Use of Anti-CLDN4 / Anti-CD137 bispecific antibody in combination with platinum-based drug for treatment of cancer

Abstract

The present invention addresses the problem of providing: an anti-CLDN4 / anti-CD137 bispecific antibody used in combination with a platinum-based drug for treatment of a target cancer; a pharmaceutical composition comprising said bispecific antibody; and a cancer treatment method involving administering, to a subject, an anti-CLDN4 / anti-CD137 bispecific antibody and a platinum-based drug.　In this invention, in a mouse bearing human CLDN4-expressing mouse cancer cells, the combined use of an anti-CLDN4 / anti-CD137 bispecific antibody and oxaliplatin exhibited a significant antitumor effect compared to the case of administering the anti-CLDN4 / anti-CD137 bispecific antibody or oxaliplatin alone. Furthermore, in a mouse bearing human CLDN4-expressing mouse cancer cells, an antitumor effect more effective than double therapy was confirmed in triple therapy in which an anti-CLDN4 / anti-CD137 bispecific antibody is additionally used with double therapy of oxaliplatin and an anti-VEGFR antibody. These results suggest that the combination of an anti-CLDN4 / anti-CD137 bispecific antibody and a platinum-based drug is effective in treatment of a CLDN4-expressing cancer.

Description

Use of anti-cldn4-anti-cd137 bispecific antibodies in combination with platinum-based drugs in cancer treatment

[0001] The present invention relates to the use of an anti-CLDN4-anti-CD137 bispecific antibody in combination with a platinum-based drug in the treatment of cancer.

[0002] Claudin-4 (CLDN4) is a four-transmembrane protein belonging to the claudin family. It is expressed in epithelial cells and endothelial cells and plays an important role as a key molecule constituting tight junctions. CLDN4 is also highly expressed in cancer tissues such as colorectal cancer, bladder cancer, and ovarian cancer, suggesting that anti-CLDN4 antibodies may be useful in cancer treatment or diagnosis (Patent Document 1, Non-Patent Document 1). Furthermore, in animal models, the combined use of anti-CLDN4 antibodies and anti-epidermal growth factor receptor (EGFR) antibodies has been shown to have an antitumor effect (Non-Patent Document 2).

[0003] Cluster of Differentiation 137 (CD137, also known as 4-1BB) is a molecule belonging to the Tumor Necrosis Factor Receptor Superfamily (TNFRSF) and has been reported to be expressed on the surface of immune cells such as T cells, B cells, natural killer (NK) cells, dendritic cells, eosinophils, and mast cells. In particular, CD137 on T cells is known to bind to the CD137 ligand on antigen-presenting cells and act as a costimulatory molecule, contributing to the activation and survival of T cells (Non-Patent Document 3). Anti-CD137 agonist antibodies have demonstrated antitumor effects in animal models via activation of immune cells in the tumor microenvironment (Non-Patent Document 4). The anti-CD137 agonist antibody urelumab has demonstrated therapeutic efficacy in clinical trials, but has also been reported to cause liver damage as a side effect (Non-Patent Document 5).

[0004] Bispecific T-cell-recruiting antibodies of various antibody formats have been reported as an innovative method for obtaining cancer cell-selective cytotoxic activity at low antibody concentrations (Non-Patent Document 6). Bispecific T-cell-recruiting antibodies are bispecific antibodies containing an antibody against tumor-associated antigens (TAA) expressed on the surface of cancer cells and an antibody that binds to T cells, and anti-CD3 antibodies are often used as the antibody that binds to T cells. Furthermore, in recent years, research has also been conducted on bispecific T-cell-recruiting antibodies against CD137 and TAA (Patent Documents 2 and 3). We have produced an anti-CLDN4-anti-CD137 bispecific antibody and demonstrated its usefulness in cancer treatment (Patent Documents 4 and 5).

[0005] Platinum-based drugs are drugs with antitumor activity that contain platinum compounds as their main ingredient. Platinum compounds bind to the DNA in cancer cells, inhibiting DNA replication and suppressing cancer cell proliferation. To date, cisplatin, carboplatin, nedaplatin, oxaliplatin, and miriplatin have been approved.

[0006] To improve the efficacy of treatment in cancer patients, clinical trials are being conducted to investigate the combined effects of bispecific antibodies and cytotoxic chemotherapeutic agents, including platinum-based drugs (Non-Patent Documents 7 and 8). However, to date, no cancer treatment methods have been reported that use anti-CLDN4-anti-CD137 bispecific antibodies in combination with platinum-based drugs.

[0007] International Publication No. WO 2008 / 114733 International Publication No. WO 2015 / 156268 International Publication No. WO 2016 / 177802 International Publication No. WO 2022 / 224997 International Publication No. WO 2024 / 085166

[0008] Cancer Science, 2009:100(9): p. 1623-1630 Oncotarget, 2018:9(100):p. 37367-37378 Cancer Science, 2020:111(5): p. 1461-1467 Cancer Immunology Immunotherapy, 2012:61(5): p. 1721-1733Clinical Cancer Research, 2017:23(8):p. 1929-1936 MAbs, 2017:9(2):p. 182-212Cell Reports Medicine, 2024:5(3):p. 101470Annals of Oncology, 2022:33(7):p. S1423

[0009] The present invention addresses the need to provide a novel combination of an anti-CLDN4-anti-CD137 bispecific antibody and a cytotoxic chemotherapeutic agent for treating cancer in a subject, or to provide a method for treating cancer comprising administering the combination to a subject.

[0010] The present inventors investigated the combination of an anti-CLDN4-anti-CD137 bispecific antibody and a cytotoxic chemotherapeutic agent for use in the treatment of CLDN4-expressing cancer. In mice bearing human CLDN4-expressing murine cancer cells, the combination of an anti-CLDN4-anti-CD137 bispecific antibody and oxaliplatin showed a significant antitumor effect compared to the administration of the anti-CLDN4-anti-CD137 bispecific antibody or oxaliplatin alone (Example 2). Furthermore, in mice bearing human CLDN4-expressing murine cancer cells, a triple-drug combination of an anti-CLDN4-anti-CD137 bispecific antibody in addition to a dual-drug combination of oxaliplatin and an anti-VEGFR antibody also showed a stronger antitumor effect than the dual-drug combination (Example 3).

[0011] That is, the present invention relates to the following [1] to

[68] , but is not limited thereto: [1] A pharmaceutical composition for use in combination with a platinum-based drug to treat cancer in a subject, comprising an anti-CLDN4-anti-CD137 bispecific antibody, wherein the bispecific antibody comprises the heavy chain variable region and light chain variable region of an anti-CLDN4 antibody and the heavy chain variable region and light chain variable region of an anti-CD137 antibody, wherein the heavy chain variable region of the anti-CLDN4 antibody comprises CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 66 of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence from amino acid numbers 99 to 112 of SEQ ID NO: 2, and the light chain variable region of the anti-CLDN4 antibody comprises CDR1 consisting of the amino acid sequence from amino acid numbers 24 to 35 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence from amino acid numbers 51 to 57 of SEQ ID NO: 4, and CDR3 consisting of the amino acid sequence from amino acid numbers 90 to 98 of SEQ ID NO: 4. [2] The pharmaceutical composition of [1], wherein the heavy chain variable region of the anti-CLDN4 antibody consists of the amino acid sequence of amino acid numbers 1 to 123 of SEQ ID NO: 2, and the light chain variable region of the anti-CLDN4 antibody consists of the amino acid sequence of amino acid numbers 1 to 109 of SEQ ID NO: 4. [3] The pharmaceutical composition of [2], wherein the anti-CLDN4-anti-CD137 bispecific antibody comprises an IgG antibody (anti-CLDN4 IgG antibody) consisting of a heavy chain comprising the heavy chain variable region of the anti-CLDN4 antibody and a light chain comprising the light chain variable region of the anti-CLDN4 antibody. [4] The pharmaceutical composition of [3], wherein the Fc region of the anti-CLDN4 IgG antibody comprises either or both of an LALA mutation (L234A and L235A) or a P331G mutation (wherein the mutation positions are amino acid positions according to the EU index in the human Igγ1 constant region).[5] The pharmaceutical composition according to any one of [1] to [4], wherein the heavy chain variable region of the anti-CD137 antibody comprises CDR1 consisting of the amino acid sequence of amino acids 625 to 629 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequence of amino acids 644 to 659 of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence of amino acids 692 to 701 of SEQ ID NO: 2, and the light chain variable region of the anti-CD137 antibody comprises CDR1 consisting of the amino acid sequence of amino acids 486 to 498 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequence of amino acids 514 to 520 of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence of amino acids 553 to 563 of SEQ ID NO: 2. [6] The pharmaceutical composition according to [5], wherein the heavy chain variable region of the anti-CD137 antibody consists of the amino acid sequence of amino acids 595 to 712 of SEQ ID NO: 2, and the light chain variable region of the anti-CD137 antibody consists of the amino acid sequence of amino acids 464 to 573 of SEQ ID NO: 2. [7] The pharmaceutical composition of [6], wherein the anti-CLDN4-anti-CD137 bispecific antibody comprises an anti-CD137 single-chain variable region fragment (anti-CD137 scFv) comprising the heavy chain variable region and light chain variable region of the anti-CD137 antibody. [8] The pharmaceutical composition of [7], wherein the anti-CD137 scFv consists of the amino acid sequence from amino acid numbers 464 to 712 of SEQ ID NO: 2. [9] The pharmaceutical composition of [8], wherein the anti-CLDN4-anti-CD137 bispecific antibody comprises an anti-CLDN4 IgG antibody and an anti-CD137 scFv, and the amino terminus of the anti-CD137 scFv is linked to the heavy chain carboxy terminus of the anti-CLDN4 IgG antibody via a linker.

[10] A pharmaceutical composition used in combination with a platinum-based drug to treat cancer in a subject, comprising an anti-CLDN4-anti-CD137 bispecific antibody, wherein the bispecific antibody comprises a heavy chain of the anti-CLDN4 antibody comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 123 of SEQ ID NO: 2 and a light chain of the anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4, and an anti-CD137 scFv comprising a light chain variable region of the anti-CD137 antibody consisting of the amino acid sequence of amino acids 464 to 573 of SEQ ID NO: 2 and a heavy chain variable region of the anti-CD137 antibody consisting of the amino acid sequence of amino acids 595 to 712 of SEQ ID NO: 2, and the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker.

[11] A pharmaceutical composition used in combination with a platinum-based drug to treat cancer in a subject, comprising an anti-CLDN4-anti-CD137 bispecific antibody, wherein the bispecific antibody comprises an anti-CLDN4 antibody heavy chain consisting of the amino acid sequence of amino acids 1 to 453 of SEQ ID NO: 2, an anti-CLDN4 antibody light chain consisting of the amino acid sequence of amino acids 1 to 215 of SEQ ID NO: 4, and an anti-CD137 scFv consisting of the amino acid sequence of amino acids 464 to 712 of SEQ ID NO: 2, and wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker.

[12] The pharmaceutical composition according to any of [9] to

[11] , wherein the linker is a GS linker.

[13] A pharmaceutical composition comprising an anti-CLDN4-anti-CD137 bispecific antibody used in combination with a platinum-based drug to treat cancer in a subject, wherein the bispecific antibody comprises a polypeptide comprising a heavy chain of an anti-CLDN4 antibody consisting of the amino acid sequence of SEQ ID NO: 2 and an anti-CD137 scFv, and a light chain of an anti-CLDN4 antibody consisting of the amino acid sequence of SEQ ID NO: 4.

[14] The pharmaceutical composition according to any of [1] to

[13] , wherein the anti-CLDN4-anti-CD137 bispecific antibody is post-translationally modified.

[15] The pharmaceutical composition according to any of [1] to

[14] , which is used in combination with a platinum-based drug simultaneously, consecutively, or sequentially.

[16] The pharmaceutical composition according to any one of [1] to

[15] , wherein the cancer is selected from the group consisting of colorectal cancer, non-small cell lung cancer, prostate cancer, ovarian cancer, breast cancer, urothelial cancer, gastric cancer, kidney cancer, pancreatic cancer, uterine cancer, cervical cancer, esophageal cancer, bile duct cancer, and head and neck cancer.

[17] The pharmaceutical composition according to any one of [1] to

[16] , wherein the platinum-based drug is oxaliplatin.

[18] The pharmaceutical composition according to any one of [1] to

[17] , wherein the pharmaceutical composition comprises an anti-CLDN4-anti-CD137 bispecific antibody and is used in combination with a platinum-based drug to treat cancer in a subject, and further comprises a VEGF pathway inhibitory antibody.

[19] An anti-CLDN4-anti-CD137 bispecific antibody used in combination with a platinum-based drug to treat a target cancer, the bispecific antibody comprising the heavy chain variable region and light chain variable region of an anti-CLDN4 antibody, and the heavy chain variable region and light chain variable region of an anti-CD137 antibody, wherein the heavy chain variable region of the anti-CLDN4 antibody comprises CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 66 of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence from amino acid numbers 99 to 112 of SEQ ID NO: 2, and the light chain variable region of the anti-CLDN4 antibody comprises CDR1 consisting of the amino acid sequence from amino acid numbers 24 to 35 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence from amino acid numbers 51 to 57 of SEQ ID NO: 4, and CDR3 consisting of the amino acid sequence from amino acid numbers 90 to 98 of SEQ ID NO: 4. A bispecific antibody.

[20] The bispecific antibody according to

[19] , wherein the heavy chain variable region of the anti-CLDN4 antibody consists of the amino acid sequence of amino acid numbers 1 to 123 of SEQ ID NO: 2, and the light chain variable region of the anti-CLDN4 antibody consists of the amino acid sequence of amino acid numbers 1 to 109 of SEQ ID NO: 4.

[21] The bispecific antibody according to

[20] , comprising an IgG antibody (anti-CLDN4 IgG antibody) consisting of a heavy chain comprising the heavy chain variable region of the anti-CLDN4 antibody and a light chain comprising the light chain variable region of the anti-CLDN4 antibody.

[22] The bispecific antibody according to

[21] , wherein the Fc region of the anti-CLDN4 IgG antibody comprises either or both of an LALA mutation (L234A and L235A) or a P331G mutation (wherein the mutation positions are amino acid positions according to the EU index in the human Igγ1 constant region).

[23] The bispecific antibody according to any of

[18] to

[22] , wherein the heavy chain variable region of the anti-CD137 antibody comprises CDR1 consisting of the amino acid sequence from amino acid numbers 625 to 629 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequence from amino acid numbers 644 to 659 of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence from amino acid numbers 692 to 701 of SEQ ID NO: 2, and the light chain variable region of the anti-CD137 antibody comprises CDR1 consisting of the amino acid sequence from amino acid numbers 486 to 498 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequence from amino acid numbers 514 to 520 of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence from amino acid numbers 553 to 563 of SEQ ID NO: 2.

[24] The bispecific antibody according to

[23] , wherein the heavy chain variable region of the anti-CD137 antibody consists of the amino acid sequence of amino acids 595 to 712 of SEQ ID NO: 2, and the light chain variable region of the anti-CD137 antibody consists of the amino acid sequence of amino acids 464 to 573 of SEQ ID NO: 2.

[25] The bispecific antibody according to

[24] , comprising an anti-CD137 single-chain variable region fragment (anti-CD137 scFv) comprising the heavy chain variable region and light chain variable region of the anti-CD137 antibody.

[26] The bispecific antibody according to

[25] , wherein the anti-CD137 scFv consists of the amino acid sequence of amino acids 464 to 712 of SEQ ID NO: 2.

[27] The bispecific antibody according to

[26] , comprising an anti-CLDN4 IgG antibody and anti-CD137 scFv, wherein the amino terminus of the anti-CD137 scFv is linked to the heavy chain carboxy terminus of the anti-CLDN4 IgG antibody via a linker.

[28] An anti-CLDN4-anti-CD137 bispecific antibody used in combination with a platinum-based drug to treat a target cancer, the bispecific antibody comprising a heavy chain of an anti-CLDN4 antibody comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 123 of SEQ ID NO: 2 and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4, and an anti-CD137 scFv comprising a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 464 to 573 of SEQ ID NO: 2 and a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 595 to 712 of SEQ ID NO: 2, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker.

[29] An anti-CLDN4-anti-CD137 bispecific antibody used in combination with a platinum-based drug to treat a target cancer, the bispecific antibody comprising an anti-CLDN4 antibody heavy chain consisting of the amino acid sequence of amino acids 1 to 453 of SEQ ID NO: 2, an anti-CLDN4 antibody light chain consisting of the amino acid sequence of amino acids 1 to 215 of SEQ ID NO: 4, and an anti-CD137 scFv consisting of the amino acid sequence of amino acids 464 to 712 of SEQ ID NO: 2, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker.

[30] The bispecific antibody according to any of

[27] to

[29] , wherein the linker is a GS linker.

[31] An anti-CLDN4-anti-CD137 bispecific antibody used in combination with a platinum-based drug to treat a target cancer, the bispecific antibody comprising a polypeptide comprising an anti-CLDN4 antibody heavy chain and an anti-CD137 scFv consisting of the amino acid sequence of SEQ ID NO: 2, and an anti-CLDN4 antibody light chain consisting of the amino acid sequence of SEQ ID NO: 4.

[32] The anti-CLDN4-anti-CD137 bispecific antibody according to any of

[19] to

[31] , wherein the anti-CLDN4-anti-CD137 bispecific antibody is post-translationally modified.

[33] The bispecific antibody according to any of

[19] to

[32] , which is used in combination with a platinum-based drug simultaneously, consecutively, or sequentially.

[34] The bispecific antibody according to any of

[19] to

[33] , wherein the cancer is selected from the group consisting of colorectal cancer, non-small cell lung cancer, prostate cancer, ovarian cancer, breast cancer, urothelial cancer, gastric cancer, kidney cancer, pancreatic cancer, uterine cancer, cervical cancer, esophageal cancer, bile duct cancer, and head and neck cancer.

[35] The bispecific antibody according to any of

[19] to

[34] , wherein the platinum-based drug is oxaliplatin.

[36] The bispecific antibody according to any of

[19] to

[35] , wherein the anti-CLDN4-anti-CD137 bispecific antibody is used in combination with a platinum-based drug to treat a target cancer, and is further used in combination with a VEGF pathway inhibitor antibody.

[37] A method for treating cancer, comprising administering to a subject a combination of an anti-CLDN4-anti-CD137 bispecific antibody and a platinum-based drug, wherein the bispecific antibody comprises the heavy chain variable region and light chain variable region of an anti-CLDN4 antibody, and the heavy chain variable region and light chain variable region of an anti-CD137 antibody, wherein the heavy chain variable region of the anti-CLDN4 antibody comprises CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 66 of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence from amino acid numbers 99 to 112 of SEQ ID NO: 2, and the light chain variable region of the anti-CLDN4 antibody comprises CDR1 consisting of the amino acid sequence from amino acid numbers 24 to 35 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence from amino acid numbers 51 to 57 of SEQ ID NO: 4, and CDR3 consisting of the amino acid sequence from amino acid numbers 90 to 98 of SEQ ID NO: 4.

[38] The treatment method according to

[37] , wherein the heavy chain variable region of the anti-CLDN4 antibody consists of the amino acid sequence from amino acid Nos. 1 to 123 of SEQ ID NO: 2, and the light chain variable region of the anti-CLDN4 antibody consists of the amino acid sequence from amino acid Nos. 1 to 109 of SEQ ID NO: 4.

[39] The treatment method according to

[38] , wherein the anti-CLDN4-anti-CD137 bispecific antibody comprises an IgG antibody (anti-CLDN4 IgG antibody) consisting of a heavy chain comprising the heavy chain variable region of the anti-CLDN4 antibody and a light chain comprising the light chain variable region of the anti-CLDN4 antibody.

[40] The treatment method according to

[39] , wherein the Fc region of the anti-CLDN4 IgG antibody comprises either or both of an LALA mutation (L234A and L235A) or a P331G mutation (wherein the mutation position is an amino acid position according to the EU index in the human Igγ1 constant region).

[41] The treatment method according to any of

[37] to

[40] , wherein the heavy chain variable region of the anti-CD137 antibody comprises CDR1 consisting of the amino acid sequence from amino acid numbers 625 to 629 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequence from amino acid numbers 644 to 659 of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence from amino acid numbers 692 to 701 of SEQ ID NO: 2, and the light chain variable region of the anti-CD137 antibody comprises CDR1 consisting of the amino acid sequence from amino acid numbers 486 to 498 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequence from amino acid numbers 514 to 520 of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence from amino acid numbers 553 to 563 of SEQ ID NO: 2.

[42] The therapeutic method according to

[41] , wherein the heavy chain variable region of the anti-CD137 antibody consists of the amino acid sequence from amino acid No. 595 to amino acid No. 712 of SEQ ID NO: 2, and the light chain variable region of the anti-CD137 antibody consists of the amino acid sequence from amino acid No. 464 to amino acid No. 573 of SEQ ID NO: 2.

[43] The therapeutic method according to

[42] , wherein the anti-CLDN4-anti-CD137 bispecific antibody comprises an anti-CD137 single-chain variable region fragment (anti-CD137 scFv) comprising the heavy chain variable region and light chain variable region of the anti-CD137 antibody.

[44] The therapeutic method according to

[43] , wherein the anti-CD137 scFv consists of the amino acid sequence from amino acid No. 464 to amino acid No. 712 of SEQ ID NO: 2.

[45] The anti-CLDN4-anti-CD137 bispecific antibody comprises an anti-CLDN4 IgG antibody and an anti-CD137 scFv, and the amino terminus of the anti-CD137 scFv is linked to the heavy chain carboxy terminus of the anti-CLDN4 IgG antibody via a linker.

[44] The therapeutic method according to.

[46] A method for treating cancer, comprising administering to a subject a combination of an anti-CLDN4-anti-CD137 bispecific antibody and a platinum-based drug, wherein the bispecific antibody comprises a heavy chain of an anti-CLDN4 antibody comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 123 of SEQ ID NO: 2 and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4, and an anti-CD137 scFv comprising a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 464 to 573 of SEQ ID NO: 2 and a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 595 to 712 of SEQ ID NO: 2, and wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker.

[47] A method for treating cancer, comprising administering to a subject a combination of an anti-CLDN4-anti-CD137 bispecific antibody and a platinum-based drug, wherein the bispecific antibody comprises an anti-CLDN4 antibody heavy chain consisting of the amino acid sequence of amino acids 1 to 453 of SEQ ID NO: 2, an anti-CLDN4 antibody light chain consisting of the amino acid sequence of amino acids 1 to 215 of SEQ ID NO: 4, and an anti-CD137 scFv consisting of the amino acid sequence of amino acids 464 to 712 of SEQ ID NO: 2, and wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker.

[48] The method for treating cancer according to any of

[45] to

[47] , wherein the linker is a GS linker.

[49] A method for treating cancer, comprising administering to a subject a combination of an anti-CLDN4-anti-CD137 bispecific antibody and a platinum-based drug, wherein the bispecific antibody comprises a heavy chain of an anti-CLDN4 antibody consisting of the amino acid sequence of SEQ ID NO: 2 and a polypeptide comprising an anti-CD137 scFv, and a light chain of an anti-CLDN4 antibody consisting of the amino acid sequence of SEQ ID NO: 4.

[50] The method of treatment according to any of

[37] to

[49] , wherein the anti-CLDN4-anti-CD137 bispecific antibody is post-translationally modified.

[51] The method of treatment according to any of

[37] to

[50] , wherein the anti-CLDN4-anti-CD137 bispecific antibody and the platinum-based drug are used in combination simultaneously, consecutively, or sequentially.

[52] The method of any one of

[37] to

[51] , wherein the cancer is selected from the group consisting of colorectal cancer, non-small cell lung cancer, prostate cancer, ovarian cancer, breast cancer, urothelial cancer, gastric cancer, kidney cancer, pancreatic cancer, uterine cancer, cervical cancer, esophageal cancer, bile duct cancer, and head and neck cancer.

[53] The method of any one of

[37] to

[52] , wherein the platinum-based drug is oxaliplatin.

[54] The method of any one of

[37] to

[53] , wherein the cancer is a cancer selected from the group consisting of colorectal cancer, non-small cell lung cancer, prostate cancer, ovarian cancer, breast cancer, urothelial cancer, gastric cancer, kidney cancer, pancreatic cancer, uterine cancer, cervical cancer, esophageal cancer, bile duct cancer, and head and neck cancer.

[55] Use of an anti-CLDN4-anti-CD137 bispecific antibody for the manufacture of a pharmaceutical composition to be used in combination with a platinum-based drug to treat cancer in a subject, wherein the bispecific antibody comprises the heavy chain variable region and light chain variable region of an anti-CLDN4 antibody, and the heavy chain variable region and light chain variable region of an anti-CD137 antibody, wherein the heavy chain variable region of the anti-CLDN4 antibody comprises CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 66 of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence from amino acid numbers 99 to 112 of SEQ ID NO: 2, and the light chain variable region of the anti-CLDN4 antibody comprises CDR1 consisting of the amino acid sequence from amino acid numbers 24 to 35 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence from amino acid numbers 51 to 57 of SEQ ID NO: 4, and CDR3 consisting of the amino acid sequence from amino acid numbers 90 to 98 of SEQ ID NO: 4.

[56] The use according to

[55] , wherein the heavy chain variable region of the anti-CLDN4 antibody consists of the amino acid sequence from amino acid Nos. 1 to 123 of SEQ ID NO: 2, and the light chain variable region of the anti-CLDN4 antibody consists of the amino acid sequence from amino acid Nos. 1 to 109 of SEQ ID NO: 4.

[57] The use according to

[56] , wherein the anti-CLDN4-anti-CD137 bispecific antibody comprises an IgG antibody (anti-CLDN4 IgG antibody) consisting of a heavy chain comprising the heavy chain variable region of the anti-CLDN4 antibody and a light chain comprising the light chain variable region of the anti-CLDN4 antibody.

[58] The use according to

[57] , wherein the Fc region of the anti-CLDN4 IgG antibody comprises either or both of an LALA mutation (L234A and L235A) or a P331G mutation (wherein the mutation positions are amino acid positions according to the EU index in the human Igγ1 constant region).

[59] The use according to any of

[55] to

[58] , wherein the heavy chain variable region of the anti-CD137 antibody comprises CDR1 consisting of the amino acid sequence from amino acid numbers 625 to 629 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequence from amino acid numbers 644 to 659 of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence from amino acid numbers 692 to 701 of SEQ ID NO: 2, and the light chain variable region of the anti-CD137 antibody comprises CDR1 consisting of the amino acid sequence from amino acid numbers 486 to 498 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequence from amino acid numbers 514 to 520 of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence from amino acid numbers 553 to 563 of SEQ ID NO: 2.

[60] The use according to

[59] , wherein the heavy chain variable region of the anti-CD137 antibody consists of the amino acid sequence from amino acid numbers 595 to 712 of SEQ ID NO: 2, and the light chain variable region of the anti-CD137 antibody consists of the amino acid sequence from amino acid numbers 464 to 573 of SEQ ID NO: 2.

[61] The use according to

[58] , wherein the anti-CLDN4-anti-CD137 bispecific antibody comprises an anti-CD137 single-chain variable region fragment (anti-CD137 scFv) comprising the heavy chain variable region and light chain variable region of the anti-CD137 antibody.

[62] The use according to

[59] , wherein the anti-CD137 scFv consists of the amino acid sequence from amino acid numbers 464 to 712 of SEQ ID NO: 2.

[63] The anti-CLDN4-anti-CD137 bispecific antibody comprises an anti-CLDN4 IgG antibody and an anti-CD137 scFv, and the amino terminus of the anti-CD137 scFv is linked to the heavy chain carboxy terminus of the anti-CLDN4 IgG antibody via a linker. Use according to

[62] .

[64] Use of an anti-CLDN4-anti-CD137 bispecific antibody for the manufacture of a pharmaceutical composition to be used in combination with a platinum-based drug to treat cancer in a subject, wherein the bispecific antibody comprises a heavy chain of an anti-CLDN4 antibody comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 123 of SEQ ID NO: 2 and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4, and an anti-CD137 scFv comprising a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 464 to 573 of SEQ ID NO: 2 and a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 595 to 712 of SEQ ID NO: 2, and the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker.

[65] Use of an anti-CLDN4-anti-CD137 bispecific antibody for the manufacture of a pharmaceutical composition to be used in combination with a platinum-based drug to treat cancer in a subject, wherein the bispecific antibody comprises an anti-CLDN4 antibody heavy chain consisting of the amino acid sequence from amino acid numbers 1 to 453 of SEQ ID NO: 2 and an anti-CLDN4 antibody light chain consisting of the amino acid sequence from amino acid numbers 1 to 215 of SEQ ID NO: 4, and an anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 464 to 712 of SEQ ID NO: 2, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker.

[66] The use according to any of

[63] to

[65] , wherein the linker is a GS linker.

[67] Use of an anti-CLDN4-anti-CD137 bispecific antibody for the manufacture of a pharmaceutical composition to be used in combination with a platinum-based drug to treat cancer in a subject, wherein the bispecific antibody comprises a polypeptide comprising an anti-CLDN4 antibody heavy chain and an anti-CD137 scFv consisting of the amino acid sequence of SEQ ID NO: 2, and an anti-CLDN4 antibody light chain consisting of the amino acid sequence of SEQ ID NO: 4.

[68] The use according to any of

[55] to

[67] , wherein the anti-CLDN4-anti-CD137 bispecific antibody is post-translationally modified.

[69] The use according to any of

[55] to

[68] , wherein the pharmaceutical composition is used in combination with a platinum-based drug simultaneously, consecutively, or sequentially.

[70] The use according to any one of

[55] to

[69] , wherein the cancer is selected from the group consisting of colorectal cancer, non-small cell lung cancer, prostate cancer, ovarian cancer, breast cancer, urothelial cancer, gastric cancer, kidney cancer, pancreatic cancer, uterine cancer, cervical cancer, esophageal cancer, bile duct cancer, and head and neck cancer.

[71] The use according to any one of

[55] to

[70] , wherein the platinum-based drug is oxaliplatin.

[72] The use according to any one of

[55] to

[71] , wherein the anti-CLDN4-anti-CD137 bispecific antibody is used in combination with a platinum-based drug to treat cancer in a subject, and the use is further used in combination with a VEGF pathway inhibitory antibody.

[0012] The anti-CLDN4-anti-CD137 bispecific antibody of the present invention binds to both CLDN4, which is highly expressed in cancer, and CD137, a T cell surface molecule, and activates immune cells surrounding the cancer cells, thereby enhancing the killing effect against the cancer cells. The combination of the anti-CLDN4-anti-CD137 bispecific antibody of the present invention with a platinum-based drug produces a significant antitumor effect compared to the administration of the anti-CLDN4-anti-CD137 bispecific antibody or the platinum-based drug alone, or the combination of the platinum-based drug with another drug that exhibits antitumor activity. Thus, the present invention provides the use of an anti-CLDN4-anti-CD137 bispecific antibody in combination with a platinum-based drug in cancer treatment.

[0013] FIG. 1 shows the antitumor effect of the test substance on B-h4-1BB mice bearing human CLDN4-expressing B16-F10 cells. FIG. 1-1 shows the average tumor volume (n = 10) at each day after the start of test substance administration. Error bars indicate the standard error of the tumor volume. The vertical axis of the figure indicates tumor volume, and the horizontal axis indicates the number of days from the first administration of the test substance. FIG. 1-2 shows the tumor volume of each individual (n = 10) on the final day of the test. The plot shows the tumor volume of each individual. The horizontal line indicates the average value. The error bars indicate the standard error. The vertical axis of the figure indicates tumor volume. The significance probability P value was determined by comparing the tumor volume of Group 4 with the tumor volumes of Groups 2 and 3 using the Student's t-test. ** in the figure indicates that the P value is less than the significance level of 0.01. Group 1: Control Group 2: hKM3900_tA2-32LH (0.3 mg / kg) Group 3: Oxaliplatin (5 mg / kg) Group 4: Combination of hKM3900_tA2-32LH and oxaliplatin Figure 2 shows the antitumor effect of the test substance on B-h4-1BB mice bearing human CLDN4-expressing B16-F10 cells. Figure 2-1 shows the average tumor volume (n = 10) at each day after the start of test substance administration. Error bars indicate the standard error of tumor volume. The vertical axis of the figure indicates tumor volume, and the horizontal axis indicates the number of days from the first administration of the test substance. Figure 2-2 shows the tumor volume of each individual (n = 10) on the final day of the test. The plot shows the tumor volume of each individual. The horizontal line indicates the average value. Error bars indicate the standard error. The vertical axis of the figure indicates tumor volume. The significance probability P value was calculated by comparing the tumor volume in Group 4 with the tumor volumes in Groups 2 and 3 using Student's t-test. ** in the figure indicates that the P value is less than the significance level of 0.01. Group 1: Control Group 2: hKM3900_tA2-32LH (0.3 mg / kg) Group 3: Combination of oxaliplatin (5 mg / kg) and anti-mouse VEGFR antibody (5 mg / kg) Group 4: Combination of hKM3900_tA2-32LH, oxaliplatin, and anti-mouse VEGFR antibody

[0014] The present invention will be described in detail below.

[0015] Terms used herein are used in the sense commonly used by those skilled in the art unless otherwise defined below.

[0016] An antibody (or immunoglobulin) is a glycoprotein whose basic structure is a four-chain structure with a symmetric Y-shape, consisting of two heavy chains with a single sequence and two light chains with a single sequence. There are five classes of antibodies: IgG, IgM, IgA, IgD, and IgE. The basic structure of an antibody molecule is common to all classes, consisting of two heavy chains with a molecular weight of 50,000 to 70,000 and two light chains with a molecular weight of 20,000 to 30,000, bound by disulfide bonds and non-covalent bonds to form an antibody molecule with a molecular weight of 150,000 to 190,000, consisting of a Y-shaped four-chain structure. The heavy chain is typically a polypeptide chain containing approximately 440 amino acids, and each class has a characteristic structure, called Igγ, Igμ, Igα, Igδ, and Igε, corresponding to IgG, IgM, IgA, IgD, and IgE, respectively. IgG is further divided into subclasses, IgG1, IgG2, IgG3, and IgG4, and the corresponding heavy chains are called Igγ1, Igγ2, Igγ3, and Igγ4. Light chains are typically polypeptide chains containing approximately 220 amino acids, and two types, λ and κ, are known, called Igλ and Igκ, respectively. The two types of light chains can pair with either type of heavy chain.

[0017] Antibody molecules have four intrachain disulfide bonds in heavy chains (five in Igμ and Igε) and two in light chains, forming a loop every 100-110 amino acid residues. The three-dimensional structures of these disulfide bonds are similar between each loop and are referred to as structural units or domains. The domain located at the amino terminus (also referred to herein as the "N-terminus") of both heavy and light chains is called the variable region. It has diverse amino acid sequences even among antibodies produced from the same class (or subclass) of the same animal species, and is known to be involved in the specific binding between the antibody and the antigen. The amino acid sequence of the C-terminal domain downstream of the variable region is nearly constant for each class or subclass and is referred to as the constant region. From the N-terminus to the carboxy-terminus (also referred to herein as the "C-terminus"), the heavy chain has a heavy chain variable region (VH) and a heavy chain constant region (CH). The CH is further divided into three domains from the N-terminus: CH1 domain, CH2 domain, and CH3 domain. Light chains have, from N- to C-terminus, a light chain variable region (VL) and a light chain constant region (CL).

[0018] The amino acid sequences of the three complementarity-determining regions (CDRs) present in VH and VL vary greatly, contributing to the variability of the variable regions. The CDRs are regions consisting of approximately 5 to 10 amino acid residues located in the order of CDR1, CDR2, and CDR3 at the N-terminus of each heavy chain and light chain, and form the antigen-binding site. On the other hand, the portions of the variable regions other than the CDRs are called framework regions (FRs), and consist of FR1 to FR4, and show relatively little variation in amino acid sequence.

[0019] When an antibody is treated with the protease papain, three antibody fragments are obtained. The two fragments on the N-terminal side are called Fab (Fragment, antigen binding) regions. As used herein, "Fab region" refers to a region consisting of the VH and CH1 domains of the heavy chain and the light chain (VL and CL), and binds to an antigen at the antigen-binding site at the tip of the Fab region. As used herein, "heavy chain fragment" refers to a fragment consisting of the VH and CH1 domains of the heavy chain that make up the Fab region. The C-terminal fragment is called "Fc (Fragment, crystallizable) region."

[0020] As used herein, the term "antigen" is used in its commonly used sense, particularly as a term referring to a molecule or a portion of a molecule to which an antigen-binding protein, such as an antibody or an antigen-binding fragment, can specifically bind. An antigen can be a protein, a nucleic acid, or other molecule. A single antigen may have one or more epitopes that can interact with different antibodies, etc.

[0021] As used herein, the term "IgG antibody" refers to an antibody having a Y-shaped structure consisting of two Fab regions and an Fc region. In one embodiment, the two Fab regions of an IgG antibody comprise identical VH and VL sequences.

[0022] As used herein, an "antigen-binding fragment" refers to a molecule containing at least one polypeptide chain having antigen-binding activity derived from an antibody. Representative antigen-binding fragments include single-chain variable region fragments (scFv), Fab fragments, Fab' fragments, and F(ab') fragments. ２Examples of such fragments include F(ab') and F(ab') fragments. scFv is a monovalent antigen-binding fragment composed of VH and VL linked by a linker. Fab fragment is a monovalent antigen-binding fragment composed of a light chain and a fragment containing the VH and CH1 domain of the heavy chain. Fab' fragment is a monovalent antigen-binding fragment composed of a light chain and a fragment containing the VH and CH1 domain of the heavy chain and a part of the hinge region, and this hinge region contains cysteine ​​residues that constituted the inter-heavy chain S-S bond. F(ab') ２ The fragment is a bivalent molecule in which the Fab' fragments are linked by disulfide bonds. Monovalent means that it contains one antigen-binding site, and bivalent means that it contains two antigen-binding sites.

[0023] As used herein, the term "bispecific antibody" refers to an antibody that can specifically bind to two different antigens. An "anti-CLDN4-anti-CD137 bispecific antibody" refers to a bispecific antibody that has binding activity for CLDN4 and binding activity for CD137.

[0024] As used herein, the term "antibody" includes full-length antibodies, antigen-binding fragments, and bispecific antibodies of any structure, unless otherwise limited by the context.

[0025] As used herein, the term "human antibody" refers to an antibody having a human immunoglobulin amino acid sequence. As used herein, the term "humanized antibody" refers to an antibody in which some, most, or all of the amino acid residues other than the CDRs have been substituted with amino acid residues derived from human immunoglobulin molecules. The method of humanization is not particularly limited, and humanized antibodies can be prepared by referring to, for example, U.S. Pat. No. 5,225,539 and U.S. Pat. No. 6,180,370.

[0026] The amino acid residue numbers of antibodies used herein can be specified according to the Kabat numbering system or the EU index (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., 1991: NIH Publication: No. 91-3242) by designating the numbering system.

[0027] As used herein, the terms "linkage," "conjugate," or "linked" mean that multiple components (e.g., an IgG antibody and an scFv) are linked together directly or via an intermediary (e.g., a peptide linker). As used herein, a "peptide linker" refers to any one or more amino acid sequences that can be introduced by genetic engineering techniques and are used to link multiple components. The length of the peptide linker used in the present invention is not particularly limited, and can be appropriately selected by those skilled in the art depending on the purpose.

[0028] As used herein, a "pharmaceutical composition" refers to a drug that contains an active ingredient and a pharmaceutically acceptable excipient (including, but not limited to, a pharmaceutical excipient or pharmaceutical carrier) and is prescribed for the purpose of treating a subject.

[0029] As used herein, "subject" refers to a human or other animal in need of disease prevention or treatment. In one embodiment, the subject is a human in need of disease prevention or treatment. In one embodiment, the subject is a human with cancer.

[0030] As used herein, "treatment" refers to any intervention, procedure, or administration of an active ingredient to a subject for the purpose of reversing, alleviating, ameliorating, suppressing, or delaying the progression, onset, severity, or recurrence of symptoms, pathology, or biochemical signs associated with a disease.

[0031] As used herein, the term "active ingredient" refers to a substance contained in a pharmaceutical composition, drug, etc. used for the prevention or treatment of a disease, which exhibits some kind of physiological activity. In one embodiment, the active ingredient is an antibody, a low molecular weight compound, a nucleic acid, a fusion protein, or a peptide. In one embodiment, the active ingredient is an antibody. In one embodiment, the active ingredient is a bispecific antibody.

[0032] As used herein, an "effective amount" or "therapeutically effective amount" of a drug refers to the dosage of the drug required to exert an effect in the subject's body. An effective amount of a drug may be determined by indicators such as reducing the severity of symptoms, increasing the frequency and duration of symptom-free periods, or promoting disease regression. An effective amount of a drug can be assessed using various methods known to those skilled in the art, for example, in human subjects during clinical trials, in animal model systems predictive of human efficacy, or by measuring physiological activity in vitro.

[0033] As used herein, the terms "concomitant use," "combination," or "use in combination" refer to the simultaneous, sequential, or sequential administration of multiple active ingredients to the same subject for the prevention or treatment of a disease. The multiple active ingredients may be contained in the same pharmaceutical composition, or may be contained separately in different pharmaceutical compositions. As used herein, "simultaneous" refers to the administration of multiple active ingredients in parallel within one administration period, "sequential" refers to the administration of one active ingredient immediately after the completion of the administration of the other active ingredient, and "sequential" refers to the administration of multiple active ingredients in order according to an administration schedule.

[0034] As used herein, "platinum compounds" refer to compounds containing platinum (Pt) in their structure, and "platinum-based drugs" refer to drugs with antitumor activity that contain platinum compounds as their main component. Platinum compounds bind to the DNA in cancer cells, thereby inhibiting DNA replication and suppressing cancer cell proliferation. As used herein, "oxaliplatin," "cisplatin," "carboplatin," "nedaplatin," and "miriplatin" refer to compounds identified by their chemical names and CAS numbers shown in Table 1.

[0035] As used herein, a "VEGF pathway inhibitory antibody" refers to an antibody that inhibits the binding of vascular endothelial growth factor (VEGF) to a VEGF receptor. In one embodiment, the VEGF pathway inhibitory antibody of the present invention is an anti-VEGF antibody or an anti-VEGFR antibody. In one embodiment, the VEGF pathway inhibitory antibody of the present invention is an anti-VEGFR antibody.

[0036] The present invention relates to the following (1) to (4): (1) a pharmaceutical composition comprising an anti-CLDN4-anti-CD137 bispecific antibody, which is used in combination with a platinum-based drug to treat cancer in a subject (also referred to herein as the "pharmaceutical composition of the present invention"); (2) an anti-CLDN4-anti-CD137 bispecific antibody used in combination with a platinum-based drug to treat cancer in a subject; (3) a method for treating cancer, which comprises administering an anti-CLDN4-anti-CD137 bispecific antibody and a platinum-based drug to a subject (also referred to herein as the "therapeutic method of the present invention"); or (4) use of an anti-CLDN4-anti-CD137 bispecific antibody for the manufacture of a pharmaceutical composition used in combination with a platinum-based drug to treat cancer in a subject.

[0037] <Anti-CLDN4-anti-CD137 bispecific antibody of the present invention> The bispecific antibody that binds to CLDN4 and CD137 used in the present invention (also referred to as the "anti-CLDN4-anti-CD137 bispecific antibody of the present invention") comprises the heavy chain variable region and light chain variable region of an anti-CLDN4 antibody, and the heavy chain variable region and light chain variable region of an anti-CD137 antibody.

[0038] As used herein, an "anti-CLDN4 antibody" is an antibody capable of binding to human CLDN4, and an "anti-CD137 antibody" is an antibody capable of binding to human CD137. Whether or not an antibody binds to human CLDN4 or human CD137 can be confirmed using a known binding activity measurement method. Methods for measuring binding activity include, for example, enzyme-linked immunosorbent assay (ELISA) and flow cytometry. ELISA and flow cytometry can be performed using methods commonly used by those skilled in the art.

[0039] The anti-CLDN4-anti-CD137 bispecific antibody of the present invention may have any structure as long as it binds to CLDN4 and CD137, and examples thereof include bispecific antibodies having the structures described in Non-Patent Document 6. In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention may be a conjugate of the Fab region of an anti-CLDN4 antibody and the Fab region of an anti-CD137 antibody, a conjugate of an IgG antibody-type anti-CLDN4 antibody (also referred to as "anti-CLDN4 IgG antibody") and an IgG antibody-type anti-CD137 antibody (also referred to as "anti-CD137 IgG antibody"), a conjugate of an antigen-binding fragment of an anti-CLDN4 IgG antibody and an anti-CD137 antibody, a conjugate of an antigen-binding fragment of an anti-CLDN4 antibody and an anti-CD137 IgG antibody, or a conjugate of an antigen-binding fragment of an anti-CLDN4 antibody and an antigen-binding fragment of an anti-CD137 antibody.

[0040] In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises a heavy chain variable region of an anti-CLDN4 antibody comprising CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 66 of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence from amino acid numbers 99 to 112 of SEQ ID NO: 2, and a light chain variable region of an anti-CLDN4 antibody comprising CDR1 consisting of the amino acid sequence from amino acid numbers 24 to 35 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence from amino acid numbers 51 to 57 of SEQ ID NO: 4, and CDR3 consisting of the amino acid sequence from amino acid numbers 90 to 98 of SEQ ID NO: 4.

[0041] In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises a heavy chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 123 of SEQ ID NO: 2 and a light chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 109 of SEQ ID NO: 4.

[0042] The anti-CLDN4 antibody contained in the anti-CLDN4-anti-CD137 bispecific antibody of the present invention may be an IgG antibody. The heavy chain constant region contained in the anti-CLDN4 antibody can be any of Igγ, Igμ, Igα, Igδ, or Igε constant regions. Igγ can be selected from, for example, Igγ1, Igγ2, Igγ3, or Igγ4. The light chain constant region contained in the anti-CLDN4 antibody contained in the anti-CLDN4-anti-CD137 bispecific antibody of the present invention can be any of Igλ or Igκ constant regions. In one embodiment, the heavy and light chains of the anti-CLDN4 antibody are human Igγ1 and Igκ, respectively. In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises a full-length anti-CLDN4 antibody. In one embodiment, the anti-CLDN4 antibody included in the anti-CLDN4-anti-CD137 bispecific antibody of the present invention is an IgG antibody (anti-CLDN4 IgG antibody) comprising the heavy chain variable region and light chain variable region of an anti-CLDN4 antibody.

[0043] When the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises an Fc region, the Fc region of the bispecific antibody may contain a mutation that reduces antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). L234A is a substitution of leucine at amino acid position 234 in the human Igγ1 constant region with alanine. L235A is a substitution of leucine at amino acid position 235 in the human Igγ1 constant region with alanine. The amino acid mutations L234A and L235A in the human Igγ1 constant region are referred to as "LALA mutations." This mutation is known to reduce the ADCC and CDC of antibodies (Mol. Immunol., 1992: Vol. 29: pp. 633-639, J. Immunol., 2000: Vol. 164(8): pp. 4178-4184). P331G or P331S is a substitution of proline at amino acid position 331 in the human Igγ1 constant region with glycine or serine. This mutation is known to reduce the CDC of antibodies (J. Immunol., 2000: Vol. 164(8): pp. 4178-4184).

[0044] In one embodiment, the anti-CLDN4 IgG antibody included in the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises an Fc region comprising the amino acid mutations L234A and L235A (LALA mutation). In one embodiment, the anti-CLDN4 IgG antibody comprises an Fc region comprising either a P331G or P331S mutation. In one embodiment, the anti-CLDN4 IgG antibody comprises an Fc region comprising the LALA mutation and either a P331G or P331S mutation. In one embodiment, the anti-CLDN4 IgG antibody comprises an Fc region comprising either or both of the LALA mutation and the P331G mutation.

[0045] In this specification, the descriptions of amino acid mutations such as the LALA mutation, P331G mutation, and P331S mutation are based on the amino acid position in the human Igγ1 constant region according to the EU index. For example, as described above, L234A is a substitution of leucine with alanine at amino acid position 234 in the human Igγ1 constant region according to the EU index.

[0046] In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention is an IgG antibody consisting of an anti-CLDN4 antibody heavy chain consisting of the amino acid sequence from amino acid numbers 1 to 453 of SEQ ID NO: 2 and an anti-CLDN4 antibody light chain consisting of the amino acid sequence from amino acid numbers 1 to 215 of SEQ ID NO: 4.

[0047] In one embodiment, the heavy chain variable region of the anti-CD137 antibody contained in the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises CDR1 consisting of the amino acid sequence from amino acid numbers 625 to 629 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequence from amino acid numbers 644 to 659 of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence from amino acid numbers 692 to 701 of SEQ ID NO: 2, and the light chain variable region of the anti-CD137 antibody comprises CDR1 consisting of the amino acid sequence from amino acid numbers 486 to 498 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequence from amino acid numbers 514 to 520 of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence from amino acid numbers 553 to 563 of SEQ ID NO: 2.

[0048] In one embodiment, the heavy chain variable region of the anti-CD137 antibody contained in the anti-CLDN4-anti-CD137 bispecific antibody consists of the amino acid sequence from amino acid numbers 595 to 712 of SEQ ID NO: 2, and the light chain variable region of the anti-CD137 antibody consists of the amino acid sequence from amino acid numbers 464 to 573 of SEQ ID NO: 2.

[0049] In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the invention comprises an scFv of an anti-CD137 antibody (also referred to herein as "anti-CD137 scFv").

[0050] In anti-CD137 scFv, the type and length of the linker linking the heavy chain variable region and light chain variable region of the anti-CD137 antibody are not particularly limited and can be appropriately selected by those skilled in the art. A peptide linker may be used as the linker. The preferred length is 5 amino acids or more (the upper limit is not particularly limited, but is usually 30 amino acids or less, preferably 20 amino acids or less), with 15 amino acids being particularly preferred. Examples of linkers that can be used include a glycine-serine linker (GS linker) and a glycine-lysine-proline-glycine-serine linker (GKPGS linker). Examples of linkers used in the present invention include the following: Ser Gly-Ser Gly-Gly-Ser Ser-Gly-Gly Gly-Gly-Gly-Ser (SEQ ID NO: 5) Ser-Gly-Gly-Gly (SEQ ID NO: 6) Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 7) Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 8) Gly-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 9) Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 10) Gly-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 11) Ser-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 12) Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 13) (Gly-Gly-Gly-Gly-Ser)n (Ser-Gly-Gly-Gly-Gly-Gly)n Gly-Lys-Pro-Gly-Ser (SEQ ID NO: 14) (Gly-Lys-Pro-Gly-Ser)n The above n represents an integer of 1 or more. In addition, in one aspect, the above n is 1 to 10, 2 to 8, or 2 to 6. The length and sequence of the linker can be appropriately selected by those skilled in the art depending on the purpose.

[0051] In one embodiment, the linker used in the anti-CD137 scFv is a GS linker of (Gly-Gly-Gly-Gly-Ser)n.

[0052] In one embodiment, the linker used in the anti-CD137 scFv is a GS linker of (Gly-Gly-Gly-Gly-Ser)4.

[0053] In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises a light chain variable region consisting of the amino acid sequence from amino acid numbers 464 to 573 of SEQ ID NO: 2 and a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 595 to 712 of SEQ ID NO: 2 linked via a GS linker. It comprises an anti-CD137 scFv.

[0054] In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises an anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 464 to 712 of SEQ ID NO:2.

[0055] In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the invention comprises an anti-CLDN4 IgG antibody and an anti-CD137 scFv.

[0056] In the anti-CLDN4-anti-CD137 bispecific antibody of the present invention, the anti-CLDN4 antibody or antigen-binding fragment thereof and the anti-CD137 antibody or antigen-binding fragment thereof (e.g., anti-CD137 scFv) may be linked via a linker. In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises an anti-CLDN4 IgG antibody and an anti-CD137 scFv, and the anti-CLDN4 IgG antibody and the anti-CD137 scFv are linked via a linker. In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises an anti-CLDN4 IgG antibody and an anti-CD137 scFv, and the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 IgG antibody via a linker. The type and length of the linker linking the anti-CLDN4 antibody or its antigen-binding fragment and the anti-CD137 antibody or its antigen-binding fragment are not particularly limited and can be appropriately selected by those skilled in the art. A peptide linker may be used as the linker. The preferred length is 5 amino acids or more (the upper limit is not particularly limited, but is usually 30 amino acids or less, preferably 20 amino acids or less), and 10 amino acids is particularly preferred. Examples of peptide linkers that can be used include a glycine-serine linker (GS linker) and a glycine-lysine-proline-glycine-serine linker (GKPGS linker). Examples of linkers used in the present invention include the following:Ser Gly-Ser Gly-Gly-Ser Ser-Gly-Gly Gly-Gly-Gly-Ser (SEQ ID NO: 5) Ser-Gly-Gly-Gly (SEQ ID NO: 6) Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 7) Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 8) Gly-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 9) Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 10) Gly-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 11) Ser-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 12) Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 13) (Gly-Gly-Gly-Gly-Ser)n (Ser-Gly-Gly-Gly-Gly-Gly)n Gly-Lys-Pro-Gly-Ser (SEQ ID NO: 14) (Gly-Lys-Pro-Gly-Ser)n The above n represents an integer of 1 or more. In addition, in one aspect, the above n is 1 to 10, 2 to 8, or 2 to 6. The length and sequence of the peptide linker can be appropriately selected by those skilled in the art depending on the purpose.

[0057] In one embodiment, the linker used as a peptide linker linking an anti-CLDN4 antibody or antigen-binding fragment thereof and an anti-CD137 antibody or antigen-binding fragment thereof (e.g., anti-CD137 scFv) is a linker consisting of the amino acid sequence of SEQ ID NO: 13.

[0058] In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises a heavy chain of an anti-CLDN4 antibody comprising a heavy chain variable region including CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 66 of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence from amino acid numbers 99 to 112 of SEQ ID NO: 2; and a light chain of an anti-CLDN4 antibody comprising a light chain variable region including CDR1 consisting of the amino acid sequence from amino acid numbers 24 to 35 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence from amino acid numbers 51 to 57 of SEQ ID NO: 4, and CDR3 consisting of the amino acid sequence from amino acid numbers 90 to 98 of SEQ ID NO: 4; and a light chain of an anti-CLDN4 antibody comprising a light chain variable region including CDR1 consisting of the amino acid sequence from amino acid numbers 625 to 629 of SEQ ID NO: 2. a CDR1 consisting of the amino acid sequence from 644 to 659 of SEQ ID NO:2, a CDR2 consisting of the amino acid sequence from 644 to 659 of SEQ ID NO:2, and a CDR3 consisting of the amino acid sequence from 692 to 701 of SEQ ID NO:2; and an anti-CD137 scFv comprising a light chain variable region of an anti-CD137 antibody comprising CDR1 consisting of the amino acid sequence from 486 to 498 of SEQ ID NO:2, a CDR2 consisting of the amino acid sequence from 514 to 520 of SEQ ID NO:2, and a CDR3 consisting of the amino acid sequence from 553 to 563 of SEQ ID NO:2, wherein the amino terminus of the anti-CD137 scFv is linked to the heavy chain carboxy terminus of the anti-CLDN4 antibody via a linker.

[0059] In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention is a bispecific antibody comprising a heavy chain of an anti-CLDN4 antibody comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 123 of SEQ ID NO: 2 and a light chain of an anti-CLDN4 antibody comprising a light chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4, and an anti-CD137 scFv comprising a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 464 to 573 of SEQ ID NO: 2 and a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 595 to 712 of SEQ ID NO: 2, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker.

[0060] In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention is a bispecific antibody comprising an anti-CLDN4 antibody heavy chain consisting of the amino acid sequence from amino acid numbers 1 to 453 of SEQ ID NO: 2, an anti-CLDN4 antibody light chain consisting of the amino acid sequence from amino acid numbers 1 to 215 of SEQ ID NO: 4, and an anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 464 to 712 of SEQ ID NO: 2, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker.

[0061] In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention is a bispecific antibody comprising a heavy chain of an anti-CLDN4 antibody consisting of the amino acid sequence of SEQ ID NO: 2 and a polypeptide comprising an anti-CD137 scFv, and a light chain of an anti-CLDN4 antibody consisting of the amino acid sequence of SEQ ID NO: 4.

[0062] As used herein, the term "post-translational modification" refers to post-translational modification of an antibody when the antibody is expressed in a cell. Examples of post-translational modifications include pyroglutamylation, glycosylation, oxidation, deamidation, glycation, and other modifications of glutamine or glutamic acid at the N-terminus of the heavy chain, and lysine deletion due to cleavage of lysine at the C-terminus of the heavy chain by carboxypeptidase. Such post-translational modifications are known to occur in various antibodies (J. Pharm. Sci., 2008: Vol. 97: pp. 2426-2447).

[0063] In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention may be post-translationally modified. In one embodiment, the post-translational modification is pyroglutamylation of the N-terminus of the heavy chain variable region and / or deletion of a heavy chain C-terminal lysine. It is known in the art that post-translational modifications such as pyroglutamylation of the N-terminus or deletion of a C-terminal lysine do not affect the activity of the antibody (Analytical Biochemistry, 2006: Vol. 348: pp. 24-39).

[0064] The anti-CLDN4-anti-CD137 bispecific antibody of the present invention binds to human CLDN4 and human CD137. Whether or not it binds to human CLDN4 or human CD137 can be confirmed using a known binding activity measurement method. Methods for measuring binding activity include, for example, enzyme-linked immunosorbent assay (ELISA) and flow cytometry.

[0065] Those skilled in the art can prepare the anti-CLDN4-anti-CD137 bispecific antibodies of the present invention using methods known in the art based on the sequence information of the heavy chain variable regions and light chain variable regions of the anti-CLDN4 antibody and anti-CD137 antibody disclosed herein. In the anti-CLDN4-anti-CD137 bispecific antibodies of the present invention, the heavy chain variable regions and light chain variable regions of the anti-CLDN4 antibody and anti-CD137 antibody may be derived from a human antibody, a humanized antibody, or a combination thereof. When preparing a humanized antibody, backmutation may be introduced as appropriate using methods well known to those skilled in the art (Bioinformatics, 2015: Vol. 31: pp. 434-435). The anti-CLDN4-anti-CD137 bispecific antibodies of the present invention are not particularly limited, and can be produced, for example, according to the method described in PCT / JP2022 / 18350. The methods for producing anti-CLDN4-anti-CD137 bispecific antibodies described in PCT / JP2022 / 18350 (including, but not limited to, <Polynucleotides of bispecific antibodies of the invention>, <Expression vectors of bispecific antibodies of the invention>, <Host cells of the invention>, <Methods for producing bispecific antibodies of the invention> and Examples) are incorporated herein by reference (Incorporation by Reference).

[0066] Pharmaceutical Compositions of the Present Invention The pharmaceutical compositions of the present invention are produced using the anti-CLDN4-anti-CD137 bispecific antibody of the present invention and comprise the anti-CLDN4-anti-CD137 bispecific antibody of the present invention and a pharmaceutically acceptable excipient. The pharmaceutical compositions of the present invention can be prepared by commonly used methods using excipients commonly used in the art, i.e., pharmaceutical excipients, pharmaceutical carriers, etc. Examples of dosage forms of these pharmaceutical compositions include parenteral preparations such as injections and infusions, which can be administered intravenously, subcutaneously, intraperitoneally, etc. When formulating the compositions, excipients, carriers, additives, etc. appropriate for these dosage forms can be used within a pharmaceutically acceptable range. Furthermore, as described above, the pharmaceutical compositions of the present invention comprise the anti-CLDN4-anti-CD137 bispecific antibody of the present invention and a pharmaceutically acceptable excipient, and in one embodiment, may further comprise a platinum-based drug.

[0067] The pharmaceutical composition of the present invention may contain a post-translationally modified form of the anti-CLDN4-anti-CD137 bispecific antibody of the present invention. For example, the present invention also includes pharmaceutical compositions containing antibodies that have undergone C-terminal lysine deletion and / or N-terminal pyroglutamylation.

[0068] In one embodiment, the pharmaceutical composition of the present invention comprises a heavy chain variable region of an anti-CLDN4 antibody comprising CDR1 consisting of the amino acid sequence of amino acids 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequence of amino acids 50 to 66 of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence of amino acids 99 to 112 of SEQ ID NO: 2; a light chain variable region of an anti-CLDN4 antibody comprising CDR1 consisting of the amino acid sequence of amino acids 24 to 35 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence of amino acids 51 to 57 of SEQ ID NO: 4, and CDR3 consisting of the amino acid sequence of amino acids 90 to 98 of SEQ ID NO: 4; and a light chain variable region of an anti-CLDN4 antibody comprising CDR1 consisting of the amino acid sequence of amino acids 625 to 629 of SEQ ID NO: 2. and a light chain variable region of an anti-CD137 antibody comprising CDR1 consisting of the amino acid sequence of amino acids 486 to 498 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequence of amino acids 514 to 520 of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence of amino acids 553 to 563 of SEQ ID NO: 2, and / or a post-translationally modified form of the bispecific antibody.

[0069] In one embodiment, the pharmaceutical composition of the present invention comprises an anti-CLDN4-anti-CD137 bispecific antibody and / or a post-translationally modified form of the bispecific antibody, comprising: a heavy chain of an anti-CLDN4 antibody comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 123 of SEQ ID NO: 2; and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4; and an anti-CD137 scFv comprising a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 464 to 573 of SEQ ID NO: 2 and a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 595 to 712 of SEQ ID NO: 2, wherein the amino terminus of the anti-CD137 scFv is linked to the heavy chain carboxy terminus of the anti-CLDN4 antibody via a linker.

[0070] In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition comprising an anti-CLDN4-anti-CD137 bispecific antibody comprising a polypeptide comprising the heavy chain of an anti-CLDN4 antibody consisting of the amino acid sequence of SEQ ID NO: 2 and an anti-CD137 scFv, and a light chain of an anti-CLDN4 antibody consisting of the amino acid sequence of SEQ ID NO: 4, and / or a post-translationally modified form of the bispecific antibody.

[0071] The amount of the anti-CLDN4-anti-CD137 bispecific antibody of the present invention added in a formulation will vary depending on the severity of symptoms and age of the patient, the dosage form of the formulation used, the binding titer of the antibody, etc., but for example, an anti-CLDN4-anti-CD137 bispecific antibody of about 0.0001 mg / kg to 1000 mg / kg, calculated as a human dose, can be used in the formulation. In one embodiment, the amount of the anti-CLDN4-anti-CD137 bispecific antibody of the present invention added in a formulation is in the range of 0.0001 mg / kg to 1000 mg / kg, calculated as a human dose. In one embodiment, the amount of the anti-CLDN4-anti-CD137 bispecific antibody of the present invention added in a formulation is in the range of 0.001 mg / kg to 100 mg / kg, calculated as a human dose. In one embodiment, the amount of the anti-CLDN4-anti-CD137 bispecific antibody of the present invention added in the formulation ranges from 0.01 mg / kg to 10 mg / kg in terms of a human dose. In one embodiment, the amount of the anti-CLDN4-anti-CD137 bispecific antibody of the present invention added in the formulation ranges from 0.065 mg to 440 mg in terms of a human dose.

[0072] <Platinum-based drug> In the present invention, a platinum-based drug is used in combination with the anti-CLDN4-anti-CD137 bispecific antibody of the present invention or the pharmaceutical composition of the present invention for the treatment of cancer in a subject. Examples of platinum-based drugs used in the present invention include oxaliplatin, cisplatin, carboplatin, nedaplatin, and miriplatin. In one embodiment, the platinum-based drug used in the present invention is oxaliplatin, cisplatin, carboplatin, nedaplatin, or miriplatin. In one embodiment, the platinum-based drug used in the present invention is oxaliplatin.

[0073] Furthermore, in the present invention, a platinum-based agent may be contained in a pharmaceutical composition comprising an anti-CLDN4-anti-CD137 bispecific antibody of the present invention, or a formulation comprising a platinum-based agent may be used in combination with a pharmaceutical composition comprising an anti-CLDN4-anti-CD137 bispecific antibody of the present invention. As used herein, a "formulation" refers to a drug that contains an active ingredient and a pharmaceutically acceptable excipient (including, but not limited to, pharmaceutical excipients and pharmaceutical carriers) and is prescribed for the purpose of treating a subject. Examples of dosage forms of these formulations include parenteral preparations such as injections and infusions, which can be administered intravenously, subcutaneously, intraperitoneally, or the like. When formulating the formulation, excipients, carriers, additives, etc. appropriate for these dosage forms can be used within a pharmaceutically acceptable range.

[0074] <Therapeutic Method of the Present Invention> The therapeutic method of the present invention is a cancer treatment method (hereinafter referred to as the "therapeutic method of the present invention") that comprises administering to a subject the anti-CLDN4-anti-CD137 bispecific antibody of the present invention and a platinum-based drug.

[0075] The protocol and results of the applicant's Phase I study of the anti-CLDN4-anti-CD137 bispecific antibody registered with ClinicalTrials.gov (https: / / clinicaltrials.gov / ) (ClinicalTrials.gov ID: NCT05719558) are incorporated by reference.

[0076] In one embodiment, the therapeutic method of the present invention is characterized in that the anti-CLDN4-anti-CD137 bispecific antibody of the present invention is administered to a subject in combination with a platinum-based drug simultaneously, sequentially, or sequentially.

[0077] In one embodiment, the therapeutic method of the present invention is characterized in that the anti-CLDN4-anti-CD137 bispecific antibody of the present invention and the platinum-based drug are (i) contained in the same pharmaceutical composition and administered simultaneously, or (ii) in separate pharmaceutical compositions and used in combination simultaneously, sequentially, or sequentially.

[0078] In one embodiment, the method of treatment of the present invention is characterized in that the anti-CLDN4-anti-CD137 bispecific antibody of the present invention and the platinum-based drug are (i) contained in the same pharmaceutical composition and administered simultaneously, or (ii) in separate pharmaceutical compositions administered on the same day.

[0079] In one embodiment, the treatment method of the present invention is characterized by sequential use, in which (a) administration of a platinum-based drug is initiated after administration of an anti-CLDN4-anti-CD137 bispecific antibody of the present invention to a subject is completed, or (b) administration of a platinum-based drug is initiated after administration of an anti-CLDN4-anti-CD137 bispecific antibody of the present invention to a subject is completed.

[0080] In one embodiment, the therapeutic method of the present invention is characterized by sequential use in which an anti-CLDN4-anti-CD137 bispecific antibody of the present invention and a platinum-based agent are administered to a subject according to a dosing regimen including administration cycles. In one embodiment, the therapeutic method of the present invention is characterized in that administration of an anti-CLDN4-anti-CD137 bispecific antibody or a pharmaceutical composition of the present invention to a subject can be initiated after administration of a platinum-based agent in at least one or all of the administration cycles. In one embodiment, the therapeutic method of the present invention is characterized in that administration of a platinum-based agent to a subject can be initiated after administration of an anti-CLDN4-anti-CD137 bispecific antibody or a pharmaceutical composition of the present invention in at least one or all of the administration cycles.

[0081] <Therapeutic Use> Cancers treated by the pharmaceutical composition and treatment method of the present invention may be either solid cancers or blood cancers. Cancers treated by the present invention may be either primary or metastatic. Cancers treated by the present invention are not particularly limited, and include, for example, various peritoneal disseminated cancers, gastric cancer, lung cancer, acute lymphoblastic leukemia, acute myeloid leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, multiple myeloma, T-cell lymphoma and other blood cancers, myelodysplastic syndrome, adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, undifferentiated carcinoma, large cell carcinoma, non-small cell lung cancer, small cell lung cancer, mesothelioma, skin cancer, cutaneous T-cell lymphoma, breast cancer, prostate cancer, bladder cancer, urothelial cancer, vaginal cancer, cervical cancer, and head and neck cancer. These include solid cancers such as uterine cancer, cervical cancer, liver cancer, gallbladder cancer, bile duct cancer, kidney cancer, pancreatic cancer, colon cancer, large intestine cancer, rectal cancer, small intestine cancer, stomach cancer, esophageal cancer, testicular cancer, ovarian cancer, and brain tumors, as well as cancers of bone tissue, cartilage tissue, adipose tissue, muscle tissue, vascular tissue, and hematopoietic tissue, as well as sarcomas such as chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, and soft tissue sarcoma, and blastomas such as glioblastoma, glioblastoma multiforme, hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma, and retinoblastoma.

[0082] In one embodiment, the cancers that are the target of treatment by the present invention are colorectal cancer, non-small cell lung cancer, small cell lung cancer, bladder cancer, urothelial cancer, ovarian cancer, breast cancer, prostate cancer, gastric cancer, kidney cancer, pancreatic cancer, uterine cancer, cervical cancer, esophageal cancer, bile duct cancer, and head and neck cancer. In one embodiment, the cancers that are the target of treatment by the present invention are cancers in which CLDN4 is highly expressed compared to normal tissue. The cancers that are the target of treatment by the present invention are cancers in which CLDN4 is highly expressed compared to normal tissue, or cancers selected from the group consisting of colorectal cancer, rectal cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, bladder cancer, ovarian cancer, breast cancer, prostate cancer, gastric cancer, kidney cancer, pancreatic cancer, uterine cancer, cervical cancer, esophageal cancer, bile duct cancer, and head and neck cancer. In one embodiment, the cancer to be treated by the present invention is colorectal cancer, non-small cell lung cancer, prostate cancer, ovarian cancer, breast cancer, urothelial cancer, gastric cancer, kidney cancer, pancreatic cancer, uterine cancer, cervical cancer, esophageal cancer, bile duct cancer, and head and neck cancer. In one embodiment, the cancer to be treated by the present invention is preferably selected from the group consisting of colorectal cancer, non-small cell lung cancer, prostate cancer, ovarian cancer, breast cancer, urothelial cancer, gastric cancer, kidney cancer, pancreatic cancer, uterine cancer, cervical cancer, esophageal cancer, bile duct cancer, and head and neck cancer.

[0083] The dose of the anti-CLDN4-anti-CD137 bispecific antibody or platinum-based drug of the present invention administered to a subject varies depending on the severity of symptoms and age of the subject, the dosage form of the antibody, pharmaceutical composition, inhibitor, etc. used, or the activity strength of the active ingredient, but can be, for example, about 0.0001 mg / kg to 1000 mg / kg. In one embodiment, the dose of the anti-CLDN4-anti-CD137 bispecific antibody of the present invention administered to a subject is 0.0001 mg / kg to 1000 mg / kg. In one embodiment, the dose of the anti-CLDN4-anti-CD137 bispecific antibody of the present invention administered to a subject is 0.001 mg / kg to 100 mg / kg. In one embodiment, the dose of the anti-CLDN4-anti-CD137 bispecific antibody of the present invention administered to a subject is 0.01 mg / kg to 10 mg / kg. In one embodiment, the dose of the anti-CLDN4-anti-CD137 bispecific antibody of the present invention administered to a subject is 0.01 mg / kg to 1000 mg. In one embodiment, the dose of an anti-CLDN4-anti-CD137 bispecific antibody of the invention administered to a subject is 0.065 mg to 440 mg. In one embodiment, the dose of an anti-CLDN4-anti-CD137 bispecific antibody of the invention administered to a subject is any of 0.065 mg, 0.55 mg, 3 mg, 10 mg, 30 mg, 100 mg, 300 mg, and 440 mg. In one embodiment, the dose of an anti-CLDN4-anti-CD137 bispecific antibody of the invention administered to a subject is 0.065 mg. In one embodiment, the dose of an anti-CLDN4-anti-CD137 bispecific antibody of the invention administered to a subject is 0.55 mg. In one embodiment, the dose of an anti-CLDN4-anti-CD137 bispecific antibody of the invention administered to a subject is 3 mg. In one embodiment, the dose of an anti-CLDN4-anti-CD137 bispecific antibody of the invention administered to a subject is 10 mg. In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the invention is administered to a subject at a dose of 30 mg. In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the invention is administered to a subject at a dose of 100 mg. In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the invention is administered to a subject at a dose of 300 mg. In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the invention is administered to a subject at a dose of 440 mg.

[0084] To provide a further understanding of the present invention, reference is now made to specific examples which are provided for purposes of illustration and not limitation.

[0085] [Example 1: Production of anti-CLDN4-anti-CD137 bispecific antibody] [Example 1-1: Production of vector encoding anti-CLDN4-anti-CD137 bispecific antibody] A vector encoding an anti-CLDN4-anti-CD137 bispecific antibody was designed following the method of Example 4 described in WO2022 / 224997. The anti-CLDN4-anti-CD137 bispecific antibody has the N-terminus of an anti-CD137 scFv linked via a GS linker to the C-terminus of the heavy chain of IgG1-type hKM3900. The designed anti-CLDN4-anti-CD137 bispecific antibody consists of an anti-CLDN4 antibody heavy chain consisting of the amino acid sequence of SEQ ID NO: 2, a polypeptide containing anti-CD137 scFv, and an anti-CLDN4 antibody light chain consisting of the amino acid sequence of SEQ ID NO: 4. A polynucleotide encoding a polypeptide containing the designed anti-CLDN4 antibody heavy chain and anti-CD137 scFv, and a polynucleotide encoding the anti-CLDN4 antibody light chain were prepared and inserted into an expression vector according to standard methods. The two prepared vectors are referred to as "anti-CLDN4-anti-CD137 bispecific antibody expression vectors."

[0086] Example 1-2: Preparation of anti-CLDN4-anti-CD137 bispecific antibody An anti-CLDN4-anti-CD137 bispecific antibody was prepared using an anti-CLDN4-anti-CD137 bispecific antibody expression vector. Specifically, the anti-CLDN4-anti-CD137 bispecific antibody expression vector was introduced into CHO-K1 cells (ATCC, CCL61), and the anti-CLDN4-anti-CD137 bispecific antibody was secreted into the culture supernatant. The anti-CLDN4-anti-CD137 bispecific antibody hKM3900_tA2-32LH was purified from the obtained culture supernatant. The anti-CLDN4-anti-CD137 bispecific antibody hKM3900_tA2-32LH prepared in Example 1 is also referred to as the "anti-CLDN4-anti-CD137 bispecific antibody" in the following examples.

[0087] Example 2: In vivo combined effect of anti-CLDN4-anti-CD137 bispecific antibody and oxaliplatin Using B-h4-1BB mice (human CD137 knock-in mice) transplanted with human CLDN4-expressing B16-F10 cells, the in vivo antitumor effect of the combined use of anti-CLDN4-anti-CD137 bispecific antibody and oxaliplatin was examined.

[0088] [Example 2-1: Construction of human CLDN4-expressing B16-F10 cells] Human CLDN4-expressing B16-F10 cells were obtained using the method described in Example 4-1 of WO2024 / 085166. The cells were cultured in Dulbecco's modified Eagle's medium (SIGMA, D6429) supplemented with a final concentration of 10% heat-inactivated fetal bovine serum (FBS) (Cytiva, SH30084.03) (hereinafter, the prepared medium is referred to as "Eagle's culture medium") at 37 ° C., 5% CO ２ CLDN4 (Myc-DDK-tagged)-Human claudin 4 (CLDN4) (ORIGENE, RC200490) was introduced into B16-F10 cells (ATCC, CRL-6475) cultured under the conditions of 1. Selection was performed in Eagle's culture medium supplemented with G418 (Nacalai Tesque, 09380-44) at a final concentration of 1 mg / mL to obtain a B16-F10 cell clone stably expressing human CLDN4 (hereinafter referred to as "human CLDN4-expressing B16-F10 cells").

[0089] [Example 2-2: Combined effect of anti-CLDN4-anti-CD137 bispecific antibody and oxaliplatin in an in vivo antitumor mouse model] B-h4-1BB male mice (C57BL / 6-Tnfrsf9tm1 (Tnfrsf9) / Bcgen; Biocytogen, 110004) (hereinafter referred to as "mice") were obtained and bred. Human CLDN4-expressing B16-F10 cells were suspended in PBS (-) (WAKO, 045-29795) and 4 × 10 ６ A cell suspension was prepared at a concentration of 2 × 10 cells / mL. The cell suspension was injected subcutaneously into the back of an 8-week-old mouse. ５ Two days after cell inoculation, the tumor diameter was measured using a caliper (Mitutoyo, CD-15AXR). ３The following formula was used to calculate the tumor volume (mm ３ ) ]=[Long axis of tumor (mm)] × [Shorter axis of tumor (mm)] ２ × 0.5

[0090] The cell-inoculated mice were divided into groups (n=10) so that tumor volumes were equal in each group, and administration of the test substance began the day after grouping. The first day of administration was defined as day 0. The test substances used were hKM3900_tA2-32LH, an anti-lysozyme antibody as an isotype control antibody, and oxaliplatin (Elplat® Intravenous Infusion, Yakult Honsha). Details of the test antibodies, dosages, administration schedules, etc. administered to the four groups are shown below. (1) Group 1: Control group. The anti-lysozyme antibody was administered intraperitoneally at 0.3 mg / kg on days 0, 4, 7, and 10. (2) Group 2: hKM3900_tA2-32LH-administered group. The hKM3900_tA2-32LH was administered intraperitoneally at 0.3 mg / kg on days 0, 4, 7, and 10. (3) Group 3: Oxaliplatin-administered group. Anti-lysozyme antibody was administered intraperitoneally at 0.3 mg / kg and oxaliplatin at 5 mg / kg on days 0, 4, 7, and 10. (4) Group 4: hKM3900_tA2-32LH and oxaliplatin-administered group. hKM3900_tA2-32LH was administered intraperitoneally at 0.3 mg / kg and oxaliplatin at 5 mg / kg on days 0, 4, 7, and 10. Tumor volume was evaluated in each group on days 4, 7, 10, and 12. Tumor volumes on day 12 in Groups 2 and 3 were compared with those in Group 4 by Student's t-test (Figure 1).

[0091] As shown in Figure 1, the tumor volume in the hKM3900_tA2-32LH and oxaliplatin combination administration group was significantly smaller than that in the hKM3900_tA2-32LH alone administration group and the oxaliplatin alone administration group. This result suggests that in the treatment of human cancer, the combination of an anti-CLDN4-anti-CD137 bispecific antibody and oxaliplatin may have a greater antitumor effect than either drug administered alone.

[0092] Example 3: Combination Effect of Anti-CLDN4-anti-CD137 Bispecific Antibody with Oxaliplatin and Anti-Mouse VEGFR Antibody in an In Vivo Antitumor Mouse Model Using the same method as in Example 2-2, the combined effect of an anti-CLDN4-anti-CD137 bispecific antibody with oxaliplatin and an anti-mouse VEGFR antibody in in vivo antitumor activity was evaluated. The test substances used were the anti-CLDN4-anti-CD137 bispecific antibody hKM3900_tA2-32LH, an isotype control antibody, an anti-lysozyme antibody, oxaliplatin, and an anti-mouse VEGFR antibody (Bio X Cell, BE0060). Details of the test antibodies administered to the four groups tested, their doses, administration schedules, etc. are shown below. (1) Group 1: Control group. The anti-lysozyme antibody was administered intraperitoneally at 0.3 mg / kg on days 0, 4, 7, and 10. (2) Group 2: hKM3900_tA2-32LH administration group: hKM3900_tA2-32LH was administered intraperitoneally at 0.3 mg / kg on days 0, 4, 7, and 10. (3) Group 3: Oxaliplatin and anti-mouse VEGFR antibody combination administration group: Anti-lysozyme antibody was administered intraperitoneally at 0.3 mg / kg, oxaliplatin at 5 mg / kg, and anti-mouse VEGFR antibody at 5 mg / kg on days 0, 4, 7, and 10. (4) Group 4: Group receiving a combination of hKM3900_tA2-32LH, oxaliplatin, and anti-mouse VEGFR antibody. hKM3900_tA2-32LH was administered intraperitoneally at 0.3 mg / kg, oxaliplatin at 5 mg / kg, and anti-mouse VEGFR antibody at 5 mg / kg on days 0, 4, 7, and 10. Tumor volume was assessed in each group on days 4, 7, 10, and 12. Tumor volumes on day 12 in Groups 2 and 3 were compared with that in Group 4 by Student's t-test (Figure 2).

[0093] As shown in Figure 2, the tumor volume in the group administered hKM3900_tA2-32LH in combination with oxaliplatin and an anti-mouse VEGFR antibody was significantly smaller than that in the group administered hKM3900_tA2-32LH alone or in combination with oxaliplatin and an anti-mouse VEGFR antibody. These results suggest that in the treatment of human cancer, the triple combination of an anti-CLDN4-anti-CD137 bispecific antibody with oxaliplatin and an anti-VEGFR antibody may have a greater antitumor effect than the administration of an anti-CLDN4-anti-CD137 bispecific antibody alone or in combination with oxaliplatin and an anti-VEGFR antibody.

[0094] The results of Examples 2 and 3 showed that in vivo mouse studies, a more potent antitumor effect can be induced by combining oxaliplatin alone or in combination with other drugs with an anti-CLDN4-anti-CD137 bispecific antibody. These results suggest that even in the treatment of human cancers, more favorable therapeutic effects may be obtained by broadly combining anti-CLDN4-anti-CD137 bispecific antibodies with oxaliplatin-based therapies.

[0095] The cancer treatment method of the present invention using an anti-CLDN4-anti-CD137 bispecific antibody in combination with a platinum-based drug is expected to be useful in cancer treatment.

[0096] SEQ ID NO: 2 is the amino acid sequence of hKM3900_tA2-32LH HC, and the nucleotide sequence shown in SEQ ID NO: 1 is a nucleotide sequence encoding the amino acid sequence of the hKM3900_tA2-32LH heavy chain shown in SEQ ID NO: 2. SEQ ID NO: 4 is the amino acid sequence of hKM3900 LC, and the nucleotide sequence shown in SEQ ID NO: 3 is a nucleotide sequence encoding the amino acid sequence of the hKM3900 light chain shown in SEQ ID NO: 4. SEQ ID NOs: 5 to 14 are the amino acid sequences of various linkers described in the detailed description of the invention.

Claims

1. A pharmaceutical composition comprising an anti-CLDN4-anti-CD137 bispecific antibody, used in combination with a platinum-based drug to treat cancer in a subject, wherein the bispecific antibody comprises the heavy and light chain variable regions of an anti-CLDN4 antibody and the heavy and light chain variable regions of an anti-CD137 antibody, wherein the heavy chain variable region of the anti-CLDN4 antibody comprises CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO:2, CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 66 of SEQ ID NO:2, and CDR3 consisting of the amino acid sequence from amino acid numbers 99 to 112 of SEQ ID NO:2, and the light chain variable region of the anti-CLDN4 antibody comprises CDR1 consisting of the amino acid sequence from amino acid numbers 24 to 35 of SEQ ID NO:4, CDR2 consisting of the amino acid sequence from amino acid numbers 51 to 57 of SEQ ID NO:4, and CDR3 consisting of the amino acid sequence from amino acid numbers 90 to 98 of SEQ ID NO:

4.

2. The pharmaceutical composition of claim 1, wherein the heavy chain variable region of the anti-CLDN4 antibody consists of the amino acid sequence from amino acid numbers 1 to 123 of SEQ ID NO: 2, and the light chain variable region of the anti-CLDN4 antibody consists of the amino acid sequence from amino acid numbers 1 to 109 of SEQ ID NO:

4.

3. The pharmaceutical composition according to claim 2, wherein the anti-CLDN4-anti-CD137 bispecific antibody comprises an IgG antibody (anti-CLDN4 IgG antibody) consisting of a heavy chain comprising the heavy chain variable region of the anti-CLDN4 antibody and a light chain comprising the light chain variable region of the anti-CLDN4 antibody.

4. The pharmaceutical composition of claim 3, wherein the Fc region of the anti-CLDN4 IgG antibody contains either or both of an LALA mutation (L234A and L235A) or a P331G mutation (wherein the mutation position is an amino acid position according to the EU index in the human Igγ1 constant region).

5. A pharmaceutical composition according to any one of claims 1 to 4, wherein the heavy chain variable region of the anti-CD137 antibody comprises CDR1 consisting of the amino acid sequence from 625 to 629 of SEQ ID NO:2, CDR2 consisting of the amino acid sequence from 644 to 659 of SEQ ID NO:2, and CDR3 consisting of the amino acid sequence from 692 to 701 of SEQ ID NO:2, and the light chain variable region of the anti-CD137 antibody comprises CDR1 consisting of the amino acid sequence from 486 to 498 of SEQ ID NO:2, CDR2 consisting of the amino acid sequence from 514 to 520 of SEQ ID NO:2, and CDR3 consisting of the amino acid sequence from 553 to 563 of SEQ ID NO:

2.

6. The pharmaceutical composition according to claim 5, wherein the heavy chain variable region of the anti-CD137 antibody consists of the amino acid sequence from amino acid numbers 595 to 712 of SEQ ID NO: 2, and the light chain variable region of the anti-CD137 antibody consists of the amino acid sequence from amino acid numbers 464 to 573 of SEQ ID NO:

2.

7. The pharmaceutical composition of claim 6, wherein the anti-CLDN4-anti-CD137 bispecific antibody comprises an anti-CD137 single-chain variable region fragment (anti-CD137 scFv) comprising the heavy chain variable region and light chain variable region of an anti-CD137 antibody.

8. The pharmaceutical composition according to claim 7, wherein the anti-CD137 scFv consists of the amino acid sequence from amino acid numbers 464 to 712 of SEQ ID NO:

2.

9. The pharmaceutical composition according to claim 8, wherein the anti-CLDN4-anti-CD137 bispecific antibody comprises an anti-CLDN4 IgG antibody and an anti-CD137 scFv, and the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 IgG antibody via a linker.

10. A pharmaceutical composition for use in combination with a platinum-based drug to treat cancer in a subject, comprising an anti-CLDN4-anti-CD137 bispecific antibody, wherein the bispecific antibody comprises a heavy chain of the anti-CLDN4 antibody comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 123 of SEQ ID NO: 2 and a light chain of the anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4, and an anti-CD137 scFv comprising a light chain variable region of the anti-CD137 antibody consisting of the amino acid sequence of amino acids 464 to 573 of SEQ ID NO: 2 and a heavy chain variable region of the anti-CD137 antibody consisting of the amino acid sequence of amino acids 595 to 712 of SEQ ID NO: 2, and wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker.

11. A pharmaceutical composition for use in combination with a platinum-based drug to treat cancer in a subject, comprising an anti-CLDN4-anti-CD137 bispecific antibody, wherein the bispecific antibody comprises an anti-CLDN4 antibody heavy chain consisting of the amino acid sequence from amino acid numbers 1 to 453 of SEQ ID NO: 2 and an anti-CLDN4 antibody light chain consisting of the amino acid sequence from amino acid numbers 1 to 215 of SEQ ID NO: 4, and an anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 464 to 712 of SEQ ID NO: 2, and wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker.

12. The pharmaceutical composition according to any one of claims 9 to 11, wherein the linker is a GS linker.

13. A pharmaceutical composition comprising an anti-CLDN4-anti-CD137 bispecific antibody used in combination with a platinum-based drug to treat cancer in a subject, wherein the bispecific antibody comprises a polypeptide comprising an anti-CLDN4 antibody heavy chain and an anti-CD137 scFv consisting of the amino acid sequence of SEQ ID NO: 2, and an anti-CLDN4 antibody light chain consisting of the amino acid sequence of SEQ ID NO:

4.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the anti-CLDN4-anti-CD137 bispecific antibody is post-translationally modified.

15. The pharmaceutical composition according to any one of claims 1 to 14, which is used in combination with a platinum-based drug simultaneously, sequentially or sequentially.

16. The pharmaceutical composition according to any one of claims 1 to 15, wherein the cancer is selected from the group consisting of colorectal cancer, non-small cell lung cancer, prostate cancer, ovarian cancer, breast cancer, urothelial cancer, gastric cancer, kidney cancer, pancreatic cancer, uterine cancer, cervical cancer, esophageal cancer, bile duct cancer, and head and neck cancer.

17. The pharmaceutical composition according to any one of claims 1 to 16, wherein the platinum-based drug is oxaliplatin.

18. An anti-CLDN4-anti-CD137 bispecific antibody for use in combination with a platinum-based drug to treat cancer in a subject, the bispecific antibody comprising the heavy and light chain variable regions of an anti-CLDN4 antibody and the heavy and light chain variable regions of an anti-CD137 antibody, wherein the heavy chain variable region of the anti-CLDN4 antibody comprises CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO:2, CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 66 of SEQ ID NO:2, and CDR3 consisting of the amino acid sequence from amino acid numbers 99 to 112 of SEQ ID NO:2, and the light chain variable region of the anti-CLDN4 antibody comprises CDR1 consisting of the amino acid sequence from amino acid numbers 24 to 35 of SEQ ID NO:4, CDR2 consisting of the amino acid sequence from amino acid numbers 51 to 57 of SEQ ID NO:4, and CDR3 consisting of the amino acid sequence from amino acid numbers 90 to 98 of SEQ ID NO:

4.

19. A method for treating cancer, comprising administering to a subject a combination of an anti-CLDN4-anti-CD137 bispecific antibody and a platinum-based drug, wherein the bispecific antibody comprises the heavy chain variable region and light chain variable region of an anti-CLDN4 antibody, and the heavy chain variable region and light chain variable region of an anti-CD137 antibody, wherein the heavy chain variable region of the anti-CLDN4 antibody comprises CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 66 of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence from amino acid numbers 99 to 112 of SEQ ID NO: 2, and the light chain variable region of the anti-CLDN4 antibody comprises CDR1 consisting of the amino acid sequence from amino acid numbers 24 to 35 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence from amino acid numbers 51 to 57 of SEQ ID NO: 4, and CDR3 consisting of the amino acid sequence from amino acid numbers 90 to 98 of SEQ ID NO:

4.

20. Use of an anti-CLDN4-anti-CD137 bispecific antibody for the manufacture of a pharmaceutical composition to be used in combination with a platinum-based drug to treat cancer in a subject, the bispecific antibody comprising the heavy and light chain variable regions of an anti-CLDN4 antibody and the heavy and light chain variable regions of an anti-CD137 antibody, wherein the heavy chain variable region of the anti-CLDN4 antibody comprises CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO:2, CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 66 of SEQ ID NO:2, and CDR3 consisting of the amino acid sequence from amino acid numbers 99 to 112 of SEQ ID NO:2, and the light chain variable region of the anti-CLDN4 antibody comprises CDR1 consisting of the amino acid sequence from amino acid numbers 24 to 35 of SEQ ID NO:4, CDR2 consisting of the amino acid sequence from amino acid numbers 51 to 57 of SEQ ID NO:4, and CDR3 consisting of the amino acid sequence from amino acid numbers 90 to 98 of SEQ ID NO:

4.

21. A pharmaceutical composition according to any one of claims 1 to 17, comprising an anti-CLDN4-anti-CD137 bispecific antibody used in combination with a platinum-based drug to treat cancer in a subject, further comprising a VEGF pathway inhibitory antibody.

22. The bispecific antibody of claim 18, which is an anti-CLDN4-anti-CD137 bispecific antibody used in combination with a platinum-based drug to treat cancer in a subject, and which is further used in combination with a VEGF pathway inhibitor antibody.

23. A method for treating cancer comprising administering to a subject an anti-CLDN4-anti-CD137 bispecific antibody in combination with a platinum-based drug, wherein the method further comprises administering to the subject in combination with a VEGF pathway inhibitory antibody.

24. The use of an anti-CLDN4-anti-CD137 bispecific antibody for the manufacture of a pharmaceutical composition for use in combination with a platinum-based drug to treat cancer in a subject, wherein the bispecific antibody is further used in combination with a VEGF pathway inhibitory antibody, as described in claim 20.

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