Use of anti-CLDN4-anti-CD137 bispecific antibody in treatment of cancer by combination with PD-1 signaling inhibitor

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 signaling inhibitor in order to treat cancer in a subject, or a pharmaceutical composition containing the bispecific antibody, or providing a cancer treatment method comprising the step of administering an anti-CLDN4-anti-CD137 bispecific antibody in combination with a PD-1 signaling inhibitor to a subject. Under the co-culture condition of CLDN4 expression cancer cells and T cells, the combination of the anti-CLDN4-anti-CD137 bispecific antibody and the PD-1 signal inhibitor shows the promotion effect and the cell killing effect of interferon-gamma brought by the T cells and shows the remarkable anti-tumor effect in CLDN4 expression cancer cell transplantation mice compared with the respective independent administration. This result shows that a combination of an anti-CLDN4-anti-CD137 bispecific antibody and a PD-1 signal inhibitor is useful in the treatment of CLDN4 expressing cancers.

Description

Technical Field

[0001] The present invention relates to the use of an anti-CLDN4-anti-CD137 bispecific antibody in cancer treatment by combining it with a PD-1 signaling inhibitor. Background Art

[0002] Claudin-4 (CLDN4) is a four-transmembrane protein belonging to the tight junction protein family. It is expressed in epithelial cells and endothelial cells and plays an important role as the main molecule constituting tight junctions. CLDN4 has also been confirmed to be highly expressed in cancer tissues such as colorectal cancer, bladder cancer, and ovarian cancer, suggesting the possibility that anti-CLDN4 antibodies can be used for the treatment or diagnosis of cancer (Patent Document 1, Non-Patent Document 1). Furthermore, the anti-tumor effect of the combination of anti-CLDN4 antibodies and anti-epidermal growth factor receptor (EGFR) antibodies was shown in animal models (Non-Patent Document 2).

[0003] Cluster of Differentiation 137 (Cluster of Differentiation 137, CD137, alias 4-1BB) is a molecule belonging to the Tumor Necrosis Factor Receptor Superfamily (TNFRSF), which is reported to be expressed on the surface of immune cells such as T cells, B cells, natural killer (NK) cells, dendritic cells, eosinophils, mast cells, etc. In particular, it is known that CD137 on T cells binds to the CD137 ligand on antigen presenting cells and participates in the activation and survival of T cells as a co-stimulatory molecule (non-patent literature 3). Anti-CD137 agonist antibodies show anti-tumor effects mediated by immune cell activation in the tumor microenvironment in animal models (non-patent literature 4). Urelumab, an anti-CD137 agonist antibody, has shown therapeutic effects in clinical trials, but has also been reported to cause liver disorders (non-patent literature 5).

[0004] As a groundbreaking method that can obtain selective cell killing activity of cancer cells at low antibody concentrations, bispecific T-cell-recruiting antibodies (bispecific T-cell-recruitingantibodies) in various antibody forms have been reported. Bispecific T-cell-recruiting antibodies are bispecific antibodies containing antibodies against tumor-associated antigens (TAA) expressed on the surface of cancer cells and antibodies that bind to T cells, and the effects of these antibodies on T cell-mediated immunotherapy are being studied (Non-patent Document 6). As antibodies that bind to T cells, anti-CD3 antibodies are mostly used, and various bispecific T-cell-recruiting antibodies for TAA are currently being studied and developed.

[0005] Furthermore, bispecific T cell recruiting antibodies against CD137 and TAAs have been actively studied in recent years. Anti-GPC3-anti-CD137 bispecific antibodies, anti-HER2-anti-CD137 bispecific antibodies, anti-PD-L1-anti-CD137 bispecific antibodies, anti-FAP-anti-CD137 bispecific antibodies, etc. that recognize Glypican 3 (GPC3), human epidermal growth factor receptor type 2 (HER2), programmed cell death ligand 1 (PD-L1), and fibroblast activation protein (FAP) as TAAs are being studied (Patent Documents 2 and 3, Non-Patent Documents 7 to 9).

[0006] Programmed cell death protein 1 (PD-1; also known as PDCD1 or CD279) is a type I transmembrane protein of 50 to 55 kDa belonging to the immunoglobulin superfamily (non-patent literature 10). In T cells, PD-1 is induced to express as it is continuously activated, and the activation of T cells is inhibitorily regulated by binding to programmed cell death ligand 1 (PD-L1; also known as PDCD1LG1, B7-H1 or CD274) or programmed cell death ligand 2 (PD-L2; also known as PDCD1LG2, B7-DC or CD273) as a ligand (non-patent literature 11). In general, such a T cell activation regulation mechanism is called an immune checkpoint, which is known as one of the negative feedback mechanisms for avoiding excessive immune responses.

[0007] In the early stages of cancer, immune cells such as T cells eliminate cancer through anti-tumor immune responses based on immune surveillance mechanisms. On the other hand, cancer obtains immune escape mechanisms by directly or indirectly inhibiting immune cells in the cancer microenvironment. As direct activated T cell inhibition mechanisms, immune checkpoint mechanisms such as the PD-1 / PD-L1 or PD-L2 (hereinafter referred to as "PD-1 signal") pathway, CTLA-4 / CD80 or CD86 pathway are known. In the tumor microenvironment of cancer, PD-1 expression in T cells and PD-L1 expression in tumors are confirmed (non-patent document 12), and it is believed that cancer shows immune escape through the activation of the PD-1 signal. It is reported that the immune escape mechanism is relieved by inhibiting the PD-1 signal in a variety of mouse tumor models, resulting in anti-tumor activity (non-patent documents 13 to 15). Furthermore, as a PD-1 signal inhibitor, efforts are made to develop PD-1 signal inhibitors such as anti-PD-1 antibodies such as nivolumab and pembrolizumab, and significant results have been achieved in melanoma, lung cancer, lymphoma, etc. In addition, as PD-1 signal inhibitors, in addition to antibodies, nucleic acid drugs and low-molecular drugs are also being studied (Non-Patent Document 16).

[0008] In order to improve the effectiveness of treatment for cancer patients, efforts are being made to promote the combination therapy of multiple cancer immunotherapy drugs and the combination trials of cancer immunotherapy drugs and existing anticancer drugs (Non-patent Documents 17 and 18). For example, the combination trials of anti-PD-1 antibodies with other immune checkpoint inhibitory antibodies, anticancer drugs, molecular targeted drugs, radiotherapy, cancer vaccines, and oncolytic viruses are being implemented.

[0009] However, to date, no cancer treatment methods based on the combination of anti-CLDN4-anti-CD137 bispecific antibodies and PD-1 signaling inhibitors have been reported.

[0010] Prior art literature

[0011] Patent Literature

[0012] Patent Document 1: International Publication No. 2008 / 114733

[0013] Patent Document 2: International Publication No. 2015 / 156268

[0014] Patent Document 3: International Publication No. 2016 / 177802

[0015] Non-patent literature

[0016] Non-patent document 1: Cancer Science, 2009: 100(9): p.1623-1630

[0017] Non-patent literature 2: Oncotarget, 2018: 9(100): p.37367-37378

[0018] Non-patent literature 3: Cancer Science, 2020: 111(5): p.1461-1467

[0019] Non-patent document 4: Cancer Immunology Immunotherapy, 2012: 61(5): p.1721-1733

[0020] Non-patent literature 5: Clinical Cancer Research, 2017: 23(8): p.1929-1936

[0021] Non-patent literature 6: MAbs, 2017: 9(2): p.182-212

[0022] Non-patent literature 7: Clinical Cancer Research, 2019: 25(19): p.5878-5889

[0023] Non-patent literature 8: Clinical Cancer Research, 2020: 26(15): p.4154-4167

[0024] Non-patent literature 9: Journal for Immunotherapy of Cancer, 2020: 8(2): e000238

[0025] Non-patent literature 10: International Immunology, 1996: Vol. 8: p. 765-772

[0026] Non-patent document 11: Annual Review of Immunology, 2008: Vol. 26: p. 677-704

[0027] Non-patent literature 12: Nature Medicine, 2002: 8: p.793-800

[0028] Non-patent literature 13: Scientific Reports, 2021: 11: p.21087-21099

[0029] Non-patent literature 14: Nature Communications, 2017: 8: p.14572-14582

[0030] Non-patent literature 15: Journal for Immunotherapy of Cancer, 2019: 7: 37: p. 1-16

[0031] Non-patent literature 16: Molecules, 2019: 24: p.2071-2100

[0032] Non-patent literature 17: Cancer Discovery, 2021: 11: p.1368-1397

[0033] Non-patent literature 18: Molecular Medicine Reports, 2021: 23: p.362-377 Summary of the invention

[0034] Problems to be solved by the invention

[0035] An object of the present invention is to provide an anti-CLDN4-anti-CD137 bispecific antibody or a pharmaceutical composition containing the bispecific antibody for use in combination with a PD-1 signaling inhibitor to treat cancer in a subject, or a cancer treatment method comprising the step of administering the anti-CLDN4-anti-CD137 bispecific antibody and a PD-1 signaling inhibitor to a subject.

[0036] Methods used to solve problems

[0037] In order to develop an antibody or pharmaceutical composition for treating cancer expressing CLDN4, the present inventors prepared an anti-CLDN4-anti-CD137 bispecific antibody based on the sequences of the well-known anti-CLDN4 antibody KM3900 and anti-CD137 antibody (Example 1). The combination of the obtained anti-CLDN4-anti-CD137 bispecific antibody and anti-PD-1 antibody or anti-PD-L1 antibody promoted the interferon γ production of T cells in vitro compared with the anti-CLDN4-anti-CD137 bispecific antibody, anti-PD-1 antibody or anti-PD-L1 antibody alone (Examples 2 and 3). Furthermore, in mice carrying mouse cancer cells expressing human CLDN4, the combination of anti-CLDN4-anti-CD137 bispecific antibody and anti-PD-1 antibody showed a more significant anti-tumor effect than when the anti-CLDN4-anti-CD137 bispecific antibody or anti-PD-1 antibody was administered alone (Example 4). This result suggests that the combination of an anti-CLDN4-anti-CD137 bispecific antibody and a PD-1 signaling inhibitor is useful in the treatment of cancers expressing CLDN4.

[0038] That is, the present invention relates to the following [1] to

[84] , but is not limited thereto.

[0039] [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 the light chain variable region of an anti-CLDN4 antibody and the heavy chain variable region and the light chain variable region of an anti-CD137 antibody, wherein the pharmaceutical composition is used in combination with a PD-1 signaling inhibitor.

[0040] [2] The pharmaceutical composition according to [1], wherein the heavy chain variable region of the anti-CLDN4 antibody comprises a CDR1 consisting of an amino acid sequence of amino acids 31 to 35 of SEQ ID NO: 2, a CDR2 consisting of an amino acid sequence of amino acids 50 to 66 of SEQ ID NO: 2, and a CDR3 consisting of an 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 a CDR1 consisting of an amino acid sequence of amino acids 24 to 35 of SEQ ID NO: 4, a CDR2 consisting of an amino acid sequence of amino acids 51 to 57 of SEQ ID NO: 4, and a CDR3 consisting of an amino acid sequence of amino acids 90 to 98 of SEQ ID NO: 4.

[0041] [3] The pharmaceutical composition according to [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.

[0042] [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) composed of a heavy chain containing the heavy chain variable region of the anti-CLDN4 antibody and a light chain containing the light chain variable region of the anti-CLDN4 antibody.

[0043] [5] The pharmaceutical composition according to [4], wherein the Fc region of the anti-CLDN4 IgG antibody contains either or both of the LALA mutation (L234A and L235A) or the P331G mutation (herein, the mutation position is an amino acid position in the human Igγ1 constant region according to the EU index).

[0044] [6] The pharmaceutical composition according to any one of [1] to [5], wherein the heavy chain variable region of the anti-CD137 antibody comprises a CDR1 consisting of an amino acid sequence of amino acids 625 to 629 of SEQ ID NO: 2, a CDR2 consisting of an amino acid sequence of amino acids 644 to 659 of SEQ ID NO: 2, and a CDR3 consisting of an 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 a CDR1 consisting of an amino acid sequence of amino acids 486 to 498 of SEQ ID NO: 2, a CDR2 consisting of an amino acid sequence of amino acids 514 to 520 of SEQ ID NO: 2, and a CDR3 consisting of an amino acid sequence of amino acids 553 to 563 of SEQ ID NO: 2.

[0045] [7] The pharmaceutical composition according to any one of [1] to [6], wherein the heavy chain variable region of the anti-CD137 antibody is composed of the amino acid sequence of amino acid numbers 595 to 712 of SEQ ID NO: 2, and the light chain variable region of the anti-CD137 antibody is composed of the amino acid sequence of amino acid numbers 464 to 573 of SEQ ID NO: 2.

[0046] [8] The pharmaceutical composition according to [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.

[0047] [9] The pharmaceutical composition according to [8], wherein the anti-CD137 scFv consists of an amino acid sequence of amino acid numbers 464 to 712 of SEQ ID NO: 2.

[0048]

[10] The pharmaceutical composition according to [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 heavy chain carboxyl terminus of the anti-CLDN4 IgG antibody via a linker.

[0049]

[11] A pharmaceutical composition comprising an anti-CLDN4-anti-CD137 bispecific antibody for treating 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 an 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 an 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 an 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 an 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 carboxyl terminus of the heavy chain of the anti-CLDN4 antibody via a linker, and the pharmaceutical composition is used in combination with a PD-1 signaling inhibitor.

[0050]

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

[0051]

[13] The pharmaceutical composition according to any one of

[10] to

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

[0052]

[14] A pharmaceutical composition comprising an anti-CLDN4-anti-CD137 bispecific antibody for treating cancer in a subject, wherein the bispecific antibody comprises a polypeptide comprising a heavy chain of an anti-CLDN4 antibody and an anti-CD137 scFv consisting of the amino acid sequence of SEQ ID NO: 2, and a light chain of an anti-CLDN4 antibody consisting of the amino acid sequence of SEQ ID NO: 4, wherein the pharmaceutical composition is used in combination with a PD-1 signaling inhibitor.

[0053]

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

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

[0054]

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

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

[0055]

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

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

[0056]

[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.

[0057]

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

[18] , wherein the PD-1 signaling 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.

[0058]

[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 cemiplizumab.

[0059]

[21] The pharmaceutical composition according to any one 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.

[0060]

[22] A bispecific antibody, which is an anti-CLDN4-anti-CD137 bispecific antibody for treating cancer in a subject, wherein the bispecific antibody contains 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 the bispecific antibody is used in combination with a PD-1 signaling inhibitor.

[0061]

[23] The bispecific antibody according to

[22] , wherein the heavy chain variable region of the anti-CLDN4 antibody comprises a CDR1 consisting of an amino acid sequence of amino acids 31 to 35 of SEQ ID NO: 2, a CDR2 consisting of an amino acid sequence of amino acids 50 to 66 of SEQ ID NO: 2, and a CDR3 consisting of an 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 a CDR1 consisting of an amino acid sequence of amino acids 24 to 35 of SEQ ID NO: 4, a CDR2 consisting of an amino acid sequence of amino acids 51 to 57 of SEQ ID NO: 4, and a CDR3 consisting of an amino acid sequence of amino acids 90 to 98 of SEQ ID NO: 4.

[0062]

[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 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.

[0063]

[25] The bispecific antibody according to any one of

[22] to

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

[0064]

[26] The bispecific antibody according to

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

[0065]

[27] The bispecific antibody according to any one of

[22] to

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

[0066]

[28] The bispecific antibody according to any one of

[22] to

[27] , wherein the heavy chain variable region of the anti-CD137 antibody is composed 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 is composed of the amino acid sequence of amino acids 464 to 573 of SEQ ID NO: 2.

[0067]

[29] The bispecific antibody according to

[27] or

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

[0068]

[30] The bispecific antibody according to

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

[0069]

[31] The bispecific antibody according to

[29] or

[30] , comprising an anti-CLDN4 IgG antibody and an anti-CD137scFv, wherein the amino terminus of the anti-CD137scFv is linked to the heavy chain carboxyl terminus of the anti-CLDN4 IgG antibody via a linker.

[0070]

[32] A bispecific antibody, which is an anti-CLDN4-anti-CD137 bispecific antibody for treating 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 an 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 an 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 an 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 an amino acid sequence of amino acids 595 to 712 of SEQ ID NO: 2, the amino terminus of the anti-CD137 scFv being linked to the heavy chain carboxyl terminus of the anti-CLDN4 antibody via a linker, and the bispecific antibody being used in combination with a PD-1 signaling inhibitor.

[0071]

[33] A bispecific antibody, which is an anti-CLDN4-anti-CD137 bispecific antibody for treating cancer in a subject, wherein the bispecific antibody comprises a heavy chain of an anti-CLDN4 antibody consisting of an amino acid sequence of amino acids 1 to 453 of SEQ ID NO: 2, a light chain of an anti-CLDN4 antibody consisting of an amino acid sequence of amino acids 1 to 215 of SEQ ID NO: 4, and an anti-CD137 scFv consisting of an amino acid sequence of amino acids 464 to 712 of SEQ ID NO: 2, the amino terminus of the anti-CD137 scFv being linked to the carboxyl terminus of the heavy chain of the anti-CLDN4 antibody via a linker, and the bispecific antibody being used in combination with a PD-1 signaling inhibitor.

[0072]

[34] The bispecific antibody according to any one of

[31] to

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

[0073]

[35] A bispecific antibody, which is an anti-CLDN4-anti-CD137 bispecific antibody for treating cancer in a subject, wherein the bispecific antibody comprises a polypeptide containing a heavy chain of an anti-CLDN4 antibody and an anti-CD137 scFv composed of the amino acid sequence of SEQ ID NO: 2, and a light chain of an anti-CLDN4 antibody composed of the amino acid sequence of SEQ ID NO: 4, wherein the bispecific antibody is used in combination with a PD-1 signaling inhibitor.

[0074]

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

[22] to

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

[0075]

[37] The bispecific antibody according to any one of

[22] to

[36] , which is used in combination with a PD-1 signaling inhibitor simultaneously, continuously or sequentially.

[0076]

[38] The bispecific antibody according to any one of

[22] to

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

[0077]

[39] The bispecific antibody according to any one of

[22] to

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

[0078]

[40] The bispecific antibody according to any one 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.

[0079]

[41] The bispecific antibody according to any one 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 cemiplizumab.

[0080]

[42] The bispecific antibody according to any one of

[22] to

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

[0081]

[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 signaling inhibitor, wherein the bispecific antibody contains 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.

[0082]

[44] The method of treatment according to

[43] , wherein the heavy chain variable region of the anti-CLDN4 antibody comprises a CDR1 consisting of an amino acid sequence of amino acids 31 to 35 of SEQ ID NO: 2, a CDR2 consisting of an amino acid sequence of amino acids 50 to 66 of SEQ ID NO: 2, and a CDR3 consisting of an 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 a CDR1 consisting of an amino acid sequence of amino acids 24 to 35 of SEQ ID NO: 4, a CDR2 consisting of an amino acid sequence of amino acids 51 to 57 of SEQ ID NO: 4, and a CDR3 consisting of an amino acid sequence of amino acids 90 to 98 of SEQ ID NO: 4.

[0083]

[45] The method of

[43] or

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

[0084]

[46] The treatment 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) composed of a heavy chain containing the heavy chain variable region of the anti-CLDN4 antibody and a light chain containing the light chain variable region of the anti-CLDN4 antibody.

[0085]

[47] The method of

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

[0086]

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

[43] to

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

[0087]

[49] A method according to any one of

[43] to

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

[0088]

[50] The treatment 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.

[0089]

[51] The treatment method according to

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

[0090]

[52] The method according to

[50] or

[51] , 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 carboxyl terminus of the anti-CLDN4 IgG antibody via a linker.

[0091]

[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 signaling inhibitor, wherein the bispecific antibody comprises a heavy chain of an anti-CLDN4 antibody comprising a heavy chain variable region consisting of an 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 an 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 an 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 an 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 carboxyl terminus of the heavy chain of the anti-CLDN4 antibody via a linker.

[0092]

[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 a heavy chain of an anti-CLDN4 antibody consisting of an amino acid sequence of amino acids 1 to 453 of SEQ ID NO: 2, a light chain of an anti-CLDN4 antibody consisting of an amino acid sequence of amino acids 1 to 215 of SEQ ID NO: 4, and an anti-CD137 scFv consisting of an amino acid sequence of amino acids 464 to 712 of SEQ ID NO: 2, and the amino terminus of the anti-CD137 scFv is linked to the carboxyl terminus of the heavy chain of the anti-CLDN4 antibody via a linker.

[0093]

[55] The treatment method according to any one of

[52] to

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

[0094]

[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 signaling inhibitor, wherein the bispecific antibody comprises a polypeptide comprising the heavy chain of an anti-CLDN4 antibody and an anti-CD137 scFv consisting of the amino acid sequence of SEQ ID NO: 2, and a light chain of an anti-CLDN4 antibody consisting of the amino acid sequence of SEQ ID NO: 4.

[0095]

[57] The treatment method according to any one of

[43] to

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

[0096]

[58] The treatment method according to any one of

[43] to

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

[0097]

[59] The treatment method according to any one of

[43] to

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

[0098]

[60] The treatment 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.

[0099]

[61] The treatment method according to any one of

[43] to

[60] , wherein the PD-1 signaling 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.

[0100]

[62] The treatment 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 cemiplizumab.

[0101]

[63] The treatment method according to any one of

[43] to

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

[0102]

[64] Use of an anti-CLDN4-anti-CD137 bispecific antibody in the manufacture of a pharmaceutical composition for use in combination with a PD-1 signaling inhibitor for treating cancer in a subject, wherein the bispecific antibody comprises the heavy chain variable region and the light chain variable region of an anti-CLDN4 antibody and the heavy chain variable region and the light chain variable region of an anti-CD137 antibody.

[0103]

[65] The use according to

[64] , wherein the heavy chain variable region of the anti-CLDN4 antibody contains a CDR1 composed of an amino acid sequence of amino acids 31 to 35 of SEQ ID NO: 2, a CDR2 composed of an amino acid sequence of amino acids 50 to 66 of SEQ ID NO: 2, and a CDR3 composed of an 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 contains a CDR1 composed of an amino acid sequence of amino acids 24 to 35 of SEQ ID NO: 4, a CDR2 composed of an amino acid sequence of amino acids 51 to 57 of SEQ ID NO: 4, and a CDR3 composed of an amino acid sequence of amino acids 90 to 98 of SEQ ID NO: 4.

[0104]

[66] The use according to

[64] or

[65] , wherein the heavy chain variable region of the anti-CLDN4 antibody is composed 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 is composed of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4.

[0105]

[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) composed of a heavy chain containing the heavy chain variable region of the anti-CLDN4 antibody and a light chain containing the light chain variable region of the anti-CLDN4 antibody.

[0106]

[68] The use according to

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

[0107]

[69] The use according to any one of

[64] to

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

[0108]

[70] The use according to any one of

[64] to

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

[0109]

[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 the light chain variable region of the anti-CD137 antibody.

[0110]

[72] The use according to

[71] , wherein the anti-CD137 scFv consists of an amino acid sequence of amino acid numbers 464 to 712 of sequence number 2.

[0111]

[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 connected to the heavy chain carboxyl terminus of the anti-CLDN4 IgG antibody via a linker.

[0112]

[74] Use of an anti-CLDN4-anti-CD137 bispecific antibody in the manufacture of a pharmaceutical composition for use in combination with a PD-1 signaling inhibitor for treating 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 an 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 an 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 an 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 an amino acid sequence of amino acids 595 to 712 of SEQ ID NO: 2, the amino terminus of the anti-CD137 scFv being linked to the carboxyl terminus of the heavy chain of the anti-CLDN4 antibody via a linker.

[0113]

[75] Use of an anti-CLDN4-anti-CD137 bispecific antibody in the manufacture of a pharmaceutical composition for use in combination with a PD-1 signal inhibitor for treating cancer in a subject, wherein the bispecific antibody comprises a heavy chain of an anti-CLDN4 antibody consisting of an amino acid sequence of amino acids 1 to 453 of SEQ ID NO: 2, a light chain of an anti-CLDN4 antibody consisting of an amino acid sequence of amino acids 1 to 215 of SEQ ID NO: 4, and an anti-CD137 scFv consisting of an amino acid sequence of amino acids 464 to 712 of SEQ ID NO: 2, the amino terminus of the anti-CD137 scFv being linked to the carboxyl terminus of the heavy chain of the anti-CLDN4 antibody via a linker.

[0114]

[76] The use according to any one of

[73] to

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

[0115]

[77] Use of an anti-CLDN4-anti-CD137 bispecific antibody in the manufacture of a pharmaceutical composition for use in combination with a PD-1 signaling inhibitor for treating cancer in a subject, wherein the bispecific antibody comprises a polypeptide comprising the heavy chain of an anti-CLDN4 antibody and an anti-CD137 scFv consisting of the amino acid sequence of SEQ ID NO: 2, and a light chain of an anti-CLDN4 antibody consisting of the amino acid sequence of SEQ ID NO: 4.

[0116]

[78] The use according to any one of

[64] to

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

[0117]

[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, continuously or sequentially.

[0118]

[80] The use according to any one of

[64] to

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

[0119]

[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.

[0120]

[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.

[0121]

[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 cemiplizumab.

[0122]

[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.

[0123] Effects of the Invention

[0124] The anti-CLDN4-anti-CD137 bispecific antibody of the present invention binds to both CLDN4, which is highly expressed in cancer, and CD137, which is a T cell surface molecule, and enhances the killing effect on cancer cells by activating immune cells around cancer cells. The combination of the anti-CLDN4-anti-CD137 bispecific antibody of the present invention and a PD-1 signal inhibitor brings a more significant anti-tumor effect compared to the single administration of the anti-CLDN4-anti-CD137 bispecific antibody or the PD-1 signal inhibitor. Therefore, the present invention provides the use of the anti-CLDN4-anti-CD137 bispecific antibody in cancer treatment by combining it with a PD-1 signal inhibitor. BRIEF DESCRIPTION OF THE DRAWINGS

[0125] Figure 1-1The interferon-γ production caused by the addition of the test antibody in the co-culture system of the human large cell lung cancer cell line LCLC-OKT3scFv cells and the expanded panT cells is shown. The vertical axis of the figure represents the interferon-γ production 4 days after the addition of the antibody, and the horizontal axis represents the antibody concentration. The symbol represents the average value of the interferon-γ production at each concentration of each antibody. The error bar represents the standard deviation.

[0126] Figure 1-2 The interferon-γ production caused by the addition of the test antibody in the co-culture system of human large cell lung cancer cell line LCLC-OKT3scFv cells and amplified panT cells is shown. The vertical axis of the figure represents the interferon-γ production 5 days after the addition of the antibody, and the horizontal axis represents the antibody concentration. The symbol represents the average value of the interferon-γ production at each concentration of the antibody. The error bar represents the standard deviation.

[0127] Figure 2 The figure shows the proliferation inhibitory effect of B16-F10 cells expressing human CLDN4 carried by B-h4-1BB mice. Figure 2 The mean values ​​of tumor volumes at different days after the start of antibody administration are shown (n=10). Error bars indicate the standard error of tumor volume. The vertical axis of the graph indicates tumor volume, and the horizontal axis indicates the number of days from the first administration of the antibody. The significance probability P value was calculated by comparing the tumor volume of the combination group with the tumor volume of the group receiving the test antibody alone using an unpaired Student's t test. It means the P value is less than the significance level of 0.01. DETAILED DESCRIPTION

[0128] The present invention is described in detail below.

[0129] Unless otherwise specifically defined below, terms in this specification are used according to the meanings commonly used by those skilled in the art in the relevant technical field.

[0130] Antibodies (or immunoglobulins) are glycoproteins with a four-chain structure that is a left-right symmetrical Y-shaped structure consisting of two heavy chains with a single sequence and two light chains with a single sequence as the basic structure. There are five types of antibodies: IgG, IgM, IgA, IgD, and IgE. The basic structure of antibody molecules is common to each type. 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 are bound by disulfide bonds and non-covalent bonds to form an antibody molecule with a molecular weight of 150,000 to 190,000 and a Y-shaped four-chain structure. The heavy chain is usually composed of a polypeptide chain containing about 440 amino acids. It has a characteristic structure in each type and is called Igγ, Igμ, Igα, Igδ, and Igε corresponding to IgG, IgM, IgA, IgD, and IgE, respectively. In addition, IgG has subclasses of IgG1, IgG2, IgG3, and IgG4, and the corresponding heavy chains are called Igγ1, Igγ2, Igγ3, and Igγ4. Light chains are usually composed of a polypeptide chain containing about 220 amino acids, and there are two known types, λ type and κ type, called Igλ and Igκ, respectively. The above two types of light chains can be paired with any type of heavy chain.

[0131] Regarding the intrachain disulfide bonds of antibody molecules, there are 4 in the heavy chain (5 in Igμ and Igε) and 2 in the light chain, and 1 ring is formed for every 100 to 110 amino acid residues. These three-dimensional structures are similar between each ring and are called structural units or domains. The domain located at the amino terminus (also referred to as "N terminus" in this specification) of both the heavy chain and the light chain is called a variable region. It is known that even antibodies produced by the same class (or subclass) of the same animal have diverse amino acid sequences and participate in the binding specificity of antibodies to antigens. The amino acid sequence of the domain on the C-terminal side downstream of the variable region is roughly constant in each class or subclass, and is called a constant region. In the heavy chain, from the N-terminus to the carboxyl terminus (also referred to as "C terminus" in this specification), there is a heavy chain variable region (VH) and a heavy chain constant region (CH). In CH, it is further divided into three domains, namely, the CH1 domain, the CH2 domain, and the CH3 domain, from the N-terminal side. The light chain has a light chain variable region (VL) and a light chain constant region (CL) from the N-terminus to the C-terminus.

[0132] The amino acid sequences of the three complementary determining regions (CDRs) present in VH and VL vary greatly, contributing to the variability of the variable region. CDR is a region consisting of about 5 to 10 amino acid residues that exist in the order of CDR1, CDR2, and CDR3 at the N-terminus of the heavy chain and light chain, respectively, forming an antigen binding site. On the other hand, the part other than the CDR of the variable region is called the framework region (FR), which is composed of FR1 to 4 and has relatively few changes in the amino acid sequence.

[0133] When the antibody is treated with the proteolytic enzyme papain, three antibody fragments are obtained. The two fragments on the N-terminal side are called Fab (Fragment, antigen binding) region. In this specification, "Fab region" refers to the region composed of the VH and CH1 domains of the heavy chain and the light chain (VL and CL), and the antigen binding site of the front end portion constituted by the Fab region binds to the antigen. In this specification, "heavy chain fragment" refers to a fragment composed of the VH and CH1 domains of the heavy chain constituting the Fab region. In addition, the fragment on the C-terminal side is called "Fc (Crystallizable fragment, Fragment, crystallizable) region".

[0134] In this specification, "antigen" is used in the commonly used sense, and is particularly used as a term to represent a molecule or a part of a molecule that an antigen-binding protein such as an antibody or antigen-binding fragment can specifically bind to. Antigens can be molecules such as proteins and nucleic acids. Sometimes, an antigen has one or more epitopes that can interact with different antibodies, etc.

[0135] In the present specification, "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 the IgG antibody contain the same VH and VL sequences.

[0136] In this specification, "antigen-binding fragment" refers to a molecule containing at least one polypeptide chain with antigen-binding activity from an antibody. As representative antigen-binding fragments, single-chain variable region fragments (scFv), Fab fragments, Fab' fragments, and F(ab')2 fragments can be cited. scFv is a monovalent antigen-binding fragment consisting of VH and VL connected by a linker. Fab fragments are monovalent antigen-binding fragments consisting of fragments of VH and CH1 domains containing light and heavy chains. Fab' fragments are monovalent antigen-binding fragments consisting of fragments of VH, CH1 domains, and a portion of the hinge region containing a cysteine ​​residue constituting an SS bond between heavy chains. F(ab')2 fragments are divalent molecules formed by connecting Fab' fragments with disulfide bonds. Monovalent refers to containing one antigen-binding site, and divalent refers to containing two antigen-binding sites.

[0137] In the present specification, a "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 to CLDN4 and binding activity to CD137.

[0138] In this specification, "antibody" is used as a term including full-length antibodies, antigen-binding fragments, and bispecific antibodies of all structures unless otherwise specified in the context.

[0139] In this specification, "human antibody" means an antibody having a human immunoglobulin amino acid sequence. In this specification, "humanized antibody" means an antibody in which a part, most or all of the amino acid residues other than CDR are replaced by amino acid residues from a human immunoglobulin molecule. The method of humanization is not particularly limited, for example, humanized antibodies can be prepared with reference to U.S. Patent No. 5,225,539, U.S. Patent No. 6,180,370, etc.

[0140] The amino acid residue numbers of the antibody used in the present specification can be specified by using the Kabat numbering or the EU index (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., 1991: NIH Publication: No. 91-3242) and following these numbering systems.

[0141] In this specification, "connection", "connector" or "connected" refers to the direct connection of multiple components (such as IgG antibodies and scFv) or the connection via an intermediary (such as a peptide linker). In this specification, "peptide linker" refers to any one or more amino acid sequences that can be introduced by genetic engineering methods for connecting multiple components. The length of the peptide linker used in the present invention is not particularly limited, and those skilled in the art can select it appropriately according to the purpose.

[0142] In this specification, "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 suffering from cancer.

[0143] As used herein, "treatment" refers to certain therapeutic interventions, treatments or administration of active ingredients to a subject in order to restore, alleviate, improve, inhibit or delay the progression, onset, aggravation or recurrence of disease symptoms, disease conditions or biochemical signs associated with the disease.

[0144] In this specification, "active ingredient" refers to a substance that exhibits a certain physiological activity in a substance contained in a pharmaceutical composition, medicine, etc. for preventing or treating a disease. 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.

[0145] In the present specification, "pharmaceutical composition" refers to a drug that contains an active ingredient and a pharmaceutically acceptable excipient (including, for example, a pharmaceutical excipient, a pharmaceutical carrier, etc., but not limited thereto) and is prescribed for the purpose of treating a subject.

[0146] In this specification, "combination", "combination" or "combined use" refers to the simultaneous, continuous or sequential administration of two or more active ingredients to the same subject for the purpose of preventing or treating a disease. The two or more active ingredients may be contained in the same pharmaceutical composition or in different pharmaceutical compositions. In this specification, "simultaneously" means that two or more active ingredients are administered in parallel during the administration of one, "continuously" means that after the administration of one active ingredient is completed, the administration of another active ingredient is carried out without interruption in time, and "successively" means that two or more active ingredients are administered in sequence according to the administration schedule.

[0147] In this specification, the "effective amount" of a drug refers to the amount of the drug required to produce physiological changes in cells or tissues to which the drug is administered.

[0148] In this specification, "PD-1 signal inhibitor" refers to an agent that can relieve the inhibition of immune cell activation caused by PD-1. PD-1 signal inhibitors can inhibit the immune checkpoint function of PD-1 by binding to PD-1 or PD-L1 or PD-L2 as its ligand and inhibiting the immunosuppressive signal. As a PD-1 signal inhibitor, it can be any substance as long as it has the effect of blocking the PD-1 signal, for example, it can be an antibody, a low molecular compound, a nucleic acid (which can include DNA or RNA, or a natural or artificial nucleic acid), a fusion protein, a peptide, etc. For example, an anti-PD-1 antibody or an anti-PD-L1 antibody or an anti-PD-L2 antibody can inhibit the PD-1 signal by inhibiting the binding of PD-1 to PD-L1 or PD-L2 (Expert Opinion on Therapeutic Patents, 2016: Vol. 26: p. 555-564).

[0149] The present invention relates to the following (1) to (4):

[0150] (1) A pharmaceutical composition containing an anti-CLDN4-anti-CD137 bispecific antibody for use in combination with a PD-1 signaling inhibitor (also referred to herein as “the pharmaceutical composition of the present invention”);

[0151] (2) an anti-CLDN4-anti-CD137 bispecific antibody used in combination with a PD-1 signaling inhibitor for treating cancer in a subject;

[0152] (3) a cancer treatment method comprising the step of administering an anti-CLDN4-anti-CD137 bispecific antibody and a PD-1 signaling inhibitor to a subject (also referred to herein as “the treatment method of the present invention”); or

[0153] (4) Use of an anti-CLDN4-anti-CD137 bispecific antibody for the manufacture of a pharmaceutical composition for use in combination with a PD-1 signaling inhibitor for treating cancer in a subject.

[0154] <Anti-CLDN4-anti-CD137 bispecific antibody of the present invention>

[0155] 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") contains 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.

[0156] In this specification, "anti-CLDN4 antibody" is an antibody that can bind to human CLDN4, and "anti-CD137 antibody" is an antibody that can bind to human CD137. Whether it binds to human CLDN4 or human CD137 can be confirmed by using a known binding activity measurement method. As a method for measuring binding activity, methods such as enzyme-linked immunosorbent assay (ELISA) method and flow cytometry method can be cited. ELISA method or flow cytometry method can be implemented using methods commonly used by those skilled in the art.

[0157] 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 linker in which the Fab region of an anti-CLDN4 antibody is linked to the Fab region of an anti-CD137 antibody, a linker 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 linker of an anti-CLDN4 IgG antibody and an antigen-binding fragment of an anti-CD137 antibody, a linker of an antigen-binding fragment of an anti-CLDN4 antibody and an anti-CD137 IgG antibody, or a linker of an antigen-binding fragment of an anti-CLDN4 antibody and an antigen-binding fragment of an anti-CD137 antibody.

[0158] 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 a CDR1 consisting of an amino acid sequence of amino acids 31 to 35 of SEQ ID NO: 2, a CDR2 consisting of an amino acid sequence of amino acids 50 to 66 of SEQ ID NO: 2, and a CDR3 consisting of an amino acid sequence of amino acids 99 to 112 of SEQ ID NO: 2, and a light chain variable region of an anti-CLDN4 antibody comprising a CDR1 consisting of an amino acid sequence of amino acids 24 to 35 of SEQ ID NO: 4, a CDR2 consisting of an amino acid sequence of amino acids 51 to 57 of SEQ ID NO: 4, and a CDR3 consisting of an amino acid sequence of amino acids 90 to 98 of SEQ ID NO: 4.

[0159] In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises the heavy chain variable region of the anti-CLDN4 antibody consisting 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 consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4.

[0160] The anti-CLDN4 antibody contained in the anti-CLDN4-anti-CD137 bispecific antibody of the present invention may be an IgG antibody. As the heavy chain constant region contained in the anti-CLDN4 antibody, any one of Igγ, Igμ, Igα, Igδ or Igε may be selected. As Igγ, for example, it may be selected from Igγ1, Igγ2, Igγ3 or Igγ4. As the light chain constant region contained in the anti-CLDN4 antibody contained in the anti-CLDN4-anti-CD137 bispecific antibody of the present invention, any one of Igλ or Igκ may be selected. In one embodiment, the heavy chain and light chain 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 contains a full-length anti-CLDN4 antibody. In one embodiment, the anti-CLDN4 antibody contained in the anti-CLDN4-anti-CD137 bispecific antibody of the present invention is an IgG antibody (anti-CLDN4 IgG antibody) containing the heavy chain variable region and light chain variable region of the anti-CLDN4 antibody.

[0161] When the anti-CLDN4-anti-CD137 bispecific antibody of the present invention contains an Fc region, the Fc region in the bispecific antibody may contain a mutation that reduces antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). L234A refers to a substitution of leucine at amino acid position 234 of the human Igγ1 constant region with alanine. L235A refers to a substitution of leucine at amino acid position 235 of the human Igγ1 constant region with alanine. The amino acid mutations of 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 the antibody (Mol. Immunol., 1992: Vol. 29: p. 633-639, J. Immunol., 2000: Vol. 164 (8): p. 4178-4184). P331G or P331S refers to substitution of proline at amino acid position 331 of 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): p. 4178-4184).

[0162] In one embodiment, the anti-CLDN4 IgG antibody contained in the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises an Fc region comprising amino acid mutations (LALA mutations) of L234A and L235A. 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 a 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 a LALA mutation and a P331G mutation.

[0163] It should be noted that in this specification, the description of amino acid mutations such as LALA mutation, P331G or P331S mutation is 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 at amino acid position 234 in the human Igγ1 constant region according to the EU index with alanine.

[0164] In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention is an IgG antibody composed of the heavy chain of an anti-CLDN4 antibody consisting of the amino acid sequence of amino acids 1 to 453 of SEQ ID NO: 2 and the light chain of an anti-CLDN4 antibody consisting of the amino acid sequence of amino acids 1 to 215 of SEQ ID NO: 4.

[0165] 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 contains a CDR1 composed of an amino acid sequence of amino acid numbers 625 to 629 of SEQ ID NO: 2, a CDR2 composed of an amino acid sequence of amino acid numbers 644 to 659 of SEQ ID NO: 2, and a CDR3 composed of an amino acid sequence of amino acid numbers 692 to 701 of SEQ ID NO: 2, and the light chain variable region of the anti-CD137 antibody contains a CDR1 composed of an amino acid sequence of amino acid numbers 486 to 498 of SEQ ID NO: 2, a CDR2 composed of an amino acid sequence of amino acid numbers 514 to 520 of SEQ ID NO: 2, and a CDR3 composed of an amino acid sequence of amino acid numbers 553 to 563 of SEQ ID NO: 2.

[0166] 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 of 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 of amino acid numbers 464 to 573 of SEQ ID NO: 2.

[0167] In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises scFv of an anti-CD137 antibody (also referred to herein as "anti-CD137 scFv").

[0168] In anti-CD137scFv, the type and length of the joint connecting the heavy chain variable region and the light chain variable region of the anti-CD137 antibody are not particularly limited, and those skilled in the art can appropriately select. As a joint, a peptide joint can be used. The preferred length is more than 5 amino acids (the upper limit is not particularly limited, usually less than 30 amino acids, preferably less than 20 amino acids), and 15 amino acids are particularly preferred. As a joint, for example, a glycine-serine joint (GS joint), a glycine-lysine-proline-glycine-serine joint (GKPGS joint) can be used. As a joint in the present invention, for example, the following joints can be cited.

[0169] Ser

[0170] Gly-Ser

[0171] Gly-Gly-Ser

[0172] Ser-Gly-Gly

[0173] Gly-Gly-Gly-Ser (serial number 5)

[0174] Ser-Gly-Gly-Gly (serial number 6)

[0175] Gly-Gly-Gly-Gly-Ser (serial number 7)

[0176] Ser-Gly-Gly-Gly-Gly (serial number 8)

[0177] Gly-Gly-Gly-Gly-Gly-Ser (serial number 9)

[0178] Ser-Gly-Gly-Gly-Gly-Gly (serial number 10)

[0179] Gly-Gly-Gly-Gly-Gly-Gly-Ser (serial number 11)

[0180] Ser-Gly-Gly-Gly-Gly-Gly-Gly (serial number 12)

[0181] Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (serial number 13)

[0182] (Gly-Gly-Gly-Gly-Ser)n

[0183] (Ser-Gly-Gly-Gly-Gly)n

[0184] Gly-Lys-Pro-Gly-Ser (serial number 14)

[0185] (Gly-Lys-Pro-Gly-Ser)n

[0186] The above n represents an integer greater than or equal to 1. In one embodiment, 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 according to the purpose.

[0187] In one embodiment, the linker used in the anti-CD137 scFv is a GS linker of (Gly-Gly-Gly-Gly-Ser)n.

[0188] In one embodiment, the linker used in the anti-CD137 scFv is a GS linker of (Gly-Gly-Gly-Gly-Ser)4.

[0189] In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises an anti-CD137 scFv comprising a light chain variable region consisting of an amino acid sequence of amino acids 464 to 573 of SEQ ID NO: 2 and a heavy chain variable region consisting of an amino acid sequence of amino acids 595 to 712 of SEQ ID NO: 2 connected via a GS linker.

[0190] 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 of amino acids 464 to 712 of SEQ ID NO:2.

[0191] 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.

[0192] In the anti-CLDN4-anti-CD137 bispecific antibody of the present invention, the anti-CLDN4 antibody or its antigen-binding fragment and the anti-CD137 antibody or its antigen-binding fragment (e.g., anti-CD137scFv) can be connected with a linker. In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention contains an anti-CLDN4 IgG antibody and an anti-CD137scFv, and the anti-CLDN4 IgG antibody and the anti-CD137scFv are connected by means of a linker. In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention contains an anti-CLDN4 IgG antibody and an anti-CD137scFv, and the amino terminus of the anti-CD137scFv is connected to the heavy chain carboxyl terminus of the anti-CLDN4 IgG antibody by means of a linker. The type and length of the linker connecting the anti-CLDN4 antibody or its antigen-binding fragment to the anti-CD137 antibody or its antigen-binding fragment are not particularly limited, and those skilled in the art can select appropriately. As a linker, a peptide linker can be used. The preferred length is 5 amino acids or more (the upper limit is not particularly limited, usually 30 amino acids or less, preferably 20 amino acids or less), particularly preferably 10 amino acids. As a peptide linker, for example, a glycine-serine linker (GS linker) and a glycine-lysine-proline-glycine-serine linker (GKPGS linker) can be used. As a linker in the present invention, for example, the following linkers can be cited.

[0193] Ser

[0194] Gly-Ser

[0195] Gly-Gly-Ser

[0196] Ser-Gly-Gly

[0197] Gly-Gly-Gly-Ser (serial number 5)

[0198] Ser-Gly-Gly-Gly (serial number 6)

[0199] Gly-Gly-Gly-Gly-Ser (serial number 7)

[0200] Ser-Gly-Gly-Gly-Gly (serial number 8)

[0201] Gly-Gly-Gly-Gly-Gly-Ser (serial number 9)

[0202] Ser-Gly-Gly-Gly-Gly-Gly (serial number 10)

[0203] Gly-Gly-Gly-Gly-Gly-Gly-Ser (serial number 11)

[0204] Ser-Gly-Gly-Gly-Gly-Gly-Gly (serial number 12)

[0205] Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (serial number 13)

[0206] (Gly-Gly-Gly-Gly-Ser)n

[0207] (Ser-Gly-Gly-Gly-Gly)n

[0208] Gly-Lys-Pro-Gly-Ser (serial number 14)

[0209] (Gly-Lys-Pro-Gly-Ser)n

[0210] The above n represents an integer greater than or equal to 1. In one embodiment, 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 according to the purpose.

[0211] In one embodiment, the linker used as a peptide linker for connecting an anti-CLDN4 antibody or an antigen-binding fragment thereof to an anti-CD137 antibody or an antigen-binding fragment thereof (eg, anti-CD137 scFv) is a linker consisting of the amino acid sequence of SEQ ID NO:13.

[0212] 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 CDR1 consisting of an amino acid sequence of amino acids 31 to 35 of SEQ ID NO: 2, a CDR2 consisting of an amino acid sequence of amino acids 50 to 66 of SEQ ID NO: 2, and a CDR3 consisting of an amino acid sequence of amino acids 99 to 112 of SEQ ID NO: 2, and a light chain of an anti-CLDN4 antibody comprising a light chain variable region comprising a CDR1 consisting of an amino acid sequence of amino acids 24 to 35 of SEQ ID NO: 4, a CDR2 consisting of an amino acid sequence of amino acids 51 to 57 of SEQ ID NO: 4, and a CDR3 consisting of an amino acid sequence of amino acids 90 to 98 of SEQ ID NO: 4, and an anti-CD137 scFv The anti-CD137 scFv comprises a heavy chain variable region of an anti-CD137 antibody comprising a CDR1 consisting of an amino acid sequence of amino acid numbers 625 to 629 of SEQ ID NO: 2, a CDR2 consisting of an amino acid sequence of amino acid numbers 644 to 659 of SEQ ID NO: 2, and a CDR3 consisting of an amino acid sequence of amino acid numbers 692 to 701 of SEQ ID NO: 2, and a light chain variable region of an anti-CD137 antibody comprising a CDR1 consisting of an amino acid sequence of amino acid numbers 486 to 498 of SEQ ID NO: 2, a CDR2 consisting of an amino acid sequence of amino acid numbers 514 to 520 of SEQ ID NO: 2, and a CDR3 consisting of an amino acid sequence of amino acid numbers 553 to 563 of SEQ ID NO: 2, and the amino terminus of the anti-CD137 scFv is connected to the heavy chain carboxyl terminus of the anti-CLDN4 antibody via a linker.

[0213] 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, a light chain of an anti-CLDN4 antibody comprising a light chain variable region of an anti-CLDN4 antibody consisting of an 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 an 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 an 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 carboxyl terminus of the heavy chain of the anti-CLDN4 antibody via a linker.

[0214] 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 an amino acid sequence of amino acids 1 to 453 of SEQ ID NO: 2, a light chain of an anti-CLDN4 antibody consisting of an amino acid sequence of amino acids 1 to 215 of SEQ ID NO: 4, and an anti-CD137 scFv consisting of an amino acid sequence of amino acids 464 to 712 of SEQ ID NO: 2, and the amino terminus of the anti-CD137 scFv is connected to the carboxyl terminus of the heavy chain of the anti-CLDN4 antibody via a linker.

[0215] In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention is a bispecific antibody comprising a polypeptide comprising the heavy chain of an anti-CLDN4 antibody and anti-CD137 scFv consisting of the amino acid sequence of SEQ ID NO: 2, and a light chain of an anti-CLDN4 antibody consisting of the amino acid sequence of SEQ ID NO: 4.

[0216] In this specification, "post-translational modification" refers to the modification of an antibody after translation when the antibody is expressed in a cell. Examples of post-translational modification include pyroglutamylation, glycosylation, oxidation, deamidation, glycation and the like of glutamine or glutamic acid at the N-terminus of the heavy chain, and lysine loss caused by cleavage of lysine at the C-terminus of the heavy chain by carboxypeptidase. Such post-translational modification is known to occur in various antibodies (J. Pharm. Sci., 2008: Vol. 97: p. 2426-2447).

[0217] In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention may be post-translationally modified. In one embodiment, the post-translation modification is pyroglutamylation of the N-terminus of the heavy chain variable region and / or deletion of lysine at the C-terminus of the heavy chain. Post-translational modification based on pyroglutamylation of the N-terminus or deletion of lysine at the C-terminus does not affect the activity of the antibody, which is known in the art (Analytical Biochemistry, 2006: Vol. 348: p. 24-39).

[0218] The anti-CLDN4-anti-CD137 bispecific antibody of the present invention binds to human CLDN4 and human CD137. Whether or not the antibody binds to human CLDN4 and human CD137 can be confirmed using a known binding activity assay method. Examples of methods for measuring binding activity include enzyme-linked immunosorbent assay (ELISA) and flow cytometry.

[0219] The anti-CLDN4-anti-CD137 bispecific antibody of the present invention can be prepared by a person skilled in the art using the sequence information of the heavy chain variable region and the light chain variable region of the anti-CLDN4 antibody and the anti-CD137 antibody disclosed in the present specification and using methods known in the art. In the anti-CLDN4-anti-CD137 bispecific antibody of the present invention, the heavy chain variable region and the light chain variable region of the anti-CLDN4 antibody and the anti-CD137 antibody can be derived from human antibodies, humanized antibodies, or a combination thereof. When making humanized antibodies, back mutations can be appropriately introduced using methods known to those skilled in the art (Bioinformatics, 2015: Vol. 31: p. 434-435). The anti-CLDN4-anti-CD137 bispecific antibody of the present invention is not particularly limited, and can be prepared, for example, according to the method described in PCT / JP2022 / 18350. The method for producing the anti-CLDN4-anti-CD137 bispecific antibody described in PCT / JP2022 / 18350 (including <Polynucleotide of the bispecific antibody of the present invention>, <Expression vector of the bispecific antibody of the present invention>, <Host cell of the present invention>, <Method for producing the bispecific antibody of the present invention> and Examples, but not limited thereto) is incorporated herein by reference (Incorporation by Reference).

[0220] <Pharmaceutical composition of the present invention>

[0221] The pharmaceutical composition of the present invention is prepared using the anti-CLDN4-anti-CD137 bispecific antibody of the present invention, and contains the anti-CLDN4-anti-CD137 bispecific antibody of the present invention and a pharmaceutically acceptable excipient. The pharmaceutical composition of the present invention can be prepared using excipients commonly used in the art, i.e., pharmaceutical excipients, pharmaceutical carriers, etc., using commonly used methods. Examples of dosage forms of these pharmaceutical compositions include non-oral preparations such as injections and drip preparations, which can be administered by intravenous administration, subcutaneous administration, intraperitoneal administration, etc. When formulated, excipients, carriers, additives, etc. corresponding to these dosage forms can be used within a pharmaceutically acceptable range. In addition, as described above, the pharmaceutical composition of the present invention contains the anti-CLDN4-anti-CD137 bispecific antibody of the present invention and a pharmaceutically acceptable excipient, but in one embodiment, it may also contain a PD-1 signal inhibitor.

[0222] 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, a pharmaceutical composition containing an antibody that has undergone both or one of C-terminal lysine deletion and N-terminal pyroglutamylation is also included in the present invention.

[0223] In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition containing an anti-CLDN4-anti-CD137 bispecific antibody and / or a post-translational modification of the bispecific antibody, wherein the anti-CLDN4-anti-CD137 bispecific antibody comprises a heavy chain variable region of an anti-CLDN4 antibody comprising a CDR1 consisting of an amino acid sequence of amino acids 31 to 35 of SEQ ID NO: 2, a CDR2 consisting of an amino acid sequence of amino acids 50 to 66 of SEQ ID NO: 2, and a CDR3 consisting of an amino acid sequence of amino acids 99 to 112 of SEQ ID NO: 2, and a CDR1 consisting of an amino acid sequence of amino acids 24 to 35 of SEQ ID NO: 4, a CDR2 consisting of an amino acid sequence of amino acids 51 to 57 of SEQ ID NO: 4, and a CDR4 consisting of an amino acid sequence of amino acids 99 to 112 of SEQ ID NO: 4. The invention also provides a light chain variable region of an anti-CLDN4 antibody comprising a CDR3 consisting of an amino acid sequence of amino acid numbers 90 to 98 of SEQ ID NO:2, a heavy chain variable region of an anti-CD137 antibody comprising a CDR1 consisting of an amino acid sequence of amino acid numbers 625 to 629 of SEQ ID NO:2, a CDR2 consisting of an amino acid sequence of amino acid numbers 644 to 659 of SEQ ID NO:2, and a CDR3 consisting of an amino acid sequence of amino acid numbers 692 to 701 of SEQ ID NO:2, and a light chain variable region of an anti-CD137 antibody comprising a CDR1 consisting of an amino acid sequence of amino acid numbers 486 to 498 of SEQ ID NO:2, a CDR2 consisting of an amino acid sequence of amino acid numbers 514 to 520 of SEQ ID NO:2, and a CDR3 consisting of an amino acid sequence of amino acid numbers 553 to 563 of SEQ ID NO:2.

[0224] In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition containing an anti-CLDN4-anti-CD137 bispecific antibody and / or a post-translational modification of the bispecific antibody, wherein the anti-CLDN4-anti-CD137 bispecific antibody comprises a heavy chain of an anti-CLDN4 antibody comprising a heavy chain variable region consisting of an 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 an 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 an 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 an amino acid sequence of amino acids 595 to 712 of SEQ ID NO: 2, and the amino terminus of the anti-CD137 scFv is connected to the carboxyl terminus of the heavy chain of the anti-CLDN4 antibody via a linker.

[0225] In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition containing an anti-CLDN4-anti-CD137 bispecific antibody and / or a post-translational modification of the bispecific antibody, wherein the anti-CLDN4-anti-CD137 bispecific antibody comprises a polypeptide containing the heavy chain of an anti-CLDN4 antibody and an anti-CD137scFv composed of the amino acid sequence of SEQ ID NO: 2 and a light chain of an anti-CLDN4 antibody composed of the amino acid sequence of SEQ ID NO: 4.

[0226] The amount of the anti-CLDN4-anti-CD137 bispecific antibody of the present invention added in the formulation varies depending on the degree of symptoms, age, dosage form of the formulation used, or antibody binding titer of the patient. For example, about 0.0001 mg / kg to about 1000 mg / kg of the anti-CLDN4-anti-CD137 bispecific antibody can be used in the formulation in terms of the dosage for humans. 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 0.0001 mg / kg to 1000 mg / kg in terms of the dosage for humans. 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 0.001 mg / kg to 100 mg / kg in terms of the dosage for humans. 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 in terms of the dosage for humans. In one embodiment, the amount of the anti-CLDN4-anti-CD137 bispecific antibody of the present invention added during formulation is preferably in the range of 0.01 mg / kg to 10 mg / kg in terms of the dosage for humans.

[0227] <PD-1 signaling inhibitors>

[0228] In the present invention, a PD-1 signaling 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 in order to treat cancer in a subject.

[0229] As long as the PD-1 signal can be blocked, the mechanism of action and treatment of the PD-1 signal inhibitor are not particularly limited. As a mechanism of action, for example, it can be a mechanism of action such as inhibiting the binding between molecules related to the PD-1 signal, reducing the expression of PD-1 signal molecules (for example, inhibiting the production of proteins or inducing the decomposition of proteins, etc.). As a treatment method, for example, it can be an antibody, a low molecular compound, a nucleic acid (which can include DNA or RNA, natural or artificial nucleic acids), a fusion protein, a peptide, or other treatment methods.

[0230] PD-1 signal inhibitors can be obtained by measuring the binding inhibition of PD-1 with one or more proteins selected from the group consisting of PD-L1 or PD-L2, and the expression reduction effect of PD-1 signal molecules as indicators. For example, in terms of PD-1 binding inhibitors with PD-L1 or PD-L2, after obtaining an inhibitor that binds to PD-1 and PD-L1 or PD-L2, the obtained inhibitor can be screened by the ability to inhibit the binding of PD-1 to PD-L1 or PD-L2. The binding of inhibitors to proteins can be evaluated using methods known to those skilled in the art, such as flow cytometry (FCM), ELISA, surface plasmon resonance (SPR), thermal shift analysis (TSA), isothermal titration calorimetry (ITC), etc. In addition, inhibitors that reduce the expression of PD-1 signal molecules such as PD-1, PD-L1 or PD-L2 can be obtained using the amount of proteins such as PD-1, PD-L1 or PD-L2 in cells as indicators. The inhibitory effect of PD-1 signaling can be confirmed by T cell proliferation, IFN-γ release, or reporter gene analysis. The effect of an inhibitor that reduces the expression level of a certain protein can be confirmed by methods known to those skilled in the art, such as ELISA, quantitative PCR, in situ hybridization, and live cell imaging.

[0231] As PD-1 signal inhibitors, for example, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies, and other antibodies that inhibit PD-1 signals can be listed. Such antibodies can be humanized antibodies, chimeric antibodies, mouse antibodies, human antibodies, and antigen-binding fragments thereof. As known anti-PD-1 antibodies, there is no limitation, for example, there are antibodies described in U.S. Patent No. 8008449, U.S. Patent No. 6808710, U.S. Patent No. 7488802, U.S. Patent No. 8168757, and U.S. Patent No. 8354509, and International Publication No. 2006 / 121168 and International Publication No. 2012 / 145493. Known anti-PD-L1 antibodies are not limited, and examples thereof include antibodies described in International Publication No. 2007 / 005874, International Publication No. 2010 / 077634, International Publication No. 2011 / 066389, International Publication No. 2013 / 079174, and U.S. Patent No. 8217149. 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, and cemiprilimab. In one embodiment, the anti-PD-L1 antibody may be an anti-PD-L1 antibody such as atezolizumab, durvalumab, or avelumab.

[0232] As PD-1 signal inhibitors, fusion proteins and low molecular weight compounds that inhibit the binding of PD-1 and PD-L1, such as AMP-224 (International Publication No. 2010 / 027827 and International Publication No. 2011 / 066342) and BMS-1166 (Oncotarget, 2017: Vol. 8: p. 72167-72181) can also be cited. Various PD-1 signal inhibitors are well known in the art (Non-Patent Document 8).

[0233] <Therapeutic method of the present invention>

[0234] The therapeutic method of the present invention is a cancer therapeutic method comprising the step of administering the anti-CLDN4-anti-CD137 bispecific antibody of the present invention and a PD-1 signaling inhibitor to a subject (referred to as “the therapeutic method of the present invention”).

[0235] 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 a PD-1 signaling inhibitor are used (administered) in combination to a subject simultaneously, continuously or sequentially.

[0236] In one embodiment, the therapeutic method of the present invention is characterized in that (i) the anti-CLDN4-anti-CD137 bispecific antibody of the present invention and the PD-1 signal inhibitor are contained in the same pharmaceutical composition and are administered to the subject at the same time; or (ii) the anti-CLDN4-anti-CD137 bispecific antibody of the present invention and the PD-1 signal inhibitor are different pharmaceutical compositions and are used in combination in the subject simultaneously, continuously or sequentially.

[0237] In one embodiment, the therapeutic method of the present invention is characterized in that (i) the anti-CLDN4-anti-CD137 bispecific antibody of the present invention and the PD-1 signal inhibitor are contained in the same pharmaceutical composition and are administered to the subject at the same time; or (ii) the anti-CLDN4-anti-CD137 bispecific antibody of the present invention and the PD-1 signal inhibitor are different pharmaceutical compositions and are administered to the subject on the same day.

[0238] In one embodiment, the therapeutic method of the present invention is characterized by (a) starting administration of a PD-1 signaling inhibitor to a subject after completion of administration of the anti-CLDN4-anti-CD137 bispecific antibody of the present invention, or (b) starting administration of the anti-CLDN4-anti-CD137 bispecific antibody of the present invention to a subject after completion of administration of the PD-1 signaling inhibitor.

[0239] 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 administered to the subject sequentially according to a dosing schedule including a dosing cycle. In one embodiment, the therapeutic method of the present invention is characterized in that, in at least one dosing cycle or all dosing cycles, the subject is started to be administered with the anti-CLDN4-anti-CD137 bispecific antibody or the pharmaceutical composition of the present invention, and then the PD-1 signal inhibitor is started. In one embodiment, the therapeutic method of the present invention is characterized in that, in at least one dosing cycle or all dosing cycles, the subject is started to be administered with the PD-1 signal inhibitor, and then the anti-CLDN4-anti-CD137 bispecific antibody or the pharmaceutical composition of the present invention is started.

[0240] <Therapeutic Use>

[0241] The cancer treated by the pharmaceutical composition and treatment method of the present invention can be any of solid cancers or blood cancers. The cancer treated by the present invention can be any of primary or metastatic. The cancer treated by the present invention is not particularly limited, and examples thereof include various peritoneal disseminated cancers, gastric cancer, lung cancer, acute lymphoblastic leukemia, acute myeloid leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, multiple myeloma, T-cell lymphoma and other blood cancers; myelodysplastic syndrome, adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, undifferentiated carcinoma, large cell carcinoma, non-small cell lung cancer, small cell lung cancer, mesothelioma, skin cancer, cutaneous T-cell lymphoma, breast cancer, prostate cancer, bladder cancer, vaginal cancer, neck cancer, head and neck cancer, uterine cancer, cervical cancer, liver cancer, gallbladder cancer, Solid cancers such as cystic cancer, bile duct cancer, kidney cancer, pancreatic cancer, colon cancer, large intestine cancer, rectal cancer, small intestine cancer, gastric cancer, esophageal cancer, testicular cancer, ovarian cancer, brain tumors; as well as cancers of bone tissue, cartilage tissue, adipose tissue, muscle tissue, vascular tissue and hematopoietic tissue, and sarcomas such as chondrosarcoma, Ewing sarcoma, malignant hemangioendothelioma, malignant Schwannoma, osteosarcoma, soft tissue sarcoma, etc.; blastomas such as glioblastoma, glioblastoma multiforme, hepatoblastoma, medulloblastoma, Wilms tumor, neuroblastoma, pancreatic blastoma, pleuropulmonary blastoma, retinoblastoma, etc.

[0242] In one embodiment, the cancer to be treated by the present invention is colorectal cancer, non-small cell lung cancer, small cell lung cancer, bladder cancer, ovarian cancer, breast cancer, and prostate cancer. In one embodiment, the cancer to be treated by the present invention is a cancer that highly expresses CLDN4 compared to normal tissues. The cancer to be treated by the present invention is preferably a cancer that highly expresses CLDN4 compared to normal tissues or a cancer selected from the group consisting of colorectal cancer, rectal cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, bladder cancer, ovarian cancer, breast cancer, and prostate cancer.

[0243] The dosage of the anti-CLDN4-anti-CD137 bispecific antibody or PD-1 signal inhibitor of the present invention to the subject varies depending on the degree of the subject's symptoms, age, dosage form of the antibody, pharmaceutical composition, inhibitor, etc. used, or the activity strength of the active ingredient, and for example, about 0.0001 mg / kg to about 1000 mg / kg can be used. In one embodiment, the dosage of the anti-CLDN4-anti-CD137 bispecific antibody of the present invention to the subject is 0.0001 mg / kg to 1000 mg / kg. In one embodiment, the dosage of the anti-CLDN4-anti-CD137 bispecific antibody of the present invention to the subject is 0.001 mg / kg to 100 mg / kg. In one embodiment, the dosage of the anti-CLDN4-anti-CD137 bispecific antibody of the present invention to the subject is 0.01 mg / kg to 10 mg / kg.

[0244] Specific examples are provided herein for reference to further understand the present invention, but these are for illustrative purposes only and are not intended to limit the present invention.

[0245] Example

[0246] [Example 1: Preparation of anti-CLDN4-anti-CD137 bispecific antibody]

[0247] [Example 1-1: Preparation of a vector encoding an anti-CLDN4-anti-CD137 bispecific antibody]

[0248] It has been reported that KM3900, an anti-CLDN4 antibody, selectively binds to CLDN4 compared to other claudin family molecules such as CLDN6 (Patent Document 1). Based on this report, a humanized antibody against KM3900 was prepared.

[0249] Specifically, based on the humanized amino acid sequence of the variable region of KM3900, a humanized antibody was designed according to the sequences of the constant region of human Igγ1 and the constant region of human Igκ. The amino acid mutations L234A, L235A and P331G were introduced into the constant region of human Igγ1 to design the anti-CLDN4 antibody sequence. The designed humanized anti-CLDN4 antibody is referred to as "hKM3900".

[0250] AlivaMab mice (Ablexis, U.S. Patent No. 9346873) were immunized by administering human CD137-human Fc fusion proteins and immune adjuvants described in Example 3 of PCT / JP2022 / 18350 multiple times. Lymphocytes were recovered from the lymph nodes of immunized mice according to conventional methods, and cell fusion was performed with mouse myeloma cells SP2 / 0 to prepare hybridomas. Single colonies of hybridomas were isolated using an automatic picking device to obtain monoclonal hybridoma cells (hereinafter referred to as "clones"). Clones that produced antibodies that bind to human CD137 were screened to obtain clones that produced anti-CD137 antibodies (hereinafter referred to as "A2-32"). cDNA was further synthesized from the cell lysate of the clone to identify the antibody base sequence. Anti-CD137scFv was designed based on the sequences of the heavy chain variable region and light chain variable region of the obtained A2-32.

[0251] Based on the amino acid sequence of hKM3900 and the amino acid sequence of the designed anti-CD137scFv, an anti-CLDN4-anti-CD137 bispecific antibody was designed. In the anti-CLDN4-anti-CD137 bispecific antibody, a GS linker was connected to the C-terminus of the heavy chain of hKM3900 of the IgG1 type, and the N-terminus of the anti-CD137scFv was bound to the C-terminus of the GS linker. The designed anti-CLDN4-anti-CD137 bispecific antibody is composed of a polypeptide containing the heavy chain of the anti-CLDN4 antibody and the anti-CD137scFv composed of the amino acid sequence of sequence number 2, and a light chain of the anti-CLDN4 antibody composed of the amino acid sequence of sequence number 4. A polynucleotide encoding a polypeptide containing the heavy chain of the anti-CLDN4 antibody and the anti-CD137scFv and a polynucleotide encoding the light chain of the anti-CLDN4 antibody were prepared and inserted into the pcDNA3.4 TOPO vector (Thermo Fisher Scientific) according to conventional methods. The two prepared vectors are referred to as "anti-CLDN4-anti-CD137 bispecific antibody expression vectors".

[0252] [Example 1-2: Preparation of anti-CLDN4-anti-CD137 bispecific antibody]

[0253] The anti-CLDN4-anti-CD137 bispecific antibody was prepared using the 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 the ExpiFectamine CHO transfection kit (Thermo Fisher Scientific, A29129), and the anti-CLDN4-anti-CD137 bispecific antibody was secreted into the culture supernatant. From the obtained culture supernatant, hKM3900_tA2-32LH as the anti-CLDN4-anti-CD137 bispecific antibody was purified by affinity purification using MabSelect SuRe (Cytiva, 17-5438-02) and size exclusion chromatography purification using HiLoad 26 / 600 superdex 200 pg (GE Healthcare, 28-9893-36).

[0254] hKM3900_tA2-32LH, which is the anti-CLDN4-anti-CD137 bispecific antibody prepared in Example 1, may also be referred to as “anti-CLDN4-anti-CD137 bispecific antibody”.

[0255] [Example 2: In vitro combined effect of anti-CLDN4-anti-CD137 bispecific antibody and anti-PD-1 antibody]

[0256] The in vitro combination effect of anti-CLDN4-anti-CD137 bispecific antibody and anti-PD-1 antibody was studied in a co-culture system using LCLC-103H cells expressing human OKT3scFv and expanded panT cells.

[0257] [Example 2-1: Preparation of expanded PanT cells]

[0258] The medium containing RPMI-1640 (Sigma, R8758) supplemented with 10% FBS (Cytiva, SH30084.03) and 1% penicillin-streptomycin (Thermo Fisher Scientific, 15070-063) was referred to as "culture medium". Anti-CD3 antibody (BioLegend, 317325) was added to a 150 mm / tissue culture dish (Tissue Culture Dish) (IWAKI, 3030-150) (hereinafter referred to as "culture dish") at a final concentration of 1 μg / mL, and the anti-CD3 antibody was immobilized. PanT Cell Isolation Kit (PanT Cell Isolation Kit, human) (Miltenyi Biotec, 130-096-535) was used to isolate PanT cells (containing both CD4-positive T cells and CD8-positive T cells, hereinafter referred to as "PanT cells") from human peripheral blood mononuclear cells (LONZA, CC-2702) according to the manufacturer's recommended protocol. The isolated PanT cells were centrifuged and the supernatant was removed, and then suspended in the culture medium. All PanT cells suspended in the culture medium were inoculated in the culture dish after the above-mentioned anti-CD3 antibody was solidified. Human IL-2 (PeproTech, 200-2) with a final concentration of 200U / mL and anti-CD28 antibody (BioLegend, 302934) with a final concentration of 4μg / mL were added and cultured in a 37°C, 5% CO2 incubator. PanT cells were recovered 3 days after the start of culture, and the recovered PanT cells were suspended in culture medium and inoculated in a culture dish. Human IL-2 was added at a final concentration of 200U / mL and cultured in a 37°C, 5% CO2 incubator. All PanT cells were recovered 4 days later, centrifuged, and resuspended in Cell Banker (Takara, CB011) after removing the supernatant, and then divided into tubes and frozen at -80°C. In this manual, the PanT cells frozen here are referred to as "expanded PanT cells".

[0259] [Example 2-2: Acquisition of LCLC-103H cells expressing human CD3 antibody single-chain variable region fragment (OKT3scFv)]

[0260] Human large cell lung cancer cell line LCLC-103H cells expressing human CLDN4 were obtained from the German Collection of Microorganisms (Deutsche Sammlung von Mikroorganismen und Zellkulturen, DSMZ, ACC384). Human large cell lung cancer cell line LCLC-103H cells were cultured in a culture medium at 37°C and 5% CO2. A polynucleotide encoding human OKT3scFv (Journal of Immunological Methods, 2010: 362: p.131-141) prepared by gene synthesis according to a conventional method was subcloned into a pcDNA3.4-TOPO vector. The prepared human OKT3scFv expression vector was transfected into LCLC-103H cells by lipofectamine LTX (Invitrogen, 15338-100) according to the manufacturer's recommended protocol. LCLC-103H cell clones stably expressing human OKT3scFv (hereinafter referred to as "LCLC-OKT3scFv cells") were obtained by selective culture using a culture medium supplemented with Geneticin Selective Antibiotic (Thermo Fisher Scientific, 10131-027) at a final concentration of 600 μg / mL and the limiting dilution method.

[0261] [Example 2-3: Effect of combined use of anti-CLDN4-anti-CD137 bispecific antibody and anti-PD-1 antibody in promoting interferon-γ production in vitro in a co-culture system of cancer cells and T cells]

[0262] Using the co-culture system of LCLC-OKT3scFv cells and expanded PanT cells, the T cell activity enhancement effect of anti-CLDN4-anti-CD137 bispecific antibody and anti-PD-1 antibody was evaluated using the interferon γ production promotion function as an indicator. LCLC-OKT3scFv cells were prepared into 2×10 5 50 μL of each was inoculated into a flat-bottom 96-well plate (IWAKI, 4020-010) and cultured in a 37°C, 5% CO2 incubator. The next day, 1.32×10 630 μL of expanded PanT cells per well / mL were inoculated in a flat-bottom 96-well plate in culture. As test antibodies, hKM3900_tA2-32LH obtained in Example 1 and nivolumab as an anti-human PD-1 antibody were used. The amino acid sequence design of nivolumab is based on the amino acid sequences of the heavy chain and light chain of nivolumab described in International Publication No. 2014 / 055648. According to the designed amino acid sequence, nivolumab was obtained according to the method described in Example 11 of International Publication No. 2021 / 241616. As an isotype control ( Figure 1-1 The anti-lysozyme antibody was prepared and used using a culture medium containing nivolumab (referred to as "isotype" in the text). As a single-dose condition, hKM3900_tA2-32LH serially diluted at about 3 times the common ratio from the maximum concentration of 50000 ng / mL with a culture medium or nivolumab serially diluted at about 3 times the common ratio from the maximum concentration of 50000 ng / mL with a culture medium was added. In addition, as a combined condition, 20 μL of hKM3900_tA2-32LH serially diluted at about 3 times the common ratio from the maximum concentration of 50000 ng / mL with a culture medium containing nivolumab at a concentration of 50000 ng / mL was added. The final concentration of nivolumab after addition was 10 μg / mL. After addition, culture was carried out by culturing in a 37°C, 5% CO2 incubator. Four days later, the amount of interferon-γ produced in the culture supernatant was measured using the AlphaLISA interferon-γ assay kit (Perkin Elmer, AL217C) according to the manufacturer's recommended protocol. Figure 1-1 The interferon-γ production amount is shown. For each condition, the mean value and standard deviation were calculated. hKM3900_tA2-32LH and nivolumab showed an interferon-γ production-promoting effect in a co-culture system of cancer cell lines expressing human CLDN4 and expanded PanT cells. The interferon-γ production-promoting effect of the combination of hKM3900_tA2-32LH and nivolumab was stronger than that of hKM3900_tA2-32LH or nivolumab alone.

[0263] [Example 3: In vitro combined effect of anti-CLDN4-anti-CD137 bispecific antibody and anti-PD-L1 antibody]

[0264] Based on the sequence of the atezolizumab antibody as an anti-PD-L1 antibody described in International Publication No. 2012 / 155019 (sequence numbers 22 and 23), an atezolizumab analogue as an anti-PD-L1 antibody using a human Igγ1 constant region sequence with a mutation introduced in the Fc region (hereinafter referred to as an "atezolizumab analogue") was designed, and the antibody was obtained according to the method described in Example 4-2 of PCT / JP2022 / 18350. In the co-culture system using LCLC-OKT3scFv cells and expanded panT cells obtained in Example 2, the in vitro combined effect of hKM3900_tA2-32LH and atezolizumab analogues was studied. Specifically, the T cell activity enhancing effect of hKM3900_tA2-32LH and atezolizumab analogues was evaluated using the amount of interferon γ produced as an indicator using the co-culture system of LCLC-OKT3scFv cells and expanded PanT cells. LCLC-OKT3scFv cells were prepared with culture medium to 2×10 5 50 μL of each was inoculated into a flat-bottom 96-well plate and cultured in a 37°C, 5% CO2 incubator. The next day, 1.33×10 6 30 μL of the expanded PanT cells were inoculated into a flat-bottom 96-well plate in culture. As test antibodies, hKM3900_tA2-32LH and atezolizumab analogs obtained in Example 1 were used. As isotype controls ( Figure 1-2 The anti-lysozyme antibody was prepared and used using a single-dose condition (referred to as "isotype" in the text). As a single-dose condition, hKM3900_tA2-32LH serially diluted at about 3 times the common ratio from the maximum concentration of 50,000 ng / mL was added to the culture medium, or an atezolizumab analogue serially diluted at about 3 times the common ratio from the maximum concentration of 50,000 ng / mL was added to the culture medium. In addition, as a combined condition, 20 μL of hKM3900_tA2-32LH serially diluted at about 3 times the common ratio from the maximum concentration of 50,000 ng / mL was added to the culture medium containing the atezolizumab analogue at a concentration of 50,000 ng / mL. The final concentration of the atezolizumab analogue after addition was 10 μg / mL. After addition, culture was carried out by culturing in a 37°C, 5% CO2 incubator. Five days later, the amount of interferon-γ produced in the culture supernatant was measured using the AlphaLISA interferon-γ assay kit (Perkin Elmer, AL217C) according to the manufacturer's recommended protocol. Figure 1-2The interferon-γ production amount is shown. For each condition, the mean and standard deviation were calculated. hKM3900_tA2-32LH and atezolizumab analogs showed an interferon-γ production-promoting effect in a co-culture system of cancer cell lines expressing human CLDN4 and expanded PanT cells. The interferon-γ production-promoting effect of the combination of hKM3900_tA2-32LH and atezolizumab analogs was stronger than that of a single agent of hKM3900_tA2-32LH or atezolizumab analogs.

[0265] [Example 4: In vivo combined effect of anti-CLDN4-anti-CD137 bispecific antibody and anti-mouse PD-1 antibody]

[0266] The in vivo antitumor effects of anti-CLDN4-anti-CD137 bispecific antibodies and anti-human PD-1 antibodies were studied using B-h4-1BB mice (human CD137 knock-in mice) transplanted with B16-F10 cells expressing human CLDN4.

[0267] [Example 4-1: Construction of B16-F10 cells expressing human CLDN4]

[0268] Mouse melanoma cell line B16-F10 cells 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) (hereinafter, the prepared medium is referred to as "Eagle's culture medium") at 37°C and 5% CO2. CLDN4 (Myc-DDK-tagged)-Human claudin4 (CLDN4) (ORIGENE, RC200490) was introduced into B16-F10 cells using jetPRIME (Polyplus-transfection, 114-15). By selecting with Eagle's culture medium supplemented with G418 (Nacalai Tesque, 09380-44) at a final concentration of 1 mg / mL, a B16-F10 cell clone stably expressing human CLDN4 (hereinafter referred to as "human CLDN4-expressing B16-F10 cell") was obtained.

[0269] [Example 4-2: Combination effect of anti-CLDN4-anti-CD137 bispecific antibody and anti-mouse PD-1 antibody in in vivo anti-tumor activity]

[0270] Obtain B-h4-1BB male mice (C57BL / 6-Tnfrsf9tm1(Tnfrsf9) / Bcgen; Biocytogen, 110004) (hereinafter referred to as "mice"), and the cells were bred. B16-F10 cells expressing human CLDN4 were suspended in PBS (-) (WAKO, 045-29795) and 4×10 6 2×10 cells / mL of cell suspension were inoculated subcutaneously on the back of 6-week-old mice. 5 cells / 50 μL of cell suspension. Three days after cell inoculation, tumor diameter was measured using a vernier caliper (Mitutoyo, CD-15AXR). Tumor volume [mm 3 ].

[0271] [Tumor volume (mm 3 )] = [long diameter of tumor (mm)] × [short diameter of tumor (mm)] 2 × 0.5

[0272] The mice inoculated with cells were grouped in such a way that the tumor volume of each group was roughly equal (n=10), and the test antibody was administered. The first administration day was defined as day 0. As the test antibody, hKM3900_tA2-32LH obtained in Example 1 as an anti-CLDN4-anti-CD137 bispecific antibody and anti-mouse PD-1 antibody (Bio XCell, BE0146) as an anti-PD-1 antibody were used. As an isotype control antibody for hKM3900_tA2-32LH, an anti-lysozyme antibody was used, and as an isotype control antibody for the anti-mouse PD-1 antibody, a rat IgG2a isotype control antibody (Bio X Cell, BE0089) was used ( Figure 2 The details of the test antibodies, dosages, and dosing schedules administered to the four groups in the experiment are shown below.

[0273] (1) Group 1: Control group

[0274] 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 / mouse on days 0, 4, 7, and 11.

[0275] (2) Group 2: hKM3900_tA2-32LH administration group

[0276] 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 / mouse on days 0, 4, 7, and 11.

[0277] (3) Group 3: Anti-mouse PD-1 antibody administration group

[0278] Anti-lysozyme antibody was intraperitoneally administered at 0.3 mg / kg on days 0 and 7, and anti-mouse PD-1 antibody was intraperitoneally administered at 100 μg / mouse on days 0, 4, 7, and 11.

[0279] (4) Group 4: Combination administration of hKM3900_tA2-32LH and anti-mouse PD-1 antibody

[0280] hKM3900_tA2-32LH was intraperitoneally administered at 0.3 mg / kg on days 0 and 7, and anti-mouse PD-1 antibody was intraperitoneally administered at 100 μg / mouse on days 0, 4, 7, and 11.

[0281] The tumor volumes of each group were evaluated on days 4, 7, 11, and 14. The tumor volumes of groups 2 and 3 on day 14 were compared with those of group 4 by unpaired Student's t-test ( Figure 2 ).

[0282] like Figure 2 As shown in the figure, the tumor volume of the hKM3900_tA2-32LH combined with anti-mouse PD-1 antibody group was significantly smaller than that of the hKM3900_tA2-32LH alone group and the anti-mouse PD-1 antibody alone group. This result suggests that in the treatment of human cancer, the combination of anti-CLDN4-anti-CD137 bispecific antibody and anti-PD-1 antibody may achieve a higher anti-tumor effect than the single administration of each.

[0283] Industrial Applicability

[0284] The cancer treatment method based on the combination of the anti-CLDN4-anti-CD137 bispecific antibody and the PD-1 signaling inhibitor of the present invention is expected to be useful in cancer treatment.

[0285] Sequence Listing Free Text

[0286] Sequence number 2 is the amino acid sequence of hKM3900_tA2-32LH HC, and the base sequence shown in sequence number 1 is the base sequence encoding the amino acid sequence of the hKM3900_tA2-32LH heavy chain shown in sequence number 2. Sequence number 4 is the amino acid sequence of hKM3900 LC, and the base sequence shown in sequence number 3 is the base sequence encoding the amino acid sequence of the hKM3900 light chain shown in sequence number 4. Sequence numbers 5 to 14 are the amino acid sequences of various linkers described in the detailed description of the invention.

Claims

1. A pharmaceutical composition comprising an anti-CLDN4-anti-CD137 bispecific antibody for treating cancer in a subject, wherein: The bispecific antibody contains the heavy chain variable region and the light chain variable region of the anti-CLDN4 antibody and the heavy chain variable region and the light chain variable region of the anti-CD137 antibody. The pharmaceutical composition is used in combination with a PD-1 signal inhibitor.

2. The pharmaceutical composition according to claim 1, wherein The heavy chain variable region of the anti-CLDN4 antibody contains a CDR1 consisting of an amino acid sequence of amino acid numbers 31 to 35 of SEQ ID NO: 2, a CDR2 consisting of an amino acid sequence of amino acid numbers 50 to 66 of SEQ ID NO: 2, and a CDR3 consisting of an amino acid sequence of amino acid numbers 99 to 112 of SEQ ID NO: 2, and the light chain variable region of the anti-CLDN4 antibody contains a CDR1 consisting of an amino acid sequence of amino acid numbers 24 to 35 of SEQ ID NO: 4, a CDR2 consisting of an amino acid sequence of amino acid numbers 51 to 57 of SEQ ID NO: 4, and a CDR3 consisting of an amino acid sequence of amino acid numbers 90 to 98 of SEQ ID NO:

4.

3. The pharmaceutical composition according to claim 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 claims 1 to 3, wherein The anti-CLDN4-anti-CD137 bispecific antibody includes an IgG antibody (anti-CLDN4 IgG antibody) composed of a heavy chain containing the heavy chain variable region of the anti-CLDN4 antibody and a light chain containing the light chain variable region of the anti-CLDN4 antibody.

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

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein The heavy chain variable region of the anti-CD137 antibody contains a CDR1 composed of an amino acid sequence of amino acid numbers 625 to 629 of SEQ ID NO: 2, a CDR2 composed of an amino acid sequence of amino acid numbers 644 to 659 of SEQ ID NO: 2, and a CDR3 composed of an amino acid sequence of amino acid numbers 692 to 701 of SEQ ID NO: 2, and the light chain variable region of the anti-CD137 antibody contains a CDR1 composed of an amino acid sequence of amino acid numbers 486 to 498 of SEQ ID NO: 2, a CDR2 composed of an amino acid sequence of amino acid numbers 514 to 520 of SEQ ID NO: 2, and a CDR3 composed of an amino acid sequence of amino acid numbers 553 to 563 of SEQ ID NO:

2.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein The heavy chain variable region of the anti-CD137 antibody consists of the amino acid sequence of 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 of amino acid numbers 464 to 573 of SEQ ID NO:

2.

8. The pharmaceutical composition according to claim 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 the light chain variable region of the anti-CD137 antibody.

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

2.

10. The pharmaceutical composition according to claim 8 or 9, wherein The anti-CLDN4-anti-CD137 bispecific antibody contains an anti-CLDN4 IgG antibody and an anti-CD137 scFv, wherein the amino terminus of the anti-CD137 scFv is connected to the heavy chain carboxyl terminus of the anti-CLDN4 IgG antibody via a linker.

11. A pharmaceutical composition comprising an anti-CLDN4-anti-CD137 bispecific antibody for treating 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 an amino acid sequence of amino acids 1 to 123 of SEQ ID NO: 2, a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of an 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 an 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 an amino acid sequence of amino acids 595 to 712 of SEQ ID NO: 2, the amino terminus of the anti-CD137 scFv being connected to the heavy chain carboxyl terminus of the anti-CLDN4 antibody via a linker, and the pharmaceutical composition is used in combination with a PD-1 signaling inhibitor.

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

13. The pharmaceutical composition according to any one of claims 10 to 12, wherein The connector is a GS connector.

14. A pharmaceutical composition comprising an anti-CLDN4-anti-CD137 bispecific antibody for treating cancer in a subject, wherein: The bispecific antibody comprises a polypeptide containing the heavy chain of an anti-CLDN4 antibody and an anti-CD137scFv composed of the amino acid sequence of SEQ ID NO: 2, and a light chain of an anti-CLDN4 antibody composed of the amino acid sequence of SEQ ID NO:

4. The pharmaceutical composition is used in combination with a PD-1 signal inhibitor.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein The anti-CLDN4-anti-CD137 bispecific antibody was post-translationally modified. 16 . The pharmaceutical composition according to claim 1 , which is used in combination with a PD-1 signaling inhibitor simultaneously, continuously or sequentially.

17. The pharmaceutical composition according to any one of claims 1 to 16, wherein (i) the anti-CLDN4-anti-CD137 bispecific antibody and the PD-1 signal inhibitor are contained in the same pharmaceutical composition and administered simultaneously; or (ii) the anti-CLDN4-anti-CD137 bispecific antibody and the PD-1 signal inhibitor are contained in different pharmaceutical compositions and used in combination simultaneously, continuously or sequentially.

18. The pharmaceutical composition according to any one of claims 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 claims 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 claims 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 cemiplizumab.

21. The pharmaceutical composition according to any one of claims 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. A bispecific antibody, which is an anti-CLDN4-anti-CD137 bispecific antibody for treating cancer in a subject, wherein: The bispecific antibody contains the heavy chain variable region and the light chain variable region of an anti-CLDN4 antibody and the heavy chain variable region and the light chain variable region of an anti-CD137 antibody, and the bispecific antibody is used in combination with a PD-1 signal inhibitor.

23. A bispecific antibody, which is an anti-CLDN4-anti-CD137 bispecific antibody for treating cancer in a subject, wherein: The bispecific antibody comprises a polypeptide comprising the heavy chain of an anti-CLDN4 antibody and an anti-CD137scFv composed of the amino acid sequence of SEQ ID NO: 2, and a light chain of an anti-CLDN4 antibody composed of the amino acid sequence of SEQ ID NO: 4, and the bispecific antibody is used in combination with a PD-1 signaling inhibitor.

24. A method for treating cancer comprising administering an anti-CLDN4-anti-CD137 bispecific antibody and a PD-1 signaling inhibitor to a subject, wherein: The anti-CLDN4-anti-CD137 bispecific antibody contains the heavy chain variable region and the light chain variable region of the anti-CLDN4 antibody and the heavy chain variable region and the light chain variable region of the anti-CD137 antibody.

25. A method for treating cancer comprising administering to a subject a combination of an anti-CLDN4-anti-CD137 bispecific antibody and a PD-1 signaling inhibitor, wherein: The bispecific antibody comprises a polypeptide comprising the heavy chain of an anti-CLDN4 antibody and anti-CD137 scFv and composed of the amino acid sequence of SEQ ID NO: 2, and a light chain of an anti-CLDN4 antibody and composed of the amino acid sequence of SEQ ID NO:

4.

26. Use of an anti-CLDN4-anti-CD137 bispecific antibody in the manufacture of a pharmaceutical composition for use in combination with a PD-1 signaling inhibitor for treating cancer in a subject, wherein: The bispecific antibody contains the heavy chain variable region and the light chain variable region of the anti-CLDN4 antibody and the heavy chain variable region and the light chain variable region of the anti-CD137 antibody.

27. Use of an anti-CLDN4-anti-CD137 bispecific antibody in the manufacture of a pharmaceutical composition for use in combination with a PD-1 signaling inhibitor for treating cancer in a subject, wherein: The bispecific antibody comprises a polypeptide comprising the heavy chain of an anti-CLDN4 antibody and anti-CD137 scFv and composed of the amino acid sequence of SEQ ID NO: 2, and a light chain of an anti-CLDN4 antibody and composed of the amino acid sequence of SEQ ID NO: 4.

Images