Anti-CLDN4-anti-CD137 dual-specific antibody

Abstract

To provide an anti-CLDN4-anti-CD137 bispecific antibody that can be used for treating cancer.SOLUTION: An anti-CLDN4-anti-CD137 bispecific antibody produced by using an anti-CLDN4 antibody bonding to CLDN4 and an anti-CD137 antibody bonding to CD137 has agonist action to CD137, promotes interferon γ production of T-cell, and shows cytotoxic activity to a cancer cell that expresses CLDN4 on a cell surface. Furthermore, the anti-CLDN4-anti-CD137 bispecific antibody can be safely administered to a monkey. Therefore, the anti-CLDN4-anti-CD137 bispecific antibody can be used for treating human cancer.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an anti-CLDN4-anti-CD137 bispecific antibody that is expected to be useful as an active ingredient in a pharmaceutical composition for the treatment of cancer. [Background technology]

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

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

[0004] Bispecific T-cell-recruiting antibodies in various antibody formats have been reported as an innovative method for achieving selective cytotoxicity to cancer cells at low antibody concentrations. Bispecific T-cell-recruiting antibodies are bispecific antibodies that contain an antibody against a tumor-associated antigen (TAA) expressed on the surface of cancer cells and an antibody that binds to T cells, and the effects of these antibodies on T-cell-mediated immunotherapy are currently being investigated (Non-Patent Document 6). Anti-CD3 antibodies are often used as antibodies that bind to T cells, and research and development of bispecific T-cell-recruiting antibodies against various TAAs is currently underway.

[0005] Furthermore, in recent years, active research has been conducted on bispecific T cell-recruiting antibodies against CD137 and TAAs, such as anti-GPC3-anti-CD137 bispecific antibodies, anti-HER2-anti-CD137 bispecific antibodies, anti-PDL1-anti-CD137 bispecific antibodies, and anti-FAP-anti-CD137 bispecific antibodies that recognize the TAAs Glypican 3 (GPC3), Human Epidermal Growth Factor Receptor Type 2 (HER2), Programmed Cell Death-Ligand 1 (PD-L1), and Fibroblast Activation Protein (FAP) (Patent Documents 2 and 3, Non-Patent Documents 7 to 9). However, to date, there have been no reports of anti-CLDN4-anti-CD137 bispecific antibodies. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2008 / 114733 [Patent Document 2] International Publication No. 2015 / 156268 [Patent Document 3] International Publication No. 2016 / 177802 [Non-patent literature]

[0007] [Non-Patent Document 1] Cancer Science, 2009:100(9):p.1623-1630 [Non-patent document 2] Oncotarget, 2018:9(100):p.37367-37378 [Non-patent document 3] Cancer Science, 2020:111(5):p.1461-1467 [Non-patent document 4] Cancer Immunology Immunotherapy, 2012:61(5):p.1721-1733 [Non-patent document 5] Clinical Cancer Research, 2017:23(8):p.1929-1936 [Non-patent document 6] mAbs, 2017:9(2):p.182-212 [Non-Patent Document 7] Clinical Cancer Research, 2019:25(19):p.5878-5889 [Non-patent document 8] Clinical Cancer Research, 2020:26(15):p.4154-4167 [Non-Patent Document 9] Journal for Immunotherapy of Cancer, 2020;8(2):e000238 Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide an anti-CLDN4-anti-CD137 bispecific antibody that can be used in the treatment of cancer. [Means for solving the problem]

[0009] The present inventors searched for antibodies that selectively bind to antigens expressed on peritoneally disseminated cancer cells and isolated the anti-CLDN4 antibody 3D11 (Examples 1 and 2). It was found that a bispecific T cell-recruiting antibody using 3D11 and an anti-CD3 antibody could not be used to treat cancer patients (Reference Example). Therefore, we attempted to isolate a bispecific T cell-recruiting antibody using 3D11 and a known anti-CLDN4 antibody, KM3900, in combination with an anti-CD137 antibody (Examples 3 and 4). The isolated anti-CLDN4-anti-CD137 bispecific antibody bound to CLDN4 and CD137 (Example 5), promoted T cell interferon-γ production in vitro, and exhibited cytotoxic activity against cancer cells expressing CLDN4 on their cell surface (Example 6). Furthermore, the isolated anti-CLDN4-anti-CD137 bispecific antibody exhibited antitumor activity in an in vivo mouse model (Example 7-1) and was confirmed to be safe for use in cynomolgus monkeys (Example 7-2). It was suggested that the anti-CLDN4-anti-CD137 bispecific antibody of the present invention is useful for cancer treatment.

[0010] That is, the present invention relates to the following [1] to

[32] . [1] An anti-CLDN4-anti-CD137 bispecific antibody comprising the heavy chain variable region and light chain variable region of an anti-CLDN4 antibody and the heavy chain variable region and light chain variable region of an anti-CD137 antibody, wherein the heavy chain variable region and light chain variable region of the anti-CLDN4 antibody are either (a) or (b) below: (a) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 2, a CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 66 of SEQ ID NO: 2, and a CDR3 consisting of the amino acid sequence from amino acid numbers 99 to 114 of SEQ ID NO: 2, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 24 to 35 of SEQ ID NO: 4, a CDR2 consisting of the amino acid sequence from amino acid numbers 51 to 57 of SEQ ID NO: 4, and a CDR3 consisting of the amino acid sequence from amino acid numbers 90 to 98 of SEQ ID NO: 4; or (b) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 6, a CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 66 of SEQ ID NO: 6, and a CDR3 consisting of the amino acid sequence from amino acid numbers 99 to 112 of SEQ ID NO: 6; and a light chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 24 to 35 of SEQ ID NO: 8, a CDR2 consisting of the amino acid sequence from amino acid numbers 51 to 57 of SEQ ID NO: 8, and a CDR3 consisting of the amino acid sequence from amino acid numbers 90 to 98 of SEQ ID NO: 8. [2] The bispecific antibody of [1], wherein the heavy chain variable region and the light chain variable region of the anti-CLDN4 antibody are either (a) or (b) below: (a) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 125 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 109 of SEQ ID NO: 4; or (b) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 123 of SEQ ID NO: 6 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 109 of SEQ ID NO: 8. [3] The bispecific antibody according to either [1] or [2], comprising an IgG antibody (anti-CLDN4 IgG antibody) consisting of a heavy chain comprising the heavy chain variable region of an anti-CLDN4 antibody and a light chain comprising the light chain variable region. [4] The bispecific antibody according to [3], which comprises LALA mutations (L234A and L235A (wherein the mutation positions are amino acid positions according to the EU index in the human Igγ1 constant region)) in the Fc region of the anti-CLDN4 IgG antibody. [5] The anti-CLDN4-anti-CD137 bispecific antibody according to [3], which comprises a P331G mutation in the Fc region of the anti-CLDN4 IgG antibody (wherein the mutation position is an amino acid position according to the EU index in the human Igγ1 constant region). [6] The bispecific antibody described in [3], which comprises a LALA mutation and a P331G mutation in the Fc region of the anti-CLDN4 IgG antibody. [7] The bispecific antibody according to any one of [1] to [6], wherein the heavy chain variable region and the light chain variable region of the anti-CD137 antibody are any of the following (a) to (d): (a) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 10, a CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 66 of SEQ ID NO: 10, and a CDR3 consisting of the amino acid sequence from amino acid numbers 99 to 107 of SEQ ID NO: 10, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 24 to 34 of SEQ ID NO: 12, a CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 56 of SEQ ID NO: 12, and a CDR3 consisting of the amino acid sequence from amino acid numbers 89 to 98 of SEQ ID NO: 12; (b) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 14, a CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 65 of SEQ ID NO: 14, and a CDR3 consisting of the amino acid sequence from amino acid numbers 98 to 107 of SEQ ID NO: 14, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 23 to 35 of SEQ ID NO: 16, a CDR2 consisting of the amino acid sequence from amino acid numbers 51 to 57 of SEQ ID NO: 16, and a CDR3 consisting of the amino acid sequence from amino acid numbers 90 to 100 of SEQ ID NO: 16; (c) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 18, a CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 65 of SEQ ID NO: 18, and a CDR3 consisting of the amino acid sequence from amino acid numbers 98 to 107 of SEQ ID NO: 18, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 23 to 35 of SEQ ID NO: 20, a CDR2 consisting of the amino acid sequence from amino acid numbers 51 to 57 of SEQ ID NO: 20, and a CDR3 consisting of the amino acid sequence from amino acid numbers 90 to 100 of SEQ ID NO: 20; or (d) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 22, a CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 65 of SEQ ID NO: 22, and a CDR3 consisting of the amino acid sequence from amino acid numbers 98 to 110 of SEQ ID NO: 22; and a light chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 23 to 35 of SEQ ID NO: 24, a CDR2 consisting of the amino acid sequence from amino acid numbers 51 to 57 of SEQ ID NO: 24, and a CDR3 consisting of the amino acid sequence from amino acid numbers 90 to 100 of SEQ ID NO: 24. [8] The bispecific antibody according to any one of [1] to [7], wherein the heavy chain variable region and the light chain variable region of the anti-CD137 antibody are any of the following (a) to (i): (a) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 10 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 109 of SEQ ID NO: 12; (b) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 14 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 16; (c) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 18 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 20; (d) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 121 of SEQ ID NO: 22 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 24 (e) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 26 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 134 to 242 of SEQ ID NO: 26; (f) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 28 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 134 to 244 of SEQ ID NO: 28; (g) a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 30 and a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 132 to 249 of SEQ ID NO: 30; (h) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 32 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 134 to 244 of SEQ ID NO: 32; or (i) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 121 of SEQ ID NO: 34 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 137 to 247 of SEQ ID NO: 34. [9] The bispecific antibody according to either [7] or [8], which comprises an anti-CD137 single-chain variable region fragment (anti-CD137scFv) comprising the heavy chain variable region and light chain variable region of an anti-CD137 antibody.

[10] The bispecific antibody according to [9], wherein the anti-CD137 scFv is any one of the following (a) to (e): (a) an anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 1 to 242 of SEQ ID NO: 26; (b) an anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 1 to 244 of SEQ ID NO: 28; (c) an anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 1 to 249 of SEQ ID NO: 30; (d) an anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 1 to 244 of SEQ ID NO: 32; or (e) An anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 1 to 247 of SEQ ID NO: 34.

[11] The bispecific antibody according to either [9] or

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

[12] Any of the following anti-CLDN4-anti-CD137 bispecific antibodies (a) to (j): (a) 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 125 of SEQ ID NO: 2 and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4, and an anti-CD137scFv comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 26 and a light chain variable region consisting of the amino acid sequence of amino acids 134 to 242 of SEQ ID NO: 26, wherein the amino terminus of the anti-CD137scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker; (b) 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 125 of SEQ ID NO: 2 and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4, and an anti-CD137scFv comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 28 and a light chain variable region consisting of the amino acid sequence of amino acids 134 to 244 of SEQ ID NO: 28, wherein the amino terminus of the anti-CD137scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker; (c) 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 125 of SEQ ID NO: 2 and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4, and an anti-CD137scFv comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 111 of SEQ ID NO: 30 and a heavy chain variable region consisting of the amino acid sequence of amino acids 132 to 249 of SEQ ID NO: 30, wherein the amino terminus of the anti-CD137scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker; (d) 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 125 of SEQ ID NO: 2 and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4, and an anti-CD137scFv comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 32 and a light chain variable region consisting of the amino acid sequence of amino acids 134 to 244 of SEQ ID NO: 32, wherein the amino terminus of the anti-CD137scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker; (e) 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 125 of SEQ ID NO: 2 and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4, and an anti-CD137scFv comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 121 of SEQ ID NO: 34 and a light chain variable region consisting of the amino acid sequence of amino acids 137 to 247 of SEQ ID NO: 34, wherein the amino terminus of the anti-CD137scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker; (f) 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: 6 and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 8, and an anti-CD137scFv comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 26 and a light chain variable region consisting of the amino acid sequence of amino acids 134 to 242 of SEQ ID NO: 26, wherein the amino terminus of the anti-CD137scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker; (g) 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: 6 and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 8, and an anti-CD137scFv comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 28 and a light chain variable region consisting of the amino acid sequence of amino acids 134 to 244 of SEQ ID NO: 28, wherein the amino terminus of the anti-CD137scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker; (h) 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: 6 and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 8, and an anti-CD137scFv comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 111 of SEQ ID NO: 30 and a heavy chain variable region consisting of the amino acid sequence of amino acids 132 to 249 of SEQ ID NO: 30, wherein the amino terminus of the anti-CD137scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker; (i) 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: 6 and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 8, and an anti-CD137scFv comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 32 and a light chain variable region consisting of the amino acid sequence of amino acids 134 to 244 of SEQ ID NO: 32, wherein the amino terminus of the anti-CD137scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker; or (j) 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: 6 and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 8, and an anti-CD137scFv comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 121 of SEQ ID NO: 34 and a light chain variable region consisting of the amino acid sequence of amino acids 137 to 247 of SEQ ID NO: 34, wherein the amino terminus of the anti-CD137scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker.

[13] The anti-CLDN4-anti-CD137 bispecific antibody according to either

[11] or

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

[14] The anti-CLDN4-anti-CD137 bispecific antibody according to

[13] , wherein the GS linker is a linker consisting of the amino acid sequence of SEQ ID NO: 54.

[15] An anti-CLDN4-anti-CD137 bispecific antibody, either (a) or (b) below: (a) a bispecific antibody comprising a polypeptide comprising an anti-CLDN4 antibody heavy chain consisting of the amino acid sequence from amino acid numbers 1 to 705 of SEQ ID NO: 36 and an anti-CD137 scFv, and an anti-CLDN4 antibody light chain consisting of the amino acid sequence from amino acid numbers 1 to 215 of SEQ ID NO: 40; or (b) A bispecific antibody comprising a polypeptide comprising an anti-CD137 scFv and a heavy chain of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 712 of SEQ ID NO: 38, and a light chain of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 215 of SEQ ID NO: 40.

[16] The post-translationally modified anti-CLDN4-anti-CD137 bispecific antibody according to any one of [1] to

[15] .

[17] The anti-CLDN4-anti-CD137 bispecific antibody according to

[16] , wherein the post-translational modification is pyroglutamylation of the N-terminus of the heavy chain variable region and / or deletion of a lysine at the C-terminus of the heavy chain.

[18] A polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region or a light chain variable region of an anti-CLDN4 antibody selected from the group consisting of the following (a) to (d): (a) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 125 of SEQ ID NO: 2; (b) a polynucleotide comprising a nucleotide sequence encoding the light chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 109 of SEQ ID NO: 4; (c) a polynucleotide comprising a nucleotide sequence encoding the heavy chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 123 of SEQ ID NO: 6; or (d) A polynucleotide comprising a base sequence encoding the light chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 109 of SEQ ID NO: 8.

[19] A polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region or a light chain variable region of an anti-CD137 antibody, selected from the group consisting of the following (a) to (r): (a) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 10; (b) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 109 of SEQ ID NO: 12; (c) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 14; (d) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 16; (e) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 18; (f) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 20; (g) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 121 of SEQ ID NO: 22; (h) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 24; (i) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 26; (j) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 134 to 242 of SEQ ID NO: 26; (k) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 28; (l) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 134 to 244 of SEQ ID NO: 28; (m) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 30; (n) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 132 to 249 of SEQ ID NO: 30; (o) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 32; (p) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 134 to 244 of SEQ ID NO: 32; (q) a polynucleotide comprising a nucleotide sequence encoding the heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 121 of SEQ ID NO: 34; or (r) A polynucleotide comprising a base sequence encoding the light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 137 to 247 of SEQ ID NO: 34.

[20] A polynucleotide comprising a nucleotide sequence encoding an anti-CD137 scFv selected from the group consisting of the following (a) to (e): (a) a polynucleotide comprising a nucleotide sequence encoding an anti-CD137scFv consisting of the amino acid sequence from amino acid numbers 1 to 242 of SEQ ID NO: 26; (b) a polynucleotide comprising a nucleotide sequence encoding an anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 1 to 244 of SEQ ID NO: 28; (c) a polynucleotide comprising a nucleotide sequence encoding an anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 1 to 249 of SEQ ID NO: 30; (d) a polynucleotide comprising a nucleotide sequence encoding an anti-CD137scFv consisting of the amino acid sequence from amino acid numbers 1 to 244 of SEQ ID NO: 32; or (e) A polynucleotide comprising a base sequence encoding an anti-CD137scFv consisting of the amino acid sequence from amino acid numbers 1 to 247 of SEQ ID NO: 34.

[21] A polynucleotide selected from the group consisting of the following (a) to (e), used for producing the anti-CLDN4-anti-CD137 bispecific antibody according to

[15] : (a) a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the heavy chain of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 705 of SEQ ID NO: 36 and an anti-CD137scFv; (b) a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the heavy chain of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 712 of SEQ ID NO: 38 and an anti-CD137scFv; (c) a polynucleotide comprising a nucleotide sequence encoding the light chain of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 215 of SEQ ID NO: 40; (d) a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an anti-CLDN4 antibody heavy chain consisting of the amino acid sequence from amino acid numbers 1 to 705 of SEQ ID NO: 36 and an anti-CD137scFv, and a polynucleotide comprising a nucleotide sequence encoding an anti-CLDN4 antibody light chain consisting of the amino acid sequence from amino acid numbers 1 to 215 of SEQ ID NO: 40; or (e) A polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an anti-CLDN4 antibody heavy chain consisting of the amino acid sequence from amino acid numbers 1 to 712 of SEQ ID NO: 38 and an anti-CD137scFv, and a polynucleotide comprising a nucleotide sequence encoding an anti-CLDN4 antibody light chain consisting of the amino acid sequence from amino acid numbers 1 to 215 of SEQ ID NO: 40.

[22] An expression vector comprising the polynucleotide according to any one of

[18] to

[21] .

[23] A host cell transformed with the expression vector described in

[22] .

[24] A host cell comprising a polynucleotide selected from the group consisting of the following (a) to (dd): (a) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 125 of SEQ ID NO: 2; (b) a polynucleotide comprising a nucleotide sequence encoding the light chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 109 of SEQ ID NO: 4; (c) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 123 of SEQ ID NO: 6; (d) A polynucleotide comprising a base sequence encoding the light chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 109 of SEQ ID NO: 8. (e) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 10; (f) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 109 of SEQ ID NO: 12; (g) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 14; (h) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 16; (i) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 18; (j) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 20; (k) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 121 of SEQ ID NO: 22; (l) A polynucleotide comprising a nucleotide sequence encoding the light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 24. (m) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 26; (n) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 134 to 242 of SEQ ID NO: 26; (o) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 28; (p) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 134 to 244 of SEQ ID NO: 28; (q) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 30; (r) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 132 to 249 of SEQ ID NO: 30; (s) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 32; (t) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 134 to 244 of SEQ ID NO: 32; (u) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 121 of SEQ ID NO: 34; (v) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 137 to 247 of SEQ ID NO: 34; (w) a polynucleotide comprising a nucleotide sequence encoding an anti-CD137scFv consisting of the amino acid sequence from amino acid numbers 1 to 242 of SEQ ID NO: 26; (x) a polynucleotide comprising a nucleotide sequence encoding an anti-CD137scFv consisting of the amino acid sequence from amino acid numbers 1 to 244 of SEQ ID NO: 28; (y) a polynucleotide comprising a nucleotide sequence encoding an anti-CD137scFv consisting of the amino acid sequence from amino acid numbers 1 to 249 of SEQ ID NO: 30; (z) a polynucleotide comprising a nucleotide sequence encoding an anti-CD137scFv consisting of the amino acid sequence from amino acid numbers 1 to 244 of SEQ ID NO: 32; (aa) A polynucleotide comprising a base sequence encoding an anti-CD137scFv consisting of the amino acid sequence from amino acid numbers 1 to 247 of SEQ ID NO: 34. (bb) a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the heavy chain of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 705 of SEQ ID NO: 36 and an anti-CD137scFv; (cc) a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the heavy chain of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 712 of SEQ ID NO: 38 and an anti-CD137 scFv; or (dd) A polynucleotide comprising a nucleotide sequence encoding the light chain of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid number 1 to amino acid number 215 of SEQ ID NO: 40.

[25] A host cell comprising either the following polynucleotide (a) or (b): (a) a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an anti-CLDN4 antibody heavy chain consisting of the amino acid sequence from amino acid numbers 1 to 705 of SEQ ID NO: 36 and an anti-CD137 scFv, and a polynucleotide comprising a nucleotide sequence encoding an anti-CLDN4 antibody light chain consisting of the amino acid sequence from amino acid numbers 1 to 215 of SEQ ID NO: 40; or (b) a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an anti-CLDN4 antibody heavy chain consisting of the amino acid sequence from amino acid numbers 1 to 712 of SEQ ID NO: 38 and an anti-CD137scFv, and a polynucleotide comprising a nucleotide sequence encoding an anti-CLDN4 antibody light chain consisting of the amino acid sequence from amino acid numbers 1 to 215 of SEQ ID NO: 40.

[26] A method for producing an anti-CLDN4-anti-CD137 bispecific antibody, the method comprising a step of culturing the host cell according to any one of

[23] to

[25] .

[27] A pharmaceutical composition comprising the anti-CLDN4-anti-CD137 bispecific antibody according to any one of [1] to

[17] and a pharmaceutically acceptable excipient.

[28] An anti-CLDN4-anti-CD137 bispecific antibody according to any one of [1] to

[17] for use in the treatment of cancer.

[29] The pharmaceutical composition according to

[27] for use in the treatment of cancer.

[30] A method for treating cancer, comprising the step of administering a therapeutically effective amount of the anti-CLDN4-anti-CD137 bispecific antibody according to any one of [1] to

[17] to a subject.

[31] Use of the anti-CLDN4-anti-CD137 bispecific antibody according to any one of [1] to

[17] in the manufacture of a pharmaceutical composition for use in the treatment of cancer. [Effects of the Invention]

[0011] The anti-CLDN4-anti-CD137 bispecific antibody of the present invention binds to both CLDN4, a cancer antigen, and CD137 expressed on the surface of immune cells such as T cells, and activates immune cells surrounding the cancer cells, thereby enhancing the killing effect against the cancer cells. The anti-CLDN4-anti-CD137 bispecific antibody of the present invention or a pharmaceutical composition containing the antibody can be used for the treatment of various cancers. [Brief explanation of the drawings]

[0012] [Figure 1-1] Figure 1-1 shows the CD137 binding activity of test antibodies measured and evaluated by ELISA. The vertical axis of the figure represents the absorbance at 450 nm minus the absorbance at 570 nm, and the horizontal axis represents the test antibody concentration. Symbols represent the average absorbance upon addition of each test antibody, and error bars represent the standard deviation. [Figure 1-2] Figure 1-2 shows the CD137 binding activity of test antibodies measured and evaluated by ELISA. The vertical axis of the figure represents the absorbance at 450 nm minus the absorbance at 570 nm, and the horizontal axis represents the test antibody concentration. The symbols represent the average absorbance at the time of addition of each test antibody. [Figure 1-3] Figure 1-3 shows the CD137 binding activity of test antibodies measured and evaluated by ELISA. The vertical axis of the figure represents the absorbance at 450 nm minus the absorbance at 570 nm, and the horizontal axis represents the test antibody concentration. The symbols represent the average absorbance upon addition of each test antibody. [Figure 1-4] Figure 1-4 shows the CD137 binding activity of test antibodies measured and evaluated by ELISA. The vertical axis of the figure represents the absorbance at 450 nm minus the absorbance at 570 nm, and the horizontal axis represents the test antibody concentration. The symbols represent the average absorbance at the time of addition of each test antibody. [Figure 2-1]Figure 2-1 shows the CD137 agonist activity of test antibodies in a co-culture test of CLDN4-expressing cancer cells (NCI-H322 cells) and 4-1BB effector cells. The vertical axis of the figure represents the relative luminescence intensity, and the horizontal axis represents the concentration of the test antibody. The symbols represent the average relative luminescence intensity upon addition of each test antibody, and the error bars represent the standard deviation. [Figure 2-2] Figure 2-2 shows the CD137 agonist activity of test antibodies in a co-culture test of CLDN4-expressing cancer cells (NCI-H322 cells) and 4-1BB effector cells. The vertical axis of the figure represents relative luminescence intensity, and the horizontal axis represents the concentration of the test antibody. The symbols indicate the average relative luminescence intensity upon addition of each test antibody. [Figure 2-3] Figure 2-3 shows the CD137 agonist activity of test antibodies in a co-culture test of CLDN4-expressing cancer cells (NCI-H322 cells) and 4-1BB effector cells. The vertical axis of the figure represents the relative light intensity, and the horizontal axis represents the concentration of the test antibody. The symbols represent the average relative light intensity upon addition of each test antibody, and the error bars represent the standard deviation. [Figure 2-4] Figure 2-4 shows the CD137 agonist activity of test antibodies in a monoculture test using 4-1BB effector cells. The vertical axis of the figure represents the relative light intensity, and the horizontal axis represents the concentration of the test antibody. The symbols represent the average relative light intensity upon addition of each test antibody, and the error bars represent the standard deviation. [Figure 2-5] Figure 2-5 shows the CD137 agonist activity of test antibodies in a monoculture test using 4-1BB effector cells. The vertical axis of the figure represents relative light intensity, and the horizontal axis represents the concentration of the test antibody. The symbols indicate the average relative light intensity upon addition of each test antibody. [Figure 2-6] Figure 2-6 shows the CD137 agonist activity of test antibodies in a monoculture test using 4-1BB effector cells. The vertical axis of the figure represents the relative light intensity, and the horizontal axis represents the concentration of the test antibody. The symbols represent the average relative light intensity upon addition of each test antibody, and the error bars represent the standard deviation. [Figure 3-1]Figure 3-1 shows the interferon-γ production promoting effect of each test antibody in a co-culture system of 60As6-Luc / GFP cells and Expanded panT cells. The vertical axis shows the amount of interferon-γ produced (pg / mL) 6 days after antibody addition, and the horizontal axis shows the antibody concentration. The symbols indicate the average amount of interferon-γ produced at each antibody concentration. [Figure 3-2] Figure 3-2 shows the interferon-γ production promoting function of each test antibody in a co-culture system of 60As6-Luc / GFP cells and Expanded panT cells. The vertical axis of the figure shows the amount of interferon-γ produced (pg / mL) four days after antibody addition, and the horizontal axis shows the antibody concentration. The symbols show the average amount of interferon-γ produced at each antibody concentration, and the error bars show the standard deviation. [Figure 3-3] Figure 3-3 shows the amount of interferon-γ produced when each test antibody was added in a monoculture system of expanded panT cells. The vertical axis of the figure shows the amount of interferon-γ produced (pg / mL) 6 days after antibody addition, and the horizontal axis shows the antibody concentration. The symbols indicate the average amount of interferon-γ produced at each antibody concentration. [Figure 3-4] Figure 3-4 shows the amount of interferon-γ produced when each test antibody was added in a monoculture system of expanded panT cells. The vertical axis of the figure shows the amount of interferon-γ produced (pg / mL) 5 days after antibody addition, and the horizontal axis shows the antibody concentration. The symbols show the average amount of interferon-γ produced at each antibody concentration, and the error bars show the standard deviation. [Figure 4] Figure 4 shows the cancer cell cytotoxicity (cancer cell growth inhibitory activity) of each test antibody in a co-culture system of 60As6-Luc / GFP cells and Expanded panT cells. The vertical axis of the figure shows the cell growth rate in the fluorescence area increased from 0 hours to 168 hours after the start of measurement, when the well without test antibody is set at 100%. The horizontal axis shows the antibody concentration. The symbols show the average cancer cell growth rate for each antibody. [Figure 5]Figure 5 shows the cancer cell cytotoxicity of each test antibody in a co-culture system of 60As6-Luc / GFP cells and Expanded panT cells. The vertical axis of the figure shows the cancer cell killing rate (%) of the chemiluminescence measured from 60As6-Luc / GFP cells 6 days after antibody addition, where the average value for no antibody addition is set to 0% killing rate and the average value for Triton-X100 addition is set to 100% killing rate. The horizontal axis shows the antibody concentration. The symbols indicate the average cancer cell killing rate at each antibody concentration, and the error bars indicate the standard deviation. [Figure 6] Figure 6 shows the change in tumor volume in PBMC-transferred mice bearing subcutaneous NCI-H322 cells. The horizontal axis of the figure shows the number of days after the first administration, the vertical axis shows the average tumor volume, and the error bars show the standard error. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention will be described in detail below. <Definition> Terms used herein are used in the sense commonly used by those skilled in the art unless otherwise defined below.

[0014] Antibodies (or immunoglobulins) are glycoproteins whose basic structure is a four-chain structure with a symmetric Y-shape, consisting of two heavy chains with a single sequence and two light chains with a single sequence. There are five classes of antibodies: IgG, IgM, IgA, IgD, and IgE. The basic structure of antibody molecules is common to all classes: two heavy chains with a molecular weight of 50,000-70,000 and two light chains with a molecular weight of 20,000-30,000 are bound by disulfide bonds and non-covalent bonds to form a Y-shaped, four-chain antibody molecule with a molecular weight of 150,000-190,000. The heavy chains are typically polypeptide chains containing approximately 440 amino acids, and each class has a characteristic structure: Igγ, Igμ, Igα, Igδ, and Igε, corresponding to IgG, IgM, IgA, IgD, and IgE, respectively. IgG is further divided into subclasses: IgG1, IgG2, IgG3, and IgG4, with corresponding heavy chains called Igγ1, Igγ2, Igγ3, and Igγ4. Light chains typically consist of a polypeptide chain containing approximately 220 amino acids, and two types, lambda and kappa, are known, called Igλ and Igκ, respectively. These two types of light chains can pair with any type of heavy chain.

[0015] Antibody molecules have four intrachain disulfide bonds in heavy chains (five in Igμ and Igε) and two in light chains, forming a loop every 100–110 amino acid residues. The three-dimensional structures of these disulfide bonds are similar between each loop and are called structural units or domains. The domain located at the N-terminus of both heavy and light chains is called the variable region. It has diverse amino acid sequences even among antibodies of the same class (or subclass) from the same animal species, and is known to be involved in the specific binding between the antibody and the antigen. The amino acid sequence of the C-terminal domain downstream of the variable region is nearly constant for each class or subclass and is called the constant region. From the N-terminus to the C-terminus, the heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region (CH). The CH is further divided into three domains from the N-terminus: CH1, CH2, and CH3. From the N-terminus to the C-terminus, the light chain contains a light chain variable region (VL) and a light chain constant region (CL).

[0016] The amino acid sequences of the three complementarity-determining regions (CDRs) present in VH and VL vary greatly, contributing to the variability of the variable regions. CDRs are regions consisting of 5 to 10 amino acid residues, and are present at the N-terminus of each heavy chain and light chain in the order of CDR1, CDR2, and CDR3, which contact the antigen to form the antibody-binding site. It is known that the heavy chain CDRs contribute more to antigen binding than the light chain CDRs, and among CDR1 to 3, CDR3 contributes most. Meanwhile, the portions of the variable regions other than the CDRs are called framework regions (FRs), which consist of FR1 to FR4 and show relatively little variation in amino acid sequence.

[0017] When an antibody is treated with the protease papain, three antibody fragments are obtained. The two N-terminal fragments are called Fab (Fragment, antigen binding) regions. The Fab region refers to the region consisting of the VH, CH1 domain, and part of the hinge region of the heavy chain, and the light chain (VL and CL), and binds to the antigen via the VH and VL (antigen binding sites) formed by the Fab region. The C-terminal fragment is called Fc (Fragment, crystallizable) region.

[0018] As used herein, the term "IgG antibody" refers to an antibody having a Y-shaped structure consisting of two Fab regions and an Fc region. In one embodiment, the two Fab regions of an IgG antibody are Fab regions consisting of the same sequence. In another embodiment, the two Fab regions of an IgG antibody are Fab regions consisting of different sequences.

[0019] As used herein, the terms "antigen-binding fragment" and "fragment antibody" can be used interchangeably and refer to a molecule comprising at least one polypeptide chain that contains the heavy chain variable region and light chain variable region of an immunoglobulin molecule and has antigen-binding activity. Representative antigen-binding fragments include single-chain variable region fragments (scFv), Fab fragments, Fab' fragments, and F(ab')2 fragments. As used herein, "scFv" refers to a monovalent antigen-binding fragment consisting of a VH and VL linked by a linker. A Fab fragment is a monovalent antigen-binding fragment consisting of a light chain and a fragment containing the VH, CH1 domain, and part of the hinge region of the heavy chain. A Fab' fragment is a monovalent antigen-binding fragment consisting of a light chain and a fragment containing the VH, CH1 domain, and part of the hinge region of the heavy chain, and this hinge region contains cysteine ​​residues that constituted the inter-heavy chain disulfide bond. A F(ab')2 fragment is a bivalent antigen-binding fragment consisting of Fab' fragments connected by disulfide bonds. Monovalent means containing one antigen-binding site; bivalent means containing two antigen-binding sites.

[0020] As used herein, the term "bispecific antibody" refers to an antibody molecule composed of two antibodies or antigen-binding fragments that specifically bind to different antigens and have binding activity for each antigen. Bispecific antibodies of various structures are known to those skilled in the art (Non-Patent Document 6). As used herein, the term "antibody" includes full-length antibodies (various immunoglobulins, particularly IgG antibodies), antigen-binding fragments, and bispecific antibodies of any structure, unless otherwise limited by the context.

[0021] Herein, an IgG-type anti-CLDN4 antibody is referred to as an "anti-CLDN4 IgG antibody."

[0022] As used herein, the term "human antibody" refers to an antibody having a human immunoglobulin amino acid sequence. As used herein, the term "humanized antibody" refers to an antibody in which some, most, or all of the amino acid residues other than the CDRs have been substituted with amino acid residues derived from human immunoglobulin molecules. The method of humanization is not particularly limited, and humanized antibodies can be prepared by referring to, for example, U.S. Patent No. 5,225,539, U.S. Patent No. 6,180,370, etc.

[0023] The amino acid residue numbers of antibodies used herein can be specified according to the Kabat numbering system or the EU index (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., 1991, NIH Publication No. 91-3242) by designating them.

[0024] As used herein, "linked" or "linked" means that multiple components (e.g., Fab regions and Fc regions) are linked together directly or via an intermediary (e.g., a peptide linker). As used herein, "peptide linker" refers to any one or more amino acid sequences that can be introduced by genetic engineering techniques and are used to link multiple components. The length of the peptide linker used in the present invention is not particularly limited, and can be appropriately selected by those skilled in the art depending on the purpose.

[0025] As used herein, "identity" refers to the Identity value obtained using EMBOSS Needle (Nucleic Acids Res., 2015, Vol. 43, pW580-W584) with the default parameters. The parameters are as follows: Gap Open Penalty = 10 Gap Extend Penalty = 0.5 Matrix = EBLOSUM62 End Gap Penalty = false

[0026] <Anti-CLDN4-anti-CD137 bispecific antibody of the present invention> The present invention provides bispecific antibodies that bind to CLDN4 and CD137 (also referred to as "anti-CLDN4-anti-CD137 bispecific antibodies"). The anti-CLDN4-anti-CD137 bispecific antibodies of the present invention comprise 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. The anti-CLDN4-anti-CD137 bispecific antibodies of the present invention include bispecific antibodies comprising an anti-CLDN4 IgG antibody and an scFv of an anti-CD137 antibody (also referred to as "anti-CD137 scFv"). In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention is a bispecific antibody comprising an anti-CLDN4 IgG antibody and an anti-CD137 scFv.

[0027] As used herein, an "anti-CLDN4 antibody" refers to an antibody capable of binding to human CLDN4, and an "anti-CD137 antibody" refers to an antibody capable of binding to human CD137. Binding to human CLDN4 or human CD137 can be confirmed using known methods for measuring binding activity. Examples of methods for measuring binding activity include enzyme-linked immunosorbent assay (ELISA) and flow cytometry. ELISA and flow cytometry can be performed using methods commonly used by those skilled in the art, such as the methods described in Example 3 or 5.

[0028] The anti-CLDN4-anti-CD137 bispecific antibody of the present invention may have any structure as long as it binds to both CLDN4 and CD137, and examples include bispecific antibodies having the structure described in Non-Patent Document 6. In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention may be a conjugate of the Fab region of an anti-CLDN4 antibody and the Fab region of an anti-CD137 antibody, a conjugate of an anti-CLDN4 IgG antibody and an IgG-type anti-CD137 antibody (also referred to as an "anti-CD137 IgG antibody"), a conjugate of an anti-CLDN4 IgG antibody and an antigen-binding fragment of an anti-CD137 antibody, a conjugate of an antigen-binding fragment of an anti-CLDN4 antibody and an anti-CD137 IgG antibody, or a conjugate of an antigen-binding fragment of an anti-CLDN4 antibody and an antigen-binding fragment of an anti-CD137 antibody.

[0029] In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises a full-length anti-CLDN4 antibody and a full-length anti-CD137 antibody. In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises an anti-CLDN4 IgG antibody and an anti-CD137 IgG antibody. The two antibodies may be linked via a linker. In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises an antigen-binding fragment of a full-length anti-CLDN4 antibody and an anti-CD137 antibody. The linking site between the two antibodies is not particularly limited, and they may be linked directly or via a linker. In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises an scFv of an anti-CLDN4 IgG antibody and an anti-CD137 antibody (referred to herein as "anti-CD137scFv"). In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises an anti-CLDN4 IgG antibody and an anti-CD137 scFv, and the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody. In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises an anti-CLDN4 IgG antibody and an anti-CD137 scFv, and the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the light chain of the anti-CLDN4 antibody. In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises an anti-CLDN4 IgG antibody and an anti-CD137 scFv, and the carboxy terminus of the anti-CD137 scFv is linked to the amino terminus of the heavy chain of the anti-CLDN4 antibody. In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises an anti-CLDN4 IgG antibody and an anti-CD137 scFv, and the carboxy terminus of the anti-CD137 scFv is linked to the amino terminus of the light chain of the anti-CLDN4 antibody. In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises an antigen-binding fragment of the anti-CLDN4 antibody and an antigen-binding fragment of anti-CD137. In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises an scFv of the anti-CLDN4 antibody and an scFv of anti-CD137.In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the invention comprises an anti-CD137 scFv.

[0030] In the anti-CLDN4-anti-CD137 bispecific antibody of the present invention, the heavy chain variable region and light chain variable region of the anti-CLDN4 antibody and anti-CD137 antibody may be derived from a human antibody, a humanized antibody, or a combination thereof. In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention is a human antibody, a humanized antibody, or an antibody consisting of a combination thereof.

[0031] The anti-CLDN4-anti-CD137 bispecific antibody of the present invention is an anti-CLDN4-anti-CD137 bispecific antibody comprising the heavy chain variable region and light chain variable region of an anti-CLDN4 antibody and the heavy chain variable region and light chain variable region of an anti-CD137 antibody, wherein the heavy chain variable region and light chain variable region of the anti-CLDN4 antibody are either (a) or (b) below: (a) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 2, a CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 66 of SEQ ID NO: 2, and a CDR3 consisting of the amino acid sequence from amino acid numbers 99 to 114 of SEQ ID NO: 2, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 24 to 35 of SEQ ID NO: 4, a CDR2 consisting of the amino acid sequence from amino acid numbers 51 to 57 of SEQ ID NO: 4, and a CDR3 consisting of the amino acid sequence from amino acid numbers 90 to 98 of SEQ ID NO: 4; or (b) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 6, a CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 66 of SEQ ID NO: 6, and a CDR3 consisting of the amino acid sequence from amino acid numbers 99 to 112 of SEQ ID NO: 6; and a light chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 24 to 35 of SEQ ID NO: 8, a CDR2 consisting of the amino acid sequence from amino acid numbers 51 to 57 of SEQ ID NO: 8, and a CDR3 consisting of the amino acid sequence from amino acid numbers 90 to 98 of SEQ ID NO: 8.

[0032] In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises the heavy chain variable region and the light chain variable region of an anti-CLDN4 antibody described in (a) or (b) below: (a) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 125 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 109 of SEQ ID NO: 4; or (b) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 123 of SEQ ID NO: 6 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 109 of SEQ ID NO: 8.

[0033] The anti-CLDN4-anti-CD137 bispecific antibody of the present invention may comprise a full-length antibody such as an IgG antibody as the anti-CLDN4 antibody, or an antigen-binding fragment such as a Fab fragment, a Fab' fragment, a F(ab')2 fragment, or an scFv.

[0034] The heavy chain constant region of the anti-CLDN4 antibody contained in the anti-CLDN4-anti-CD137 bispecific antibody of the present invention can be any of Igγ, Igμ, Igα, Igδ, or Igε constant regions. Igγ can be selected from, for example, Igγ1, Igγ2, Igγ3, or Igγ4. The light chain constant region of the anti-CLDN4 antibody contained in the anti-CLDN4-anti-CD137 bispecific antibody of the present invention can be any of Igλ or Igκ constant regions. When the anti-CLDN4 antibody contained in the anti-CLDN4-anti-CD137 bispecific antibody of the present invention is an IgG antibody, in one embodiment, the heavy and light chains of the anti-CLDN4 antibody are human Igγ1 and Igκ, respectively. In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises a full-length anti-CLDN4 antibody. In one embodiment, the anti-CLDN4 antibody included in the anti-CLDN4-anti-CD137 bispecific antibody of the present invention is an IgG antibody comprising the heavy chain variable region and light chain variable region of an anti-CLDN4 antibody (anti-CLDN4 IgG antibody).

[0035] When the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises an Fc region, the Fc region of the bispecific antibody may contain a mutation that reduces antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). L234A is a substitution of leucine at amino acid position 234 in the human Igγ1 constant region with alanine. L235A is a substitution of leucine at amino acid position 235 in the human Igγ1 constant region with alanine. The amino acid mutations L234A and L235A in the human Igγ1 constant region are referred to as "LALA mutations." Here, positions 234 and 235 are amino acid positions in the human Igγ1 constant region according to the EU index. This mutation is known to reduce the ADCC and CDC of antibodies (Mol. Immunol., 1992, Vol. 29, pp. 633-639; J. Immunol., 2000, Vol. 164, pp. 4178-4184). P331G or P331S refers to a substitution of proline at amino acid position 331 in the human Igγ1 constant region with glycine or serine. Position 331 is the amino acid position in the human Igγ1 constant region according to the EU index. This mutation is known to reduce the CDC of antibodies (J. Immunol., 2000, Vol. 164(8), pp. 4178-4184).

[0036] In one embodiment, the anti-CLDN4 IgG antibody included in the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises an Fc region comprising amino acid mutations L234A and L235A (LALA mutations). In one embodiment, the anti-CLDN4 IgG antibody comprises an Fc region comprising either a P331G or P331S mutation. In one embodiment, the anti-CLDN4 IgG antibody comprises an Fc region comprising the LALA mutation and either a P331G or P331S mutation.

[0037] The present inventors have created novel anti-CD137 antibodies or their binding fragments (scFv) in the process of creating the anti-CLDN4-anti-CD137 bispecific antibodies of the present invention. The present inventors have identified anti-CLDN4-anti-CD137 bispecific antibodies that do not transduce CD137 signaling when bound to CD137 alone, but transduce CD137 signaling only when bound to both CD137 and CLDN4 simultaneously. These antibodies do not exhibit agonist activity when not bound to CLDN4. On the other hand, binding of anti-CLDN4-anti-CD137 bispecific antibodies to CLDN4 and CD137 leads to CD137 signaling and T cell activation. This effect is expected to reduce the hepatotoxicity and other adverse effects observed in clinical trials of urelumab, and is therefore expected to have a favorable profile for anti-CD137 antibodies to be used in bispecific antibodies.

[0038] In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises the heavy chain variable region and the light chain variable region of an anti-CD137 antibody described in any one of the following (a) to (d): (a) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 10, a CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 66 of SEQ ID NO: 10, and a CDR3 consisting of the amino acid sequence from amino acid numbers 99 to 107 of SEQ ID NO: 10, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 24 to 34 of SEQ ID NO: 12, a CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 56 of SEQ ID NO: 12, and a CDR3 consisting of the amino acid sequence from amino acid numbers 89 to 98 of SEQ ID NO: 12; (b) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 14, a CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 65 of SEQ ID NO: 14, and a CDR3 consisting of the amino acid sequence from amino acid numbers 98 to 107 of SEQ ID NO: 14, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 23 to 35 of SEQ ID NO: 16, a CDR2 consisting of the amino acid sequence from amino acid numbers 51 to 57 of SEQ ID NO: 16, and a CDR3 consisting of the amino acid sequence from amino acid numbers 90 to 100 of SEQ ID NO: 16; (c) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 18, a CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 65 of SEQ ID NO: 18, and a CDR3 consisting of the amino acid sequence from amino acid numbers 98 to 107 of SEQ ID NO: 18, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 23 to 35 of SEQ ID NO: 20, a CDR2 consisting of the amino acid sequence from amino acid numbers 51 to 57 of SEQ ID NO: 20, and a CDR3 consisting of the amino acid sequence from amino acid numbers 90 to 100 of SEQ ID NO: 20; or (d) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 22, a CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 65 of SEQ ID NO: 22, and a CDR3 consisting of the amino acid sequence from amino acid numbers 98 to 110 of SEQ ID NO: 22; and a light chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 23 to 35 of SEQ ID NO: 24, a CDR2 consisting of the amino acid sequence from amino acid numbers 51 to 57 of SEQ ID NO: 24, and a CDR3 consisting of the amino acid sequence from amino acid numbers 90 to 100 of SEQ ID NO: 24.

[0039] In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises the heavy chain variable region and the light chain variable region of an anti-CD137 antibody described in any one of the following (a) to (i): (a) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 10 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 109 of SEQ ID NO: 12; (b) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 14 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 16; (c) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 18 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 20; (d) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 121 of SEQ ID NO: 22 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 24 (e) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 26 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 134 to 242 of SEQ ID NO: 26; (f) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 28 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 134 to 244 of SEQ ID NO: 28; (g) a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 30 and a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 132 to 249 of SEQ ID NO: 30; (h) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 32 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 134 to 244 of SEQ ID NO: 32; or (i) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 121 of SEQ ID NO: 34 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 137 to 247 of SEQ ID NO: 34.

[0040] The anti-CLDN4-anti-CD137 bispecific antibody of the present invention may comprise, as the anti-CD137 antibody, a full-length antibody such as an IgG antibody, or an antigen-binding fragment such as a Fab fragment, a Fab' fragment, a F(ab')2 fragment, or an scFv. In one embodiment, the anti-CD137 antibody contained in the anti-CLDN4-anti-CD137 bispecific antibody is an antigen-binding fragment of the anti-CD137 antibody. In one embodiment, the antigen-binding fragment of the anti-CD137 antibody contained in the anti-CLDN4-anti-CD137 bispecific antibody is an anti-CD137 scFv.

[0041] In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises an anti-CD137 scFv comprising the heavy chain variable region and the light chain variable region of an anti-CD137 antibody described in (a) to (m) below: (a) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 10, a CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 66 of SEQ ID NO: 10, and a CDR3 consisting of the amino acid sequence from amino acid numbers 99 to 107 of SEQ ID NO: 10, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 24 to 34 of SEQ ID NO: 12, a CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 56 of SEQ ID NO: 12, and a CDR3 consisting of the amino acid sequence from amino acid numbers 89 to 98 of SEQ ID NO: 12; (b) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 14, a CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 65 of SEQ ID NO: 14, and a CDR3 consisting of the amino acid sequence from amino acid numbers 98 to 107 of SEQ ID NO: 14, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 23 to 35 of SEQ ID NO: 16, a CDR2 consisting of the amino acid sequence from amino acid numbers 51 to 57 of SEQ ID NO: 16, and a CDR3 consisting of the amino acid sequence from amino acid numbers 90 to 100 of SEQ ID NO: 16; (c) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 18, a CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 65 of SEQ ID NO: 18, and a CDR3 consisting of the amino acid sequence from amino acid numbers 98 to 107 of SEQ ID NO: 18, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 23 to 35 of SEQ ID NO: 20, a CDR2 consisting of the amino acid sequence from amino acid numbers 51 to 57 of SEQ ID NO: 20, and a CDR3 consisting of the amino acid sequence from amino acid numbers 90 to 100 of SEQ ID NO: 20; (d) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 22, a CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 65 of SEQ ID NO: 22, and a CDR3 consisting of the amino acid sequence from amino acid numbers 98 to 110 of SEQ ID NO: 22, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 23 to 35 of SEQ ID NO: 24, a CDR2 consisting of the amino acid sequence from amino acid numbers 51 to 57 of SEQ ID NO: 24, and a CDR3 consisting of the amino acid sequence from amino acid numbers 90 to 100 of SEQ ID NO: 24; (e) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 10 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 109 of SEQ ID NO: 12; (f) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 14 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 16; (g) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 18 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 20; (h) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 121 of SEQ ID NO: 22 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 24 (i) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 26 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 134 to 242 of SEQ ID NO: 26; (j) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 28 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 134 to 244 of SEQ ID NO: 28; (k) a light chain variable region consisting of the amino acid sequence of amino acids 1 to 111 of SEQ ID NO: 30 and a heavy chain variable region consisting of the amino acid sequence of amino acids 132 to 249 of SEQ ID NO: 30; (l) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 32 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 134 to 244 of SEQ ID NO: 32; or (m) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 121 of SEQ ID NO: 34 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 137 to 247 of SEQ ID NO: 34.

[0042] In anti-CD137scFv, the type and length of the linker linking the heavy chain variable region and light chain variable region of the anti-CD137 antibody are not particularly limited and can be appropriately selected by those skilled in the art. A peptide linker may be used as the linker. The preferred length is 5 amino acids or more (the upper limit is not particularly limited, but is usually 30 amino acids or less, preferably 20 amino acids or less), with 15 amino acids being particularly preferred. Examples of linkers that can be used include a glycine-serine linker (GS linker) and a glycine-lysine-proline-glycine-serine linker (GKPGS linker). Examples of such linkers include the following: Ser Gly-Ser Gly-Gly-Ser Ser-Gly-Gly Gly-Gly-Gly-Ser (SEQ ID NO: 46) Ser-Gly-Gly-Gly (SEQ ID NO: 47) Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 48) Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 49) Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 50) Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 51) Gly-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 52) Ser-Gly-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 53) Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 54) (Gly-Gly-Gly-Gly-Ser)n (Ser-Gly-Gly-Gly-Gly)n Gly-Lys-Pro-Gly-Ser (SEQ ID NO: 55) (Gly-Lys-Pro-Gly-Ser)n The above n represents an integer of 1 or greater. In one aspect, the above n is 1 to 10, 2 to 8, or 2 to 6. The length and sequence of the linker can be appropriately selected by those skilled in the art depending on the purpose. In one embodiment, the linker used in the anti-CD137scFv is a GS linker. In one embodiment, the linker used in the anti-CD137scFv is a GS linker of (Gly-Gly-Gly-Gly-Ser)n, where n is either 3 or 4.

[0043] In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises any one of the following anti-CD137 scFvs (a) to (e): (a) an anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 1 to 242 of SEQ ID NO: 26; (b) an anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 1 to 244 of SEQ ID NO: 28; (c) an anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 1 to 249 of SEQ ID NO: 30; (d) an anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 1 to 244 of SEQ ID NO: 32; or (e) An anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 1 to 247 of SEQ ID NO: 34.

[0044] 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 may be linked via a linker. The type and length of the linker linking the anti-CLDN4 antibody or its antigen-binding fragment and the anti-CD137 antibody or its antigen-binding fragment are not particularly limited and can be appropriately selected by those skilled in the art. A peptide linker may be used as the linker. The linker preferably has a length of 5 amino acids or more (the upper limit is not particularly limited, but is usually 30 amino acids or less, preferably 20 amino acids or less), and particularly preferably 10 amino acids. Examples of peptide linkers that can be used include a glycine-serine linker (GS linker) and a glycine-lysine-proline-glycine-serine linker (GKPGS linker). Examples of such linkers include the following: Ser Gly-Ser Gly-Gly-Ser Ser-Gly-Gly Gly-Gly-Gly-Ser (SEQ ID NO: 46) Ser-Gly-Gly-Gly (SEQ ID NO: 47) Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 48) Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 49) Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 50) Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 51) Gly-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 52) Ser-Gly-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 53) Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 54) (Gly-Gly-Gly-Gly-Ser)n (Ser-Gly-Gly-Gly-Gly)n Gly-Lys-Pro-Gly-Ser (SEQ ID NO: 55) (Gly-Lys-Pro-Gly-Ser)n The above-mentioned n represents an integer of 1 or greater. In one aspect, the above-mentioned n is 1 to 10, 2 to 8, or 2 to 6. The length and sequence of the peptide linker can be appropriately selected by those skilled in the art depending on the purpose. In one embodiment, the linker used to link an anti-CLDN4 antibody or antigen-binding fragment thereof with an anti-CD137 antibody or antigen-binding fragment thereof is a GS linker or a GKPGS linker, and in one embodiment, is a linker consisting of the amino acid sequence of SEQ ID NO: 54.

[0045] In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises an anti-CLDN4 IgG antibody and an anti-CD137scFv, and has a structure in which the anti-CLDN4 IgG antibody and the anti-CD137scFv are linked via a linker. The linking site between the anti-CLDN4 IgG antibody and the anti-CD137scFv is not particularly limited, and the anti-CLDN4-anti-CD137 bispecific antibody of the present invention can have various structures, for example, in which the amino terminus of the heavy chain of the anti-CLDN4 IgG antibody is linked to the carboxy terminus of the anti-CD137scFv, the amino terminus of the light chain of the anti-CLDN4 IgG antibody is linked to the carboxy terminus of the anti-CD137scFv, the amino terminus of the anti-CD137scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 IgG antibody, or the amino terminus of the anti-CD137scFv is linked to the carboxy terminus of the light chain of the anti-CLDN4 IgG antibody. In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention has the amino terminus of an anti-CD137 scFv linked to the carboxy terminus of the heavy chain of an anti-CLDN4 IgG antibody, or the amino terminus of an anti-CD137 scFv linked to the carboxy terminus of the light chain of an anti-CLDN4 IgG antibody. In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention has the amino terminus of an anti-CD137 scFv linked to the carboxy terminus of the heavy chain of an anti-CLDN4 IgG antibody.

[0046] 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, in which the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain or light chain of the anti-CLDN4 IgG antibody via a linker, and the heavy chain variable region and light chain variable region of the anti-CD137 scFv are any of the following (a) to (m): (a) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 10, a CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 66 of SEQ ID NO: 10, and a CDR3 consisting of the amino acid sequence from amino acid numbers 99 to 107 of SEQ ID NO: 10, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 24 to 34 of SEQ ID NO: 12, a CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 56 of SEQ ID NO: 12, and a CDR3 consisting of the amino acid sequence from amino acid numbers 89 to 98 of SEQ ID NO: 12; (b) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 14, a CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 65 of SEQ ID NO: 14, and a CDR3 consisting of the amino acid sequence from amino acid numbers 98 to 107 of SEQ ID NO: 14, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 23 to 35 of SEQ ID NO: 16, a CDR2 consisting of the amino acid sequence from amino acid numbers 51 to 57 of SEQ ID NO: 16, and a CDR3 consisting of the amino acid sequence from amino acid numbers 90 to 100 of SEQ ID NO: 16; (c) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 18, a CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 65 of SEQ ID NO: 18, and a CDR3 consisting of the amino acid sequence from amino acid numbers 98 to 107 of SEQ ID NO: 18, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 23 to 35 of SEQ ID NO: 20, a CDR2 consisting of the amino acid sequence from amino acid numbers 51 to 57 of SEQ ID NO: 20, and a CDR3 consisting of the amino acid sequence from amino acid numbers 90 to 100 of SEQ ID NO: 20; (d) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 22, a CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 65 of SEQ ID NO: 22, and a CDR3 consisting of the amino acid sequence from amino acid numbers 98 to 110 of SEQ ID NO: 22, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 23 to 35 of SEQ ID NO: 24, a CDR2 consisting of the amino acid sequence from amino acid numbers 51 to 57 of SEQ ID NO: 24, and a CDR3 consisting of the amino acid sequence from amino acid numbers 90 to 100 of SEQ ID NO: 24; (e) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 10 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 109 of SEQ ID NO: 12; (f) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 14 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 16; (g) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 18 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 20; (h) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 121 of SEQ ID NO: 22 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 24 (i) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 26 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 134 to 242 of SEQ ID NO: 26; (j) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 28 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 134 to 244 of SEQ ID NO: 28; (k) a light chain variable region consisting of the amino acid sequence of amino acids 1 to 111 of SEQ ID NO: 30 and a heavy chain variable region consisting of the amino acid sequence of amino acids 132 to 249 of SEQ ID NO: 30; (l) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 32 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 134 to 244 of SEQ ID NO: 32; or (m) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 121 of SEQ ID NO: 34 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 137 to 247 of SEQ ID NO: 34.

[0047] 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, in which the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain or light chain of the anti-CLDN4 IgG antibody via a linker, and the anti-CD137 scFv is any of the following (a) to (e): (a) an anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 1 to 242 of SEQ ID NO: 26; (b) an anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 1 to 244 of SEQ ID NO: 28; (c) an anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 1 to 249 of SEQ ID NO: 30; (d) an anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 1 to 244 of SEQ ID NO: 32; or (e) An anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 1 to 247 of SEQ ID NO: 34.

[0048] In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention is any of the following anti-CLDN4-anti-CD137 scFv bispecific antibodies (a) to (h): (a) a heavy chain of an anti-CLDN4 antibody comprising a heavy chain variable region including CDR1 consisting of the amino acid sequence from 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequence from 50 to 66 of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence from 99 to 114 of SEQ ID NO: 2; a light chain of an anti-CLDN4 antibody comprising a light chain variable region including CDR1 consisting of the amino acid sequence from 24 to 35 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence from 51 to 57 of SEQ ID NO: 4, and CDR3 consisting of the amino acid sequence from 90 to 98 of SEQ ID NO: 4; and a light chain of an anti-CLDN4 antibody comprising a light chain variable region including CDR1 consisting of the amino acid sequence from 31 to 35 of SEQ ID NO: 10, CDR2 consisting of the amino acid sequence from 50 to 66 of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence from 99 to 114 of SEQ ID NO: 2; a bispecific antibody comprising an anti-CD137scFv comprising a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence from 50 to 66 of SEQ ID NO: 10, a CDR2 consisting of the amino acid sequence from 50 to 66 of SEQ ID NO: 10, and a CDR3 consisting of the amino acid sequence from 99 to 107 of SEQ ID NO: 10, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence from 24 to 34 of SEQ ID NO: 12, a CDR2 consisting of the amino acid sequence from 50 to 56 of SEQ ID NO: 12, and a CDR3 consisting of the amino acid sequence from 89 to 98 of SEQ ID NO: 12, wherein the amino terminus of the anti-CD137scFv is linked to the heavy chain carboxy terminus of the anti-CLDN4 antibody via a linker; (b) a heavy chain of an anti-CLDN4 antibody comprising a heavy chain variable region including CDR1 consisting of the amino acid sequence from 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequence from 50 to 66 of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence from 99 to 114 of SEQ ID NO: 2; a light chain of an anti-CLDN4 antibody comprising a light chain variable region including CDR1 consisting of the amino acid sequence from 24 to 35 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence from 51 to 57 of SEQ ID NO: 4, and CDR3 consisting of the amino acid sequence from 90 to 98 of SEQ ID NO: 4; and a light chain of an anti-CLDN4 antibody comprising a light chain variable region including CDR1 consisting of the amino acid sequence from 31 to 35 of SEQ ID NO: 14, CDR2 consisting of the amino acid sequence from 51 to 57 of SEQ ID NO: 4, and CDR3 consisting of the amino acid sequence from 90 to 98 of SEQ ID NO: 4. a bispecific antibody comprising an anti-CD137scFv comprising a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid nos. 50 to 65 of SEQ ID NO: 14, a CDR2 consisting of the amino acid sequence from amino acid nos. 51 to 57 of SEQ ID NO: 16, and a CDR3 consisting of the amino acid sequence from amino acid nos. 90 to 100 of SEQ ID NO: 16, wherein the amino terminus of the anti-CD137scFv is linked to the heavy chain carboxy terminus of the anti-CLDN4 antibody via a linker; (c) a heavy chain of an anti-CLDN4 antibody comprising a heavy chain variable region including CDR1 consisting of the amino acid sequence from 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequence from 50 to 66 of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence from 99 to 114 of SEQ ID NO: 2; a light chain of an anti-CLDN4 antibody comprising a light chain variable region including CDR1 consisting of the amino acid sequence from 24 to 35 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence from 51 to 57 of SEQ ID NO: 4, and CDR3 consisting of the amino acid sequence from 90 to 98 of SEQ ID NO: 4; and a light chain of an anti-CLDN4 antibody comprising a light chain variable region including CDR1 consisting of the amino acid sequence from 31 to 35 of SEQ ID NO: 18, CDR2 consisting of the amino acid sequence from 51 to 57 of SEQ ID NO: 4, and CDR3 consisting of the amino acid sequence from 90 to 98 of SEQ ID NO: 4. a bispecific antibody comprising an anti-CD137scFv comprising a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid nos. 50 to 65 of SEQ ID NO: 18, a CDR2 consisting of the amino acid sequence from amino acid nos. 51 to 57 of SEQ ID NO: 20, and a CDR3 consisting of the amino acid sequence from amino acid nos. 90 to 100 of SEQ ID NO: 20, wherein the amino terminus of the anti-CD137scFv is linked to the heavy chain carboxy terminus of the anti-CLDN4 antibody via a linker; (d) a heavy chain of an anti-CLDN4 antibody comprising a heavy chain variable region including CDR1 consisting of the amino acid sequence from 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequence from 50 to 66 of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence from 99 to 114 of SEQ ID NO: 2; a light chain of an anti-CLDN4 antibody comprising a light chain variable region including CDR1 consisting of the amino acid sequence from 24 to 35 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence from 51 to 57 of SEQ ID NO: 4, and CDR3 consisting of the amino acid sequence from 90 to 98 of SEQ ID NO: 4; and a light chain of an anti-CLDN4 antibody comprising a light chain variable region including CDR1 consisting of the amino acid sequence from 31 to 35 of SEQ ID NO: 22, CDR2 consisting of the amino acid sequence from 51 to 57 of SEQ ID NO: 4, and CDR3 consisting of the amino acid sequence from 90 to 98 of SEQ ID NO: 4. a bispecific antibody comprising an anti-CD137scFv comprising a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence from 50 to 65 of SEQ ID NO: 22, a CDR2 consisting of the amino acid sequence from 50 to 65 of SEQ ID NO: 22, and a CDR3 consisting of the amino acid sequence from 98 to 110 of SEQ ID NO: 22, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence from 23 to 35 of SEQ ID NO: 24, a CDR2 consisting of the amino acid sequence from 51 to 57 of SEQ ID NO: 24, and a CDR3 consisting of the amino acid sequence from 90 to 100 of SEQ ID NO: 24, wherein the amino terminus of the anti-CD137scFv is linked to the heavy chain carboxy terminus of the anti-CLDN4 antibody via a linker; (e) a heavy chain of an anti-CLDN4 antibody comprising a heavy chain variable region including CDR1 consisting of the amino acid sequence from 31 to 35 of SEQ ID NO: 6, CDR2 consisting of the amino acid sequence from 50 to 66 of SEQ ID NO: 6, and CDR3 consisting of the amino acid sequence from 99 to 112 of SEQ ID NO: 6; a light chain of an anti-CLDN4 antibody comprising a light chain variable region including CDR1 consisting of the amino acid sequence from 24 to 35 of SEQ ID NO: 8, CDR2 consisting of the amino acid sequence from 51 to 57 of SEQ ID NO: 8, and CDR3 consisting of the amino acid sequence from 90 to 98 of SEQ ID NO: 8; and a light chain of an anti-CLDN4 antibody comprising a light chain variable region including CDR1 consisting of the amino acid sequence from 31 to 35 of SEQ ID NO: 10, CDR2 consisting of the amino acid sequence from 50 to 66 of SEQ ID NO: 6, and CDR3 consisting of the amino acid sequence from 99 to 112 of SEQ ID NO: 6. a bispecific antibody comprising an anti-CD137scFv comprising a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence from 50 to 66 of SEQ ID NO: 10, a CDR2 consisting of the amino acid sequence from 50 to 66 of SEQ ID NO: 10, and a CDR3 consisting of the amino acid sequence from 99 to 107 of SEQ ID NO: 10, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence from 24 to 34 of SEQ ID NO: 12, a CDR2 consisting of the amino acid sequence from 50 to 56 of SEQ ID NO: 12, and a CDR3 consisting of the amino acid sequence from 89 to 98 of SEQ ID NO: 12, wherein the amino terminus of the anti-CD137scFv is linked to the heavy chain carboxy terminus of the anti-CLDN4 antibody via a linker; (f) a heavy chain of an anti-CLDN4 antibody comprising a heavy chain variable region including CDR1 consisting of the amino acid sequence of amino acids 31 to 35 of SEQ ID NO: 6, CDR2 consisting of the amino acid sequence of amino acids 50 to 66 of SEQ ID NO: 6, and CDR3 consisting of the amino acid sequence of amino acids 99 to 112 of SEQ ID NO: 6; a light chain of an anti-CLDN4 antibody comprising a light chain variable region including CDR1 consisting of the amino acid sequence of amino acids 24 to 35 of SEQ ID NO: 8, CDR2 consisting of the amino acid sequence of amino acids 51 to 57 of SEQ ID NO: 8, and CDR3 consisting of the amino acid sequence of amino acids 90 to 98 of SEQ ID NO: 8; and a light chain of an anti-CLDN4 antibody comprising a light chain variable region including CDR1 consisting of the amino acid sequence of amino acids 31 to 35 of SEQ ID NO: 14, CDR2 consisting of the amino acid sequence of amino acids 51 to 57 of SEQ ID NO: 8, and CDR3 consisting of the amino acid sequence of amino acids 90 to 98 of SEQ ID NO: 8. a bispecific antibody comprising an anti-CD137scFv comprising a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid nos. 50 to 65 of SEQ ID NO: 14, a CDR2 consisting of the amino acid sequence from amino acid nos. 51 to 57 of SEQ ID NO: 16, and a CDR3 consisting of the amino acid sequence from amino acid nos. 90 to 100 of SEQ ID NO: 16, wherein the amino terminus of the anti-CD137scFv is linked to the heavy chain carboxy terminus of the anti-CLDN4 antibody via a linker; (g) a heavy chain of an anti-CLDN4 antibody comprising a heavy chain variable region including CDR1 consisting of the amino acid sequence of amino acids 31 to 35 of SEQ ID NO: 6, CDR2 consisting of the amino acid sequence of amino acids 50 to 66 of SEQ ID NO: 6, and CDR3 consisting of the amino acid sequence of amino acids 99 to 112 of SEQ ID NO: 6; a light chain of an anti-CLDN4 antibody comprising a light chain variable region including CDR1 consisting of the amino acid sequence of amino acids 24 to 35 of SEQ ID NO: 8, CDR2 consisting of the amino acid sequence of amino acids 51 to 57 of SEQ ID NO: 8, and CDR3 consisting of the amino acid sequence of amino acids 90 to 98 of SEQ ID NO: 8; and a light chain of an anti-CLDN4 antibody comprising a light chain variable region including CDR1 consisting of the amino acid sequence of amino acids 31 to 35 of SEQ ID NO: 18. a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid nos. 50 to 65 of SEQ ID NO: 18, a CDR2 consisting of the amino acid sequence from amino acid nos. 51 to 57 of SEQ ID NO: 20, and a CDR3 consisting of the amino acid sequence from amino acid nos. 90 to 100 of SEQ ID NO: 20, and an anti-CD137 scFv comprising a light chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid nos. 23 to 35 of SEQ ID NO: 20, a CDR2 consisting of the amino acid sequence from amino acid nos. 51 to 57 of SEQ ID NO: 20, and a CDR3 consisting of the amino acid sequence from amino acid nos. 90 to 100 of SEQ ID NO: 20, wherein the amino terminus of the anti-CD137 scFv is linked to the heavy chain carboxy terminus of the anti-CLDN4 antibody via a linker; or (h) a heavy chain of an anti-CLDN4 antibody comprising a heavy chain variable region including CDR1 consisting of the amino acid sequence of amino acids 31 to 35 of SEQ ID NO: 6, CDR2 consisting of the amino acid sequence of amino acids 50 to 66 of SEQ ID NO: 6, and CDR3 consisting of the amino acid sequence of amino acids 99 to 112 of SEQ ID NO: 6; a light chain of an anti-CLDN4 antibody comprising a light chain variable region including CDR1 consisting of the amino acid sequence of amino acids 24 to 35 of SEQ ID NO: 8, CDR2 consisting of the amino acid sequence of amino acids 51 to 57 of SEQ ID NO: 8, and CDR3 consisting of the amino acid sequence of amino acids 90 to 98 of SEQ ID NO: 8; and a light chain of an anti-CLDN4 antibody comprising a light chain variable region including CDR1 consisting of the amino acid sequence of amino acids 31 to 35 of SEQ ID NO: 22, CDR2 consisting of the amino acid sequence of amino acids 51 to 57 of SEQ ID NO: 8, and CDR3 consisting of the amino acid sequence of amino acids 90 to 98 of SEQ ID NO: 8. a heavy chain variable region comprising: CDR1 consisting of the amino acid sequence from amino acid nos. 50 to 65 of SEQ ID NO: 22; CDR2 consisting of the amino acid sequence from amino acid nos. 51 to 57 of SEQ ID NO: 24; and CDR3 consisting of the amino acid sequence from amino acid nos. 90 to 100 of SEQ ID NO: 24; and an anti-CD137 scFv comprising a light chain variable region comprising: CDR1 consisting of the amino acid sequence from amino acid nos. 23 to 35 of SEQ ID NO: 24; CDR2 consisting of the amino acid sequence from amino acid nos. 51 to 57 of SEQ ID NO: 24; and CDR3 consisting of the amino acid sequence from amino acid nos. 90 to 100 of SEQ ID NO: 24, wherein the amino terminus of the anti-CD137 scFv is linked to the heavy chain carboxy terminus of the anti-CLDN4 antibody via a linker.

[0049] In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention is any of the following anti-CLDN4-anti-CD137 scFv bispecific antibodies (a) to (j): (a) 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 125 of SEQ ID NO: 2 and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4, and an anti-CD137scFv comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 26 and a light chain variable region consisting of the amino acid sequence of amino acids 134 to 242 of SEQ ID NO: 26, wherein the amino terminus of the anti-CD137scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker; (b) 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 125 of SEQ ID NO: 2 and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4, and an anti-CD137scFv comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 28 and a light chain variable region consisting of the amino acid sequence of amino acids 134 to 244 of SEQ ID NO: 28, wherein the amino terminus of the anti-CD137scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker; (c) 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 125 of SEQ ID NO: 2 and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4, and an anti-CD137scFv comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 111 of SEQ ID NO: 30 and a heavy chain variable region consisting of the amino acid sequence of amino acids 132 to 249 of SEQ ID NO: 30, wherein the amino terminus of the anti-CD137scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker; (d) 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 125 of SEQ ID NO: 2 and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4, and an anti-CD137scFv comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 32 and a light chain variable region consisting of the amino acid sequence of amino acids 134 to 244 of SEQ ID NO: 32, wherein the amino terminus of the anti-CD137scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker; (e) 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 125 of SEQ ID NO: 2 and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4, and an anti-CD137scFv comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 121 of SEQ ID NO: 34 and a light chain variable region consisting of the amino acid sequence of amino acids 137 to 247 of SEQ ID NO: 34, wherein the amino terminus of the anti-CD137scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker; (f) 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: 6 and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 8, and an anti-CD137scFv comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 26 and a light chain variable region consisting of the amino acid sequence of amino acids 134 to 242 of SEQ ID NO: 26, wherein the amino terminus of the anti-CD137scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker; (g) 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: 6 and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 8, and an anti-CD137scFv comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 28 and a light chain variable region consisting of the amino acid sequence of amino acids 134 to 244 of SEQ ID NO: 28, wherein the amino terminus of the anti-CD137scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker; (h) 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: 6 and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 8, and an anti-CD137scFv comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 111 of SEQ ID NO: 30 and a heavy chain variable region consisting of the amino acid sequence of amino acids 132 to 249 of SEQ ID NO: 30, wherein the amino terminus of the anti-CD137scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker; (i) 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: 6 and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 8, and an anti-CD137scFv comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 32 and a light chain variable region consisting of the amino acid sequence of amino acids 134 to 244 of SEQ ID NO: 32, wherein the amino terminus of the anti-CD137scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker; (j) 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: 6 and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 8, and an anti-CD137scFv comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 121 of SEQ ID NO: 34 and a light chain variable region consisting of the amino acid sequence of amino acids 137 to 247 of SEQ ID NO: 34, wherein the amino terminus of the anti-CD137scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker; (k) 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 125 of SEQ ID NO: 2 and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4, and an anti-CD137scFv comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 26 and a light chain variable region consisting of the amino acid sequence of amino acids 134 to 242 of SEQ ID NO: 26, wherein the amino terminus of the anti-CD137scFv is linked to the carboxy terminus of the light chain of the anti-CLDN4 antibody via a linker; (l) 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 125 of SEQ ID NO: 2 and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4, and an anti-CD137scFv comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 28 and a light chain variable region consisting of the amino acid sequence of amino acids 134 to 244 of SEQ ID NO: 28, wherein the amino terminus of the anti-CD137scFv is linked to the carboxy terminus of the light chain of the anti-CLDN4 antibody via a linker; (m) 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 125 of SEQ ID NO: 2 and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4, and an anti-CD137scFv comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 111 of SEQ ID NO: 30 and a heavy chain variable region consisting of the amino acid sequence of amino acids 132 to 249 of SEQ ID NO: 30, wherein the amino terminus of the anti-CD137scFv is linked to the carboxy terminus of the light chain of the anti-CLDN4 antibody via a linker; (n) 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 125 of SEQ ID NO: 2 and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4, and an anti-CD137scFv comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 32 and a light chain variable region consisting of the amino acid sequence of amino acids 134 to 244 of SEQ ID NO: 32, wherein the amino terminus of the anti-CD137scFv is linked to the carboxy terminus of the light chain of the anti-CLDN4 antibody via a linker; (o) 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 125 of SEQ ID NO: 2 and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4, and an anti-CD137scFv comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 121 of SEQ ID NO: 34 and a light chain variable region consisting of the amino acid sequence of amino acids 137 to 247 of SEQ ID NO: 34, wherein the amino terminus of the anti-CD137scFv is linked to the carboxy terminus of the light chain of the anti-CLDN4 antibody via a linker; (p) 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: 6 and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 8, and an anti-CD137scFv comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 26 and a light chain variable region consisting of the amino acid sequence of amino acids 134 to 242 of SEQ ID NO: 26, wherein the amino terminus of the anti-CD137scFv is linked to the carboxy terminus of the light chain of the anti-CLDN4 antibody via a linker; (q) 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: 6 and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 8, and an anti-CD137scFv comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 28 and a light chain variable region consisting of the amino acid sequence of amino acids 134 to 244 of SEQ ID NO: 28, wherein the amino terminus of the anti-CD137scFv is linked to the carboxy terminus of the light chain of the anti-CLDN4 antibody via a linker; (r) 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: 6 and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 8, and an anti-CD137scFv comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 111 of SEQ ID NO: 30 and a heavy chain variable region consisting of the amino acid sequence of amino acids 132 to 249 of SEQ ID NO: 30, wherein the amino terminus of the anti-CD137scFv is linked to the carboxy terminus of the light chain of the anti-CLDN4 antibody via a linker; (s) 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: 6 and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 8, and an anti-CD137scFv comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 32 and a light chain variable region consisting of the amino acid sequence of amino acids 134 to 244 of SEQ ID NO: 32, wherein the amino terminus of the anti-CD137scFv is linked to the carboxy terminus of the light chain of the anti-CLDN4 antibody via a linker; or (t) 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: 6 and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 8, and an anti-CD137scFv comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 121 of SEQ ID NO: 34 and a light chain variable region consisting of the amino acid sequence of amino acids 137 to 247 of SEQ ID NO: 34, wherein the amino terminus of the anti-CD137scFv is linked to the carboxy terminus of the light chain of the anti-CLDN4 antibody via a linker.

[0050] In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention is an anti-CLDN4-anti-CD137 scFv bispecific antibody of the following (a) or (b): (a) a bispecific antibody comprising a polypeptide comprising an anti-CLDN4 antibody heavy chain consisting of the amino acid sequence from amino acid numbers 1 to 705 of SEQ ID NO: 36 and an anti-CD137 scFv, and a polypeptide comprising an anti-CLDN4 antibody light chain consisting of the amino acid sequence from amino acid numbers 1 to 215 of SEQ ID NO: 40; or (b) A bispecific antibody comprising a polypeptide comprising an anti-CD137 scFv and a heavy chain of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 712 of SEQ ID NO: 38, and a light chain of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 215 of SEQ ID NO: 40.

[0051] As used herein, the term "post-translational modification" refers to post-translational modification of an antibody when the antibody is expressed in a cell. Examples of post-translational modifications include pyroglutamylation, glycosylation, oxidation, deamidation, glycation, and other modifications of glutamine or glutamic acid at the N-terminus of the heavy chain, and lysine deletion due to cleavage of lysine at the C-terminus of the heavy chain by carboxypeptidase. Such post-translational modifications are known to occur in various antibodies (J. Pharm. Sci., 2008, Vol. 97, pp. 2426-2447).

[0052] 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 lysine deletion at the C-terminus of the heavy chain. It is known in the art that post-translational modifications such as pyroglutamylation of the N-terminus or lysine deletion at the C-terminus do not affect the activity of the antibody (Analytical Biochemistry, 2006, Vol. 348, pp. 24-39).

[0053] The anti-CLDN4-anti-CD137 bispecific antibodies of the present invention bind to human CLDN4 and human CD137. Whether or not they bind to human CLDN4 and human CD137 can be confirmed using known methods for measuring binding activity. Methods for measuring binding activity include, for example, ELISA and flow cytometry. ELISA and flow cytometry can be performed using methods commonly used by those skilled in the art, such as the methods described in Example 3 or 5.

[0054] The anti-CLDN4-anti-CD137 bispecific antibodies of the present invention can be easily produced by those skilled in the art using antibody production techniques known in the art, using human CLDN4 and human CD137 as antigens, or can be easily produced by those skilled in the art using methods known in the art based on the sequence information of the heavy chain variable regions and light chain variable regions of the anti-CLDN4 antibody and anti-CD137 antibody disclosed herein, etc.

[0055] The anti-CLDN4-anti-CD137 bispecific antibody of the present invention can be produced, for example, according to the method described below in <Method for producing bispecific antibody of the present invention>, without particular limitation.

[0056] <Polynucleotides of bispecific antibodies of the present invention> The present invention also provides polynucleotides that can be used to produce the anti-CLDN4-anti-CD137 bispecific antibodies of the present invention (also referred to as "polynucleotides of the bispecific antibodies of the present invention").

[0057] In one embodiment, the polynucleotide for a bispecific antibody of the present invention is a polynucleotide comprising a nucleotide sequence encoding the heavy chain variable region or light chain variable region of an anti-CLDN4 antibody selected from the group consisting of the following (a) to (d): (a) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 125 of SEQ ID NO: 2; (b) a polynucleotide comprising a nucleotide sequence encoding the light chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 109 of SEQ ID NO: 4; (c) a polynucleotide comprising a nucleotide sequence encoding the heavy chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 123 of SEQ ID NO: 6; or (d) A polynucleotide comprising a base sequence encoding the light chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 109 of SEQ ID NO: 8.

[0058] In one embodiment, the polynucleotide of the bispecific antibody of the present invention is a polynucleotide comprising a nucleotide sequence encoding the heavy chain variable region and the light chain variable region of an anti-CLDN4 antibody selected from the following (a) or (b): (a) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence of amino acids 1 to 125 of SEQ ID NO: 2, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4; or (b) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 123 of SEQ ID NO: 6, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 109 of SEQ ID NO: 8.

[0059] In one embodiment, the polynucleotide for a bispecific antibody of the present invention is a polynucleotide comprising a nucleotide sequence encoding the heavy chain variable region or light chain variable region of an anti-CD137 antibody selected from the group consisting of the following (a) to (r): (a) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 10; (b) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 109 of SEQ ID NO: 12; (c) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 14; (d) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 16; (e) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 18; (f) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 20; (g) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 121 of SEQ ID NO: 22; (h) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 24; (i) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 26; (j) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 134 to 242 of SEQ ID NO: 26; (k) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 28; (l) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 134 to 244 of SEQ ID NO: 28; (m) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 30; (n) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 132 to 249 of SEQ ID NO: 30; (o) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 32; (p) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 134 to 244 of SEQ ID NO: 32; (q) a polynucleotide comprising a nucleotide sequence encoding the heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 121 of SEQ ID NO: 34; or (r) A polynucleotide comprising a base sequence encoding the light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 137 to 247 of SEQ ID NO: 34.

[0060] In one embodiment, the polynucleotide of a bispecific antibody of the present invention is a polynucleotide comprising a nucleotide sequence encoding the heavy chain variable region and the light chain variable region of an anti-CD137 antibody selected from the group consisting of the following (a) to (i): (a) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 10, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 12; (b) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 14, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 111 of SEQ ID NO: 16; (c) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 18, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 111 of SEQ ID NO: 20; (d) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 121 of SEQ ID NO: 22, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 111 of SEQ ID NO: 24; (e) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 26, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 134 to 242 of SEQ ID NO: 26; (f) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 28, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 134 to 244 of SEQ ID NO: 28; (g) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 111 of SEQ ID NO: 30, and a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 132 to 249 of SEQ ID NO: 30; (h) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 32, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 134 to 244 of SEQ ID NO: 32; or (i) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 121 of SEQ ID NO: 34, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 137 to 247 of SEQ ID NO: 34.

[0061] In one embodiment, the polynucleotide of the bispecific antibody of the present invention is a polynucleotide comprising a nucleotide sequence encoding an anti-CD137scFv selected from the group consisting of the following (a) to (e): (a) a polynucleotide comprising a nucleotide sequence encoding an anti-CD137scFv consisting of the amino acid sequence from amino acid numbers 1 to 242 of SEQ ID NO: 26; (b) a polynucleotide comprising a nucleotide sequence encoding an anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 1 to 244 of SEQ ID NO: 28; (c) a polynucleotide comprising a nucleotide sequence encoding an anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 1 to 249 of SEQ ID NO: 30; (d) a polynucleotide comprising a nucleotide sequence encoding an anti-CD137scFv consisting of the amino acid sequence from amino acid numbers 1 to 244 of SEQ ID NO: 32; or (e) A polynucleotide comprising a base sequence encoding an anti-CD137scFv consisting of the amino acid sequence from amino acid numbers 1 to 247 of SEQ ID NO: 34.

[0062] In one embodiment, the polynucleotide of the bispecific antibody of the present invention is a polynucleotide selected from the group consisting of the following (a) to (e): (a) a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the heavy chain of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 705 of SEQ ID NO: 36 and an anti-CD137scFv; (b) a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the heavy chain of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 712 of SEQ ID NO: 38 and an anti-CD137scFv; (c) a polynucleotide comprising a nucleotide sequence encoding the light chain of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 215 of SEQ ID NO: 40; (d) a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an anti-CLDN4 antibody heavy chain consisting of the amino acid sequence from amino acid numbers 1 to 705 of SEQ ID NO: 36 and an anti-CD137scFv, and a polynucleotide comprising a nucleotide sequence encoding an anti-CLDN4 antibody light chain consisting of the amino acid sequence from amino acid numbers 1 to 215 of SEQ ID NO: 40; or (e) A polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an anti-CLDN4 antibody heavy chain consisting of the amino acid sequence from amino acid numbers 1 to 712 of SEQ ID NO: 38 and an anti-CD137scFv, and a polynucleotide comprising a nucleotide sequence encoding an anti-CLDN4 antibody light chain consisting of the amino acid sequence from amino acid numbers 1 to 215 of SEQ ID NO: 40.

[0063] Polynucleotides of bispecific antibodies of the present invention can be easily prepared by those skilled in the art based on their nucleotide sequences using methods known in the art. For example, polynucleotides of bispecific antibodies of the present invention can be synthesized using gene synthesis methods known in the art. Various methods known to those skilled in the art can be used for such gene synthesis, such as the antibody gene synthesis method described in WO 90 / 07861.

[0064] <Expression vector for bispecific antibodies of the present invention> The present invention also provides expression vectors comprising polynucleotides of the bispecific antibodies of the present invention (also referred to as "expression vectors of the bispecific antibodies of the present invention"). These polynucleotides may be contained in separate vectors, or multiple polynucleotides may be contained in a single vector.

[0065] In one embodiment, the expression vector for a bispecific antibody of the present invention comprises a polynucleotide selected from the group consisting of the following (a) to (aa): (a) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 125 of SEQ ID NO: 2; (b) a polynucleotide comprising a nucleotide sequence encoding the light chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 109 of SEQ ID NO: 4; (c) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 123 of SEQ ID NO: 6; (d) A polynucleotide comprising a base sequence encoding the light chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 109 of SEQ ID NO: 8. (e) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 10; (f) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 109 of SEQ ID NO: 12; (g) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 14; (h) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 16; (i) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 18; (j) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 20; (k) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 121 of SEQ ID NO: 22; (l) A polynucleotide comprising a nucleotide sequence encoding the light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 24. (m) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 26; (n) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 134 to 242 of SEQ ID NO: 26; (o) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 28; (p) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 134 to 244 of SEQ ID NO: 28; (q) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 30; (r) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 132 to 249 of SEQ ID NO: 30; (s) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 32; (t) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 134 to 244 of SEQ ID NO: 32; (u) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 121 of SEQ ID NO: 34; (v) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 137 to 247 of SEQ ID NO: 34; (w) a polynucleotide comprising a nucleotide sequence encoding an anti-CD137scFv consisting of the amino acid sequence from amino acid numbers 1 to 242 of SEQ ID NO: 26; (x) a polynucleotide comprising a nucleotide sequence encoding an anti-CD137scFv consisting of the amino acid sequence from amino acid numbers 1 to 244 of SEQ ID NO: 28; (y) a polynucleotide comprising a nucleotide sequence encoding an anti-CD137scFv consisting of the amino acid sequence from amino acid numbers 1 to 249 of SEQ ID NO: 30; (z) a polynucleotide comprising a nucleotide sequence encoding an anti-CD137scFv consisting of the amino acid sequence from amino acid numbers 1 to 244 of SEQ ID NO: 32; or (aa) A polynucleotide comprising a base sequence encoding an anti-CD137scFv consisting of the amino acid sequence from amino acid numbers 1 to 247 of SEQ ID NO: 34.

[0066] In one embodiment, the expression vector for a bispecific antibody of the present invention comprises a polynucleotide selected from the group consisting of the following (a) to (k): (a) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence of amino acids 1 to 125 of SEQ ID NO: 2, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4; (b) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence of amino acids 1 to 123 of SEQ ID NO: 6, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 8; (c) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 10, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 12; (d) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 14, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 111 of SEQ ID NO: 16; (e) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 18, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 111 of SEQ ID NO: 20; (f) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 121 of SEQ ID NO: 22, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 111 of SEQ ID NO: 24; (g) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 26, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 134 to 242 of SEQ ID NO: 26; (h) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 28, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 134 to 244 of SEQ ID NO: 28; (i) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 111 of SEQ ID NO: 30, and a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 132 to 249 of SEQ ID NO: 30; (j) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 32, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 134 to 244 of SEQ ID NO: 32; or (k) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 121 of SEQ ID NO: 34, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 137 to 247 of SEQ ID NO: 34.

[0067] In one embodiment, the expression vector for a bispecific antibody of the present invention comprises a polynucleotide selected from the group consisting of the following (a) to (c): (a) a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the heavy chain of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 705 of SEQ ID NO: 36 and an anti-CD137scFv; (b) a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the heavy chain of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 712 of SEQ ID NO: 38 and an anti-CD137 scFv; or (c) A polynucleotide comprising a base sequence encoding the light chain of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 215 of SEQ ID NO: 40.

[0068] In one embodiment, the expression vector for a bispecific antibody of the invention comprises a polynucleotide selected from the following (a) or (b): (a) a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an anti-CLDN4 antibody heavy chain consisting of the amino acid sequence from amino acid numbers 1 to 705 of SEQ ID NO: 36 and an anti-CD137 scFv, and a polynucleotide comprising a nucleotide sequence encoding an anti-CLDN4 antibody light chain consisting of the amino acid sequence from amino acid numbers 1 to 215 of SEQ ID NO: 40; or (b) a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an anti-CLDN4 antibody heavy chain consisting of the amino acid sequence from amino acid numbers 1 to 712 of SEQ ID NO: 38 and an anti-CD137scFv, and a polynucleotide comprising a nucleotide sequence encoding an anti-CLDN4 antibody light chain consisting of the amino acid sequence from amino acid numbers 1 to 215 of SEQ ID NO: 40.

[0069] The expression vector for the bispecific antibody of the present invention is not particularly limited, as long as it is capable of producing the polynucleotide of the present invention in various host cells, such as eukaryotic cells (e.g., animal cells, insect cells, plant cells, yeast) and / or prokaryotic cells (e.g., Escherichia coli). Examples of such expression vectors include plasmid vectors and viral vectors. Examples of plasmid vectors that can be used include the pcDNA series (Thermo Fisher Scientific), pALTER®-MAX (Promega), pHEK293 Ultra Expression Vector (Takara Bio), pEE6.4, and pEE12.4 (Lonza Biologics). Examples of viral vectors that can be used include lentivirus, adenovirus, retrovirus, and adeno-associated virus. For example, when a lentivirus is used to introduce the polynucleotide of the present invention into cells, the lentivirus may be the pLVSIN-CMV / EF1α vector (Takara Bio), pLenti vector (Thermo Fisher Scientific), or the like. In one embodiment, the vectors used for expression of the bispecific antibodies of the present invention are pcDNA3.4-TOPO® (Thermo Fisher Scientific) and pcDNA3.1 (Thermo Fisher Scientific).

[0070] Expression vectors for bispecific antibodies of the present invention may contain a promoter operably linked to a bispecific antibody polynucleotide of the present invention. Examples of promoters for expressing bispecific antibody polynucleotides of the present invention in animal cells include promoters derived from viruses such as CMV, RSV, and SV40, as well as the actin promoter, the EF (elongation factor) 1α promoter, and heat shock promoters. Examples of promoters for expressing bispecific antibody polynucleotides of the present invention in bacteria (e.g., Escherichia) include the trp promoter, lac promoter, λPL promoter, and tac promoter. Examples of promoters for expressing bispecific antibody polynucleotides of the present invention in yeast include the GAL1 promoter, GAL10 promoter, PH05 promoter, PGK promoter, GAP promoter, and ADH promoter.

[0071] When animal cells, insect cells, or yeast cells are used as host cells, expression vectors for bispecific antibodies of the present invention may contain an initiation codon and a stop codon. In this case, they may also contain an enhancer sequence, 5'- and 3'-untranslated regions of the gene encoding the antibody of the present invention or its heavy chain or light chain, a secretion signal sequence, a splicing junction, a polyadenylation site, or a replication unit. When Escherichia coli is used as a host cell, the expression vectors of the present invention may contain an initiation codon, a stop codon, a terminator region, and a replication unit. The expression vectors of the present invention may also contain a commonly used drug selection marker gene (e.g., a tetracycline resistance gene, an ampicillin resistance gene, a kanamycin resistance gene, a neomycin resistance gene, or a dihydrofolate reductase gene) depending on the purpose.

[0072] <Host cells of the present invention> The present invention also provides host cells (also referred to as "host cells of the present invention") transformed with a polynucleotide of a bispecific antibody of the present invention or an expression vector of a bispecific antibody of the present invention (also referred to as "polynucleotides of the present invention"). The host cells of the present invention contain the polynucleotides of the present invention intracellularly. The introduced polynucleotides of the present invention may or may not be integrated into the genomic DNA of the host cell. Cells used as host cells may be either cells that can be cultured in vitro or cells within a living organism. When the host cells are cells that can be cultured in vitro, the host cells of the present invention can be produced by introducing the polynucleotides of the present invention into the cells in vitro. The method for transforming host cells is not particularly limited, and methods commonly used by those skilled in the art, such as the calcium phosphate method, electroporation, or lipofection, can be used. When the host cells are cells within a living organism, the method for introducing the polynucleotides of the present invention into the host cells is not particularly limited, and a nucleic acid delivery carrier (such as a cationic carrier or a non-cationic carrier (including, but not limited to, liposomes and lipid nanoparticles (LNPs))) can be used.

[0073] Cells that can be cultured in vitro are not particularly limited, as long as they can be transformed by the expression vector used or a method such as electroporation and can express an antibody or polypeptide. Examples of cells that can be cultured in vitro include various cells, such as conventional cells commonly used in the technical field of the present invention or artificially established cells (e.g., animal cells (e.g., CHO-K1 cells, ExpiCHO-S (registered trademark) cells, CHOK1SV cells, CHO-DG44 cells, HEK293 cells, Expi293F cells, NS0 cells), insect cells (e.g., Sf9), bacteria (e.g., Escherichia spp.), yeast (e.g., Saccharomyces spp., Pichia spp.), etc.). In one embodiment, the host cells of the present invention are Expi293F cells, CHO-K1 cells, or ExpiCHO-S cells.

[0074] Host cells transformed in vitro can be selected by methods commonly used by those skilled in the art, such as a drug selection method using a drug selection marker gene and a drug such as tetracycline, ampicillin, neomycin, or hygromycin, or a cell isolation method such as limiting dilution, single-cell sorting, or colony picking.

[0075] In one embodiment of the host cell of the present invention, the host cell comprises a polynucleotide selected from the group consisting of: (a) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 125 of SEQ ID NO: 2; (b) a polynucleotide comprising a nucleotide sequence encoding the light chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 109 of SEQ ID NO: 4; (c) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 123 of SEQ ID NO: 6; (d) A polynucleotide comprising a base sequence encoding the light chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 109 of SEQ ID NO: 8. (e) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 10; (f) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 109 of SEQ ID NO: 12; (g) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 14; (h) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 16; (i) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 18; (j) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 20; (k) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 121 of SEQ ID NO: 22; (l) A polynucleotide comprising a nucleotide sequence encoding the light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 24. (m) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 26; (n) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 134 to 242 of SEQ ID NO: 26; (o) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 28; (p) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 134 to 244 of SEQ ID NO: 28; (q) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 30; (r) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 132 to 249 of SEQ ID NO: 30; (s) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 32; (t) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 134 to 244 of SEQ ID NO: 32; (u) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 1 to 121 of SEQ ID NO: 34; (v) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid numbers 137 to 247 of SEQ ID NO: 34; (w) a polynucleotide comprising a nucleotide sequence encoding an anti-CD137scFv consisting of the amino acid sequence from amino acid numbers 1 to 242 of SEQ ID NO: 26; (x) a polynucleotide comprising a nucleotide sequence encoding an anti-CD137scFv consisting of the amino acid sequence from amino acid numbers 1 to 244 of SEQ ID NO: 28; (y) a polynucleotide comprising a nucleotide sequence encoding an anti-CD137scFv consisting of the amino acid sequence from amino acid numbers 1 to 249 of SEQ ID NO: 30; (z) a polynucleotide comprising a nucleotide sequence encoding an anti-CD137scFv consisting of the amino acid sequence from amino acid numbers 1 to 244 of SEQ ID NO: 32; or (aa) A polynucleotide comprising a base sequence encoding an anti-CD137scFv consisting of the amino acid sequence from amino acid numbers 1 to 247 of SEQ ID NO: 34.

[0076] In one embodiment, the host cell comprises a polynucleotide selected from the group consisting of: (a) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence of amino acids 1 to 125 of SEQ ID NO: 2, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4; (b) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence of amino acids 1 to 123 of SEQ ID NO: 6, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 8; (c) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 10, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 12; (d) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 14, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 111 of SEQ ID NO: 16; (e) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 18, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 111 of SEQ ID NO: 20; (f) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 121 of SEQ ID NO: 22, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 111 of SEQ ID NO: 24; (g) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 26, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 134 to 242 of SEQ ID NO: 26; (h) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 28, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 134 to 244 of SEQ ID NO: 28; (i) a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 111 of SEQ ID NO: 30, and a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 132 to 249 of SEQ ID NO: 30; (j) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 32, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 134 to 244 of SEQ ID NO: 32; or (k) a polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 121 of SEQ ID NO: 34, and a polynucleotide comprising a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 137 to 247 of SEQ ID NO: 34.

[0077] In one embodiment of the host cell of the present invention, the host cell comprises a polynucleotide selected from the group consisting of: (a) a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the heavy chain of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 705 of SEQ ID NO: 36 and an anti-CD137scFv; (b) a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the heavy chain of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 712 of SEQ ID NO: 38 and an anti-CD137 scFv; or (c) A polynucleotide comprising a base sequence encoding the light chain of an anti-CLDN4 antibody consisting of the amino acid sequence from amino acid numbers 1 to 215 of SEQ ID NO: 40.

[0078] In one embodiment, the host cell comprises a polynucleotide selected from (a) or (b): (a) a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an anti-CLDN4 antibody heavy chain consisting of the amino acid sequence from amino acid numbers 1 to 705 of SEQ ID NO: 36 and an anti-CD137 scFv, and a polynucleotide comprising a nucleotide sequence encoding an anti-CLDN4 antibody light chain consisting of the amino acid sequence from amino acid numbers 1 to 215 of SEQ ID NO: 40; or (b) a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an anti-CLDN4 antibody heavy chain consisting of the amino acid sequence from amino acid numbers 1 to 712 of SEQ ID NO: 38 and an anti-CD137scFv, and a polynucleotide comprising a nucleotide sequence encoding an anti-CLDN4 antibody light chain consisting of the amino acid sequence from amino acid numbers 1 to 215 of SEQ ID NO: 40.

[0079] In one embodiment of the host cell of the present invention, the host cell comprises the following polypeptides (a) to (j): (a) a polynucleotide comprising a nucleotide sequence encoding a polypeptide 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 125 of SEQ ID NO: 2, and an anti-CD137 scFv comprising a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 26 and a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 134 to 242 of SEQ ID NO: 26, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker, and a polynucleotide comprising a nucleotide sequence encoding a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4; (b) a polynucleotide comprising a nucleotide sequence encoding a polypeptide 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 125 of SEQ ID NO: 2, and an anti-CD137 scFv comprising a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 28 and a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 134 to 244 of SEQ ID NO: 28, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker, and a polynucleotide comprising a nucleotide sequence encoding a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4; (c) a polynucleotide comprising a nucleotide sequence encoding a polypeptide 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 125 of SEQ ID NO: 2, a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 111 of SEQ ID NO: 30, and an anti-CD137 scFv comprising a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 132 to 249 of SEQ ID NO: 30, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker, and a polynucleotide comprising a nucleotide sequence encoding a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4; (d) a polynucleotide comprising a nucleotide sequence encoding a polypeptide 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 125 of SEQ ID NO: 2, and an anti-CD137 scFv comprising a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 32 and a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 134 to 244 of SEQ ID NO: 32, in which the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker, and a polynucleotide comprising a nucleotide sequence encoding a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4; (e) a polynucleotide comprising a nucleotide sequence encoding a polypeptide 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 125 of SEQ ID NO: 2, and an anti-CD137 scFv comprising a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 121 of SEQ ID NO: 34 and a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 137 to 247 of SEQ ID NO: 34, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker, and a polynucleotide comprising a nucleotide sequence encoding a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4; (f) a polynucleotide comprising a nucleotide sequence encoding a polypeptide 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: 6, and an anti-CD137 scFv comprising a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 26 and a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 134 to 242 of SEQ ID NO: 26, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker, and a polynucleotide comprising a nucleotide sequence encoding a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 8; (g) a polynucleotide comprising a nucleotide sequence encoding a polypeptide 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: 6, and an anti-CD137 scFv comprising a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 28 and a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 134 to 244 of SEQ ID NO: 28, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker, and a polynucleotide comprising a nucleotide sequence encoding a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 8; (h) a polynucleotide comprising a nucleotide sequence encoding a polypeptide 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: 6, a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 111 of SEQ ID NO: 30, and an anti-CD137 scFv comprising a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 132 to 249 of SEQ ID NO: 30, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker, and a polynucleotide comprising a nucleotide sequence encoding a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 8; (i) a polynucleotide comprising a nucleotide sequence encoding a polypeptide 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: 6, and an anti-CD137 scFv comprising a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 32 and a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 134 to 244 of SEQ ID NO: 32, in which the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker, and a polynucleotide comprising a nucleotide sequence encoding a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 8; or (j) A polynucleotide comprising a nucleotide sequence encoding a polypeptide 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: 6, and an anti-CD137 scFv comprising a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 121 of SEQ ID NO: 34 and a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 137 to 247 of SEQ ID NO: 34, in which the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker, and a polynucleotide comprising a nucleotide sequence encoding the light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 8.

[0080] In one embodiment of the host cell of the present invention, the host cell is a host cell of (a) or (b) below: (a) a host cell comprising a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an anti-CLDN4 antibody heavy chain consisting of the amino acid sequence of amino acids 1 to 705 of SEQ ID NO: 36 and an anti-CD137scFv, and a polynucleotide comprising a nucleotide sequence encoding an anti-CLDN4 antibody light chain consisting of the amino acid sequence of amino acids 1 to 215 of SEQ ID NO: 40; or (b) A host cell comprising a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an anti-CLDN4 antibody heavy chain consisting of the amino acid sequence from amino acid numbers 1 to 712 of SEQ ID NO: 38 and an anti-CD137scFv, and a polynucleotide comprising a nucleotide sequence encoding an anti-CLDN4 antibody light chain consisting of the amino acid sequence from amino acid numbers 1 to 215 of SEQ ID NO: 40.

[0081] In one embodiment of the host cell of the present invention, the host cell comprises a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an anti-CLDN4 antibody heavy chain consisting of the amino acid sequence from amino acid numbers 1 to 712 of SEQ ID NO: 38 and an anti-CD137scFv, and a polynucleotide comprising a nucleotide sequence encoding an anti-CLDN4 antibody light chain consisting of the amino acid sequence from amino acid numbers 1 to 215 of SEQ ID NO: 40.

[0082] <Method for producing bispecific antibodies of the present invention> The present invention also provides methods for producing the anti-CLDN4-anti-CD137 bispecific antibodies of the present invention (also referred to as "production methods of the present invention"). The production methods of the present invention may include the aforementioned methods for producing polynucleotides of bispecific antibodies of the present invention, methods for producing expression vectors for bispecific antibodies of the present invention, and methods for producing host cells of the present invention. Furthermore, the production methods of the present invention may also include the steps of culturing the host cells described above in <Host cells of the present invention> and expressing the antibody in the cells or the culture supernatant, and methods for recovering, isolating, and purifying the antibody. However, the production methods of the present invention are not limited to these methods, as long as the anti-CLDN4-anti-CD137 bispecific antibodies of the present invention are produced.

[0083] In one embodiment, the production method of the present invention comprises the step of culturing the following host cells (a) to (j): (a) a host cell comprising a polynucleotide comprising a nucleotide sequence encoding a polypeptide 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 125 of SEQ ID NO: 2, a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 26, and an anti-CD137 scFv comprising a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 134 to 242 of SEQ ID NO: 26, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker, and a polynucleotide comprising a nucleotide sequence encoding a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4; (b) a host cell comprising a polynucleotide comprising a nucleotide sequence encoding a polypeptide 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 125 of SEQ ID NO: 2, a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 28, and an anti-CD137 scFv comprising a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 134 to 244 of SEQ ID NO: 28, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker, and a polynucleotide comprising a nucleotide sequence encoding a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4; (c) a host cell comprising a polynucleotide comprising a nucleotide sequence encoding a polypeptide 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 125 of SEQ ID NO: 2, a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 111 of SEQ ID NO: 30, and an anti-CD137 scFv comprising a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 132 to 249 of SEQ ID NO: 30, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker, and a polynucleotide comprising a nucleotide sequence encoding a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4; (d) a host cell comprising a polynucleotide comprising a nucleotide sequence encoding a polypeptide 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 125 of SEQ ID NO: 2, and an anti-CD137 scFv comprising a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 32 and a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 134 to 244 of SEQ ID NO: 32, in which the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker, and a polynucleotide comprising a nucleotide sequence encoding a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4; (e) a host cell comprising a polynucleotide comprising a nucleotide sequence encoding a polypeptide 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 125 of SEQ ID NO: 2, and an anti-CD137 scFv comprising a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 121 of SEQ ID NO: 34 and a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 137 to 247 of SEQ ID NO: 34, in which the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker, and a polynucleotide comprising a nucleotide sequence encoding a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4; (f) a host cell comprising a polynucleotide comprising a nucleotide sequence encoding a polypeptide 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: 6, and an anti-CD137 scFv comprising a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 26 and a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 134 to 242 of SEQ ID NO: 26, in which the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker, and a polynucleotide comprising a nucleotide sequence encoding a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 8; (g) a host cell comprising a polynucleotide comprising a nucleotide sequence encoding a polypeptide 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: 6, and an anti-CD137 scFv comprising a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 28 and a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 134 to 244 of SEQ ID NO: 28, in which the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker, and a polynucleotide comprising a nucleotide sequence encoding a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 8; (h) a host cell comprising a polynucleotide comprising a nucleotide sequence encoding a polypeptide 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: 6, a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 111 of SEQ ID NO: 30, and an anti-CD137 scFv comprising a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 132 to 249 of SEQ ID NO: 30, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker, and a polynucleotide comprising a nucleotide sequence encoding the light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 8; (i) a host cell comprising a polynucleotide comprising a nucleotide sequence encoding a polypeptide 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: 6, and an anti-CD137 scFv comprising a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 32 and a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 134 to 244 of SEQ ID NO: 32, in which the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker, and a polynucleotide comprising a nucleotide sequence encoding a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 8; or (j) A host cell comprising a polynucleotide comprising a nucleotide sequence encoding a polypeptide 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: 6; a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 1 to 121 of SEQ ID NO: 34; and an anti-CD137 scFv comprising a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence of amino acids 137 to 247 of SEQ ID NO: 34, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker; and a polynucleotide comprising a nucleotide sequence encoding the light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 8.

[0084] In one embodiment, the production method of the present invention comprises the step of culturing a host cell of (a) or (b) below: (a) a host cell comprising a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an anti-CLDN4 antibody heavy chain consisting of the amino acid sequence from amino acid numbers 1 to 705 of SEQ ID NO: 36 and an anti-CD137 scFv, and a polynucleotide comprising a nucleotide sequence encoding an anti-CLDN4 antibody light chain consisting of the amino acid sequence from amino acid numbers 1 to 215 of SEQ ID NO: 40; or (b) A host cell comprising a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an anti-CLDN4 antibody heavy chain consisting of the amino acid sequence from amino acid numbers 1 to 712 of SEQ ID NO: 38 and an anti-CD137scFv, and a polynucleotide comprising a nucleotide sequence encoding an anti-CLDN4 antibody light chain consisting of the amino acid sequence from amino acid numbers 1 to 215 of SEQ ID NO: 40.

[0085] The host cells of the present invention can be cultured by known methods. Culture conditions, such as temperature, medium pH, and culture time, can be appropriately selected by those skilled in the art. When the host cells are animal cells, examples of media that can be used include MEM medium containing about 5 to 20% fetal bovine serum (Science, 1959, Vol. 130, pp. 432-437), D-MEM medium (Virol., 1959, Vol. 8, p. 396), RPMI-1640 medium (J. Am. Med. Assoc., 1967, Vol. 199, p. 519), and 199 medium (Exp. Biol. Med., 1950, Vol. 73, pp. 1-8). The pH of the medium is, for example, about 6 to 8, and the culture is typically performed at about 30 to 40°C for about 15 to 336 hours, with aeration and stirring as necessary. When the host cells are insect cells, the culture medium can be, for example, Grace's medium containing fetal bovine serum (Proc. Natl. Acad. Sci. USA., 1985, Vol. 82, p. 8404). The pH of the medium is, for example, about 5 to 8, and the culture is typically performed at about 20 to 40°C for about 15 to 100 hours, with aeration or stirring as necessary. When the host cells are Escherichia coli or yeast, the culture medium can be, for example, a liquid medium containing nutrient sources. The nutrient medium contains, for example, a carbon source, inorganic nitrogen source, or organic nitrogen source necessary for the growth of the transformed host cells. Carbon sources include, for example, glucose, dextran, soluble starch, and sucrose. Inorganic and organic nitrogen sources include, for example, ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, soybean meal, and potato extract. If desired, the medium may contain other nutrients (for example, inorganic salts (e.g., calcium chloride, sodium dihydrogen phosphate, magnesium chloride), vitamins), antibiotics (e.g., tetracycline, neomycin, ampicillin, kanamycin), etc. The pH of the medium is, for example, about 5 to 8. When the host cell is Escherichia coli, the medium may be, for example, LB medium or M9 medium (Molecular Cloning, Cold Spring Harbor Laboratory, Vol. 3, A2.2). The culture is usually carried out at about 14 to 43°C for about 3 to 24 hours, with aeration and stirring as necessary.When the host cells are yeast, the medium that can be used may be, for example, Burkholder's minimal medium (Proc. Natl. Acad. Sci. USA., 1980, Vol. 77, p. 4505). Culturing is typically carried out at about 20 to 35°C for about 14 to 144 hours, with aeration and stirring as necessary. By culturing as described above, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention can be expressed.

[0086] The production method of the present invention can include the steps of culturing the host cells of the present invention to express the anti-CLDN4-anti-CD137 bispecific antibody, as well as the step of recovering, isolating, or purifying the anti-CLDN4-anti-CD137 bispecific antibody from the host cells. Examples of isolation or purification methods include methods that utilize solubility, such as salting out and solvent precipitation; methods that utilize differences in molecular weight, such as dialysis, ultrafiltration, and gel filtration; methods that utilize charge, such as ion exchange chromatography and hydroxyapatite chromatography; methods that utilize specific affinity, such as affinity chromatography; methods that utilize differences in hydrophobicity, such as reverse-phase high-performance liquid chromatography; and methods that utilize differences in isoelectric point, such as isoelectric focusing. In one embodiment, the antibody secreted into the culture supernatant can be purified by various types of chromatography, for example, column chromatography using a protein A column or a protein G column.

[0087] The anti-CLDN4-anti-CD137 bispecific antibody and anti-CLDN4-anti-CD137scFv bispecific antibody produced by the production method of the present invention are included in the anti-CLDN4-anti-CD137 bispecific antibody of the present invention.

[0088] <Pharmaceutical composition of the present invention> The present invention also provides pharmaceutical compositions, etc., comprising the anti-CLDN4-anti-CD137 bispecific antibody of the present invention (also referred to as the "pharmaceutical composition of the present invention"). Pharmaceutical compositions of the present invention include those comprising the anti-CLDN4-anti-CD137 bispecific antibody of the present invention and a pharmaceutically acceptable excipient. The pharmaceutical compositions of the present invention can be prepared by commonly used methods using excipients commonly used in the art, i.e., pharmaceutical excipients and pharmaceutical carriers. Examples of dosage forms of these pharmaceutical compositions include parenteral preparations such as injections and infusions, which can be administered by appropriate methods such as intravenous, subcutaneous, intraperitoneal, and intratumoral administration. When formulating the pharmaceutical compositions, excipients, carriers, additives, etc. appropriate for these dosage forms can be used within pharmaceutically acceptable limits.

[0089] The pharmaceutical compositions of the present invention may contain post-translationally modified forms of the anti-CLDN4-anti-CD137 bispecific antibodies of the present invention. For example, pharmaceutical compositions containing antibodies that have undergone C-terminal lysine deletion and / or N-terminal pyroglutamylation are also included in the present invention.

[0090] In one embodiment, the pharmaceutical composition of the present invention comprises any of the following anti-CLDN4-anti-CD137 bispecific antibodies of the present invention (a) to (j) and / or post-translationally modified forms of the antibodies: (a) an anti-CLDN4-anti-CD137 bispecific antibody and / or a post-translationally modified form of the 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 125 of SEQ ID NO: 2; a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4; and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 26 and a light chain variable region consisting of the amino acid sequence of amino acids 134 to 242 of SEQ ID NO: 26, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker; (b) an anti-CLDN4-anti-CD137 bispecific antibody and / or a post-translationally modified form of the 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 125 of SEQ ID NO: 2; a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4; and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 28 and a light chain variable region consisting of the amino acid sequence of amino acids 134 to 244 of SEQ ID NO: 28, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker; (c) an anti-CLDN4-anti-CD137 bispecific antibody and / or a post-translationally modified form of the 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 125 of SEQ ID NO: 2 and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4, and an anti-CD137 scFv comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 111 of SEQ ID NO: 30 and a heavy chain variable region consisting of the amino acid sequence of amino acids 132 to 249 of SEQ ID NO: 30, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker; (d) an anti-CLDN4-anti-CD137 bispecific antibody and / or post-translationally modified form of the 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 125 of SEQ ID NO: 2; a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4; and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 32 and a light chain variable region consisting of the amino acid sequence of amino acids 134 to 244 of SEQ ID NO: 32, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker; (e) an anti-CLDN4-anti-CD137 bispecific antibody and / or post-translationally modified form of the 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 125 of SEQ ID NO: 2; a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4; and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 121 of SEQ ID NO: 34 and a light chain variable region consisting of the amino acid sequence of amino acids 137 to 247 of SEQ ID NO: 34, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker; (f) an anti-CLDN4-anti-CD137 bispecific antibody and / or post-translationally modified form of the 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: 6; a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 8; and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 26 and a light chain variable region consisting of the amino acid sequence of amino acids 134 to 242 of SEQ ID NO: 26, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker; (g) an anti-CLDN4-anti-CD137 bispecific antibody and / or post-translationally modified form of the 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: 6; a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 8; and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 28 and a light chain variable region consisting of the amino acid sequence of amino acids 134 to 244 of SEQ ID NO: 28, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker; (h) an anti-CLDN4-anti-CD137 bispecific antibody and / or a post-translationally modified form of the 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: 6; a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 8; and an anti-CD137 scFv comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 111 of SEQ ID NO: 30 and a heavy chain variable region consisting of the amino acid sequence of amino acids 132 to 249 of SEQ ID NO: 30, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker; (i) an anti-CLDN4-anti-CD137 bispecific antibody and / or a post-translationally modified form of the 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: 6; a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 8; and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 32 and a light chain variable region consisting of the amino acid sequence of amino acids 134 to 244 of SEQ ID NO: 32, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker; or (j) An anti-CLDN4-anti-CD137 bispecific antibody and / or post-translationally modified form of the 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: 6; a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 8; and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 121 of SEQ ID NO: 34 and a light chain variable region consisting of the amino acid sequence of amino acids 137 to 247 of SEQ ID NO: 34, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker.

[0091] In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition comprising the following anti-CLDN4-anti-CD137 bispecific antibody of the present invention (a) or (b) and / or a post-translationally modified form of the antibody: (a) an anti-CLDN4-anti-CD137 bispecific antibody and / or a post-translationally modified form of said antibody, comprising a polypeptide comprising an anti-CLDN4 antibody heavy chain consisting of the amino acid sequence of amino acids 1 to 705 of SEQ ID NO: 36 and an anti-CD137 scFv, and an anti-CLDN4 antibody light chain consisting of the amino acid sequence of amino acids 1 to 215 of SEQ ID NO: 40; or (b) An anti-CLDN4-anti-CD137 bispecific antibody and / or a post-translationally modified form of said antibody, comprising a polypeptide comprising an anti-CLDN4 antibody heavy chain consisting of the amino acid sequence of amino acids 1 to 712 of SEQ ID NO: 38 and an anti-CD137 scFv, and an anti-CLDN4 antibody light chain consisting of the amino acid sequence of amino acids 1 to 215 of SEQ ID NO: 40.

[0092] The amount of the anti-CLDN4-anti-CD137 bispecific antibody of the present invention and / or post-translationally modified form of said antibody to be added in the formulation varies depending on the severity of symptoms and age of the patient, the dosage form of the formulation used, the binding titer of the antibody, etc., but can be, for example, approximately 0.001 mg / kg to 100 mg / kg.

[0093] <Medicinal uses of the anti-CLDN4-anti-CD137 bispecific antibody of the present invention> The anti-CLDN4-anti-CD137 bispecific antibodies of the present invention and pharmaceutical compositions containing them can be used to treat cancer in a subject. The present invention also includes a method for treating cancer, comprising administering a therapeutically effective amount of an anti-CLDN4-anti-CD137 bispecific antibody of the present invention to a subject. The present invention also includes an anti-CLDN4-anti-CD137 bispecific antibody of the present invention for use in cancer treatment. The present invention also includes use of the anti-CLDN4-anti-CD137 bispecific antibody of the present invention in the manufacture of a pharmaceutical composition for cancer treatment. Cancers that can be treated by the present invention are not particularly limited, but include, for example, various peritoneal disseminated cancers, gastric cancer, lung cancer, blood cancers such as acute lymphoblastic leukemia, acute myeloid leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, multiple myeloma, and T-cell lymphoma, 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, cervical cancer, and head and neck cancer. Examples of cancers that can be treated by the present invention include solid cancers such as uterine cancer, cervical cancer, liver cancer, gallbladder cancer, bile duct cancer, kidney cancer, pancreatic cancer, colon cancer, colorectal cancer, rectal cancer, small intestine cancer, stomach cancer, esophageal cancer, testicular cancer, ovarian cancer, and brain tumors, as well as cancers of bone tissue, cartilage tissue, adipose tissue, muscle tissue, vascular tissue, and hematopoietic tissue, as well as sarcomas such as chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, and soft tissue sarcoma, and blastomas such as glioblastoma, glioblastoma multiforme, hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma, and retinoblastoma. In one embodiment, cancers that can be treated by the present invention are colorectal cancer, non-small cell lung cancer, small cell lung cancer, bladder cancer, ovarian cancer, breast cancer, and prostate cancer. In one embodiment, cancers that can be treated by the present invention are cancers in which CLDN4 is highly expressed compared to normal tissues. The cancer to be treated by the present invention is preferably a cancer in which CLDN4 is highly expressed 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.

[0094] <Anti-CLDN4 antibody of the present invention> The present invention also provides any of the following anti-CLDN4 antibodies (a) to (c) or antigen-binding fragments thereof: (a) an anti-CLDN4 antibody or an antigen-binding fragment thereof, comprising: a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of amino acids 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequence of amino acids 50 to 66 of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence of amino acids 99 to 114 of SEQ ID NO: 2; and a light chain variable region comprising CDR1 consisting of the amino acid sequence of amino acids 24 to 35 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence of amino acids 51 to 57 of SEQ ID NO: 4, and CDR3 consisting of the amino acid sequence of amino acids 90 to 98 of SEQ ID NO: 4; (b) an anti-CLDN4 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 125 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4; or (c) An anti-CLDN4 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 123 of SEQ ID NO: 6 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 109 of SEQ ID NO: 8.

[0095] In one embodiment, the anti-CLDN4 antibody or antigen-binding fragment thereof of the present invention is an anti-CLDN4 antibody or antigen-binding fragment thereof comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 125 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 4, or an anti-CLDN4 antibody or antigen-binding fragment thereof comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 123 of SEQ ID NO: 6 and a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 8. In one embodiment, the anti-CLDN4 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 125 of SEQ ID NO: 2. In another embodiment, the anti-CLDN4 antibody or antigen-binding fragment thereof of the present invention is an anti-CLDN4 antibody or antigen-binding fragment thereof comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 123 of SEQ ID NO: 6 and a light chain variable region consisting of the amino acid sequence of amino acids 1 to 109 of SEQ ID NO: 8.

[0096] The heavy chain constant region of the anti-CLDN4 antibody of the present invention can be any of Igγ, Igμ, Igα, Igδ, or Igε constant regions. Igγ can be selected from, for example, Igγ1, Igγ2, Igγ3, or Igγ4. In one embodiment, the heavy chain constant region is an Igγ1 constant region, for example, a human Igγ1 constant region. The heavy chain constant region of the anti-CLDN4 antibody of the present invention may also contain amino acid mutations such as LALA, P331G, or P331S mutations to reduce ADCC or CDC. The light chain constant region of the anti-CLDN4 antibody of the present invention can be any of Igλ or Igκ constant regions. In one embodiment, the light chain constant region is an Igκ constant region, for example, a human Igκ constant region.

[0097] In one embodiment, the antigen-binding fragment of the anti-CLDN4 antibody of the present invention is scFv, Fab, Fab', or F(ab')2.

[0098] In one embodiment, the anti-CLDN4 antibody of the present invention is an anti-CLDN4 antibody comprising a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 and a light chain comprising a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 4, or an anti-CLDN4 antibody comprising a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 6 and a light chain comprising a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 8. In one embodiment, the anti-CLDN4 antibody of the present invention is an anti-CLDN4 antibody comprising a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 and a light chain comprising a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 4. In one embodiment, the anti-CLDN4 antibody of the present invention is an anti-CLDN4 antibody comprising a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 6 and a light chain comprising a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 8.

[0099] The present invention also provides anti-CLDN4 antibodies of the present invention or antigen-binding fragments thereof (also referred to as "conjugates of the present invention") conjugated with carbohydrates, lipids, metals (including radioisotopes), organic compounds (including toxins, near-infrared fluorescent dyes, chelating agents), etc. (also referred to as "modifying agents"). As used herein, "modifying agents" refer to non-peptide substances that are bound to antibodies or antigen-binding fragments thereof directly or via linkers, etc. The modifying agents used in the conjugates of the present invention are not particularly limited, and examples thereof include polyethylene glycol, sugar chains, phospholipids, radioisotopes (e.g., zirconium-89 ( 89 Zr), Yttrium-90( 90 Y), Indium-111( 111 In), astatine-211( 211 At), Actinium-225( 225Examples of the modifying agent include fluorophores, fluorophores (e.g., IRDye®), organic compounds, toxins, near-infrared fluorescent dyes (e.g., IRDye®), chelating agents, etc. The modifying agent used in the conjugate may be directly bound to the anti-CLDN4 antibody or antigen-binding fragment thereof of the present invention, or may be bound via any linker. In one embodiment, the conjugate of the present invention is an antibody drug conjugate (ADC) of the anti-CLDN4 antibody or antigen-binding fragment thereof. The drug and linker used in the ADC can be selected from drugs and linkers commonly used by those skilled in the art.

[0100] The present invention also provides cells (e.g., chimeric antigen receptor-T cells; CAR-T cells) expressing the anti-CLDN4 antibody of the present invention or an antigen-binding fragment thereof on the cell surface. Such cells can be produced by those skilled in the art using a polynucleotide encoding the anti-CLDN4 antibody of the present invention or an antigen-binding fragment thereof. Various immune cells (T cells, NK cells, NKT cells, etc.) can be used as cells expressing the anti-CLDN4 antibody of the present invention or an antigen-binding fragment thereof.

[0101] In one embodiment, the anti-CLDN4 antibody or antigen-binding fragment thereof of the present invention, the conjugate of the present invention, and the antibody or antigen-binding fragment thereof in a cell expressing the anti-CLDN4 antibody or antigen-binding fragment thereof on its surface 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 a lysine residue at the C-terminus of the heavy chain.

[0102] The anti-CLDN4 antibodies or antigen-binding fragments thereof of the present invention, the conjugates of the present invention, and cells expressing the anti-CLDN4 antibodies or antigen-binding fragments thereof on their cell surface can be prepared by those skilled in the art using methods known in the art, based on the VH and VL sequence information of the anti-CLDN4 antibodies or antigen-binding fragments thereof disclosed herein and information on modifying agents used in the conjugates of the present invention. The anti-CLDN4 antibodies or antigen-binding fragments thereof of the present invention can be produced, for example, by the methods described in the section "Methods for Producing Bispecific Antibodies of the Present Invention," without particular limitation.

[0103] The present invention also provides a pharmaceutical composition comprising an anti-CLDN4 antibody or antigen-binding fragment thereof of the present invention, a conjugate of the present invention, and a cell expressing the anti-CLDN4 antibody or antigen-binding fragment thereof on its surface (collectively referred to hereinafter in this section as the "anti-CLDN4 antibody, etc. of the present invention"), and a pharmaceutically acceptable excipient. The pharmaceutical composition can be used for the treatment of cancer. The present invention also provides a method for treating cancer comprising administering a therapeutically effective amount of the anti-CLDN4 antibody, etc. of the present invention to a subject; the anti-CLDN4 antibody, etc. of the present invention for use in the treatment of cancer; and the use of the anti-CLDN4 antibody, etc. of the present invention in the manufacture of a pharmaceutical composition for the treatment of cancer. The pharmaceutical use of the anti-CLDN4 antibody, etc. of the present invention can be carried out by those skilled in the art in accordance with the description in the above section "Pharmaceutical Composition, etc. of the Present Invention." Cancers that can be treated using the anti-CLDN4 antibody, etc. of the present invention include those described in the above section "Pharmaceutical Composition, etc. of the Present Invention."

[0104] <Anti-CD137 antibody of the present invention> The present invention also provides an anti-CD137 antibody or an antigen-binding fragment thereof comprising the heavy chain variable region and the light chain variable region of any of the anti-CD137 antibodies (a) to (d) below: (a) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 10, a CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 66 of SEQ ID NO: 10, and a CDR3 consisting of the amino acid sequence from amino acid numbers 99 to 107 of SEQ ID NO: 10, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 24 to 34 of SEQ ID NO: 12, a CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 56 of SEQ ID NO: 12, and a CDR3 consisting of the amino acid sequence from amino acid numbers 89 to 98 of SEQ ID NO: 12; (b) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 14, a CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 65 of SEQ ID NO: 14, and a CDR3 consisting of the amino acid sequence from amino acid numbers 98 to 107 of SEQ ID NO: 14, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 23 to 35 of SEQ ID NO: 16, a CDR2 consisting of the amino acid sequence from amino acid numbers 51 to 57 of SEQ ID NO: 16, and a CDR3 consisting of the amino acid sequence from amino acid numbers 90 to 100 of SEQ ID NO: 16; (c) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 18, a CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 65 of SEQ ID NO: 18, and a CDR3 consisting of the amino acid sequence from amino acid numbers 98 to 107 of SEQ ID NO: 18, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 23 to 35 of SEQ ID NO: 20, a CDR2 consisting of the amino acid sequence from amino acid numbers 51 to 57 of SEQ ID NO: 20, and a CDR3 consisting of the amino acid sequence from amino acid numbers 90 to 100 of SEQ ID NO: 20; or (d) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 22, a CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 65 of SEQ ID NO: 22, and a CDR3 consisting of the amino acid sequence from amino acid numbers 98 to 110 of SEQ ID NO: 22; and a light chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 23 to 35 of SEQ ID NO: 24, a CDR2 consisting of the amino acid sequence from amino acid numbers 51 to 57 of SEQ ID NO: 24, and a CDR3 consisting of the amino acid sequence from amino acid numbers 90 to 100 of SEQ ID NO: 24.

[0105] In one embodiment, the anti-CD137 antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain variable region and the light chain variable region of any of the following anti-CD137 antibodies (a) to (i): (a) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 10 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 109 of SEQ ID NO: 12; (b) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 14 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 16; (c) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 18 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 20; (d) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 121 of SEQ ID NO: 22 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 24; (e) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 26 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 134 to 242 of SEQ ID NO: 26; (f) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 28 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 134 to 244 of SEQ ID NO: 28; (g) a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 30 and a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 132 to 249 of SEQ ID NO: 30; (h) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 32 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 134 to 244 of SEQ ID NO: 32; or (i) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 121 of SEQ ID NO: 34 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 137 to 247 of SEQ ID NO: 34.

[0106] In one embodiment, the antigen-binding fragment of an anti-CD137 antibody of the present invention is an scFv. In one embodiment, the antigen-binding fragment of an anti-CD137 antibody of the present invention is any of the following (a) to (e): (a) an anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 1 to 242 of SEQ ID NO: 26; (b) an anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 1 to 244 of SEQ ID NO: 28; (c) an anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 1 to 249 of SEQ ID NO: 30; (d) an anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 1 to 244 of SEQ ID NO: 32; or (e) An anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 1 to 247 of SEQ ID NO: 34.

[0107] The anti-CD137 antibody or antigen-binding fragment thereof of the present invention can be prepared by one skilled in the art with reference to the descriptions herein, such as "anti-CLDN4-anti-CD137 bispecific antibody of the present invention." The anti-CD137 antibody or antigen-binding fragment thereof of the present invention can be used in a bispecific antibody with an antibody against any TAA (anti-TAA antibody) used, for example, in cancer treatment. That is, the present invention also provides a bispecific antibody comprising the anti-CD137 antibody or antigen-binding fragment thereof of the present invention and an anti-TAA antibody or antigen-binding fragment thereof (also referred to as the "anti-TAA-anti-CD137 bispecific antibody of the present invention"). The TAA is not particularly limited as long as it is expressed on the surface of tumor cells, and examples that can be used include HER2, EGFR, EpCAM, CEA, BCMA, PSMA, CD19, CD20, CD22, CD33, CD37, CD38, CD123, CD276 (B7-H3), GPC2, GPC3, GPRC5D, WT-1, NY-ESO-1, CLDN4, CLDN6, CLDN18.2, and TSPAN8. In one embodiment, the anti-TAA-anti-CD137 bispecific antibody of the present invention comprises an scFv of an anti-CD137 antibody. In one embodiment, the anti-TAA-anti-CD137 bispecific antibody of the present invention comprises an scFv of an anti-CD137 antibody that is any of the following (a) to (e): (a) an anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 1 to 242 of SEQ ID NO: 26; (b) an anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 1 to 244 of SEQ ID NO: 28; (c) an anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 1 to 249 of SEQ ID NO: 30; (d) an anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 1 to 244 of SEQ ID NO: 32; or (e) An anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 1 to 247 of SEQ ID NO: 34.

[0108] In one embodiment, the anti-TAA-anti-CD137 bispecific antibody of the invention comprises an anti-CLDN4 antibody or an antigen-binding fragment thereof, and in one embodiment, the anti-TAA-anti-CD137 bispecific antibody of the invention comprises an IgG type anti-CLDN4 antibody.

[0109] To provide a further understanding of the present invention, reference is now made to specific examples which are provided for purposes of illustration and not limitation. [Example]

[0110] Example 1: Obtaining antibodies that selectively bind to antigens expressed in peritoneally disseminated cancer cells [1-1. Acquisition of patient-derived peritoneal disseminated cancer cells] Peritoneal disseminated cancer cells were obtained from patients according to the method described in the paper "Establishment of peritoneal metastatic cancer (gastric, pancreatic, ovarian cancer, etc.) cell lines" by Senwaki Fumiko and Sasaki Hiroki (Sasaki Hiroki ed., Practical Guide to Cancer Research Using Patient-Derived Cancer Models, Yodosha, 2019, pp. 28-37). Ascites collected from patients was centrifuged at 430 × g for 3 minutes at room temperature. The supernatant was removed, and the precipitate was then hemolyzed by adding hemolysis buffer. After centrifugation, the supernatant was removed, and 50 mL of Dulbecco's PBS(-) (Nissui Pharmaceutical Co., Ltd., 05913, hereafter referred to as "PBS(-)") was added to wash the cells. The cells were then collected by centrifugation at 430 × g for 3 minutes at room temperature. All cells in the collected ascites were resuspended in RPMI-1640 (L-glutamine-containing) medium (Fujifilm Wako Pure Chemical Industries, Ltd., 189-02025) containing 10% FBS (Thermo Fisher Scientific, 10270-106) and 1× Antibiotic-Antimycotic (Thermo Fisher Scientific, 15240062) (hereinafter, the medium supplemented with FBS, etc. in Example 1 will be referred to as "RPMI-1640 medium"). The suspension was diluted and plated onto a 100 mm collagen-coated dish (hereinafter referred to as "dish") (IWAKI, 4020-010) at a density of 5 × 10 cells. 6 ~1×10 7The cells were seeded at 10 mL each and cultured at 37°C in a 5% CO2 incubator. Ascites contains both adherent and suspension cells, including not only cancer cells but also non-cancer cells (e.g., fibroblasts and peritoneal mesothelial cells). Because non-cancer cells tend to detach more quickly than cancer cells, these cells were separated and removed from the total cells in the ascites. After removing the non-cancer cells, the cancer cells were allowed to grow to approximately 80% confluence, and then half of the total cells were passaged onto a new dish. Cells that had been passaged five or more times were designated as adherent cancer cells. For suspension cells, 5 mL of culture supernatant and 5 mL of RPMI-1640 medium were seeded onto a new 100 mm dish and passaged. Cells that had been passaged five or more times were designated as suspension cancer cells. When peritoneal disseminated cancer cells from a single patient continued to grow while containing both adherent and suspension cancer cells, they were designated as mixed cancer cells. In this specification, the adherent cancer cells, suspension cancer cells, and mixed cancer cells isolated by the above-mentioned methods are collectively referred to as "peritoneally disseminated cancer cells." Six cells obtained (NSC-9C, NSC-15CF, NSC-16C, NSC-20C, NSC-22C, and NSC-32C (hereinafter also referred to as "six types of peritoneally disseminated cancer cells")) were used in the following studies.

[0111] [1-2: Generation of anti-gastric cancer antigen antibody-producing hybridomas] VelocImmune mice, created using the human monoclonal antibody development technology "VelocImmune" (VelocImmune® antibody technology; Regeneron (US Patent No. 6,596,541)), were immunized with peritoneal disseminated cancer cells to obtain antibodies against cancer antigens expressed in peritoneal disseminated cancer cells. Among the peritoneally disseminated cancer cells obtained in Example 1-1, NSC-9C, NSC-15CF, and NSC-16C cells or NSC-20C, NSC-22C, and NSC-32C cells were mixed and suspended in TiterMax® Gold ADJUVANT (Merck, T2684) or PBS(-) to prepare a peritoneally disseminated cancer cell suspension. This suspension was administered several times to Velocimmune mice for immunization. Lymphocytes were collected from the lymph nodes of immunized mice using standard methods and fused with mouse myeloma cells SP2 / 0 (ATCC, CRL-1581) to produce hybridomas. Single hybridoma colonies were isolated using an automated picking device to obtain monoclonal hybridoma cells (hereinafter referred to as "clones"). The isolated clones were cultured at 37°C in an 8% CO2 incubator, and the supernatant after about 14 days was collected into a 96-well plate and used in the following experiments.

[0112] [1-3: Selection of antibodies that selectively bind to peritoneal disseminated cancer cells] (1) Confirmation of binding to peritoneal disseminated cancer cells and EpCAM-expressing cells The cell supernatant of the clone obtained in Example 1-2 contains an antibody (hereinafter referred to as "antibody contained in the clone supernatant"). First, the binding of antibodies contained in the clone supernatants to the six types of peritoneal disseminated cancer cells obtained in Example 1-1 was measured by flow cytometry, and clones producing antibodies that strongly bound to peritoneal disseminated cancer cells were selected. BV421 Goat Anti-Mouse Ig (Becton, Dickinson and Company, 563846) was used for flow cytometry. To exclude clones that produce antibodies binding to the cancer antigen EpCAM, we measured the binding of antibodies contained in the clone supernatants to human EpCAM-Myc-DDK-expressing CHO-K1 cells. Human EpCAM-Myc-DDK-expressing CHO-K1 cells were generated by transfecting CHO-K1 cells (ATCC, CCL-61) with EPCAM (Myc-DDK-tagged)-Human epithelial cell adhesion molecule (EPCAM) (ORIGENE, RC201989). Binding of antibodies contained in the clone supernatants to these cells was measured by flow cytometry. BV421 Goat Anti-Mouse Ig was used for flow cytometry. To select clones that produce supernatants that do not bind to EpCAM-expressing cells, clones that bind to these cells were excluded. CD326 (EpCAM) monoclonal antibody (1B7) (eBioscience, 14-9326) was used as a positive control. Furthermore, to select clones that selectively bind to peritoneally disseminated cancer cells, clones that bind to cultured human peritoneal mesothelial cells were excluded. Human Mesothelial Cells (Zenbio, MES-F, Lot. MESM050311A) (hereinafter also referred to as "cultured human peritoneal mesothelial cells") were used as cultured human peritoneal mesothelial cells, and the binding of the clones to cultured human peritoneal mesothelial cells cultured in Mesothelial Cell Growth Medium (Zenbio, MSO-1) was measured using flow cytometry. BV421 Goat Anti-Mouse Ig was used for flow cytometry. Only clones that did not bind to cultured human peritoneal mesothelial cells were selected. From the above experiments, the 3D11 clone was obtained as a clone that provides an antibody that binds to at least five of six types of peritoneal disseminated cancer cells, but does not bind to human EpCAM or cultured peritoneal mesothelial cells.

[0113] (2) Antibody purification from hybridoma supernatant The 3D11 clone was cultured in CD Hybridoma Medium (Thermo Fisher Scientific, 11279023). The 3D11 antibody was purified from the culture supernatant using MabSelectSuRe (GE Healthcare, 17-5438-02) (hereinafter referred to as "3D11"). The antibody was purified according to standard methods.

[0114] [1-4: Identification of the antigen molecule recognized by 3D11] (1) Identification of antigen candidate molecules by LC-MS / MS measurement We identified candidate antigen molecules for 3D11. 1D10 and 10B5 were used as control antibodies in this experiment. These antibodies showed different binding patterns to peritoneal disseminated cancer cells than 3D11 during the process of obtaining 3D11. NSC-15CF cell lysates were prepared. 3D11 and one of two control antibodies (1D10 and 10B5) were added to the cell lysate. Dynabeads Protein G (Life Technologies, 10003D) was then added, stirred, and washed. Proteins bound to Dynabeads Protein G were digested with Trypsin / LysC (Promega, V5072) to obtain a peptide mixture. The resulting peptide mixture was subjected to LC-MS / MS analysis using an UltiMate 3000 RSLC nano (Thermo Fisher Scientific) and an Orbitrap Fusion (Thermo Fisher Scientific). The resulting LC-MS / MS data were analyzed using Progenesis QI for Proteomics (Waters) and Mascot (Matrix Science) software for comparative quantitative analysis and peptide / protein identification, and binding proteins were identified. By comparing the binding proteins of 3D11 and a control antibody, CLDN4 was identified as a candidate antigen molecule for 3D11.

[0115] (2) Antigen determination of 3D11 To determine whether CLDN4, identified in (1), is the antigen of 3D11, we performed a binding experiment with human CLDN4-Myc-DDK-expressing CHO-K1 cells. Human CLDN4-Myc-DDK-expressing CHO-K1 cells were generated by transfecting CHO-K1 cells with CLDN4 (Myc-DDK-tagged)-human claudin 4 (CLDN4) (ORIGENE, RC200490). As a result, 3D11 bound to human CLDN4-Myc-DDK-expressing CHO-K1 cells, confirming that 3D11 recognizes CLDN4 as an antigen.

[0116] Example 2: Sequencing of 3D11 and production of human antibodies The genes encoding the heavy and light chains of 3D11 were cloned according to standard methods, and the nucleotide and amino acid sequences of 3D11 were determined. The amino acid sequence of the heavy chain variable region of 3D11 is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4. Antibodies produced using Velocimine mice are antibodies in which the variable regions of endogenous immunoglobulin heavy and light chains have been replaced with the corresponding human variable regions. In other words, antibodies obtained using Velocimine technology are antibodies that have the variable regions of a human antibody and the constant regions of a mouse antibody (also called "chimeric antibodies").

[0117] [Reference example: Anti-CLDN4-anti-CD3 bispecific antibody used to treat peritoneal dissemination of cancer] Various types of drugs are known as therapeutic agents against TAAs selectively expressed in cancer cells, including antibodies, antibody-drug conjugates (ADCs), and chimeric antigen receptor T-cell therapy (CAR-T). Among these, bispecific T-cell-recruiting antibodies are known as an innovative method that can achieve cancer cell-selective cytotoxic activity at low antibody concentrations. Therefore, we investigated the generation of bispecific T-cell-recruiting antibodies using 3D11.

[0118] (1) Generation of humanized anti-CLDN4 antibodies A human anti-CLDN4-anti-CD3scFv bispecific antibody was produced by the following method. The human anti-CLDN4 antibody moiety was designed by linking the human IgG1 heavy chain constant region to the heavy chain variable region of 3D11 and the human κ light chain constant region to the light chain variable region, according to standard methods. Furthermore, the following mutations were introduced: LALA mutations (L234A and L235A) at amino acids 238 and 239 (EU index: 234 and 235) in the heavy chain, where leucine (L) is substituted with alanine (A); knobs-into-holes mutations (threonine (T) to serine (S), L to A, and tyrosine (Y) to valine (V)) at amino acids 370, 372, and 411 (EU index: 366, 368, and 407), where threonine (T) is substituted with serine (S), L to A, and tyrosine (Y) to valine (V), respectively; and asparagine (N) to A at amino acid 301 (EU index: 297). The humanized anti-CLDN4 antibody thus produced is designated 3D11.1. A polynucleotide encoding the amino acid sequence of 3D11.1 was synthesized according to standard methods and inserted into the pcDNA3.4 TOPO vector (Thermo Fisher Scientific).

[0119] (2) Preparation of anti-CD3 scFv The anti-CD3 scFv portion was constructed based on the sequences of the heavy and light chain variable regions of the mouse anti-CD3 antibody described in Japanese Patent No. 5686953. Humanization of the anti-CD3 scFv was performed according to the method described in the literature (Front Biosci., 2008, Vol. 13, pp. 1619-1633). Backmutations were introduced during this process. The three-dimensional structure information (PDB Code: 5FCS) was analyzed using the integrated computational chemistry system MOE provided by MOLSIS Inc. to determine the positions for introducing backmutations within the framework region. The amino acid sequence of the humanized anti-CD3 scFv is shown in SEQ ID NO: 41. A polynucleotide encoding the amino acid sequence described in SEQ ID NO: 41 was synthesized according to standard methods and inserted into the pcDNA3.1(+) vector (Thermo Fisher Scientific, V79020).

[0120] (3) Preparation of anti-CLDN4 (3D11)-anti-CD3 scFv bispecific antibody A bispecific antibody composed of the Fab region of an anti-CLDN4 antibody and the scFv and Fc regions of an anti-CD3 antibody was produced using a vector encoding 3D11.1 and anti-CD3 scFv, according to the method described in Example 7 of PCT / JP2021 / 41839. The produced bispecific antibody is referred to as the anti-CLDN4 (3D11)-anti-CD3 scFv bispecific antibody.

[0121] (4) Evaluation of the efficacy of anti-CLDN4 (3D11)-anti-CD3 scFv bispecific antibody The in vitro redirected T cell cytotoxicity (RTCC) activity of the anti-CLDN4(3D11)-anti-CD3scFv bispecific antibody was measured according to the method described in Example 9 of PCT / JP2021 / 41839. The anti-CLDN4(3D11)-anti-CD3scFv bispecific antibody was confirmed to have RTCC activity. The in vivo antitumor activity of the anti-CLDN4(3D11)-anti-CD3scFv bispecific antibody was also evaluated according to the method described in Example 11 of PCT / JP2021 / 41839. The anti-CLDN4(3D11)-anti-CD3scFv bispecific antibody demonstrated antitumor activity in an in vivo mouse model of peritoneal dissemination of gastric cancer. These results suggest that the anti-CLDN4(3D11)-anti-CD3scFv bispecific antibody is effective in cancer treatment. Furthermore, to evaluate the safety of the anti-CLDN4(3D11)-anti-CD3scFv bispecific antibody, a single intravenous injection of the anti-CLDN4(3D11)-anti-CD3scFv bispecific antibody was administered to cynomolgus monkeys. Death and moribund cases were observed in the monkeys administered the anti-CLDN4(3D11)-anti-CD3scFv bispecific antibody. Plasma IL-6 levels were significantly elevated 1 and 6 hours after administration in the dead monkeys, and 1, 6, and 24 hours after administration in the moribund monkeys. These results suggest that the anti-CLDN4(3D11)-anti-CD3scFv bispecific antibody exerts therapeutic effects in vitro and in vivo, but raise concerns about serious adverse reactions, possibly due to increased cytokine production. Therefore, we concluded that the anti-CLDN4(3D11)-anti-CD3scFv bispecific antibody is unsuitable for use in the treatment of human cancer.

[0122] Example 3: Preparation of anti-CLDN4 (3D11)-anti-CD137 scFv bispecific antibody A bispecific antibody was produced using 3D11 and anti-CD137scFv (hereinafter referred to as "anti-CLDN4(3D11)-anti-CD137scFv bispecific antibody") and investigated.

[0123] [3-1. Obtaining anti-CD137 antibodies] (1) Preparation of CD137-Fc fusion protein A polynucleotide encoding the extracellular region of human CD137 consisting of the amino acid sequence from amino acid no. 1 to 186 of SEQ ID NO: 42 or the extracellular region of monkey CD137 consisting of the amino acid sequence from amino acid no. 1 to 186 of SEQ ID NO: 43 was inserted into the pFUSE-hIgG1-Fc1 vector (InvivoGen, pfuse-hg1fc1) to prepare expression vectors encoding fusion proteins of the extracellular region of CD137 and human Fc (hereinafter referred to as the "human CD137-human Fc fusion protein" and the "monkey CD137-human Fc fusion protein," respectively). The constructed vectors were transfected into Expi293F cells (Thermo Fisher Scientific, A14527) or ExpiCHO-S cells (Thermo Fisher Scientific, A29133) using the Expi293 Expression System Kit (Thermo Fisher Scientific, A14635) or ExpiCHO Expression System Kit (Thermo Fisher Scientific, A29133), and then cultured. Proteins secreted into the culture supernatant were purified using MabSelect SuRe or HiTrap MabSelect SuRe (GE Healthcare, 11-0034-94), and human CD137-human Fc fusion proteins and monkey CD137-human Fc fusion proteins were obtained.

[0124] (2) Preparation of monkey CD137-His tag-3×FLAG tag fusion protein A polynucleotide encoding the monkey CD137 extracellular domain consisting of amino acid sequence 1 to 183 of SEQ ID NO: 43, a His tag of SEQ ID NO: 44, and a 3xFLAG tag of SEQ ID NO: 45 was inserted in tandem into the pcDNA3.4 TOPO vector to prepare a vector. The prepared vector was introduced into ExpiCHO-S cells using the ExpiFectamine CHO Transfection Kit (Thermo Fisher Scientific, A29129) and cultured. The protein secreted into the culture supernatant was purified using HisTrap excel (GE Healthcare Biosciences, 17-3712-06). The resulting fusion protein is referred to as "monkey CD137-His tag-3xFLAG tag fusion protein."

[0125] (3) Generation and screening of anti-CD137 antibody-producing hybridoma clones Velocimmune mice or AlivaMab mice (Ablexis, U.S. Patent No. 9346873) were immunized with human CD137-human Fc fusion protein or human CD137-His tag protein (R&D SYSTEMS, 9220-4B) and an immunoadjuvant several times. Lymphocytes were collected from the lymph nodes of the immunized mice and fused with mouse myeloma cells SP2 / 0 according to standard methods to produce hybridomas. Single hybridoma colonies were isolated using an automated picking device to obtain monoclonal hybridoma cells (hereinafter referred to as "clones"). Each clone was cultured for several days, and the culture supernatant was obtained. The binding of antibodies contained in the culture supernatant to CD137 was confirmed using the following method. Binding to CD137 was evaluated by ELISA. Plates coated with human CD137-His tag protein, human CD137-human Fc fusion protein, monkey CD137-human Fc fusion protein, or human Fc protein were used, and Goat Anti-Mouse IgG, Human ads-HRP (SouthernBiotech, 1030-05) was used as the secondary antibody. Using this method, multiple clones that bound to both human and monkey CD137 were obtained.

[0126] (4) Obtaining anti-CD137 antibodies and confirming their agonistic activity The clones obtained in (3) were cultured in Hybridoma-SFM (Thermo Fisher Scientific, 12045-076), and anti-CD137 antibodies were purified from the culture supernatant using MabSelect SuRe. The purified anti-CD137 antibodies were tested for CD137 binding and CD137 agonist activity. The binding of anti-CD137 antibodies to CD137 was confirmed using the ELISA method described in (3). The CD137 agonist activity of anti-CD137 antibodies was measured using the 4-1BB Bioassay Kit (Promega, JA2351) as follows. FcγRIIb CHO-K1 cells (Promega, JA2251) were seeded into a 384-well plate (Greiner Bio-One, 781080) and cultured overnight at 37°C and 5% CO2. The culture supernatant was removed, and purified antibodies from each clone and 4-1BB Effector Cells (Promega, JA2351) were added. The plate was then incubated at 37°C and 5% CO2 for 6 hours. Bio-Glo Reagent (Promega, JA2351) was added, and chemiluminescence due to the induction of luciferase expression in response to CD137 agonist activity was measured. To evaluate CD137 agonist activity in the absence of FcγRIIb CHO-K1 cells, the same experiment as above was performed in the absence of FcγRIIb CHO-K1 cells. Among the anti-CD137 antibodies evaluated, 3-34 derived from Velocimmune mice and A2-32, A2-48, and A2-73 derived from AlivaMab mice had strong binding activity to human and monkey CD137 and exhibited CD137 agonist activity dependent on the presence of FcγRIIb CHO-K1 cells.

[0127] (5) Identification of the gene sequence of the anti-CD137 antibody According to standard methods, cell lysates were prepared from the clones that provide the anti-CD137 antibodies (3-34, A2-32, A2-48, and A2-73), cDNA was synthesized, and the antibody nucleotide sequences were identified. The sequence numbers of the identified nucleotide sequences are shown in Table 1. [Table 1]

[0128] 3-2. Construction of anti-CLDN4 (3D11)-anti-CD137 (3-34) scFv bispecific antibody An anti-CLDN4 (3D11)-anti-CD137 (3-34) scFv bispecific antibody (hereinafter referred to as "3D11_3-34-j6_S") was designed based on the sequences of the heavy and light chain variable regions of 3D11 and 3-34. The resulting 3D11_3-34-j6_S antibody contains a GS linker linked to the heavy chain carboxy terminus (also referred to as the "C terminus") of a human IgG1κ-type anti-CLDN4 (3D11) antibody, and the amino terminus of an anti-CD137 (3-34) scFv (SEQ ID NO: 26), which contains the heavy and light chain variable regions of an anti-CD137 antibody, is attached to the C terminus of the GS linker. Furthermore, amino acid mutations L234A, L235A, and P331S were introduced into the human IgG1 constant region of 3D11. A polynucleotide encoding the designed antibody was prepared and inserted into the pcDNA3.4 TOPO vector according to standard procedures. The expression vector was transfected into ExpiCHO-S cells, and 3D11_3-34-j6_S was purified from the culture supernatant. The resulting 3D11_3-34-j6_S was confirmed to have anti-CD137 binding activity and CD137 agonistic activity in vitro, suggesting its potential as a cancer therapeutic agent.

[0129] Example 4: Preparation of anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibody 4-1. Generation of humanized anti-CLDN4 (hKM3900) antibody It has been reported that the anti-CLDN4 antibody KM3900 selectively binds to CLDN4 compared to other claudin family molecules such as CLDN6 (Patent Document 1). Based on this report, a humanized KM3900 antibody was constructed. Specifically, a humanized antibody was designed based on the humanized amino acid sequences of the KM3900 variable regions and the sequences of the human Igγ1 constant region and human Igκ constant region. Amino acid mutations L234A, L235A, and P331G, or L234A, L235A, and P331S, were introduced into the human Igγ1 constant region. A humanized anti-CLDN4 (hKM3900) antibody was constructed according to standard methods and used in subsequent studies. The antibody having amino acid mutations L234A, L235A, and P331G is referred to as "hKM3900," and the antibody having amino acid mutations L234A, L235A, and P331S is referred to as "hKM3900_S." The amino acid sequence of the heavy chain variable region of hKM3900 is shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8.

[0130] 4-2. Construction of anti-CLDN4-anti-CD137 scFv bispecific antibodies Anti-CD137 scFv were designed from the anti-CD137 antibodies (3-34, A2-32, A2-48, and A2-73) obtained in Example 3, and bispecific antibodies were produced between hKM3900 and anti-CD137 scFv (Table 2). The produced antibodies are collectively referred to as "anti-CLDN4 (hKM3900)-anti-CD137 scFv."

[0131] (1) Construction of a vector encoding the anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibody An anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibody was designed based on the hKM3900 sequence and the sequences of the heavy and light chain variable regions of an anti-CD137 antibody. The anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibody is constructed by linking a GS linker to the C-terminus of the heavy or light chain of an IgG1-type anti-CLDN4 antibody (hKM3900), and the amino terminus of an anti-CD137 scFv containing the heavy and light chain variable regions of an anti-CD137 antibody is attached to the C-terminus of the GS linker. Furthermore, amino acid mutations L234A, L235A, and P331G or P331S were introduced into the human IgG1 constant region of hKM3900. Information about the designed antibody is listed in Table 2. Polynucleotides encoding the designed antibodies were prepared and inserted into the pcDNA3.4 TOPO vector according to standard methods. The prepared vectors are collectively referred to as "anti-CLDN4 (hKM3900)-anti-CD137scFv bispecific antibody expression vectors."

[0132] [Table 2]

[0133] (2) Preparation of anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibody The 11 anti-CLDN4(hKM3900)-anti-CD137scFv bispecific antibody expression vectors listed in Table 2 were produced using the anti-CLDN4(hKM3900)-anti-CD137scFv bispecific antibody expression vectors. Specifically, the anti-CLDN4(hKM3900)-anti-CD137scFv bispecific antibody expression vectors were introduced into ExpiCHO-S cells using the ExpiFectamine CHO Transfection Kit, and the anti-CLDN4-anti-CD137scFv bispecific antibody was secreted into the culture supernatant. The anti-CLDN4(hKM3900)-anti-CD137scFv bispecific antibody was purified from the resulting culture supernatant by affinity purification using MabSelect SuRe. Furthermore, a portion of the anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibody was further purified by size-exclusion chromatography using HiLoad 26 / 600 superdex 200 pg (GE Healthcare, 28-9893-36) or Superdex 200 Increase 10 / 300 GL (GE Healthcare, 28-9909-44). Hereinafter, the anti-CLDN4 (3D11)-anti-CD137 (3-34) scFv bispecific antibody and the anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibody prepared in Examples 3 and 4 may be collectively referred to as the "anti-CLDN4-anti-CD137 scFv bispecific antibody."

[0134] Example 5: Evaluation of binding activity and agonistic activity of anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibody The binding activity of the anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibody to CLDN4 and CD137, as well as its CD137 agonistic activity, were evaluated.

[0135] 5-1. Flow cytometry analysis of the binding activity of the anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibody to CLDN4. The binding activity of the anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibody to CLDN4 was evaluated using flow cytometry. The CLDN4 gene, prepared from CLDN4 (Myc-DDK-tagged)-Human Claudin 4, was inserted into the pCMV6-AC-GFP Mammalian Expression Vector (ORIGENE, PS100010) to generate a human CLDN4 expression vector. The human CLDN4 expression vector was then transfected into CHO-K1 cells to generate human CLDN4-expressing CHO-K1 cells. The test antibodies used were hKM3900 and two anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibodies (hKM3900_tA2-32LH and hKM3900_3-34-j6). The test antibodies were serially diluted in a 3:1 ratio in Stain Buffer (FBS) (BD Bioscience, 554656) from a maximum concentration of 100 μg / mL to 5.08 ng / mL, and human CLDN4-expressing CHO-K1 cells were stained with each test antibody dilution. The secondary antibody used was Allophycocyanin (APC) AffiniPure F(ab')2 Fragment Goat Anti-Human IgG, Fcγ fragment specific (Jackson ImmunoResearch, 109-136-170). Fluorescence intensity was measured using a FACS Canto II (BD Bioscience). The measurement results were analyzed using GraphPad Prism, and the apparent dissociation constants (KD) of human CLDN4 and the anti-CLDN4 (hKM3900)-anti-CD137scFv bispecific antibody were calculated. The apparent KD values ​​are shown in Table 3. Two types of anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibodies showed binding activity to human CLDN4 similar to that of the humanized anti-CLDN4 (hKM3900) antibody. Other anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibodies are also expected to show equivalent CLDN4 binding activity.

[0136] [Table 3]

[0137] 5-2. Evaluation of the binding activity of anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibody to CD137 by ELISA The binding activity of the anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibody to CD137 was detected using ELISA. ELISA was performed according to standard methods. 20 ng / 20 μL / well of human CD137-His tagged protein (R&D SYSTEMS, 9220-4B) was immobilized on a 384-well plate (Thermo Fisher Scientific, 464718). The 11 anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibodies listed in Table 2 and the ctrl IgG_L-3-34-j6 antibody were used as test antibodies. The ctrl IgG_L-3-34-j6 antibody was an isotype control antibody in which the anti-CD137 scFv (3-34-j6) antibody was linked to the carboxy terminus of the light chain. The test antibodies were serially diluted in TBS-T (Nippon Gene, 310-07375) containing 5% Blocking One (Nacalai Tesque, 03953-95) and added at 20 μL / well. The final antibody concentrations are shown in Figures 1-1 to 1-4. The secondary antibody used was Goat Anti-Human IgG Fc, Multi-Species SP ads-HRP (SouthernBiotech, 2014-05). The control antibody was hKM3900. Absorbance was measured at 450 nm and 570 nm using an Infinite M200PRO (TECAN). The results are shown in Figures 1-1 to 1-4. Both anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibodies bound to human CD137-His tagged protein in a concentration-dependent manner. These results demonstrated that the linking position of anti-CD137scFv does not affect the binding activity to human CD137.

[0138] [5-3. Evaluation of binding activity of anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibody to CD137 by surface plasmon resonance] The binding activity of the anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibody to CD137 was detected using surface plasmon resonance (SPR). SPR analysis was performed using a Biacore T200 (GE Healthcare Japan). Two anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibodies (hKM3900-3-34-j6 and hKM3900_tA2-32LH) immobilized on a Series S Sensor Chip Protein A (GE Healthcare, 29-1275-56) were used as ligands. Human CD137-His tag or monkey CD137-His tag-3xFLAG tag fusion protein prepared in 3-1(2) was used as analyte, serially diluted in HBS-EP+ at a common ratio of 2:1 from a maximum concentration of 200 nM to 0.0977 nM. The binding and dissociation of the anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibody to CD137 were measured by adding the analyte to the ligand-bound channel at 50 μL / min for 2 minutes, followed by adding HBS-EP+ at 50 μL / min for 10 minutes. The binding rate constant (ka), dissociation rate constant (kd), and dissociation constant (KD) of the anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibody to human CD137 were calculated using the data analysis software (BIA Evaluation). Table 4 shows the ka, kd, and KD values. As a result, it was revealed that the anti-CLDN4 (hKM3900)-anti-CD137scFv bispecific antibody exhibited equivalent binding activity to both human CD137 and monkey CD137 antibody.

[0139] [Table 4]

[0140] [5-4. Evaluation of CD137 agonist activity of anti-CLDN4-anti-CD137 scFv bispecific antibodies in the presence of CLDN4-expressing cancer cells] The specific CD137 agonist activity of the anti-CLDN4-anti-CD137 scFv bispecific antibody via CLDN4-expressing cancer cells was evaluated using the 4-1BB Bioassay kit. NCI-H322 cells (ECACC, 95111734) were suspended in RPMI-1640 medium (Thermo Fisher Scientific, 11875-093) containing 10% FBS (Cytiva, SH30070.03) to prepare a suspension. NCI-H322 expresses CLDN4. The NCI-H322 suspension was added to a 384-well plate (Greiner Bio-One, 781080) at a concentration of 1 × 10 4 The cells were added at a concentration of 1000 / well and cultured overnight at 37°C in 5% CO2. The medium was removed the next day. Test antibodies diluted with RPMI-1640 medium containing 1% FBS were added at 12.5 μL / well. The final antibody concentrations are shown in Figures 2-1 to 2-3. The test antibodies used were 3D11_3-34-j6_S, an anti-CLDN4 (hKM3900)-anti-CD137scFv bispecific antibody listed in Table 2. Control antibodies used were hKM3900, ctrl IgG_L-3-34-j6 antibody, and urelumab (BMS, SEQ ID NO: 3 and SEQ ID NO: 6 listed in WO 2005 / 035584). Each vial of 4-1BB Effector Cells included in the 4-1BB Bioassay Kit was suspended in 7 mL of RPMI-1640 containing 1% FBS, and 12.5 μL was added to each well. The plate was incubated at 37°C and 5% CO2 for 6 hours. Bio-Glo Reagent was added to the plate at 25 μL / well, and chemiluminescence was measured using an EnVision 2103 (Perkin Elmer). The results are shown in Figures 2-1 to 2-3. The anti-CLDN4-anti-CD137 scFv bispecific antibody exhibited significant CD137 agonist activity against 4-1BB Effector Cells in the presence of CLDN4-expressing cancer cells. To evaluate CD137 agonist activity in the absence of CLDN4-expressing cancer cells, we performed the same experiment as above in the absence of NCI-H322. As a result, no CD137 agonist activity was observed in the absence of CLDN4-expressing cancer cells (Figures 2-4 to 2-6). These results indicate that the anti-CLDN4-anti-CD137 scFv bispecific antibody exerts CD137 agonist activity only in the presence of CLDN4-expressing cancer cells. On the other hand, urelumab showed CD137 agonist activity in the presence of CLDN4-expressing cancer cells, but its activity was weaker than that of the anti-CLDN4-anti-CD137 bispecific antibody.Urelumab also showed CD137 agonist activity in the absence of CLDN4-expressing cancer cells. These results suggest that the anti-CLDN4-anti-CD137 bispecific antibody has lower toxicity concerns than urelumab and is expected to exhibit specific and potent CD137 agonistic activity in tumor tissues with high CLDN4 expression.

[0141] Example 6: In vitro efficacy evaluation of anti-CLDN4-anti-CD137 scFv bispecific antibody The anti-CLDN4-anti-CD137scFv bispecific antibody was evaluated for its interferon-γ production-promoting activity and cancer cell cytotoxicity.

[0142] [6-1. Generation and cryopreservation of expanded PanT cells] (1) Preparation of Expanded PanT Cells In Example 6, RPMI-1640 (Sigma, R8758) supplemented with 10% FBS (Cytiva, SH30084.03) and 1% penicillin-streptomycin (Thermo Fisher Scientific, 15070-063) is referred to as the "culture medium." The culture medium may also contain MEM Non-essential Amino Acid (Merck, M7145), sodium pyruvate (Merck, S8636), GlutaMAX I (Thermo Fisher Scientific, 35050-061), and HEPES (Thermo Fisher Scientific, 15630-080) at final concentrations of 1%. Anti-CD3 antibody (BioLegend, 317325) diluted to a final concentration of 1-3 μg / mL was added to the dish for immobilization. PanT cells (including both CD4 and CD8 T cells, hereafter referred to as "PanT cells") were isolated from human peripheral blood mononuclear cells (LONZA, CC-2702) using a PanT Cell Isolation Kit, human (Miltenyi Biotec, 130-096-535) according to the manufacturer's recommended protocol. The isolated PanT cells were centrifuged, the supernatant removed, and then suspended in culture medium. The entire amount of PanT cells suspended in culture medium was seeded onto a dish coated with the aforementioned anti-CD3 antibody. Human IL-2 (PeproTech, 200-2) at a final concentration of 100-200 U / mL and anti-CD28 antibody (BioLegend, 302923) at a final concentration of 1-4 μg / mL were then added, and the cells were cultured at 37°C in a 5% CO2 incubator. After 3 days, the cells were harvested, suspended in culture medium, and seeded onto dishes. Human IL-2 was added at a final concentration of 100-200 U / mL, and the cells were cultured in a 37°C, 5% CO2 incubator. After 4 days, the entire cell mass was harvested and centrifuged. After the supernatant was removed, the cells were resuspended in Cellbanker (Takara, CB011) or Bambanker (GS Lymphotec, CS-02-001), aliquoted into tubes, and stored frozen at -80°C.The cryopreserved cells are referred to herein as "Expanded PanT cells."

[0143] [6-2.60As6-Luc / GFP expressing cells] A lentiviral solution containing Luc / GFP was prepared using L293T cells (Thermo Fisher Scientific, K4975-00) according to standard procedures. The pCDH-CMV-GL3-EF1a-GFP-T2A-puro modified vector (a gift from Associate Professor Ryo Takahashi, Laboratory of Cellular and Molecular Biology, Graduate School of Biomedical Sciences, Hiroshima University (PLoS One, 2015, Vol. 10, e0123407)) was transfected into L293T cells using Opti-MEM Reduced Serum Medium, GlutaMAX Supplement (Thermo Fisher Scientific, 51985-034), MISSION® Lentiviral Packaging Mix (Sigma, SHP001), and Lipofectamin LTX (Thermo Fisher Scientific, 15338100). GFP-expressing lentivirus propagated in L293T cells was collected using a 45 μm Millex®-HV filter (Merck Millipore, SLHV033RS). The virus solution was dispensed into cryovials (Nalgene, 5000-0020) in 1 mL aliquots and stored frozen at -80°C. 60As6-Luc / GFP-expressing lentivirus was used to generate 60As6 cells (a CLDN4-expressing cancer cell line, provided by Dr. Ikichi Yanagihara, Biomaterials Research Institute) by standard methods. Polybrene (SantaCruz, sc-134220) was used to increase infection efficiency. RPMI-1640 medium containing 10% FBS and 2 μg / mL Puromaycin (Thermo Fisher Scientific, A-11138-02) was used. The established cells are referred to herein as "60As6-Luc / GFP cells." High endogenous CLDN4 expression was confirmed by flow cytometry. The constructed cells were used in subsequent experiments.

[0144] [6-3. Evaluation of the interferon-γ production-promoting function of anti-CLDN4 (3D11)-anti-CD137 scFv bispecific antibody in a co-culture system of cancer cells and T cells] The interferon-γ production-promoting function of the anti-CLDN4 (3D11)-anti-CD137 scFv bispecific antibody was evaluated in a co-culture system of 60As6-Luc / GFP cells and Expanded PanT cells. Anti-CD3 antibody was added to a flat-bottom 96-well plate (IWAKI, 3860-096) at 0.1 μg / 100 μL / well to immobilize the anti-CD3 antibody on the plate. 5 × 10 60As6-Luc / GFP cells were then plated. 4 The cells were seeded at 50 μL / well and cultured at 37°C in a 5% CO incubator. 5Expanded PanT cells were seeded at 30 μL / well. Test antibodies and isotype controls were serially diluted in culture medium at 5-fold common ratios starting from a maximum concentration of 50,000 ng / mL, and 20 μL of each solution was added. Urelumab and anti-CLDN4 (3D11)-anti-CD137 scFv bispecific antibody (3D11_3-34-j6_S) were used as test antibodies, and IgG (lysozyme)-scFv (CD137) (prepared in-house) was used as an isotype control. After 6 days, interferon-γ production in the supernatant was measured using an AlphaLISA interferon-γ assay kit (Perkin Elmer, AL217C) according to the manufacturer's recommended protocol. Figure 3-1 shows the response curve between interferon-γ production and antibody concentration. The anti-CLDN4(3D11)-anti-CD137scFv bispecific antibody promoted interferon-γ production in a co-culture system of CLDN4-expressing cancer cell line 60As6-Luc / GFP cells and Expanded PanT cells. The anti-CLDN4(3D11)-anti-CD137scFv bispecific antibody's promotion of interferon-γ production was significantly stronger than that of urelumab.

[0145] [6-4. Evaluation of the interferon-γ production-promoting function of anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibody in a co-culture system of cancer cells and T cells] The interferon-γ production-promoting function of the anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibody in a co-culture system of 60As6-Luc / GFP cells and Expanded PanT cells was evaluated in the same manner as in 6-3. Anti-CD3 antibody was immobilized on a 96-well plate at 0.03 μg / 100 μL / well. 2 × 10 60As6-Luc / GFP cells were cultured in a 96-well plate at 0.03 μg / 100 μL / well. 4 Expanded PanT cells were seeded at 8 × 10 cells / 50 μL / well. 4Cells were seeded at 30 μL per well. Urelumab or anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibody (hKM3900_3-34-j6 or hKM3900_tA2-32LH), serially diluted at 2-fold common ratios starting from a maximum concentration of 5000 ng / mL, was used as the test antibody. 20 μL of test antibody and isotype control (lysozyme antibody) were added. Interferon-γ production in the supernatant was measured after 4 days. Figure 3-2 shows the response curve between interferon-γ production and antibody concentration. The anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibody promoted interferon-γ production in a coculture system of 60As6-Luc / GFP cells and Expanded PanT cells. The interferon-γ production promotion function of these bispecific antibodies was significantly stronger than that of urelumab.

[0146] [6-5. Evaluation of the interferon-γ production promoting function of anti-CLDN4 (3D11)-anti-CD137 scFv bispecific antibody in monoculture of T cells] The interferon-γ production-promoting function of the anti-CLDN4 (3D11)-anti-CD137 scFv bispecific antibody was evaluated in a monoculture system of expanded PanT cells. Anti-CD3 antibody was added to a flat-bottom 96-well plate at 0.1 μg / 100 μL / well to immobilize the anti-CD3 antibody on the plate. 2 × 10 5Expanded PanT cells were seeded at 80 μL / well. The test antibodies were urelumab and anti-CLDN4 (3D11)-anti-CD3 scFv bispecific antibody (3D11_3-34-j6_S), and the isotype control was IgG (lysozyme)-scFv (CD137) (prepared in-house). The test antibodies and isotype control were serially diluted in culture medium at a 5-fold common ratio starting from a maximum concentration of 50,000 ng / mL, and 20 μL each was added. After 6 days, the amount of interferon-γ produced in the supernatant was measured using the AlphaLISA interferon-γ assay kit, as described in 6-3. Figure 3-3 shows the response curve between interferon-γ production and antibody concentration. The anti-CLDN4(3D11)-anti-CD137scFv bispecific antibody did not promote interferon-γ production in a monoculture system of T cells, but urelumab did promote interferon-γ production. This result indicates that the anti-CLDN4(3D11)-anti-CD137scFv bispecific antibody does not enhance T cell activity in an environment where CLDN4-expressing cancer cell lines are not present.

[0147] [6-6. Evaluation of the interferon-γ production-promoting function of anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibody in monoculture of T cells] The interferon-γ production-promoting function of the anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibody in a monoculture system of expanded PanT cells was evaluated using the same method as in 6-5. Anti-CD3 antibody was immobilized on a 96-well plate at 0.03 μg / 100 μL / well. Expanded PanT cells were cultured in a monoculture system of 8 × 10 4The test antibodies were urelumab and anti-CLDN4 (hKM3900)-anti-CD137scFv bispecific antibodies (hKM3900_3-34-j6, hKM3900_tA2-32LH). The test antibodies and isotype control (lysozyme antibody) were serially diluted at 2-fold common ratios starting from a maximum concentration of 5000 ng / mL and added in 20 μL aliquots. Figure 3-4 shows the response curve between interferon-γ production and antibody concentration. The anti-CLDN4 (hKM3900)-anti-CD137scFv bispecific antibody did not promote interferon-γ production in a monoculture system of T cells, whereas urelumab did. The results of 6-5 and 6-6 indicate that the anti-CLDN4-anti-CD137 scFv bispecific antibody does not enhance T cell activity in the absence of CLDN4-expressing cancer cell lines, suggesting the possibility that the systemic immunostimulatory effect, which has been reported as a side effect of anti-CD137 antibodies, may be reduced.

[0148] [6-7. Evaluation of anti-CLDN4 (3D11)-anti-CD137 scFv bispecific antibody cytotoxicity in a co-culture system of cancer cells and T cells] The anti-CLDN4 (3D11)-anti-CD137 scFv bispecific antibody was evaluated for its cytotoxicity (cancer cell growth inhibitory effect) in a co-culture system of 60As6-Luc / GFP cells and Expanded PanT cells. 60As6-Luc / GFP cells were cultured in a flat-bottom 96-well plate at 1 × 10 4 The cells were seeded at 50 μL / well and cultured at 37°C in a 5% CO2 incubator. After 2 hours, 1 × 10 Expanded panT cells were added to the 96-well plate. 5Cells were seeded at 50 μL per well. Urelumab and anti-CLDN4 (3D11)-anti-CD137 scFv bispecific antibodies were used as test antibodies. These antibodies were serially diluted in culture medium at a 10-fold common ratio starting from a maximum concentration of 40,000 ng / mL, and 50 μL of each was added. Furthermore, 50 μL of 40 ng / mL anti-CD3 antibody was added, and measurement of the fluorescence (GFP) area in each well was initiated using an IncuCyte® ZOOM (Sartorius) in a 37°C, 5% CO2 incubator. The increase in fluorescence area from 0 hours after the start of measurement was used as an index of cell proliferation. Figure 4 shows the cancer cell cytotoxicity (cancer cell proliferation rate) of the test antibodies. The vertical axis of Figure 4 indicates the relative value of the increase in fluorescence area from 0 hours to 168 hours, with the well without test antibody set at 100%. As shown in Figure 4, the anti-CLDN4(3D11)-anti-CD137scFv bispecific antibody exhibited cytotoxicity in a coculture system of 60As6-Luc / GFP cells and Expanded PanT cells. The cytotoxic activity of this bispecific antibody was significantly stronger than that of urelumab.

[0149] [6-8. Evaluation of anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibody cytotoxicity in a co-culture system of cancer cells and T cells] The cancer cell killing activity of the anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibody was evaluated in a co-culture system of 60As6-Luc / GFP cells and Expanded PanT cells. Anti-CD3 antibody was added at 1 ng / 100 μL / well to a flat-bottom 96-well plate (Perkin Elmer, 6005680) and immobilized on the plate. After washing the plate twice with PBS(-), 2 × 10 4 60As6-Luc / GFP cells were seeded at 50 μL / well and cultured at 37°C in a 5% CO2 incubator. The next day, 4.0 × 10 4Expanded PanT cells were seeded at 30 μL / well. 20 μL each of test antibodies and isotype controls at concentrations ranging from 5000 ng / mL to 0.844 ng / mL were added. Test antibodies included urelumab and anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibodies (hKM3900_3-34-j6, hKM3900_tA2-32LH). Measurements were performed 6 days later. To kill the cells, 11 μL of 1% Triton-X100 (Sigma-Aldrich, T-9284) was added to some wells 30 minutes before measurement. The luciferase activity of 60As6-Luc / GFP cells in each well was measured using an ARVO X-3 (Perkin Elmer) with the ONE-Glo Luciferase Assay Kit (Promega, E6120) according to the manufacturer's recommended protocol. The tumor killing rate is expressed as a relative value, with the mean luciferase chemiluminescence of wells without antibody set to 0% and the mean value of wells with Triton-X100 set to 100%. An anti-lysozyme antibody (prepared in-house) was used as an isotype control. Figure 5 shows the cell killing rate curve for 60As6-Luc / GFP cells. In a coculture system of 60As6-Luc / GFP cells and expanded PanT cells, the addition of anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibody significantly increased the 60As6-Luc / GFP cell killing rate. On the other hand, the anti-CD137 specific antibody urelumab did not increase the cell killing rate of the 60As6-Luc / GFP cell line.

[0150] Example 7: In vivo evaluation of anti-CLDN4-anti-CD137 scFv bispecific antibodies [7-1. In vivo antitumor effect of anti-CLDN4-anti-CD137 scFv bispecific antibody] NCI-H322 cells were suspended in PBS(-) (WAKO, 045-29795) and diluted to 5.0 × 10 6 The cell suspension was subcutaneously inoculated into 7-week-old female mice (NOG, Invivo Sciences) at a concentration of 100 μL. Six days after inoculation, 3.4 × 10 6Human peripheral blood mononuclear cells (Lonza, CC-2702) were injected via the tail vein at a rate of 200 μL per cell. 15 days after the human peripheral blood mononuclear cell transplantation, mice were divided into groups and administration of the test antibody was initiated. Each group consisted of 10 mice, and the mice were divided so that tumor volumes were approximately equal between groups (day 0). hKM3900_tA2-32LH was used as the test antibody. The test antibody and isotype control (anti-lysozyme antibody) were mixed with PBS and administered intravenously. Administration was performed on days 0, 4, and 8, and tumor diameter and body weight were measured at the time of administration. The following formula was used to calculate tumor volume. [Tumor volume (mm 3 )] = [Tumor long diameter (mm)] x [Tumor short diameter (mm)] 2 × 0.5 As shown in Figure 6, significant tumor volume reduction was observed in the groups administered 0.1, 1, or 10 mg / kg of hKM3900_tA2-32LH. The vertical axis represents tumor volume, and the horizontal axis represents the number of days after the first administration. The solid and dashed lines represent the time course of the mean tumor volume in each group, and the error bars represent the standard error of the mean. The significance probability P value (*: P<0.05, **: P<0.01) was calculated by comparing the tumor volumes on day 12 between the isotype control-administered group and the hKM3900_tA2-32LH-administered group using Dunnett's multiple comparison test. The number of cases on day 12 in the hKM3900_tA2-32LH 0.1 mg / kg-administered group was 9.

[0151] [7-2. Safety study of anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibody in cynomolgus monkeys] To evaluate the safety of the anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibody (hKM3900_3-34-j6 and hKM3900_tA2-32LH), a single dose of the anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibody was administered intravenously to cynomolgus monkeys. No significant abnormalities were observed in serum cytokine concentrations, such as IL-6 and TNF-α, in monkeys administered the anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibody. Therefore, the anti-CLDN4 (hKM3900)-anti-CD137 scFv bispecific antibody was deemed suitable for use in cancer therapy from a safety perspective.

[0152] Example 8: Cancer treatment with anti-CLDN4-anti-CD137 bispecific antibodies [8-1. Confirmation of CLDN4 expression level] To investigate the versatility of CLDN4 as a therapeutic target for cancer, we investigated the expression levels of CLDN4 RNA in cancer tissues using The Cancer Genome Atlas (TCGA). We confirmed that CLDN4 expression was elevated in cancer tissues, including colorectal cancer, bladder cancer, and lung cancer. This suggests that CLDN4 may be a therapeutic target not only for peritoneal dissemination but also for other cancer types. Furthermore, we investigated the expression levels of CLDN4 RNA in normal tissues using The Genotype-Tissue Expression (GTEx). We found that CLDN4 was expressed in several normal tissues. However, the expression levels of CLDN4 in normal tissues were lower than in cancer tissues. These findings suggest that cancer therapeutic drugs targeting CLDN4 may have a wide safety margin and may have fewer side effects. [Industrial Applicability]

[0153] The anti-CLDN4-anti-CD137 bispecific antibody of the present invention is expected to be useful in the treatment of cancer, and the polynucleotide, expression vector, transformed host cell, and antibody production method of the present invention are useful for producing the anti-CLDN4-anti-CD137 bispecific antibody. [Sequence List Free Text]

[0154] Numerical headings in the sequence listing below <223> An explanation of "Artificial Sequence" is provided below. Specifically, SEQ ID NO: 1 in the Sequence Listing is the nucleotide sequence of the heavy chain variable region of 3D11, and SEQ ID NO: 2 is the amino acid sequence of the heavy chain variable region of 3D11. SEQ ID NO: 3 is the nucleotide sequence of the light chain variable region of 3D11, and SEQ ID NO: 4 is the amino acid sequence of the light chain variable region of 3D11. SEQ ID NO: 5 is the nucleotide sequence of the heavy chain variable region of hKM3900, and SEQ ID NO: 6 is the amino acid sequence of the heavy chain variable region of hKM3900. SEQ ID NO: 7 is the nucleotide sequence of the light chain variable region of hKM3900, and SEQ ID NO: 8 is the amino acid sequence of the light chain variable region of hKM3900. SEQ ID NO: 9 is the nucleotide sequence of the heavy chain variable region of 3-34, and SEQ ID NO: 10 is the amino acid sequence of the heavy chain variable region of 3-34. SEQ ID NO: 11 is the nucleotide sequence of the light chain variable region of 3-34, and SEQ ID NO: 12 is the amino acid sequence of the light chain variable region of 3-34. SEQ ID NO: 13 is the nucleotide sequence of the heavy chain variable region of A2-32, and SEQ ID NO: 14 is the amino acid sequence of the heavy chain variable region of A2-32. SEQ ID NO: 15 is the nucleotide sequence of the light chain variable region of A2-32, and SEQ ID NO: 16 is the amino acid sequence of the light chain variable region of A2-32. SEQ ID NO: 17 is the nucleotide sequence of the heavy chain variable region of A2-48, and SEQ ID NO: 18 is the amino acid sequence of the heavy chain variable region of A2-48. SEQ ID NO: 19 is the nucleotide sequence of the light chain variable region of A2-48, and SEQ ID NO: 20 is the amino acid sequence of the light chain variable region of A2-48. SEQ ID NO: 21 is the nucleotide sequence of the heavy chain variable region of A2-73, and SEQ ID NO: 22 is the amino acid sequence of the heavy chain variable region of A2-73. SEQ ID NO: 23 is the nucleotide sequence of the light chain variable region of A2-73, and SEQ ID NO: 24 is the amino acid sequence of the light chain variable region of A2-73. SEQ ID NO: 25 is the nucleotide sequence of 3-34-j6 scFv, and SEQ ID NO: 26 is the amino acid sequence of 3-34-j6 scFv. SEQ ID NO: 27 is the nucleotide sequence of tA2-32 scFv, and SEQ ID NO: 28 is the amino acid sequence of tA2-32 scFv. SEQ ID NO: 29 is the nucleotide sequence of tA2-32LH scFv, and SEQ ID NO: 30 is the amino acid sequence of tA2-32LH scFv. SEQ ID NO: 31 is the nucleotide sequence of tA2-48 scFv, and SEQ ID NO: 32 is the amino acid sequence of tA2-48 scFv.SEQ ID NO: 33 is the nucleotide sequence of tA2-73 scFv, and SEQ ID NO: 34 is the amino acid sequence of tA2-73 scFv. SEQ ID NO: 35 is the nucleotide sequence of the heavy chain of hKM3900_3-34-j6, and SEQ ID NO: 36 is the amino acid sequence of the heavy chain of hKM3900_3-34-j6. SEQ ID NO: 37 is the nucleotide sequence of the heavy chain of hKM3900_tA2-32LH, and SEQ ID NO: 38 is the amino acid sequence of the heavy chain of hKM3900_tA2-32LH. SEQ ID NO: 39 is the nucleotide sequence of the light chain of hKM3900, and SEQ ID NO: 40 is the amino acid sequence of the light chain of hKM3900. SEQ ID NO: 41 is the amino acid sequence of an anti-CD3 scFv-human Fc fusion protein. SEQ ID NO: 42 is the amino acid sequence of human CD137. SEQ ID NO: 43 is the amino acid sequence of monkey CD137. SEQ ID NO: 44 is the amino acid sequence of the His-tagged protein. SEQ ID NO: 45 is the amino acid sequence of the 3xFLAG tag. SEQ ID NOs: 46 to 54 are the amino acid sequences of the GS linker. SEQ ID NO: 55 is the amino acid sequence of the GKPGS linker.

Claims

1. An anti-CD137 antibody or an antigen-binding fragment thereof comprising the heavy chain variable region and the light chain variable region of any of the anti-CD137 antibodies (a) to (d) below: (a) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 10, a CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 66 of SEQ ID NO: 10, and a CDR3 consisting of the amino acid sequence from amino acid numbers 99 to 107 of SEQ ID NO: 10, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 24 to 34 of SEQ ID NO: 12, a CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 56 of SEQ ID NO: 12, and a CDR3 consisting of the amino acid sequence from amino acid numbers 89 to 98 of SEQ ID NO: 12; (b) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 14, a CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 65 of SEQ ID NO: 14, and a CDR3 consisting of the amino acid sequence from amino acid numbers 98 to 107 of SEQ ID NO: 14, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 23 to 35 of SEQ ID NO: 16, a CDR2 consisting of the amino acid sequence from amino acid numbers 51 to 57 of SEQ ID NO: 16, and a CDR3 consisting of the amino acid sequence from amino acid numbers 90 to 100 of SEQ ID NO: 16; (c) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 18, a CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 65 of SEQ ID NO: 18, and a CDR3 consisting of the amino acid sequence from amino acid numbers 98 to 107 of SEQ ID NO: 18, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid numbers 23 to 35 of SEQ ID NO: 20, a CDR2 consisting of the amino acid sequence from amino acid numbers 51 to 57 of SEQ ID NO: 20, and a CDR3 consisting of the amino acid sequence from amino acid numbers 90 to 100 of SEQ ID NO: 20; or (d) a heavy chain variable region comprising CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 22, CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 65 of SEQ ID NO: 22, and CDR3 consisting of the amino acid sequence from amino acid numbers 98 to 110 of SEQ ID NO: 22; and a light chain variable region comprising CDR1 consisting of the amino acid sequence from amino acid numbers 23 to 35 of SEQ ID NO: 24, CDR2 consisting of the amino acid sequence from amino acid numbers 51 to 57 of SEQ ID NO: 24, and CDR3 consisting of the amino acid sequence from amino acid numbers 90 to 100 of SEQ ID NO:

24.

2. The anti-CD137 antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region and the light chain variable region of the anti-CD137 antibody are any of the following (a) to (i): (a) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 10 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 109 of SEQ ID NO: 12; (b) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 14 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 16; (c) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 18 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 20; (d) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 121 of SEQ ID NO: 22 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 24; (e) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 26 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 134 to 242 of SEQ ID NO: 26; (f) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 28 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 134 to 244 of SEQ ID NO: 28; (g) a light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 30 and a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 132 to 249 of SEQ ID NO: 30; (h) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 32 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 134 to 244 of SEQ ID NO: 32; or (i) a heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 121 of SEQ ID NO: 34 and a light chain variable region consisting of the amino acid sequence from amino acid numbers 137 to 247 of SEQ ID NO:

34.

3. The anti-CD137 antibody of claim 1, which is an IgG antibody consisting of a heavy chain comprising the heavy chain variable region of the anti-CD137 antibody and a light chain comprising the light chain variable region.

4. The antigen-binding fragment of claim 1, which is an anti-CD137 single-chain variable region fragment (anti-CD137 scFv) comprising the heavy chain variable region and light chain variable region of an anti-CD137 antibody.

5. The antigen-binding fragment of claim 1, which is an anti-CD137 scFv consisting of any one of the following sequences (a) to (e): (a) an anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 1 to 242 of SEQ ID NO: 26; (b) an anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 1 to 244 of SEQ ID NO: 28; (c) an anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 1 to 249 of SEQ ID NO: 30; (d) an anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 1 to 244 of SEQ ID NO: 32; or (e) An anti-CD137 scFv consisting of the amino acid sequence from amino acid numbers 1 to 247 of SEQ ID NO:

34.

6. The anti-CD137 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, which is post-translationally modified.

7. An anti-TAA-anti-CD137 bispecific antibody comprising an antibody against any TAA (anti-TAA antibody) and an anti-CD137 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

8. An anti-TAA-anti-CD137 bispecific antibody comprising an antibody against any TAA (anti-TAA antibody) and the antigen-binding fragment of claim 5.

9. The anti-TAA-anti-CD137 bispecific antibody of claim 7, which is post-translationally modified.

10. An anti-CD137 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 for use in the treatment of cancer.

11. A pharmaceutical composition comprising the anti-CD137 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 and a pharmaceutically acceptable excipient.

12. 12. The pharmaceutical composition of claim 11 for use in the treatment of cancer.

13. The anti-TAA-anti-CD137 bispecific antibody of claim 7 for use in the treatment of cancer.

14. A pharmaceutical composition comprising the anti-TAA-anti-CD137 bispecific antibody of claim 7 and a pharmaceutically acceptable excipient.

15. 15. The pharmaceutical composition of claim 14 for use in the treatment of cancer.

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