecule.

19. A method for conferring signal transduction or blocking in a cell, comprising the step of contacting a cell with an antigen-binding molecule or the step of administering an antigen-binding molecule to a subject, wherein the antigen-binding molecule: (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; Including, 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 or a tumor cell, and The first antigen is (a) splicing factor 3B subunit 3 (SF3B3); and (b) Proliferating cell nuclear antigen (PCNA) selected from the group consisting of method.

20. A method for activating or inhibiting an immune response, comprising the step of contacting a cell with an antigen-binding molecule or administering an antigen-binding molecule to a subject, wherein the antigen-binding molecule: (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; Including, 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 or a tumor cell, and The first antigen is (a) splicing factor 3B subunit 3 (SF3B3); and (b) Proliferating cell nuclear antigen (PCNA) selected from the group consisting of method.

21. A bispecific anti-phosphatidylserine (PS) anti-CD3 antibody comprising a heavy chain of the IgG1 isotype and a light chain of either the κ or λ isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers.

22. 21. The bispecific antibody or pharmaceutical composition of claim 20 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

23. A bispecific anti-phosphatidylserine (PS) anti-CD3 antibody comprising a heavy chain of the IgG2 isotype and a light chain of either the κ or λ isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers.

24. 24. The bispecific antibody or pharmaceutical composition of claim 23 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

25. A bispecific anti-phosphatidylserine (PS) anti-CD3 antibody comprising a heavy chain of the IgG3 isotype and a light chain of either the κ or λ isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers.

26. 26. The bispecific antibody or pharmaceutical composition of claim 25 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

27. A bispecific anti-phosphatidylserine (PS) anti-CD3 antibody comprising a heavy chain of the IgG4 isotype and a light chain of either the κ or λ isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers.

28. 28. The bispecific antibody or pharmaceutical composition of claim 27 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

29. A bispecific anti-phosphatidylserine (PS) anti-CD137 antibody comprising a heavy chain of the IgG1 isotype and a light chain of either the κ or λ isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers.

30. 30. The bispecific antibody or pharmaceutical composition of claim 29 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

31. A bispecific anti-phosphatidylserine (PS) anti-CD137 antibody comprising a heavy chain of the IgG2 isotype and a light chain of either the kappa or lambda isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers.

32. 32. The bispecific antibody or pharmaceutical composition of claim 31 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

33. A bispecific anti-phosphatidylserine (PS) anti-CD137 antibody comprising a heavy chain of the IgG3 isotype and a light chain of either the kappa or lambda isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers.

34. 34. The bispecific antibody or pharmaceutical composition of claim 33 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

35. A bispecific anti-phosphatidylserine (PS) anti-CD137 antibody comprising a heavy chain of the IgG4 isotype and a light chain of either the kappa or lambda isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers.

36. 36. The bispecific antibody or pharmaceutical composition of claim 35 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

37. A bispecific anti-F-actin anti-CD3 antibody comprising a heavy chain of the IgG1 isotype and a light chain of the κ or λ isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers.

38. 38. The bispecific antibody or pharmaceutical composition of claim 37 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

39. A bispecific anti-F-actin anti-CD3 antibody comprising a heavy chain of the IgG2 isotype and a light chain of the κ or λ isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers.

40. 40. The bispecific antibody or pharmaceutical composition of claim 39 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

41. 1. A bispecific anti-F-actin, anti-CD3 antibody comprising a heavy chain of the IgG3 isotype and a light chain of the κ or λ isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers.

42. 42. The bispecific antibody or pharmaceutical composition of claim 41 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

43. 1. A bispecific anti-F-actin, anti-CD3 antibody comprising a heavy chain of the IgG4 isotype and a light chain of either the κ or λ isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers.

44. 44. The bispecific antibody or pharmaceutical composition of claim 43 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

45. A bispecific anti-F-actin anti-CD137 antibody comprising a heavy chain of the IgG1 isotype and a light chain of the κ or λ isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers.

46. 46. The bispecific antibody or pharmaceutical composition of claim 45 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

47. A bispecific anti-F-actin anti-CD137 antibody comprising a heavy chain of the IgG2 isotype and a light chain of the κ or λ isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers.

48. 48. The bispecific antibody or pharmaceutical composition of claim 47 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

49. 1. A bispecific anti-F-actin anti-CD137 antibody comprising a heavy chain of the IgG3 isotype and a light chain of either the κ or λ isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers.

50. 50. The bispecific antibody or pharmaceutical composition of claim 49 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

51. 1. A bispecific anti-F-actin anti-CD137 antibody comprising a heavy chain of the IgG4 isotype and a light chain of either the κ or λ isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers.

52. 52. The bispecific antibody or pharmaceutical composition of claim 51 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

53. A bispecific antigen-binding molecule that is an anti-CD3 antibody comprising a Clec9A polypeptide, wherein the bispecific antigen-binding molecule is an IgG-type antibody comprising a heavy chain of the IgG1 isotype and a light chain of the κ or λ isotype, or a pharmaceutical composition comprising the bispecific antigen-binding molecule and one or more pharmaceutically acceptable carriers.

54. 54. The bispecific molecule or pharmaceutical composition of claim 53 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

55. A bispecific antigen-binding molecule that is an anti-CD3 antibody comprising a Clec9A polypeptide, wherein the bispecific antigen-binding molecule is an IgG type antibody comprising a heavy chain of an IgG2 isotype and a light chain of a κ or λ isotype, or a pharmaceutical composition comprising the bispecific antigen-binding molecule and one or more pharmaceutically acceptable carriers.

56. 56. The bispecific molecule or pharmaceutical composition of claim 55 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

57. A bispecific antigen-binding molecule that is an anti-CD3 antibody comprising a Clec9A polypeptide, wherein the bispecific antigen-binding molecule is an IgG-type antibody comprising a heavy chain of the IgG3 isotype and a light chain of either the κ isotype or the λ isotype, or a pharmaceutical composition comprising the bispecific antigen-binding molecule and one or more pharmaceutically acceptable carriers.

58. 58. The bispecific molecule or pharmaceutical composition of claim 57, for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

59. A bispecific antigen-binding molecule that is an anti-CD3 antibody comprising a Clec9A polypeptide, wherein the bispecific antigen-binding molecule is an IgG-type antibody comprising a heavy chain of an IgG4 isotype and a light chain of a κ or λ isotype, or a pharmaceutical composition comprising the bispecific antigen-binding molecule and one or more pharmaceutically acceptable carriers.

60. 60. The bispecific molecule or pharmaceutical composition of claim 59 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

61. A bispecific antigen-binding molecule that is an anti-CD137 antibody comprising a Clec9A polypeptide, wherein the bispecific antigen-binding molecule is an IgG type antibody comprising a heavy chain of IgG1 isotype and a light chain of κ or λ isotype, or a pharmaceutical composition comprising the bispecific antigen-binding molecule and one or more pharmaceutically acceptable carriers.

62. 62. The bispecific molecule or pharmaceutical composition of claim 61 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

63. A bispecific antigen-binding molecule that is an anti-CD137 antibody comprising a Clec9A polypeptide, wherein the bispecific antigen-binding molecule is an IgG type antibody comprising a heavy chain of an IgG2 isotype and a light chain of a κ or λ isotype, or a pharmaceutical composition comprising the bispecific antigen-binding molecule and one or more pharmaceutically acceptable carriers.

64. 64. The bispecific molecule or pharmaceutical composition of claim 63 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

65. A bispecific antigen-binding molecule that is an anti-CD137 antibody comprising a Clec9A polypeptide, wherein the bispecific antigen-binding molecule is an IgG-type antibody comprising a heavy chain of an IgG3 isotype and a light chain of a κ or λ isotype, or a pharmaceutical composition comprising the bispecific antigen-binding molecule and one or more pharmaceutically acceptable carriers.

66. 66. The bispecific molecule or pharmaceutical composition of claim 65 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

67. A bispecific antigen-binding molecule that is an anti-CD137 antibody comprising a Clec9A polypeptide, wherein the bispecific antigen-binding molecule is an IgG-type antibody comprising a heavy chain of an IgG4 isotype and a light chain of a κ or λ isotype, or a pharmaceutical composition comprising the bispecific antigen-binding molecule and one or more pharmaceutically acceptable carriers.

68. 68. The bispecific molecule or pharmaceutical composition of claim 67 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

69. A bispecific anti-splice factor 3B subunit 3 (SF3B3) anti-CD3 antibody comprising a heavy chain of the IgG1 isotype and a light chain of the kappa or lambda isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers.

70. 70. The bispecific antibody or pharmaceutical composition of claim 69 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

71. A bispecific anti-splice factor 3B subunit 3 (SF3B3) anti-CD3 antibody comprising a heavy chain of the IgG2 isotype and a light chain of the kappa or lambda isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers.

72. 72. The bispecific antibody or pharmaceutical composition of claim 71 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

73. A bispecific anti-splice factor 3B subunit 3 (SF3B3) anti-CD3 antibody comprising a heavy chain of the IgG3 isotype and a light chain of the kappa or lambda isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers.

74. 74. The bispecific antibody or pharmaceutical composition of claim 73 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

75. 1. A bispecific anti-splice factor 3B subunit 3 (SF3B3) anti-CD3 antibody comprising a heavy chain of the IgG4 isotype and a light chain of the kappa or lambda isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers.

76. 76. The bispecific antibody or pharmaceutical composition of claim 75 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

77. A bispecific anti-splice factor 3B subunit 3 (SF3B3) anti-CD137 antibody comprising a heavy chain of the IgG1 isotype and a light chain of the kappa or lambda isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers.

78. 78. The bispecific antibody or pharmaceutical composition of claim 77 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

79. 1. A bispecific anti-splice factor 3B subunit 3 (SF3B3) anti-CD137 antibody comprising a heavy chain of the IgG2 isotype and a light chain of the kappa or lambda isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers.

80. 80. The bispecific antibody or pharmaceutical composition of claim 79 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

81. 1. A bispecific anti-splice factor 3B subunit 3 (SF3B3) anti-CD137 antibody comprising a heavy chain of the IgG3 isotype and a light chain of the kappa or lambda isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers.

82. 82. The bispecific antibody or pharmaceutical composition of claim 81 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

83. 1. A bispecific anti-splice factor 3B subunit 3 (SF3B3) anti-CD137 antibody comprising a heavy chain of the IgG4 isotype and a light chain of the kappa or lambda isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers.

84. 84. The bispecific antibody or pharmaceutical composition of claim 83 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

85. A bispecific antigen-binding molecule that is an anti-CD3 antibody comprising a Clec4e polypeptide, wherein the bispecific antigen-binding molecule is an IgG-type antibody comprising a heavy chain of the IgG1 isotype and a light chain of the κ or λ isotype, or a pharmaceutical composition comprising the bispecific antigen-binding molecule and one or more pharmaceutically acceptable carriers.

86. 86. The bispecific molecule or pharmaceutical composition of claim 85 for use in a method for treating or preventing a cancer disease, wherein preferably the cancer disease is a solid tumor.

87. A bispecific antigen-binding molecule that is an anti-CD3 antibody comprising a Clec4e polypeptide, wherein the bispecific antigen-binding molecule is an IgG-type antibody comprising a heavy chain of the IgG2 isotype and a light chain of the κ or λ isotype, or a pharmaceutical composition comprising the bispecific antigen-binding molecule and one or more pharmaceutically acceptable carriers.

88. 88. The bispecific molecule or pharmaceutical composition of claim 87 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

89. A bispecific antigen-binding molecule that is an anti-CD3 antibody comprising a Clec4e polypeptide, wherein the bispecific antigen-binding molecule is an IgG-type antibody comprising a heavy chain of the IgG3 isotype and a light chain of the κ or λ isotype, or a pharmaceutical composition comprising the bispecific antigen-binding molecule and one or more pharmaceutically acceptable carriers.

90. 90. The bispecific molecule or pharmaceutical composition of claim 89 for use in a method for treating or preventing a cancer disease, wherein preferably said cancer disease is a solid tumor.

91. A bispecific antigen-binding molecule that is an anti-CD3 antibody comprising a Clec4e polypeptide, wherein the bispecific antigen-binding molecule is an IgG-type antibody comprising a heavy chain of the IgG4 isotype and a light chain of the κ or λ isotype, or a pharmaceutical composition comprising the bispecific antigen-binding molecule and one or more pharmaceutically acceptable carriers.

92. 92. The bispecific molecule or pharmaceutical composition of claim 91 for use in a method for treating or preventing a cancer disease, wherein preferably said cancer disease is a solid tumor.

93. A bispecific antigen-binding molecule that is an anti-CD137 antibody comprising a Clec4e polypeptide, wherein the bispecific antigen-binding molecule is an IgG type antibody comprising a heavy chain of IgG1 isotype and a light chain of κ or λ isotype, or a pharmaceutical composition comprising the bispecific antigen-binding molecule and one or more pharmaceutically acceptable carriers.

94. 94. The bispecific molecule or pharmaceutical composition of claim 93 for use in a method for treating or preventing a cancer disease, wherein preferably said cancer disease is a solid tumor.

95. A bispecific antigen-binding molecule that is an anti-CD137 antibody comprising a Clec4e polypeptide, wherein the bispecific antigen-binding molecule is an IgG type antibody comprising a heavy chain of an IgG2 isotype and a light chain of a κ or λ isotype, or a pharmaceutical composition comprising the bispecific antigen-binding molecule and one or more pharmaceutically acceptable carriers.

96. 96. The bispecific molecule or pharmaceutical composition of claim 95 for use in a method for treating or preventing a cancer disease, wherein preferably the cancer disease is a solid tumor.

97. A bispecific antigen-binding molecule that is an anti-CD137 antibody comprising a Clec4e polypeptide, wherein the bispecific antigen-binding molecule is an IgG type antibody comprising a heavy chain of IgG3 isotype and a light chain of κ or λ isotype, or a pharmaceutical composition comprising the bispecific antigen-binding molecule and one or more pharmaceutically acceptable carriers.

98. 98. The bispecific molecule or pharmaceutical composition of claim 97 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

99. A bispecific antigen-binding molecule that is an anti-CD137 antibody comprising a Clec4e polypeptide, wherein the bispecific antigen-binding molecule is an IgG-type antibody comprising a heavy chain of the IgG4 isotype and a light chain of the κ or λ isotype, or a pharmaceutical composition comprising the bispecific antigen-binding molecule and one or more pharmaceutically acceptable carriers.

100. 100. The bispecific molecule or pharmaceutical composition of claim 99 for use in a method for treating or preventing a cancer disease, preferably wherein said cancer disease is a solid tumor.

101. A bispecific anti-proliferating cell nuclear antigen (PCNA) anti-CD3 antibody comprising a heavy chain of the IgG1 isotype and a light chain of either the κ or λ isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers.

102. 102. The bispecific antibody or pharmaceutical composition of claim 101 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

103. A bispecific anti-proliferating cell nuclear antigen (PCNA) anti-CD3 antibody comprising a heavy chain of the IgG2 isotype and a light chain of either the kappa or lambda isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers.

104. 104. The bispecific antibody or pharmaceutical composition of claim 103 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

105. A bispecific anti-proliferating cell nuclear antigen (PCNA) anti-CD3 antibody comprising a heavy chain of the IgG3 isotype and a light chain of either the kappa or lambda isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers.

106. 106. The bispecific antibody or pharmaceutical composition of claim 105 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

107. 1. A bispecific anti-proliferating cell nuclear antigen (PCNA) anti-CD3 antibody comprising a heavy chain of the IgG4 isotype and a light chain of either the kappa or lambda isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers.

108. 108. The bispecific antibody or pharmaceutical composition of claim 107 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

109. A bispecific anti-proliferating cell nuclear antigen (PCNA) anti-CD137 antibody comprising a heavy chain of the IgG1 isotype and a light chain of either the kappa or lambda isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers.

110. 110. The bispecific molecule or pharmaceutical composition of claim 109 for use in a method for treating or preventing a cancer disease, wherein preferably said cancer disease is a solid tumor.

111. A bispecific anti-proliferating cell nuclear antigen (PCNA) anti-CD137 antibody comprising a heavy chain of the IgG2 isotype and a light chain of either the kappa or lambda isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers.

112. 112. The bispecific antibody or pharmaceutical composition of claim 111 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

113. 1. A bispecific anti-proliferating cell nuclear antigen (PCNA) anti-CD137 antibody comprising a heavy chain of the IgG3 isotype and a light chain of either the kappa or lambda isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers.

114. 114. The bispecific antibody or pharmaceutical composition of claim 113 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

115. 1. A bispecific anti-proliferating cell nuclear antigen (PCNA) anti-CD137 antibody comprising a heavy chain of the IgG4 isotype and a light chain of either the kappa or lambda isotype, or a pharmaceutical composition comprising said bispecific antibody and one or more pharmaceutically acceptable carriers.

116. 116. The bispecific antibody or pharmaceutical composition of claim 115 for use in a method for treating or preventing a cancer disease, preferably wherein the cancer disease is a solid tumor.

117. (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 or a tumor cell, and the molecule comprises at least one third moiety that binds to a third antigen; the third antigen is different from the first antigen; preferably, the third antigen is a membrane protein of an immune cell or a tumor cell; and The first antigen is (a) F-actin; (b) phosphatidylserine (PS); (c) splicing factor 3B subunit 3 (SF3B3); and (d) Proliferating cell nuclear antigen (PCNA) selected from the group consisting of Antigen-binding molecules.

118. 118. The antigen-binding molecule of claim 117, wherein (i) the third antigen is different from the first and second antigens, or (ii) the third antigen is different from the first antigen but the same as the second antigen.

119. The first part is (A) an Ig-type binding moiety; or (B) A binding polypeptide, particularly a non-Ig binding moiety.

119. The antigen-binding molecule of any one of claims 117 to 118, selected from the group consisting of:

120. (A) if the first antigen is splicing factor 3B subunit 3 (SF3B3), the first moiety is an Ig-type binding moiety; or (B) when the first antigen is splicing factor 3B subunit 3 (SF3B3), the first moiety is a Clec4e polypeptide; or (C) if the first antigen is proliferating cell nuclear antigen (PCNA), the first moiety is an Ig-type binding moiety; or (D) when the first antigen is F-actin, the first moiety is an Ig-type binding moiety; or (E) when the first antigen is F-actin, the first moiety is a Clec9A polypeptide; or (F) When the first antigen is PS, the first moiety is an Ig-type binding moiety; The antigen-binding molecule of claim 119.

121. the second and / or third 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 these, the second and / or third 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 117 to 120.

122. The antigen-binding molecule of any one of claims 117 to 121, wherein the second and / or third antigen is a membrane protein of a tumor cell.

123. The antigen-binding molecule of any one of claims 117 to 122, wherein the second and / or third moiety is an Ig-type binding moiety.

124. The antigen-binding molecule of any one of claims 117 to 123, which is an antibody, particularly an IgG type antibody.

125. the antibody further comprises at least one binding polypeptide capable of binding to a membrane protein of an immune cell or a tumor cell, and preferably linked to an Fc region; In particular, the binding polypeptide is selected from a Clec9A polypeptide and a Clec4e polypeptide; Among others, the antibody an anti-CD3 antibody comprising a Clec9A polypeptide; an anti-CD137 antibody comprising a Clec9A polypeptide; an anti-CD3 antibody comprising a Clec4e polypeptide, and Anti-CD137 Antibodies Containing Clec4e Polypeptides selected from the group consisting of The antigen-binding molecule of claim 124.

126. the antibody is a trispecific antibody; In particular, the antigen-binding molecule is (i) against membrane proteins of immune cells and against membrane proteins of tumor cells; and (a) F-actin; (b) phosphatidylserine (PS); (c) splicing factor 3B subunit 3 (SF3B3); and (d) proliferating cell nuclear antigen (PCNA), or (ii) to a first membrane protein of an immune cell and a second membrane protein of an immune cell that is different from the first membrane protein; and (a) F-actin; (b) phosphatidylserine (PS); (c) splicing factor 3B subunit 3 (SF3B3); and (d) proliferating cell nuclear antigen (PCNA), or (iii) to a first membrane protein of a tumor cell and a second membrane protein of a tumor cell that is different from the first membrane protein; and (a) F-actin; (b) phosphatidylserine (PS); (c) splicing factor 3B subunit 3 (SF3B3); and (d) proliferating cell nuclear antigen (PCNA). It is a trispecific antibody, Among others, the antibody trispecific anti-SF3B3 anti-CD3 anti-CD137 antibody, trispecific anti-PCNA anti-CD3 anti-CD137 antibody, trispecific anti-PS anti-CD3 anti-CD137 antibody, Trispecific anti-F-actin, anti-CD3, and anti-CD137 antibodies a bispecific anti-CD3 anti-CD137 antibody comprising a Clec9A polypeptide, and Bispecific anti-CD3 anti-CD137 antibodies containing Clec4e polypeptides selected from the group consisting of The antigen-binding molecule of claim 125.

127. A method for screening an antigen-binding molecule, comprising the steps of: (a) selecting one or more nucleic acids encoding at least one first portion, wherein the first portion binds to a first antigen, and the first antigen is a damage-associated molecular pattern (DAMP), the first antigen being selected from the group consisting of: a) F-actin; b) phosphatidylserine (PS); c) splicing factor 3B subunit 3 (SF3B3); and d) proliferating cell nuclear antigen (PCNA); (b) selecting one or more nucleic acids encoding at least one second portion, wherein the second portion binds to a second antigen and the second antigen is different from the first antigen, preferably the second antigen is a membrane protein of an immune cell or a tumor cell; (b1) providing one or more nucleic acids encoding at least one third portion, wherein the third portion binds to a third antigen, the third antigen being different from the first antigen, and preferably the third antigen being a membrane protein of an immune cell or a tumor cell; (c) obtaining one or more nucleic acids encoding the antigen-binding molecule, wherein at least one first portion provided in (a), at least one second portion provided in (b), and at least one third portion provided in (b1) are linked; and (d) producing an antigen-binding molecule using one or more nucleic acids prepared in (c), thereby obtaining the antigen-binding molecule.

128. A method for producing an antigen-binding molecule, comprising the steps of: (a) providing one or more nucleic acids encoding at least one first portion, wherein the first portion binds to a first antigen, and the first antigen is a damage-associated molecular pattern (DAMP); the first antigen is selected from the group consisting of: a) F-actin; b) phosphatidylserine (PS); c) splicing factor 3B subunit 3 (SF3B3); and d) proliferating cell nuclear antigen (PCNA); (b) providing one or more nucleic acids encoding at least one second portion, wherein the second portion binds to a second antigen and the second antigen is different from the first antigen, preferably the second antigen is a membrane protein of an immune cell or a tumor cell; (b1) providing one or more nucleic acids encoding at least one third portion, wherein the third portion binds to a third antigen, the third antigen being different from the first antigen, and preferably the third antigen being a membrane protein of an immune cell or a tumor cell; (c) obtaining one or more nucleic acids encoding the antigen-binding molecule, wherein at least one first portion provided in (a), at least one second portion provided in (b), and at least one third portion provided in (b1) are linked; and (d) producing an antigen-binding molecule using one or more nucleic acids prepared in (c), thereby obtaining the antigen-binding molecule.

129. A method for conferring signal transduction or blocking in a cell, comprising the step of contacting a cell with an antigen-binding molecule or the step of administering an antigen-binding molecule to a subject, wherein the antigen-binding molecule: (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; Including, 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 or a tumor cell, and the molecule comprises at least one third moiety that binds to a third antigen; the third antigen is different from the first antigen; preferably, the third antigen is a membrane protein of an immune cell or a tumor cell; and The first antigen is (a) F-actin; (b) phosphatidylserine (PS); (c) splicing factor 3B subunit 3 (SF3B3); and (d) Proliferating cell nuclear antigen (PCNA) selected from the group consisting of method.

130. A method for activating or inhibiting an immune response, comprising the step of contacting a cell with an antigen-binding molecule or administering an antigen-binding molecule to a subject, wherein the antigen-binding molecule: (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; Including, 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 or a tumor cell, and the molecule comprises at least one third moiety that binds to a third antigen; the third antigen is different from the first antigen; preferably, the third antigen is a membrane protein of an immune cell or a tumor cell; and The first antigen is (a) F-actin; (b) phosphatidylserine (PS); (c) splicing factor 3B subunit 3 (SF3B3); and (d) Proliferating cell nuclear antigen (PCNA) selected from the group consisting of method.

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