Use of Anti-CLDN4-Anti-CD137 bispecific antibody combined with PD-1 signal inhibitor for cancer treatment

Abstract

The present invention addresses the problem of: providing an anti-CLDN4-anti-CD137 bispecific antibody to be used in combination with a PD-1 signal inhibitor for treatment of a cancer, or a pharmaceutical composition comprising said bispecific antibody; and providing a cancer treatment method comprising administering, to a subject, an anti-CLDN4-anti-CD137 bispecific antibody in combination with a PD-1 signal inhibitor.　In the present invention, a combined use of an anti-CLDN4-anti-CD137 bispecific antibody and a PD-1 signal inhibitor under a condition for co-culturing CLDN4-expressing cancer cells and T-cells, demonstrated, due to the T-cells, a higher interferon-γ production promotion action and cytotoxic action and also demonstrated a more significant antitumor action in CLDN4-expressing cancer cell-transplanted mice, compared with single administrations of each. The foregoing results suggest that the combination of the anti-CLDN4-anti-CD137 bispecific antibody and the PD-1 signal inhibitor is effective in treatment of CLDN4-expressing cancer.

Description

Use of anti-cldn4-anti-cd137 bispecific antibodies in combination with pd-1 signal inhibitors in cancer treatment

[0001] The present invention relates to the use of an anti-CLDN4-anti-CD137 bispecific antibody in combination with a PD-1 signal inhibitor in cancer treatment.

[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 in various antibody formats have been reported as an innovative method for obtaining cancer cell-selective cytotoxic activity at low antibody concentrations. Bispecific T-cell-recruiting antibodies are bispecific antibodies that contain an antibody against a tumor-associated antigen (TAA) expressed on the surface of cancer cells and an antibody that binds to T cells, and the effects of these antibodies on T-cell-mediated immunotherapy are currently being investigated (Non-Patent Document 6). Anti-CD3 antibodies are often used as antibodies that bind to T cells, and research and development of bispecific T-cell-recruiting antibodies against various TAAs is currently being conducted.

[0005] Furthermore, in recent years, bispecific T cell-recruiting antibodies against CD137 and TAAs have been actively studied, including anti-GPC3-anti-CD137 bispecific antibodies, anti-HER2-anti-CD137 bispecific antibodies, anti-PD-L1-anti-CD137 bispecific antibodies, and anti-FAP-anti-CD137 bispecific antibodies that recognize the TAAs Glypican 3 (GPC3), Human Epidermal Growth Factor Receptor Type 2 (HER2), Programmed Cell Death-Ligand 1 (PD-L1), and Fibroblast Activation Protein (FAP) (Patent Documents 2 and 3, Non-Patent Documents 7 to 9).

[0006] Programmed cell death-1 (PD-1; also known as PDCD1 or CD279) is a 50-55 kDa type I transmembrane protein belonging to the immunoglobulin superfamily (Non-Patent Document 10). PD-1 expression is induced in T cells with sustained activation, and by binding to its ligands, programmed death-ligand 1 (PD-L1; also known as PDCD1LG1, B7-H1, or CD274) or programmed death-ligand 2 (PD-L2; also known as PDCD1LG2, B7-DC, or CD273), it suppresses T cell activation (Non-Patent Document 11). Generally, such mechanisms for controlling T cell activation are called immune checkpoints and are known as negative feedback mechanisms that prevent excessive immune responses.

[0007] In the early stages of cancer development, immune cells such as T cells eliminate cancer through an anti-tumor immune response via immune surveillance. Meanwhile, cancer acquires immune escape mechanisms by directly or indirectly suppressing immune cells in the tumor microenvironment. Known immune checkpoint mechanisms directly suppress activated T cells, such as the PD-1 / PD-L1 or PD-L2 (hereinafter referred to as "PD-1 signal") pathway and the CTLA-4 / CD80 or CD86 pathway, have been shown to suppress immune escape. Expression of PD-1 on T cells and PD-L1 on tumors has been confirmed within the cancer tumor microenvironment (Non-Patent Document 12), and activation of this PD-1 signal is thought to cause cancer to evade the immune system. Inhibition of PD-1 signaling has been reported in several mouse tumor-bearing models, resulting in anti-tumor activity by disabling the immune escape mechanism (Non-Patent Documents 13-15). Furthermore, PD-1 signal inhibitors such as anti-PD-1 antibodies, such as nivolumab and pembrolizumab, have been vigorously developed and have achieved significant results in the treatment of melanoma, lung cancer, lymphoma, etc. In addition to antibodies, research is also being conducted on nucleic acid drugs and small molecule drugs as PD-1 signal inhibitors (Non-Patent Document 16).

[0008] In order to improve the effectiveness of treatment in cancer patients, studies are being actively conducted on combination therapies using multiple cancer immunotherapies and on combinations of cancer immunotherapies with existing anticancer drugs (Non-Patent Documents 17 and 18). For example, studies are being conducted on combinations of anti-PD-1 antibodies with other immune checkpoint inhibitor antibodies, anticancer drugs, molecular targeted drugs, radiation therapy, cancer vaccines, and oncolytic viruses. However, to date, no cancer treatment methods have been reported that use an anti-CLDN4-anti-CD137 bispecific antibody in combination with a PD-1 signal inhibitor.

[0009] International Publication No. WO 2008 / 114733 International Publication No. WO 2015 / 156268 International Publication No. WO 2016 / 177802

[0010] 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. 182-212 Clinical Cancer Research, 2019: 25 (19): p. 5878-5889Clinical Cancer Research, 2020:26(15):p. 4154-4167 Journal for Immunotherapy of Cancer, 2020: 8 (2): e000238 International Immunology, 1996: Vol. 8:p. 765-772 Annual Review of Immunology, 2008: Vol. 26:p. 677-704 Nature Medicine, 2002:8:p. 793-800Scientific Reports, 2021:11:p. 21087-21099 Nature Communications, 2017:8:p. 14572-14582 Journal for Immunotherapy of Cancer, 2019:7:37:p. 1-16 Molecules, 2019:24:p. 2071-2100 Cancer Discovery, 2021:11:p. 1368-1397Molecular Medicine Reports, 2021:23:p. 362-377

[0011] An object of the present invention is to provide an anti-CLDN4-anti-CD137 bispecific antibody to be used in combination with a PD-1 signal inhibitor for the treatment of cancer in a subject, or a pharmaceutical composition comprising the bispecific antibody, or to provide a method for treating cancer comprising administering an anti-CLDN4-anti-CD137 bispecific antibody and a PD-1 signal inhibitor to a subject.

[0012] The present inventors aimed to create an antibody or pharmaceutical composition for use in the treatment of CLDN4-expressing cancers, and prepared an anti-CLDN4-anti-CD137 bispecific antibody based on the sequences of the known anti-CLDN4 antibody KM3900 and anti-CD137 antibodies (Example 1). The combined use of the obtained anti-CLDN4-anti-CD137 bispecific antibody with an anti-PD-1 antibody or an anti-PD-L1 antibody promoted T cell interferon-γ production in vitro compared to the use of the anti-CLDN4-anti-CD137 bispecific antibody, anti-PD-1 antibody, or anti-PD-L1 antibody alone (Examples 2 and 3). Furthermore, in mice bearing human CLDN4-expressing mouse cancer cells, the combined use of the anti-CLDN4-anti-CD137 bispecific antibody with an anti-PD-1 antibody showed a significant antitumor effect compared to the use of the anti-CLDN4-anti-CD137 bispecific antibody or anti-PD-1 antibody alone (Example 4). These results suggest that the combination of an anti-CLDN4-anti-CD137 bispecific antibody and a PD-1 signal inhibitor is useful for treating CLDN4-expressing cancer.

[0013] That is, the present invention relates to the following [1] to

[84] , but is not limited thereto: [1] A pharmaceutical composition for treating 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, and is used in combination with a PD-1 signal inhibitor. [2] The pharmaceutical composition of [1], wherein the heavy chain variable region of the anti-CLDN4 antibody comprises 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, and the light chain variable region of the anti-CLDN4 antibody comprises 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. [3] The pharmaceutical composition of [1] or [2], wherein the heavy chain variable region of the anti-CLDN4 antibody consists of the amino acid sequence of amino acids 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 acids 1 to 109 of SEQ ID NO: 4. [4] The pharmaceutical composition according to any one of [1] to [3], 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. [5] The pharmaceutical composition according to [4], wherein 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) in the Fc region.[6] The pharmaceutical composition according to any one of [1] to [5], 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. [7] The pharmaceutical composition according to any one of [1] to [6], 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. [8] The pharmaceutical composition of [6] or [7], 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. [9] The pharmaceutical composition of [8], wherein the anti-CD137 scFv consists of the amino acid sequence from amino acid numbers 464 to 712 of SEQ ID NO: 2.

[10] The pharmaceutical composition of [8] or [9], 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.

[11] A pharmaceutical composition for treating 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 heavy chain carboxy terminus of the anti-CLDN4 antibody via a linker, and the pharmaceutical composition is used in combination with a PD-1 signal inhibitor.

[12] A pharmaceutical composition for treating 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 consisting of the amino acid sequence of amino acids 1 to 453 of SEQ ID NO: 2, a light chain of the anti-CLDN4 antibody 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, the pharmaceutical composition being used in combination with a PD-1 signal inhibitor.

[13] The pharmaceutical composition according to any of

[10] to

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

[14] A pharmaceutical composition for treating cancer in a subject, comprising an anti-CLDN4-anti-CD137 bispecific antibody, 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, and is used in combination with a PD-1 signal inhibitor.

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

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

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

[15] , which is used in combination with a PD-1 signal inhibitor simultaneously, sequentially, or successively.

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

[16] , wherein the anti-CLDN4-anti-CD137 bispecific antibody and the PD-1 signal inhibitor are (i) contained in the same pharmaceutical composition and administered simultaneously, or (ii) contained in separate pharmaceutical compositions and used in combination simultaneously, sequentially, or sequentially.

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

[17] , wherein the cancer is selected from the group consisting of colorectal cancer, bladder cancer, and lung cancer.

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

[18] , wherein the PD-1 signal inhibitor is an antibody or an antigen-binding fragment thereof that binds to one or more proteins selected from the group consisting of PD-1, PD-L1, and PD-L2.

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

[19] , wherein the PD-1 signal inhibitor is an anti-PD-1 antibody selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, spartalizumab, and cemiplimab.

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

[19] , wherein the PD-1 signal inhibitor is an anti-PD-L1 antibody selected from the group consisting of atezolizumab, durvalumab, and avelumab.

[22] An anti-CLDN4-anti-CD137 bispecific antibody for treating cancer in a subject, 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, and used in combination with a PD-1 signal inhibitor.

[23] The bispecific antibody according to

[22] , 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.

[24] The bispecific antibody according to

[22] or

[23] , 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.

[25] The bispecific antibody according to any of

[22] to

[24] , 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.

[26] The bispecific antibody according to

[25] , 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).

[27] The bispecific antibody of any of

[22] to

[26] , 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.

[28] The bispecific antibody of any of

[22] to

[27] , 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.

[29] The bispecific antibody according to

[27] or

[28] , which 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.

[30] The bispecific antibody according to

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

[31] The bispecific antibody according to

[29] or

[30] , comprising an anti-CLDN4 IgG antibody and an anti-CD137 scFv, wherein 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.

[32] An anti-CLDN4-anti-CD137 bispecific antibody for treating 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, and used in combination with a PD-1 signal inhibitor.

[33] An anti-CLDN4-anti-CD137 bispecific antibody for treating 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, the bispecific antibody being used in combination with a PD-1 signal inhibitor.

[34] The bispecific antibody according to any of

[31] to

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

[35] An anti-CLDN4-anti-CD137 bispecific antibody used to treat a target cancer, the 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, and used in combination with a PD-1 signal inhibitor.

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

[22] to

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

[37] The bispecific antibody according to any of

[22] to

[36] , wherein the bispecific antibody is used in combination with a PD-1 signal inhibitor simultaneously, sequentially, or sequentially.

[38] The bispecific antibody according to any of

[22] to

[37] , wherein the anti-CLDN4-anti-CD137 bispecific antibody and the PD-1 signal inhibitor are (i) contained in the same pharmaceutical composition and administered simultaneously, or (ii) in separate pharmaceutical compositions and administered simultaneously, sequentially, or sequentially.

[39] The bispecific antibody according to any of

[22] to

[38] , wherein the cancer is selected from the group consisting of colorectal cancer, bladder cancer, and lung cancer.

[40] The bispecific antibody according to any of

[22] to

[39] , wherein the PD-1 signal inhibitor is an antibody or an antigen-binding fragment thereof that binds to one or more proteins selected from the group consisting of PD-1, PD-L1, and PD-L2.

[41] The bispecific antibody according to any of

[22] to

[40] , wherein the PD-1 signal inhibitor is an anti-PD-1 antibody selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, spartalizumab, and cemiplimab.

[42] The bispecific antibody according to any of

[22] to

[40] , wherein the PD-1 signal inhibitor is an anti-PD-L1 antibody selected from the group consisting of atezolizumab, durvalumab, and avelumab.

[43] A method for treating cancer, comprising administering to a subject a combination of an anti-CLDN4-anti-CD137 bispecific antibody and a PD-1 signal inhibitor, 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.

[44] The therapeutic method according to

[43] , wherein the heavy chain variable region of the anti-CLDN4 antibody comprises 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, and the light chain variable region of the anti-CLDN4 antibody comprises 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.

[45] The therapeutic method according to

[43] or

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

[46] The therapeutic method according to any one of

[43] to

[45] , 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.

[47] The therapeutic method according to

[46] , wherein 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) in the Fc region.

[48] ​​A method of treatment according to any one of

[43] to

[47] , 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.

[49] The therapeutic method according to any one of

[43] to

[48] , 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.

[50] The therapeutic method according to

[48] or

[49] , 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.

[51] The therapeutic method according to

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

[52] 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.

[50] or

[51] The therapeutic method according to.

[53] A method for treating cancer, comprising administering to a subject a combination of an anti-CLDN4-anti-CD137 bispecific antibody and a PD-1 signal inhibitor, 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 acid numbers 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 acid numbers 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 acid numbers 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 acid numbers 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.

[54] A method for treating cancer, comprising administering to a subject a combination of an anti-CLDN4-anti-CD137 bispecific antibody and a PD-1 signal inhibitor, 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, 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.

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

[52] to

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

[56] A method for treating cancer, comprising administering to a subject a combination of an anti-CLDN4-anti-CD137 bispecific antibody and a PD-1 signal inhibitor, 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.

[57] The method of treatment according to any one of

[43] to

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

[58] The method of treatment according to any one of

[43] to

[57] , wherein the anti-CLDN4-anti-CD137 bispecific antibody and the PD-1 signal inhibitor are used in combination simultaneously, consecutively, or sequentially.

[59] The therapeutic method according to any one of

[43] to

[58] , wherein the anti-CLDN4-anti-CD137 bispecific antibody and the PD-1 signal inhibitor are (i) contained in the same pharmaceutical composition and administered simultaneously, or (ii) in separate pharmaceutical compositions and used in combination simultaneously, consecutively, or sequentially.

[60] The therapeutic method according to any one of

[43] to

[58] , wherein the cancer is selected from the group consisting of colorectal cancer, bladder cancer, and lung cancer.

[61] The therapeutic method according to any one of

[43] to

[60] , wherein the PD-1 signal inhibitor is an antibody or an antigen-binding fragment thereof that binds to one or more proteins selected from the group consisting of PD-1, PD-L1, and PD-L2.

[62] The therapeutic method according to any one of

[43] to

[61] , wherein the PD-1 signal inhibitor is an anti-PD-1 antibody selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, spartalizumab, and cemiplimab.

[63] The therapeutic method according to any one of

[43] to

[61] , wherein the PD-1 signal inhibitor is an anti-PD-L1 antibody selected from the group consisting of atezolizumab, durvalumab, and avelumab.

[64] Use of an anti-CLDN4-anti-CD137 bispecific antibody for the manufacture of a pharmaceutical composition to be used in combination with a PD-1 signal inhibitor to treat cancer in a subject, 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.

[65] The use according to

[64] , 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.

[66] The use according to

[64] or

[65] , 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.

[67] The use according to any one of

[64] to

[66] , 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 an anti-CLDN4 antibody and a light chain comprising the light chain variable region of an anti-CLDN4 antibody.

[68] The use according to

[67] , wherein 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) in the Fc region.

[69] The use according to any of

[64] to

[68] , wherein the heavy chain variable region of the anti-CD137 antibody comprises a CDR1 consisting of the amino acid sequence from 625 to 629 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 the light chain variable region of the anti-CD137 antibody comprises a 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.

[70] The use according to any of

[64] to

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

[71] The use according to

[69] or

[70] , 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.

[72] The use according to

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

[73] The use according to

[71] or

[72] , 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.

[74] Use of an anti-CLDN4-anti-CD137 bispecific antibody for the manufacture of a pharmaceutical composition used in combination with a PD-1 signal inhibitor 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.

[75] Use of an anti-CLDN4-anti-CD137 bispecific antibody for the manufacture of a pharmaceutical composition to be used in combination with a PD-1 signal inhibitor 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.

[76] The use according to any of

[73] to

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

[77] Use of an anti-CLDN4-anti-CD137 bispecific antibody for the manufacture of a pharmaceutical composition to be used in combination with a PD-1 signal inhibitor 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.

[78] The use according to any of

[64] to

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

[79] The use according to any one of

[64] to

[78] , wherein the pharmaceutical composition is used in combination with a PD-1 signal inhibitor simultaneously, sequentially, or sequentially.

[80] The use according to any one of

[64] to

[79] , wherein the anti-CLDN4-anti-CD137 bispecific antibody and the PD-1 signal inhibitor are (i) contained in the same pharmaceutical composition and administered simultaneously, or (ii) in separate pharmaceutical compositions and administered simultaneously, sequentially, or sequentially.

[81] The use according to any one of

[64] to

[80] , wherein the cancer is selected from the group consisting of colorectal cancer, bladder cancer, and lung cancer.

[82] The use according to any one of

[64] to

[81] , wherein the PD-1 signal inhibitor is an antibody or an antigen-binding fragment thereof that binds to one or more proteins selected from the group consisting of PD-1, PD-L1, and PD-L2.

[83] The use according to any one of

[64] to

[82] , wherein the PD-1 signal inhibitor is an anti-PD-1 antibody selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, spartalizumab, and cemiplimab.

[84] The use according to any one of

[64] to

[82] , wherein the PD-1 signal inhibitor is an anti-PD-L1 antibody selected from the group consisting of atezolizumab, durvalumab, and avelumab.

[0014] 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 PD-1 signal inhibitor produces a significant antitumor effect compared to the administration of either the anti-CLDN4-anti-CD137 bispecific antibody or the PD-1 signal inhibitor alone. Thus, the present invention provides use of an anti-CLDN4-anti-CD137 bispecific antibody in combination with a PD-1 signal inhibitor in cancer treatment.

[0015] Figure 1-1 shows the amount of interferon-γ produced by the addition of a test antibody in a co-culture system of human large cell lung cancer cell line LCLC-OKT3scFv cells and Expanded panT cells. The vertical axis of the figure shows the amount of interferon-γ produced 4 days after addition of the antibody, and the horizontal axis shows the antibody concentration. Symbols indicate the average amount of interferon-γ produced at each antibody concentration. Error bars indicate standard deviation. Figure 1-2 shows the amount of interferon-γ produced by the addition of a test antibody in a co-culture system of human large cell lung cancer cell line LCLC-OKT3scFv cells and Expanded panT cells. The vertical axis of the figure shows the amount of interferon-γ produced 5 days after addition of the antibody, and the horizontal axis shows the antibody concentration. Symbols indicate the average amount of interferon-γ produced at each antibody concentration. Error bars indicate standard deviation. Figure 2 shows the growth inhibitory effect of human CLDN4-expressing B16-F10 cells bearing tumors in B-h4-1BB mice. Figure 2 shows the average tumor volume (n = 10) at each day after the start of antibody 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 antibody administration date. The significance probability P value was determined by comparing the tumor volume of the combination group with the tumor volume of the test antibody single-agent administration group using an unpaired Student's t-test. ** in the figure indicates that the P value is less than the significance level of 0.01.

[0016] The present invention will be described in detail below.

[0017] Terms used herein are used in the sense commonly used by those skilled in the art unless otherwise defined below.

[0018] 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.

[0019] 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).

[0020] 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.

[0021] 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."

[0022] 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.

[0023] 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.

[0024] 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. ２ Examples 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] As used herein, an "effective amount" of an agent refers to the amount of agent necessary to produce a physiological change in a cell or tissue to which it is administered.

[0036] As used herein, the term "PD-1 signal inhibitor" refers to a drug that reverses the suppression of immune cell activation by PD-1. PD-1 signal inhibitors can inhibit the function of the PD-1 immune checkpoint by binding to PD-1 or its ligands, PD-L1 or PD-L2, and inhibiting immunosuppressive signals. The PD-1 signal inhibitor may be any substance that has the effect of blocking PD-1 signaling, such as an antibody, a low-molecular-weight compound, a nucleic acid (which may include DNA or RNA, or natural or artificial nucleic acid), a fusion protein, or a peptide. For example, an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody can inhibit PD-1 signaling by inhibiting the binding of PD-1 to PD-L1 or PD-L2 (Expert Opinion on Therapeutic Patents, 2016: Vol. 26: pp. 555-564).

[0037] The present invention relates to the following (1) to (4): (1) a pharmaceutical composition used in combination with a PD-1 signal inhibitor, comprising an anti-CLDN4-anti-CD137 bispecific antibody (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 PD-1 signal inhibitor to treat cancer in a subject; (3) a method for treating cancer, comprising administering an anti-CLDN4-anti-CD137 bispecific antibody and a PD-1 signal inhibitor 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 PD-1 signal inhibitor to treat cancer in a subject.

[0038] <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.

[0039] 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.

[0040] 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 structure 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.

[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 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.

[0042] 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.

[0043] 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.

[0044] 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).

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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").

[0051] 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.

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

[0053] In one embodiment, the linker used in the anti-CD137 scFv is a GS linker of (Gly-Gly-Gly-Gly-Ser)4.

[0054] 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.

[0055] 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.

[0056] In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the invention comprises an anti-CLDN4 IgG antibody and an anti-CD137 scFv.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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).

[0064] 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).

[0065] 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.

[0066] 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).

[0067] 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 PD-1 signal inhibitor.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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 is in the range of 0.01 mg / kg to 10 mg / kg, equivalent to a human dose. In one embodiment, the amount of the anti-CLDN4-anti-CD137 bispecific antibody of the present invention added in the formulation is preferably in the range of 0.01 mg / kg to 10 mg / kg, equivalent to a human dose.

[0073] <PD-1 Signal Inhibitor> In the present invention, a PD-1 signal inhibitor is used in combination with the anti-CLDN4-anti-CD137 bispecific antibody of the present invention or the pharmaceutical composition of the present invention to treat a target cancer. The mechanism of action and therapeutic modality of the PD-1 signal inhibitor are not particularly limited, as long as they block PD-1 signaling. The mechanism of action may be, for example, inhibition of binding between molecules involved in PD-1 signaling, or reduction in the expression level of PD-1 signal molecules (e.g., inhibition of protein production or induction of protein degradation). The therapeutic modality may be, for example, an antibody, a small molecule compound, a nucleic acid (DNA or RNA, which may include natural or artificial nucleic acids), a fusion protein, a peptide, or other therapeutic modality.

[0074] PD-1 signal inhibitors can be obtained by measuring the inhibitory effect of binding of one or more proteins selected from the group consisting of PD-1 and PD-L1 or PD-L2, or the expression-reducing effect using the expression level of PD-1 signal molecules as an indicator. For example, inhibitors of binding between PD-1 and PD-L1 or PD-L2 can be obtained by first obtaining an inhibitor that binds to PD-1 and PD-L1 or PD-L2, and then selecting the obtained inhibitor based on its ability to inhibit the binding of PD-1 to PD-L1 or PD-L2. The binding of an inhibitor to a protein can be evaluated using methods well known to those skilled in the art, such as flow cytometry (FCM), ELISA, surface plasmon resonance (SPR), thermal shift assay (TSA), and isothermal titration calorimetry (ITC). Furthermore, inhibitors that reduce the expression level of PD-1 signal molecules, such as PD-1, PD-L1, or PD-L2, can be obtained using the amount of PD-1, PD-L1, PD-L2, or other protein in cells as an indicator. The inhibitory effect of PD-1 signaling can be confirmed by the effects of T cell proliferation, IFN-γ release, reporter assay, etc. The effect of an inhibitor that reduces the expression level of a certain protein can be confirmed using methods well known to those skilled in the art, such as ELISA, quantitative PCR, in situ hybridization, and live cell imaging.

[0075] PD-1 signal inhibitors include antibodies that inhibit PD-1 signaling, such as anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-PD-L2 antibodies. Such antibodies may be humanized antibodies, chimeric antibodies, mouse antibodies, human antibodies, and antigen-binding fragments thereof. Known anti-PD-1 antibodies include, but are not limited to, the antibodies described in U.S. Pat. Nos. 8,008,449, 6,808,710, 7,488,802, 8,168,757, and 8,354,509, as well as WO 2006 / 121168 and WO 2012 / 145493. Known anti-PD-L1 antibodies include, but are not limited to, the antibodies described in, for example, WO 2007 / 005874, WO 2010 / 077634, WO 2011 / 066389, WO 2013 / 079174, and U.S. Pat. No. 8,217,149. In one embodiment, the PD-1 signal inhibitor used in the present invention is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody, or an antigen-binding fragment thereof. In one embodiment, the PD-1 signal inhibitor used in the present invention is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody. In one embodiment, the anti-PD-1 antibody may be an anti-PD-1 antibody such as nivolumab, pembrolizumab, pidilizumab, spartalizumab, or cemiplimab. In one embodiment, the anti-PD-L1 antibody may be an anti-PD-L1 antibody such as atezolizumab, durvalumab, or avelumab.

[0076] Examples of PD-1 signal inhibitors include fusion proteins and small molecule compounds that inhibit the binding of PD-1 to PD-L1, such as AMP-224 (WO 2010 / 027827 and WO 2011 / 066342) and BMS-1166 (Oncotarget, 2017: Vol. 8: pp. 72167-72181). Various PD-1 signal inhibitors are known in the art (Non-Patent Document 8).

[0077] <Therapeutic Method of the Present Invention> The therapeutic method of the present invention is a cancer treatment method (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 PD-1 signal inhibitor.

[0078] 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 PD-1 signal inhibitor simultaneously, sequentially, or sequentially.

[0079] 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 PD-1 signal inhibitor 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.

[0080] In one embodiment, the treatment method of the present invention is characterized in that the anti-CLDN4-anti-CD137 bispecific antibody of the present invention and the PD-1 signal inhibitor are (i) contained in the same pharmaceutical composition and administered simultaneously, or (ii) as separate pharmaceutical compositions administered on the same day.

[0081] In one embodiment, the treatment method of the present invention is characterized by the sequential use of (a) administering a PD-1 signal inhibitor to a subject after completing administration of the anti-CLDN4-anti-CD137 bispecific antibody of the present invention, or (b) administering a PD-1 signal inhibitor to a subject after completing administration of the anti-CLDN4-anti-CD137 bispecific antibody of the present invention.

[0082] 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 PD-1 signal inhibitor 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 PD-1 signal inhibitor in at least one administration cycle or all administration cycles. In one embodiment, the therapeutic method of the present invention is characterized in that administration of a PD-1 signal inhibitor 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 administration cycle or all administration cycles.

[0083] <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 cancer, 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, vaginal cancer, cervical cancer, head and neck cancer, and uterine cancer. Examples of cancers include solid cancers such as uterine cancer, cervical cancer, liver cancer, gallbladder cancer, bile duct cancer, kidney cancer, pancreatic cancer, colon cancer, colorectal 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.

[0084] In one embodiment, the cancer to be treated by the present invention is colon cancer, non-small cell lung cancer, small cell lung cancer, bladder cancer, ovarian cancer, breast cancer, or prostate cancer. In one embodiment, the cancer to be treated by the present invention is a cancer in which CLDN4 is highly expressed compared to normal tissue. The cancer to be treated by the present invention is preferably a cancer in which CLDN4 is highly expressed compared to normal tissue, or a cancer selected from the group consisting of colon cancer, rectal cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, bladder cancer, ovarian cancer, breast cancer, and prostate cancer.

[0085] The dose of the anti-CLDN4-anti-CD137 bispecific antibody or PD-1 signal inhibitor 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.

[0086] 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.

[0087] [Example 1: Preparation of anti-CLDN4-anti-CD137 bispecific antibody] [Example 1-1: Preparation of vector encoding anti-CLDN4-anti-CD137 bispecific antibody] It has been reported that the anti-CLDN4 antibody KM3900 selectively binds to CLDN4 compared to other claudin family molecules such as CLDN6 (Patent Document 1). Based on this report, a humanized antibody of KM3900 was prepared. Specifically, based on the humanized amino acid sequence of the variable region of KM3900, a humanized antibody was designed from the sequences of the human Igγ1 constant region and the human Igκ constant region. The amino acid mutations L234A, L235A, and P331G were introduced into the human Igγ1 constant region to design an anti-CLDN4 antibody sequence. The designed humanized anti-CLDN4 antibody is referred to as "hKM3900." AlivaMab mice (Ablexis, U.S. Patent No. 9,346,873) were immunized with the human CD137-human Fc fusion protein and immune adjuvant described in Example 3 of PCT / JP2022 / 18350 by multiple administrations. Lymphocytes were collected from the lymph nodes of the immunized mice and fused with mouse myeloma cells SP2 / 0 according to standard methods to produce hybridomas. Single hybridoma colonies were isolated using an automated picking device to obtain monoclonal hybridoma cells (hereinafter referred to as "clones"). Screening for clones producing antibodies that bind to human CD137 was performed, and a clone producing an anti-CD137 antibody (hereinafter referred to as "A2-32") was obtained. Furthermore, cDNA was synthesized from the cell lysate of this clone, and the antibody nucleotide sequence was identified. An anti-CD137 scFv was designed based on the sequences of the heavy chain variable region and light chain variable region of the obtained A2-32. An anti-CLDN4-anti-CD137 bispecific antibody was designed based on the amino acid sequence of hKM3900 and the amino acid sequence of the designed anti-CD137 scFv. The anti-CLDN4-anti-CD137 bispecific antibody has a GS linker linked to the C-terminus of the heavy chain of IgG1 type hKM3900, and the N-terminus of the anti-CD137 scFv is bound to the C-terminus of the GS linker.The designed anti-CLDN4-anti-CD137 bispecific antibody consists of a polypeptide comprising the heavy chain of the anti-CLDN4 antibody and anti-CD137 scFv consisting of the amino acid sequence of SEQ ID NO: 2, and a light chain of the anti-CLDN4 antibody consisting of the amino acid sequence of SEQ ID NO: 4. A polynucleotide encoding the polypeptide comprising the heavy chain and anti-CD137 scFv of the designed anti-CLDN4 antibody, and a polynucleotide encoding the light chain of the anti-CLDN4 antibody were prepared and each was inserted into a pcDNA3.4 TOPO vector (Thermo Fisher Scientific) according to standard methods. The two prepared vectors are referred to as "anti-CLDN4-anti-CD137 bispecific antibody expression vectors."

[0088] [Example 1-2: Production of anti-CLDN4-anti-CD137 bispecific antibody] An anti-CLDN4-anti-CD137 bispecific antibody was produced using an anti-CLDN4-anti-CD137 bispecific antibody expression vector. Specifically, the anti-CLDN4-anti-CD137 bispecific antibody expression vector was introduced into ExpiCHO-S cells (Thermo Fisher Scientific, A29127) using an ExpiFectamine CHO Transfection Kit (Thermo Fisher Scientific, A29129), 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 by affinity purification using MabSelect SuRe (Cytiva, 17-5438-02) and further by size exclusion chromatography purification using HiLoad 26 / 600 Superdex 200 pg (GE Healthcare, 28-9893-36). The anti-CLDN4-anti-CD137 bispecific antibody hKM3900_tA2-32LH prepared in Example 1 is sometimes referred to as "anti-CLDN4-anti-CD137 bispecific antibody."

[0089] [Example 2: In vitro combined effect of anti-CLDN4-anti-CD137 bispecific antibody and anti-PD-1 antibody] In a co-culture system using human OKT3scFv-expressing LCLC-103H cells and Expanded pan T cells, the in vitro combined effect of anti-CLDN4-anti-CD137 bispecific antibody and anti-PD-1 antibody was examined.

[0090] Example 2-1: Preparation of Expanded PanT Cells RPMI-1640 (Sigma, R8758) supplemented with 10% FBS (Cytiva, SH30084.03) and 1% penicillin-streptomycin (Thermo Fisher Scientific, 15070-063) was referred to as the "culture medium." Anti-CD3 antibody (BioLegend, 317325) was added to a 150 mm Tissue Culture Dish (IWAKI, 3030-150) (hereinafter referred to as the "dish") to a final concentration of 1 μg / mL, and the anti-CD3 antibody was immobilized. PanT cells (including both CD4+ T cells and CD8+ T cells, hereinafter referred to as "PanT cells") were isolated from human peripheral blood mononuclear cells (LONZA, CC-2702) using a PanT Cell Isolation Kit, human (Miltenyi Biotec, 130-096-535) according to the manufacturer's recommended protocol. The isolated PanT cells were centrifuged, the supernatant was removed, and then the cells were suspended in culture medium. The entire amount of PanT cells suspended in culture medium was seeded onto the aforementioned dish on which the anti-CD3 antibody had been immobilized. Furthermore, human IL-2 (PeproTech, 200-2) at a final concentration of 200 U / mL and anti-CD28 antibody (BioLegend, 302934) at a final concentration of 4 μg / mL were added, and the mixture was incubated at 37°C, 5% CO ２ The cells were cultured in an incubator. Three days after the start of culture, PanT cells were collected, suspended in culture medium, and seeded onto a dish. Human IL-2 was then added at a final concentration of 200 U / mL, and the cells were cultured at 37°C, 5% CO ２The cells were cultured in an incubator. After 4 days, all Pan T cells were collected and centrifuged. After removing the supernatant, the cells were resuspended in Cell Banker (Takara, CB011), aliquoted into tubes, and cryopreserved at -80°C. The cryopreserved Pan T cells are referred to herein as "Expanded Pan T cells."

[0091] [Example 2-2: Obtaining LCLC-103H cells expressing human CD3 antibody single chain variable region fragment (OKT3scFv)] LCLC-103H cells, a human large cell lung cancer cell line expressing human CLDN4, were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, ACC384). LCLC-103H cells, a human large cell lung cancer cell line, were cultured in culture medium at 37 ° C., 5% CO ２ The cells were cultured under the conditions of . A polynucleotide encoding human OKT3scFv (Journal of Immunological Methods, 2010:362:pp. 131-141) prepared by gene synthesis according to standard methods was subcloned into the pcDNA3.4-TOPO vector. The prepared human OKT3scFv expression vector was lipofected into LCLC-103H cells using Lipofectamine LTX (Invitrogen, 15338-100) according to the manufacturer's recommended protocol. An LCLC-103H cell clone stably expressing human OKT3scFv (hereinafter referred to as "LCLC-OKT3scFv cells") was obtained by selective culture in a culture medium supplemented with Geneticin Selective Antibiotic (Thermo Fisher Scientific, 10131-027) at a final concentration of 600 μg / mL and limiting dilution.

[0092] [Example 2-3: Combined effect of anti-CLDN4-anti-CD137 bispecific antibody and anti-PD-1 antibody on in vitro interferon-γ production promotion in a co-culture system of cancer cells and T cells] The T cell activity enhancing effect of the anti-CLDN4-anti-CD137 bispecific antibody and anti-PD-1 antibody was evaluated using the interferon-γ production promotion function as an index in a co-culture system of LCLC-OKT3scFv cells and Expanded PanT cells. LCLC-OKT3scFv cells were cultured at 2 × 10 ５ The cells were adjusted to a concentration of 50 μL / mL, and 50 μL of each was seeded into a flat-bottom 96-well plate (IWAKI, 4020-010). ２ The cells were cultured in an incubator. The next day, 1.32 × 10 ６ Expanded Pan T cells prepared at a concentration of cells / mL were seeded in 30 μL aliquots onto a flat-bottom 96-well plate during culture. hKM3900_tA2-32LH obtained in Example 1 and the anti-human PD-1 antibody nivolumab were used as test antibodies. The amino acid sequence of nivolumab was designed based on the amino acid sequences of the heavy and light chains of nivolumab described in WO 2014 / 055648. Nivolumab was obtained from the designed amino acid sequence according to the method described in Example 11 of WO 2021 / 241616. An anti-lysozyme antibody was prepared and used as an isotype control (referred to as "isotype" in Figure 1-1). For single agent conditions, hKM3900_tA2-32LH serially diluted in culture medium at approximately 3-fold common ratios from a maximum concentration of 50,000 ng / mL, or nivolumab serially diluted in culture medium at approximately 3-fold common ratios from a maximum concentration of 50,000 ng / mL, was added. For combination conditions, 20 μL of hKM3900_tA2-32LH serially diluted in culture medium at approximately 3-fold common ratios from a maximum concentration of 50,000 ng / mL containing nivolumab at a concentration of 50,000 ng / mL was added. The final concentration of nivolumab after addition was 10 μg / mL. After addition, the cells were incubated at 37°C, 5% CO ２The cells were cultured in an incubator. After 4 days, the amount of interferon-γ produced in the culture supernatant was measured using an AlphaLISA Interferon-γ Measurement Kit (Perkin-Elmer, AL217C) according to the manufacturer's recommended protocol. Figure 1-1 shows the amount of interferon-γ produced. The mean and standard deviation were calculated for each condition. hKM3900_tA2-32LH and nivolumab promoted interferon-γ production in a co-culture system of human CLDN4-expressing cancer cell lines and Expanded PanT cells. The combined use of hKM3900_tA2-32LH and nivolumab promoted interferon-γ production more strongly than hKM3900_tA2-32LH or nivolumab alone.

[0093] Example 3 In Vitro Combination Effect of Anti-CLDN4-Anti-CD137 Bispecific Antibody and Anti-PD-L1 Antibody Based on the sequence of the atezolizumab antibody, an anti-PD-L1 antibody described in WO 2012 / 155019 (SEQ ID NOs: 22 and 23), an atezolizumab analog (hereinafter referred to as "atezolizumab analog") was designed as an anti-PD-L1 antibody using a sequence of a human Igγ1 constant region with a mutation in the Fc region, and the antibody was obtained according to the method described in Example 4-2 of PCT / JP2022 / 18350. In a co-culture system using LCLC-OKT3scFv cells and Expanded panT cells obtained in Example 2, the in vitro combination effect of hKM3900_tA2-32LH and the atezolizumab analog was examined. Specifically, the T cell activity enhancing effect of hKM3900_tA2-32LH and atezolizumab analogs was evaluated using the amount of interferon-γ produced as an index in a co-culture system of LCLC-OKT3scFv cells and Expanded PanT cells. ５ The cells were prepared at a concentration of 1000 cells / mL, and 50 μL of each was seeded into a flat-bottom 96-well plate. ２ The cells were cultured in an incubator. The next day, 1.33 × 10 ６Expanded Pan T cells prepared at a concentration of 100 cells / mL were seeded in 30 μL aliquots onto a flat-bottom 96-well plate during culture. hKM3900_tA2-32LH obtained in Example 1 and an atezolizumab analog were used as test antibodies. An anti-lysozyme antibody was prepared and used as an isotype control (referred to as "isotype" in Figures 1-2). For single-agent conditions, hKM3900_tA2-32LH serially diluted at approximately 3-fold common ratios from a maximum concentration of 50,000 ng / mL was added to the culture medium, or an atezolizumab analog serially diluted at approximately 3-fold common ratios from a maximum concentration of 50,000 ng / mL was added to the culture medium. For the combined use conditions, 20 μL of hKM3900_tA2-32LH serially diluted at approximately 3-fold common ratio from the highest concentration of 50,000 ng / mL was added to a culture medium containing an atezolizumab analog at a concentration of 50,000 ng / mL. The final concentration of the atezolizumab analog after addition was 10 μg / mL. After addition, the cells were incubated at 37°C, 5% CO ２ The cells were cultured in an incubator. After 5 days, the amount of interferon-γ produced in the culture supernatant was measured using an AlphaLISA Interferon-γ Measurement Kit (Perkin-Elmer, AL217C) according to the manufacturer's recommended protocol. Figure 1-2 shows the amount of interferon-γ produced. The mean and standard deviation were calculated for each condition. hKM3900_tA2-32LH and atezolizumab analogs promoted interferon-γ production in a co-culture system of human CLDN4-expressing cancer cell lines and Expanded PanT cells. The interferon-γ production promotion effect of the combination of hKM3900_tA2-32LH and atezolizumab analogs was stronger than that of hKM3900_tA2-32LH or atezolizumab analogs alone.

[0094] Example 4: In vivo combined effect of anti-CLDN4-anti-CD137 bispecific antibody and anti-mouse PD-1 antibody The in vivo antitumor effect of anti-CLDN4-anti-CD137 bispecific antibody and anti-human PD-1 antibody was examined using B-h4-1BB mice (human CD137 knock-in mice) transplanted with human CLDN4-expressing B16-F10 cells.

[0095] [Example 4-1: Construction of human CLDN4-expressing B16-F10 cells] B16-F10 cells, a mouse melanoma cell line, were obtained from the American Type Culture Collection (ATCC, CRL-6475). They were cultured in Dulbecco's modified Eagle's medium (SIGMA, D6429) supplemented with a final concentration of 10% inactivated fetal bovine serum (FBS) (Cytiva, SH30084.03) (the prepared medium is hereinafter referred to as "Eagle's culture medium") at 37 ° C. and 5% CO ２ The cells were cultured under the conditions of CLDN4 (Myc-DDK-tagged)-Human claudin 4 (CLDN4) (ORIGENE, RC200490) was introduced into B16-F10 cells using jetPRIME (Polyplus-transfection, 114-15). A B16-F10 cell clone stably expressing human CLDN4 (hereinafter referred to as "human CLDN4-expressing B16-F10 cells") was obtained by selection in Eagle's culture medium supplemented with G418 (Nacalai Tesque, 09380-44) at a final concentration of 1 mg / mL.

[0096] [Example 4-2: Combined effect of anti-CLDN4-anti-CD137 bispecific antibody and anti-mouse PD-1 antibody in in vivo antitumor activity] 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 a 6-week-old mouse. ５ The tumor diameter was measured using a caliper (Mitutoyo, CD-15AXR) three days after cell inoculation. ３ The following formula was used to calculate the tumor volume (mm ３ ) ]=[Long axis of tumor (mm)] × [Shorter axis of tumor (mm)] ２ × 0.5

[0097] The cell-inoculated mice were divided into groups (n = 10) so that the tumor volume was equal in each group, and administration of the test antibody was initiated. The first day of administration was defined as day 0. The test antibody was hKM3900_tA2-32LH, an anti-CLDN4-anti-CD137 bispecific antibody obtained in Example 1, and the anti-mouse PD-1 antibody (Bio X Cell, BE0146) was used as the anti-PD-1 antibody. An anti-lysozyme antibody was used as the isotype control antibody for hKM3900_tA2-32LH, and a rat IgG2a isotype control antibody (Bio X Cell, BE0089) (referred to as "control" in Figure 2) was used as the isotype control antibody for the anti-mouse PD-1 antibody. Details of the test antibodies administered to the four groups tested, the dosage, administration schedule, etc. are shown below. (1) Group 1: Control group: Anti-lysozyme antibody was intraperitoneally administered at 0.3 mg / kg on days 0 and 7, and rat IgG2a isotype control antibody was intraperitoneally administered at 100 μg / head on days 0, 4, 7, and 11. (2) Group 2: hKM3900_tA2-32LH-administered group: hKM3900_tA2-32LH was intraperitoneally administered at 0.3 mg / kg on days 0 and 7, and rat IgG2a isotype control antibody was intraperitoneally administered at 100 μg / head on days 0, 4, 7, and 11. (3) Group 3: Anti-mouse PD-1 antibody administration group. Anti-lysozyme antibody was administered intraperitoneally at 0.3 mg / kg on days 0 and 7, and anti-mouse PD-1 antibody was administered intraperitoneally at 100 μg / head on days 0, 4, 7, and 11. (4) Group 4: hKM3900_tA2-32LH and anti-mouse PD-1 antibody co-administration group. hKM3900_tA2-32LH was administered intraperitoneally at 0.3 mg / kg on days 0 and 7, and anti-mouse PD-1 antibody was administered intraperitoneally at 100 μg / head on days 0, 4, 7, and 11. Tumor volume was assessed in each group on days 4, 7, 11, and 14. Tumor volumes on day 14 in groups 2 and 3 were compared with those in group 4 by unpaired Student's t-test (Figure 2).

[0098] As shown in Figure 2, the tumor volume in the group administered the combination of hKM3900_tA2-32LH and the anti-mouse PD-1 antibody was significantly smaller than that in the group administered hKM3900_tA2-32LH alone and the group administered the anti-mouse PD-1 antibody alone. This result suggests that in the treatment of human cancer, the combination of the anti-CLDN4-anti-CD137 bispecific antibody and the anti-PD-1 antibody may have a greater antitumor effect than either antibody administered alone.

[0099] The cancer treatment method of the present invention using an anti-CLDN4-anti-CD137 bispecific antibody in combination with a PD-1 signal inhibitor is expected to be useful in cancer treatment.

[0100] 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

**Claim 1** A pharmaceutical composition for treating cancer in a subject, comprising an anti-CLDN4-anti-CD137 bispecific antibody, wherein the bispecific antibody comprises a heavy chain variable region and a light chain variable region of an anti-CLDN4 antibody, and a heavy chain variable region and a light chain variable region of an anti-CD137 antibody, the heavy chain variable region of the anti-CLDN4 antibody comprising a CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 2, a CDR2 consisting of the amino acid sequence from amino acid number 50 to 66 of SEQ ID NO: 2, and a CDR3 consisting of the amino acid sequence from amino acid number 99 to 112 of SEQ ID NO: 2, the light chain variable region of the anti-CLDN4 antibody comprising a CDR1 consisting of the amino acid sequence from amino acid number 24 to 35 of SEQ ID NO: 4, a CDR2 consisting of the amino acid sequence from amino acid number 51 to 57 of SEQ ID NO: 4, and a CDR3 consisting of the amino acid sequence from amino acid number 90 to 98 of SEQ ID NO: 4, and is used in combination with a PD-1 signaling inhibitor. **Claim 2** The pharmaceutical composition according to claim 1, wherein the heavy chain variable region of the anti-CLDN4 antibody consists of the amino acid sequence from amino acid number 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 number 1 to 109 of SEQ ID NO:

4. **Claim 3** The pharmaceutical composition according to claim 1 or 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. **Claim 4** The pharmaceutical composition according to claim 3, wherein the Fc region of the anti-CLDN4 IgG antibody comprises either or both of the LALA mutations (L234A and L235A) or the P331G mutation (wherein the mutation positions are amino acid positions according to the EU index in the human Igγ1 constant region). **Claim 5** The pharmaceutical composition according to claim 1, wherein the heavy chain variable region of the anti-CD137 antibody comprises CDR1 consisting of the amino acid sequence from amino acid number 625 to 629 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequence from amino acid number 644 to 659 of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence from amino acid number 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 number 486 to 498 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequence from amino acid number 514 to 520 of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence from amino acid number 553 to 563 of SEQ ID NO:

2.

6. The pharmaceutical composition according to claim 1, wherein the heavy chain variable region of the anti-CD137 antibody consists of the amino acid sequence from amino acid number 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 number 464 to 573 of SEQ ID NO:

2.

7. The pharmaceutical composition according to claim 5 or 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 the light chain variable region of the 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 number 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 via a linker to the carboxy terminus of the heavy chain of the anti-CLDN4 IgG antibody.

10. A pharmaceutical composition for treating cancer in a subject, comprising an anti-CLDN4-anti-CD137 bispecific antibody, 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 from amino acid number 1 to 123 of SEQ ID NO: 2, a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence from amino acid number 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 from amino acid number 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 from amino acid number 595 to 712 of SEQ ID NO: 2, and the amino terminus of the anti-CD137 scFv is linked via a linker to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody, and is used in combination with a PD-1 signal inhibitor.

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

12. The pharmaceutical composition according to claim 10 or 11, wherein the linker is a GS linker.

13. A pharmaceutical composition for treating cancer in a subject, comprising 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, and is used in combination with a PD-1 signal inhibitor.

14. The pharmaceutical composition according to claim 13, wherein the anti-CLDN4-anti-CD137 bispecific antibody is post-translationally modified.

15. The pharmaceutical composition according to claim 14, which is used in combination with a PD-1 signal inhibitor simultaneously, continuously or sequentially.

16. The pharmaceutical composition according to claim 15, wherein the anti-CLDN4-anti-CD137 bispecific antibody and the PD-1 signal inhibitor are (i) included in the same pharmaceutical composition and administered simultaneously, or (ii) included in separate pharmaceutical compositions and used in combination simultaneously, continuously, or sequentially.

17. The pharmaceutical composition according to claim 16, wherein the cancer is selected from the group consisting of colorectal cancer, bladder cancer, and lung cancer.

18. The pharmaceutical composition according to any one of claims 1-2, 5-6, 10-11, and 13-17, wherein the PD-1 signal inhibitor is an antibody or an antigen-binding fragment thereof that binds to one or more proteins selected from the group consisting of PD-1, PD-L1, and PD-L2.

19. The pharmaceutical composition according to claim 18, wherein the PD-1 signal inhibitor is an anti-PD-1 antibody selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, spartalizumab, and semiprimab.

20. The pharmaceutical composition according to claim 18, wherein the PD-1 signal inhibitor is an anti-PD-L1 antibody selected from the group consisting of atezolizumab, durvalumab, and avelumab.

21. An anti-CLDN4-anti-CD137 bispecific antibody used for treating cancer in a subject, the 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 the bispecific antibody is used in combination with a PD-1 signal inhibitor.

22. Use of an anti-CLDN4-anti-CD137 bispecific antibody for the manufacture of a pharmaceutical composition used in combination with a PD-1 signal inhibitor for treating cancer in a subject, the 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.