Anti-CLDN4-Anti-CD137 bispecific 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] This invention relates to an anti-CLDN4-anti-CD137 bispecific antibody that is expected to be useful as an active ingredient in pharmaceutical compositions used for the treatment of cancer. [Background technology]

[0002] Claudin-4 (CLDN4) is a four-pass transmembrane protein belonging to the claudin family. It is expressed in epithelial and endothelial cells and plays an important role as a major molecule in the formation of tight junctions. High expression of CLDN4 has been observed in cancer tissues such as colorectal cancer, bladder cancer, and ovarian cancer, suggesting that anti-CLDN4 antibodies may be applicable to the treatment or diagnosis of cancer (Patent Document 1, Non-Patent Document 1). Furthermore, antitumor effects have been demonstrated in animal models by the combined use of anti-CLDN4 antibodies and anti-Epidermal growth factor receptor (EGFR) antibodies (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 CD137 ligands on antigen-presenting cells and is involved in T cell activation and survival as a co-stimulatory molecule (Non-Patent Literature 3). Anti-CD137 agonist antibodies have shown antitumor effects in animal models through the activation of immune cells in the tumor microenvironment (Non-Patent Literature 4). Urelumab, an anti-CD137 agonist antibody, showed therapeutic efficacy in clinical trials, but it has also been reported to cause liver damage as a side effect (Non-Patent Literature 5).

[0004] As a groundbreaking method for obtaining cancer cell-selective cytotoxic activity at low antibody concentrations, bispecific T-cell-recruiting antibodies in various antibody formats have been reported. Bispecific T-cell-recruiting antibodies are bispecific antibodies that include antibodies against tumor-associated antigens (TAAs) expressed on the surface of cancer cells and antibodies that bind to T cells, and the effects of these antibodies on T-cell-mediated immunotherapy are currently being investigated (Non-patent Literature 6). Anti-CD3 antibodies are frequently 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, research on bispecific T cell recruitment antibodies against CD137 and TAAs has become increasingly active. Studies have been conducted on 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 TAAs such as 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 7-9). However, to date, there are no reports of bispecific antibodies against CLDN4 and CD137. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] International Publication Number 2008 / 114733 [Patent Document 2] International Publication Number 2015 / 156268 [Patent Document 3] International Publication Number 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 [Overview of the project] [Problems that the invention aims to solve]

[0008] The 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 inventors searched for an antibody that selectively binds to an antigen expressed on peritoneal disseminated cancer cells and obtained 3D11, an anti-CLDN4 antibody (Examples 1 and 2). It was found that bispecific T cell-recruiting antibodies using 3D11 and an anti-CD3 antibody cannot be used for the treatment of cancer patients (Reference Example). Therefore, an attempt was made to obtain a bispecific T cell-recruiting antibody with an anti-CD137 antibody using 3D11 and the KM3900 antibody, a known anti-CLDN4 antibody (Examples 3 and 4). The obtained anti-CLDN4-anti-CD137 bispecific antibody bound to CLDN4 and CD137 (Example 5), promoted interferon-γ production of T cells in vitro, and exhibited cytotoxic activity against cancer cells expressing CLDN4 on the cell surface (Example 6). Furthermore, the obtained anti-CLDN4-anti-CD137 bispecific antibody exerted an antitumor effect in an in vivo mouse model (Example 7-1), and it was confirmed that it can be safely used in cynomolgus monkeys as well (Example 7-2). It was suggested that the anti-CLDN4-anti-CD137 bispecific antibody of the present invention is useful for the treatment of cancer.

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

[10] The bispecific antibody described in [9], wherein the anti-CD137scFv is one of the following (a) to (e): (a) Anti-CD137scFv consisting of the amino acid sequence from amino acid number 1 to 242 of SEQ ID NO: 26; (b) Anti-CD137scFv consisting of the amino acid sequence of amino acid numbers 1 to 244 of SEQ ID NO: 28; (c) Anti-CD137scFv consisting of the amino acid sequence from amino acid number 1 to 249 of SEQ ID NO: 30; (d) Anti-CD137scFv consisting of the amino acid sequence from amino acid number 1 to 244 of SEQ ID NO. 32; or (e) Anti-CD137scFv consisting of the amino acid sequence of amino acid numbers 1 to 247 of SEQ ID NO: 34.

[11] A 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 heavy or light chain carboxyl terminus 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) a heavy chain of an anti-CLDN4 antibody containing a heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 125 of SEQ ID NO: 2; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences from amino acid numbers 1 to 109 of SEQ ID NO: 4; and an anti-CD137scFv containing a heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 118 of SEQ ID NO: 26 and a light chain variable region consisting of amino acid sequences from amino acid numbers 134 to 242 of SEQ ID NO: 26, wherein the amino terminus of the anti-CD137scFv is linked to the carboxyl 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 containing a heavy chain variable region consisting of amino acid sequences 1 to 125 of SEQ ID NO: 2; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 4; and an anti-CD137scFv containing a heavy chain variable region consisting of amino acid sequences 1 to 118 of SEQ ID NO: 28 and a light chain variable region consisting of amino acid sequences 134 to 244 of SEQ ID NO: 28, wherein the amino terminus of the anti-CD137scFv is linked to the carboxyl 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 containing a heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 125 of SEQ ID NO: 2; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences from amino acid numbers 1 to 109 of SEQ ID NO: 4; and an anti-CD137scFv containing a light chain variable region consisting of amino acid sequences from amino acid numbers 1 to 111 of SEQ ID NO: 30 and a heavy chain variable region consisting of amino acid sequences from amino acid numbers 132 to 249 of SEQ ID NO: 30, wherein the amino terminus of the anti-CD137scFv is linked to the carboxyl 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 containing a heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 125 of SEQ ID NO: 2; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences from amino acid numbers 1 to 109 of SEQ ID NO: 4; and an anti-CD137scFv containing a heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 118 of SEQ ID NO: 32 and a light chain variable region consisting of amino acid sequences from amino acid numbers 134 to 244 of SEQ ID NO: 32, wherein the amino terminus of the anti-CD137scFv is linked to the carboxyl 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 containing a heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 125 of SEQ ID NO: 2; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences from amino acid numbers 1 to 109 of SEQ ID NO: 4; and an anti-CD137scFv containing a heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 121 of SEQ ID NO: 34 and a light chain variable region consisting of amino acid sequences from amino acid numbers 137 to 247 of SEQ ID NO: 34, 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 bispecific antibody comprising: a heavy chain of an anti-CLDN4 antibody containing a heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 123 of SEQ ID NO: 6; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences from amino acid numbers 1 to 109 of SEQ ID NO: 8; and an anti-CD137scFv containing a heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 118 of SEQ ID NO: 26 and a light chain variable region consisting of amino acid sequences from amino acid numbers 134 to 242 of SEQ ID NO: 26, 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 bispecific antibody comprising: a heavy chain of an anti-CLDN4 antibody containing a heavy chain variable region consisting of amino acid sequences 1 to 123 of SEQ ID NO: 6; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 8; and an anti-CD137scFv containing a heavy chain variable region consisting of amino acid sequences 1 to 118 of SEQ ID NO: 28 and a light chain variable region consisting of amino acid sequences 134 to 244 of SEQ ID NO: 28, wherein the amino terminus of the anti-CD137scFv is linked to the carboxyl 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 containing a heavy chain variable region consisting of amino acid sequences 1 to 123 of SEQ ID NO: 6; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 8; and an anti-CD137scFv containing a light chain variable region consisting of amino acid sequences 1 to 111 of SEQ ID NO: 30 and a heavy chain variable region consisting of amino acid sequences 132 to 249 of SEQ ID NO: 30, wherein the amino terminus of the anti-CD137scFv is linked to the carboxyl terminus of the heavy chain of the anti-CLDN4 antibody via a linker; (i) A bispecific antibody comprising: (i) a heavy chain of an anti-CLDN4 antibody containing a heavy chain variable region consisting of amino acid sequences 1 to 123 of SEQ ID NO: 6; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 8; and an anti-CD137scFv containing a heavy chain variable region consisting of amino acid sequences 1 to 118 of SEQ ID NO: 32 and a light chain variable region consisting of amino acid sequences 134 to 244 of SEQ ID NO: 32, wherein the amino terminus of the anti-CD137scFv is linked to the carboxyl 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 containing a heavy chain variable region consisting of amino acid sequences 1 to 123 of SEQ ID NO: 6; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 8; and an anti-CD137scFv containing a heavy chain variable region consisting of amino acid sequences 1 to 121 of SEQ ID NO: 34 and a light chain variable region consisting of amino acid sequences 137 to 247 of SEQ ID NO: 34, wherein the amino terminus of the anti-CD137scFv is linked to the heavy chain carboxy terminus of the anti-CLDN4 antibody via a linker.

[13] An anti-CLDN4-anti-CD137 bispecific antibody as described in either

[11] or

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

[14] The anti-CLDN4-anti-CD137 bispecific antibody described in

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

[15] Either (a) or (b) below, an anti-CLDN4-anti-CD137 bispecific antibody: (a) A bispecific antibody comprising a heavy chain of an anti-CLDN4 antibody consisting of amino acid sequences 1 to 705 of SEQ ID NO: 36 and a polypeptide containing anti-CD137scFv, and a light chain of an anti-CLDN4 antibody consisting of amino acid sequences 1 to 215 of SEQ ID NO: 40; or (b) A bispecific antibody comprising 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 polypeptide containing anti-CD137scFv, 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] Post-translation modified anti-CLDN4-anti-CD137 bispecific antibody as described in any of [1] to

[15] .

[17] The anti-CLDN4-anti-CD137 bispecific antibody described in

[16] , wherein the post-translational 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.

[18] A polynucleotide selected from the group consisting of (a) to (d) below, which contains a base sequence encoding the heavy chain variable region or light chain variable region of an anti-CLDN4 antibody: (a) A polynucleotide containing a nucleotide sequence that encodes the 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 containing a nucleotide sequence that encodes 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 containing a base sequence that encodes the light chain variable region of the anti-CLDN4 antibody, consisting of the amino acid sequence from amino acid number 1 to 109 of SEQ ID NO: 8.

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

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

[21]

[15] Polynucleotides selected from the group consisting of (a) to (e) below for use in the production of the anti-CLDN4-anti-CD137 bispecific antibody: (a) A polynucleotide comprising a nucleotide sequence encoding a polypeptide containing the heavy chain of an anti-CLDN4 antibody and anti-CD137scFv, consisting of the amino acid sequence from amino acid numbers 1 to 705 of SEQ ID NO: 36; (b) A polynucleotide comprising a nucleotide sequence encoding a polypeptide containing the heavy chain of an anti-CLDN4 antibody and anti-CD137scFv, consisting of the amino acid sequence from amino acid number 1 to 712 of SEQ ID NO. 38; (c) A polynucleotide containing the base sequence encoding the light chain of the anti-CLDN4 antibody, consisting of the amino acid sequence from amino acid number 1 to 215 of SEQ ID NO: 40; (d) A polynucleotide comprising a nucleotide sequence encoding a polypeptide containing the heavy chain of an anti-CLDN4 antibody consisting of amino acid sequences 1 to 705 of SEQ ID NO: 36 and anti-CD137scFv, and a polynucleotide comprising a nucleotide sequence encoding the light chain of an anti-CLDN4 antibody consisting of amino acid sequences 1 to 215 of SEQ ID NO: 40; or (e) A polynucleotide comprising a base sequence encoding the heavy chain of an anti-CLDN4 antibody consisting of amino acid sequences from amino acid numbers 1 to 712 of SEQ ID NO: 38 and a polypeptide containing anti-CD137scFv, and a polynucleotide comprising a base sequence encoding the light chain of an anti-CLDN4 antibody consisting of amino acid sequences from amino acid numbers 1 to 215 of SEQ ID NO: 40. An expression vector containing a polynucleotide as described in any of

[22]

[18] to

[21] .

[23] Host cells transformed with the expression vector described in

[22] .

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

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

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

[23] to

[25] . A pharmaceutical composition comprising an anti-CLDN4-anti-CD137 bispecific antibody as described in any of [1] to

[17] and a pharmaceutically acceptable excipient.

[28] An anti-CLDN4-anti-CD137 bispecific antibody as described in any of [1] to

[17] , for use in the treatment of cancer.

[29] The pharmaceutical composition described in

[27] for use in the treatment of cancer. A method for treating cancer, comprising the step of administering a therapeutically effective dose of an anti-CLDN4-anti-CD137 bispecific antibody as described in any of [1] to

[17] .

[31] Use of an anti-CLDN4-anti-CD137 bispecific antibody as described in any of [1] to

[17] in the manufacture of a pharmaceutical composition for 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, which is expressed on the surface of immune cells such as T cells, thereby enhancing the killing effect on cancer cells by activating immune cells surrounding 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 drawing]

[0012] [Figure 1-1] Figure 1-1 shows the CD137 binding activity of the test antibody 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 concentration of the test antibody. The symbols represent the average absorbance when each test antibody is added, and the error bars represent the standard deviation. [Figure 1-2] Figure 1-2 shows the CD137 binding activity of the test antibody 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 concentration of the test antibody. The symbols indicate the average absorbance when each test antibody is added. [Figure 1-3] Figure 1-3 shows the CD137 binding activity of the 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 concentration of the test antibody. The symbols indicate the average absorbance when each test antibody is added. [Figure 1-4] Figures 1-4 show the CD137 binding activity of the 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 concentration of the test antibody. The symbols indicate the average absorbance when each test antibody is added. [Figure 2-1]Figure 2-1 shows the CD137 agonist activity of the test antibody in a co-culture study of CLDN4-expressing cancer cells (NCI-H322 cells) and 4-1BB Effector Cells. The vertical axis of the figure represents relative luminescence, and the horizontal axis represents the concentration of the test antibody. The symbols represent the average relative luminescence when each test antibody is added, and the error bars represent the standard deviation. [Figure 2-2] Figure 2-2 shows the CD137 agonist activity of the test antibody in a co-culture study of CLDN4-expressing cancer cells (NCI-H322 cells) and 4-1BB Effector Cells. The vertical axis of the figure represents relative luminescence, and the horizontal axis represents the concentration of the test antibody. The symbols indicate the average relative luminescence when each test antibody is added. [Figure 2-3] Figure 2-3 shows the CD137 agonist activity of the test antibody in a co-culture study of CLDN4-expressing cancer cells (NCI-H322 cells) and 4-1BB Effector Cells. The vertical axis of the figure represents relative luminescence, and the horizontal axis represents the concentration of the test antibody. The symbols represent the average relative luminescence when each test antibody is added, and the error bars represent the standard deviation. [Figure 2-4] Figure 2-4 shows the CD137 agonist activity of the test antibody in a single-cell culture test of 4-1BB Effector Cells. The vertical axis of the figure represents relative luminescence, and the horizontal axis represents the concentration of the test antibody. The symbols represent the average relative luminescence when each test antibody is added, and the error bars represent the standard deviation. [Figure 2-5] Figure 2-5 shows the CD137 agonist activity of the test antibody in a single-cell culture test of 4-1BB Effector Cells. The vertical axis of the figure represents relative luminescence, and the horizontal axis represents the concentration of the test antibody. The symbols indicate the average relative luminescence value when each test antibody is added. [Figure 2-6] Figure 2-6 shows the CD137 agonist activity of the test antibody in a single-cell culture test of 4-1BB Effector Cells. The vertical axis of the figure represents relative luminescence, and the horizontal axis represents the concentration of the test antibody. The symbols represent the average relative luminescence when each test antibody is added, and the error bars represent the standard deviation. [Figure 3-1]Figure 3-1 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 represents the amount of interferon-γ produced (pg / mL) 6 days after antibody addition, and the horizontal axis represents 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 represents the amount of interferon-γ produced (pg / mL) 4 days after antibody addition, and the horizontal axis represents the antibody concentration. The symbols represent the average amount of interferon-γ produced at each antibody concentration, and the error bars represent the standard deviation. [Figure 3-3] Figure 3-3 shows the interferon-γ production in expanded panT cell monoculture systems upon addition of each test antibody. The vertical axis of the figure represents the interferon-γ production (pg / mL) 6 days after antibody addition, and the horizontal axis represents the antibody concentration. The symbols indicate the average interferon-γ production at each antibody concentration. [Figure 3-4] Figures 3-4 show the interferon-γ production in expanded panT cell monoculture systems upon addition of each test antibody. The vertical axis of the figure represents interferon-γ production (pg / mL) 5 days after antibody addition, and the horizontal axis represents antibody concentration. The symbols represent the average interferon-γ production at each antibody concentration, and the error bars represent the standard deviation. [Figure 4] Figure 4 shows the cancer cytotoxic activity (inhibitory effect on cancer cell proliferation) 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 proliferation rate when the well without the test antibody is set to 100% based on the increase in fluorescence area from 0 to 168 hours after the start of measurement. The horizontal axis shows the antibody concentration. The symbols indicate the average value of cancer cell proliferation rate for each antibody. [Figure 5]Figure 5 shows the cancer cell cytotoxic 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 cancer cell cytotoxicity (%) measured by chemiluminescence of 60As6-Luc / GFP cells 6 days after antibody addition, with the mean value without antibody addition set as 0% cytotoxicity and the mean value with Triton-X100 added as 100% cytotoxicity. The horizontal axis shows the antibody concentration. The symbols represent the mean value of cancer cell cytotoxicity at each antibody concentration, and the error bars represent the standard deviation. [Figure 6] Figure 6 shows the change in tumor volume in PBMC-transplanted mice subcutaneously carrying NCI-H322 cells. The horizontal axis of the figure represents the number of days after the first administration, the vertical axis represents the mean tumor volume, and the error bars represent the standard error. [Modes for carrying out the invention]

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

[0014] Antibodies (or immunoglobulins) are glycoproteins with a basic structure consisting of four chains with a symmetrical Y-shaped configuration, comprising 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 the antibody molecule is common to all classes, with two heavy chains with a molecular weight of 50,000 to 70,000 and two light chains with a molecular weight of 20,000 to 30,000 linked by disulfide bonds and non-covalent bonds, forming an antibody molecule with a Y-shaped four-chain structure and a molecular weight of 150,000 to 190,000. The heavy chains usually consist of polypeptide chains containing about 440 amino acids, and each class has a characteristic structure, corresponding to IgG, IgM, IgA, IgD, and IgE, which are called Igγ, Igμ, Igα, Igδ, and Igε, respectively. Furthermore, IgG has subclasses IgG1, IgG2, IgG3, and IgG4, and the corresponding heavy chains are called Igγ1, Igγ2, Igγ3, and Igγ4, respectively. The light chain usually consists of a polypeptide chain containing about 220 amino acids, and two types are known: λ-type and κ-type, called Igλ and Igκ, respectively. These two types of light chains can be paired with any type of heavy chain.

[0015] Antibody molecules have four disulfide bonds in the heavy chain (five in Igμ and Igε) and two in the light chain, forming one loop every 100-110 amino acid residues. Their three-dimensional structures are similar across loops and are called structural units or domains. The N-terminal domain in both the heavy and light chains is called the variable region. Even antibodies produced from the same class (or subclass) of the same animal species exhibit diverse amino acid sequences and are known to be involved in antibody-antigen binding specificity. 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. The heavy chain has a heavy chain variable region (VH) and a heavy chain constant region (CH) extending from the N-terminus to the C-terminus. The CH is further divided into three domains from the N-terminus: the CH1 domain, the CH2 domain, and the CH3 domain. The light chain has a light chain variable region (VL) and a light chain constant region (CL) extending from the N-terminus to the C-terminus.

[0016] The amino acid sequences of the three complementarity-determining regions (CDRs) present in the VH and VL regions change significantly, contributing to the variability of the variable region. CDRs are regions consisting of 5 to 10 amino acid residues, located at the N-terminuses of the heavy and light chains in the order of CDR1, CDR2, and CDR3, respectively, and form antibody-binding sites upon contact with the antigen. The CDRs on the heavy chain contribute more to antigen binding than those on the light chain, and among CDR1-3, CDR3 is known to have the greatest contribution. On the other hand, the portion of the variable region other than the CDRs is called the framework region (FR), consisting of FR1-FR4, and its amino acid sequence changes relatively little.

[0017] When an antibody is treated with the protease papain, three antibody fragments are obtained. The two fragments at the N-terminus are called Fab (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, as well as the light chain (VL and CL). The VH and VL (antigen binding sites) that make up the Fab region bind to the antigen. The fragment at the C-terminus is called Fc (crystallizable) region.

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

[0019] In this specification, "antigen-binding fragment" and "fragment antibody" are interchangeable and refer to a molecule containing at least one polypeptide chain that includes 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. In this specification, "scFv" refers to a monovalent antigen-binding fragment composed of VH and VL linked by a linker. A Fab fragment is a monovalent antigen-binding fragment composed 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 composed of a light chain and a fragment containing the VH, CH1 domain and part of the hinge region of the heavy chain, and the hinge region contains cysteine ​​residues that constituted the inter-heavy chain disulfide bond. A F(ab')2 fragment is a bivalent antigen-binding fragment in which a Fab' fragment is linked by a disulfide bond. Monovalent means that it contains one antigen-binding site, while bivalent means that it contains two antigen-binding sites.

[0020] In this specification, “bispecific antibody” means an antibody molecule composed of two antibodies or antigen-binding fragments that specifically bind to different antigens and have binding activity to each antigen. Various structures of bispecific antibodies are known to those skilled in the art (Non-Patent Literature 6). As used herein, the term “antibody” includes, unless otherwise specified in the context, full-length antibodies (various immunoglobulins, particularly IgG antibodies), antigen-binding fragments, and bispecific antibodies of any structure.

[0021] In this specification, IgG-type anti-CLDN4 antibodies are referred to as "anti-CLDN4 IgG antibodies."

[0022] In this specification, "human antibody" refers to an antibody having a human immunoglobulin amino acid sequence. In this specification, "humanized antibody" refers to an antibody in which some, most, or all of the amino acid residues other than the CDR are replaced with amino acid residues derived from a human immunoglobulin molecule. The method of humanization is not particularly limited, but humanized antibodies can be produced 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 or EU index (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed, 1991, NIH Publication No. 91-3242) by designating these numbering systems.

[0024] In this specification, “linked” or “linked” means that multiple components (e.g., Fab regions and Fc regions) are linked directly or via an intermediary (e.g., a peptide linker). In this specification, “peptide linker” means one or more arbitrary amino acid sequences that can be introduced by genetic engineering techniques and 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] In this specification, "identity" refers to the Identity value obtained using the 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] <The anti-CLDN4-anti-CD137 bispecific antibody of the present invention> The present invention provides a bispecific antibody that binds to CLDN4 and CD137 (also referred to as "anti-CLDN4-anti-CD137 bispecific antibody"). The anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises a heavy chain variable region and a light chain variable region of an anti-CLDN4 antibody, and a heavy chain variable region and a light chain variable region of an anti-CD137 antibody. The anti-CLDN4-anti-CD137 bispecific antibody of the present invention includes a bispecific antibody comprising an anti-CLDN4 IgG antibody and an scFv of an anti-CD137 antibody (also referred to as "anti-CD137scFv"). 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-CD137scFv.

[0027] In this specification, "anti-CLDN4 antibody" refers to an antibody capable of binding to human CLDN4, and "anti-CD137 antibody" refers to an antibody capable of binding to human CD137. Whether or not an antibody binds to human CLDN4 or human CD137 can be confirmed using known binding activity measurement methods. Examples of methods for measuring binding activity include enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The ELISA or flow cytometry method can be carried out using methods commonly used by those skilled in the art, for example, the methods described in Examples 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 CLDN4 and CD137, for example, a bispecific antibody having the structure described in Non-Patent Literature 6. In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention may be a conjugate in which the Fab region of an anti-CLDN4 antibody and the Fab region of an anti-CD137 antibody are linked, a conjugate of an anti-CLDN4 IgG antibody and an IgG-type anti-CD137 antibody (also referred to as "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 of the two antibodies is not particularly limited and may be linked directly or via a linker. In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises an anti-CLDN4 IgG antibody and an anti-CD137 antibody scFv (hereinafter referred to as "anti-CD137 scFv"). In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises an anti-CLDN4 IgG antibody and an anti-CD137scFv, wherein the amino terminus of anti-CD137scFv is ligated to the heavy chain carboxy terminus 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-CD137scFv, wherein the amino terminus of anti-CD137scFv is ligated to the light chain carboxy terminus 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-CD137scFv, wherein the carboxy terminus of anti-CD137scFv is ligated 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, wherein the carboxyl terminus of the anti-CD137 scFv is ligated 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 present invention comprises anti-CD137scFv.

[0030] In the anti-CLDN4-anti-CD137 bispecific antibody of the present invention, the heavy chain variable region and the light chain variable region of the anti-CLDN4 antibody and the anti-CD137 antibody 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 an antibody consisting of a human antibody, a humanized antibody, or 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 CDR1 consisting of amino acid sequences 31 to 35 of SEQ ID NO: 2, CDR2 consisting of amino acid sequences 50 to 66 of SEQ ID NO: 2, and CDR3 consisting of amino acid sequences 99 to 114 of SEQ ID NO: 2, and a light chain variable region comprising CDR1 consisting of amino acid sequences 24 to 35 of SEQ ID NO: 4, CDR2 consisting of amino acid sequences 51 to 57 of SEQ ID NO: 4, and CDR3 consisting of amino acid sequences 90 to 98 of SEQ ID NO: 4; or (b) A heavy chain variable region comprising CDR1 consisting of amino acid sequences 31 to 35 of SEQ ID NO: 6, CDR2 consisting of amino acid sequences 50 to 66 of SEQ ID NO: 6, and CDR3 consisting of amino acid sequences 99 to 112 of SEQ ID NO: 6, and a light chain variable region comprising CDR1 consisting of amino acid sequences 24 to 35 of SEQ ID NO: 8, CDR2 consisting of amino acid sequences 51 to 57 of SEQ ID NO: 8, and CDR3 consisting of amino acid sequences 90 to 98 of SEQ ID NO: 8.

[0032] In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises a heavy chain variable region and a light chain variable region of the anti-CLDN4 antibody described in (a) or (b) below: (a) The heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 125 of SEQ ID NO: 2 and the 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 contain a full-length antibody such as an IgG antibody as the anti-CLDN4 antibody, and may also contain an antigen-binding fragment such as a Fab fragment, Fab' fragment, F(ab')2 fragment, or 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 selected from any of the constant regions of Igγ, Igμ, Igα, Igδ, or Igε. For example, Igγ can be selected from 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 selected from any of the constant regions of Igλ or Igκ. 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 chain and light chain of the anti-CLDN4 antibody are human Igγ1 and Igκ, respectively. In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention contains a full-length anti-CLDN4 antibody. In one embodiment, the anti-CLDN4 antibody included in the anti-CLDN4-anti-CD137 bispecific antibody of the present invention is an IgG antibody (anti-CLDN4 IgG antibody) containing the heavy chain variable region and the light chain variable region of the anti-CLDN4 antibody.

[0035] If the anti-CLDN4-anti-CD137 bispecific antibody of the present invention includes an Fc region, the Fc region in the bispecific antibody may include mutations that reduce antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). L234A is a substitution of leucine to alanine at amino acid position 234 of the human Igγ1 constant region. L235A is a substitution of leucine to alanine at amino acid position 235 of the human Igγ1 constant region. The amino acid mutations of L234A and L235A in the human Igγ1 constant region are called "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 is a substitution of proline to glycine or serine at amino acid position 331 of the human Igγ1 constant region. Here, position 331 is an amino acid position according to the EU index in the human Igγ1 constant region. 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 contained in the anti-CLDN4-anti-CD137 bispecific antibody of the present invention includes an Fc region containing L234A and L235A amino acid mutations (LALA mutations). In one embodiment, the anti-CLDN4 IgG antibody includes an Fc region containing either a P331G or P331S mutation. In one embodiment, the anti-CLDN4 IgG antibody includes an Fc region containing an LALA mutation and either a P331G or P331S mutation.

[0037] In the process of creating the anti-CLDN4-anti-CD137 bispecific antibody of the present invention, the inventors created a novel anti-CD137 antibody or its binding fragment (scFv). The inventors identified an anti-CLDN4-anti-CD137 bispecific antibody that does not transmit the CD137 signal when bound only to CD137, but transmits the CD137 signal only when simultaneously bound to CD137 and CLDN4. These antibodies do not exhibit agonist activity when not bound to CLDN4. On the other hand, binding of the anti-CLDN4-anti-CD137 bispecific antibody to CLDN4 and CD137 leads to CD137 signal transduction and T cell activation. This action is expected to mitigate hepatotoxicity and other side effects observed in clinical trials of urelumab, and is therefore expected to have a favorable profile as an anti-CD137 antibody used in bispecific antibodies.

[0038] In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises a heavy chain variable region and a light chain variable region of an anti-CD137 antibody described in any of (a) to (d) below: (a) A heavy chain variable region comprising CDR1 consisting of amino acid sequences 31 to 35 of SEQ ID NO: 10, CDR2 consisting of amino acid sequences 50 to 66 of SEQ ID NO: 10, and CDR3 consisting of amino acid sequences 99 to 107 of SEQ ID NO: 10, and a light chain variable region comprising CDR1 consisting of amino acid sequences 24 to 34 of SEQ ID NO: 12, CDR2 consisting of amino acid sequences 50 to 56 of SEQ ID NO: 12, and CDR3 consisting of amino acid sequences 89 to 98 of SEQ ID NO: 12; (b) A heavy chain variable region comprising CDR1 consisting of amino acid sequences 31 to 35 of SEQ ID NO: 14, CDR2 consisting of amino acid sequences 50 to 65 of SEQ ID NO: 14, and CDR3 consisting of amino acid sequences 98 to 107 of SEQ ID NO: 14, and a light chain variable region comprising CDR1 consisting of amino acid sequences 23 to 35 of SEQ ID NO: 16, CDR2 consisting of amino acid sequences 51 to 57 of SEQ ID NO: 16, and CDR3 consisting of amino acid sequences 90 to 100 of SEQ ID NO: 16; (c) A heavy chain variable region comprising CDR1 consisting of amino acid sequences 31 to 35 of SEQ ID NO: 18, CDR2 consisting of amino acid sequences 50 to 65 of SEQ ID NO: 18, and CDR3 consisting of amino acid sequences 98 to 107 of SEQ ID NO: 18, and a light chain variable region comprising CDR1 consisting of amino acid sequences 23 to 35 of SEQ ID NO: 20, CDR2 consisting of amino acid sequences 51 to 57 of SEQ ID NO: 20, and CDR3 consisting of amino acid sequences 90 to 100 of SEQ ID NO: 20; or (d) A heavy chain variable region comprising CDR1 consisting of amino acid sequences from amino acid numbers 31 to 35 of SEQ ID NO: 22, CDR2 consisting of amino acid sequences from amino acid numbers 50 to 65 of SEQ ID NO: 22, and CDR3 consisting of amino acid sequences from amino acid numbers 98 to 110 of SEQ ID NO: 22, and a light chain variable region comprising CDR1 consisting of amino acid sequences from amino acid numbers 23 to 35 of SEQ ID NO: 24, CDR2 consisting of amino acid sequences from amino acid numbers 51 to 57 of SEQ ID NO: 24, and CDR3 consisting of amino acid sequences 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 a heavy chain variable region and a light chain variable region of an anti-CD137 antibody described in any of (a) to (i) below: (a) The heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 118 of SEQ ID NO: 10 and the light chain variable region consisting of amino acid sequences from amino acid numbers 1 to 109 of SEQ ID NO: 12; (b) The heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 14 and the light chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 111 of SEQ ID NO: 16; (c) Heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 118 of SEQ ID NO: 18 and light chain variable region consisting of amino acid sequences from amino acid numbers 1 to 111 of SEQ ID NO: 20; (d) Heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 121 of SEQ ID NO: 22 and light chain variable region consisting of amino acid sequences from amino acid numbers 1 to 111 of SEQ ID NO: 24 (e) The heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 26 and the light chain variable region consisting of the amino acid sequence from amino acid numbers 134 to 242 of SEQ ID NO: 26; (f) The heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 28 and the light chain variable region consisting of the amino acid sequence from amino acid numbers 134 to 244 of SEQ ID NO: 28; (g) The light chain variable region consisting of amino acid sequences from amino acid numbers 1 to 111 of SEQ ID NO: 30 and the heavy chain variable region consisting of amino acid sequences from amino acid numbers 132 to 249 of SEQ ID NO: 30; (h) Heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 118 of SEQ ID NO: 32 and light chain variable region consisting of amino acid sequences from amino acid numbers 134 to 244 of SEQ ID NO: 32; or (i) A heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 121 of SEQ ID NO: 34 and a light chain variable region consisting of amino acid sequences 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 contain a full-length antibody such as an IgG antibody as the anti-CD137 antibody, or it may contain an antigen-binding fragment such as a Fab fragment, Fab' fragment, F(ab')2 fragment, or 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 anti-CD137scFv.

[0041] In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises an anti-CD137scFv including the heavy chain variable region and the light chain variable region of the anti-CD137 antibody described in (a) to (m) below: (a) A heavy chain variable region comprising CDR1 consisting of amino acid sequences 31 to 35 of SEQ ID NO: 10, CDR2 consisting of amino acid sequences 50 to 66 of SEQ ID NO: 10, and CDR3 consisting of amino acid sequences 99 to 107 of SEQ ID NO: 10, and a light chain variable region comprising CDR1 consisting of amino acid sequences 24 to 34 of SEQ ID NO: 12, CDR2 consisting of amino acid sequences 50 to 56 of SEQ ID NO: 12, and CDR3 consisting of amino acid sequences 89 to 98 of SEQ ID NO: 12; (b) A heavy chain variable region comprising CDR1 consisting of amino acid sequences 31 to 35 of SEQ ID NO: 14, CDR2 consisting of amino acid sequences 50 to 65 of SEQ ID NO: 14, and CDR3 consisting of amino acid sequences 98 to 107 of SEQ ID NO: 14, and a light chain variable region comprising CDR1 consisting of amino acid sequences 23 to 35 of SEQ ID NO: 16, CDR2 consisting of amino acid sequences 51 to 57 of SEQ ID NO: 16, and CDR3 consisting of amino acid sequences 90 to 100 of SEQ ID NO: 16; (c) A heavy chain variable region comprising CDR1 consisting of amino acid sequences 31 to 35 of SEQ ID NO: 18, CDR2 consisting of amino acid sequences 50 to 65 of SEQ ID NO: 18, and CDR3 consisting of amino acid sequences 98 to 107 of SEQ ID NO: 18, and a light chain variable region comprising CDR1 consisting of amino acid sequences 23 to 35 of SEQ ID NO: 20, CDR2 consisting of amino acid sequences 51 to 57 of SEQ ID NO: 20, and CDR3 consisting of amino acid sequences 90 to 100 of SEQ ID NO: 20; (d) A heavy chain variable region comprising CDR1 consisting of amino acid sequences from amino acid numbers 31 to 35 of SEQ ID NO: 22, CDR2 consisting of amino acid sequences from amino acid numbers 50 to 65 of SEQ ID NO: 22, and CDR3 consisting of amino acid sequences from amino acid numbers 98 to 110 of SEQ ID NO: 22, and a light chain variable region comprising CDR1 consisting of amino acid sequences from amino acid numbers 23 to 35 of SEQ ID NO: 24, CDR2 consisting of amino acid sequences from amino acid numbers 51 to 57 of SEQ ID NO: 24, and CDR3 consisting of amino acid sequences from amino acid numbers 90 to 100 of SEQ ID NO: 24; (e) The heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 118 of SEQ ID NO: 10 and the light chain variable region consisting of amino acid sequences from amino acid numbers 1 to 109 of SEQ ID NO: 12; (f) The heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 118 of SEQ ID NO: 14 and the light chain variable region consisting of amino acid sequences from amino acid numbers 1 to 111 of SEQ ID NO: 16; (g) The heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 118 of SEQ ID NO: 18 and the light chain variable region consisting of amino acid sequences from amino acid numbers 1 to 111 of SEQ ID NO: 20; (h) Heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 121 of SEQ ID NO: 22 and light chain variable region consisting of amino acid sequences from amino acid numbers 1 to 111 of SEQ ID NO: 24 (i) The heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 26 and the light chain variable region consisting of the amino acid sequence from amino acid numbers 134 to 242 of SEQ ID NO: 26; (j) Heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 118 of SEQ ID NO: 28 and light chain variable region consisting of amino acid sequences from amino acid numbers 134 to 244 of SEQ ID NO: 28; (k) Light chain variable region consisting of amino acid sequences from amino acid numbers 1 to 111 of SEQ ID NO: 30 and heavy chain variable region consisting of amino acid sequences from amino acid numbers 132 to 249 of SEQ ID NO: 30; (l) The heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 32 and the light chain variable region consisting of the amino acid sequence from amino acid numbers 134 to 244 of SEQ ID NO: 32; or (m) Heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 121 of SEQ ID NO: 34 and light chain variable region consisting of amino acid sequences from amino acid numbers 137 to 247 of SEQ ID NO: 34.

[0042] In anti-CD137scFv, the type and length of the linker connecting the heavy chain variable region and the 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. A preferred length is 5 amino acids or more (the upper limit is not particularly limited, but is usually 30 amino acids or less, preferably 20 amino acids or less), and particularly preferably 15 amino acids. For example, a glycine-serine linker (GS linker) or a glycine-lysine-proline-glycine-serine linker (GKPGS linker) can be used as the linker. Examples of such linkers include the following. Ser Gly-Ser Gly-Gly-Ser Ser-Gly-Gly Gly-Gly-Gly-Ser (Sequence ID 46) Ser-Gly-Gly-Gly (SEQ ID NO: 47) Gly-Gly-Gly-Gly-Ser (Sequence ID 48) Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 49) Gly-Gly-Gly-Gly-Gly-Ser (Sequence ID 50) Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 51) Gly-Gly-Gly-Gly-Gly-Gly-Ser (Sequence ID 52) Ser-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 53) Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (Sequence ID 54) (Gly-Gly-Gly-Gly-Ser)n (Ser-Gly-Gly-Gly-Gly)n Gly-Lys-Pro-Gly-Ser (Sequence ID 55) (Gly-Lys-Pro-Gly-Ser)n The above n represents an integer of 1 or more. In some embodiments, the above n is 1 to 10, 2 to 8, or 2 to 6. The length and arrangement of the linkers 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 (Gly-Gly-Gly-Gly-Ser)n GS linker, where n is either 3 or 4.

[0043] In one embodiment, the anti-CLDN4-anti-CD137 bispecific antibody of the present invention comprises any of the following anti-CD137scFv (a) to (e): (a) Anti-CD137scFv consisting of the amino acid sequence from amino acid number 1 to 242 of SEQ ID NO: 26; (b) Anti-CD137scFv consisting of the amino acid sequence of amino acid numbers 1 to 244 of SEQ ID NO: 28; (c) Anti-CD137scFv consisting of the amino acid sequence from amino acid number 1 to 249 of SEQ ID NO: 30; (d) Anti-CD137scFv consisting of the amino acid sequence from amino acid number 1 to 244 of SEQ ID NO. 32; or (e) Anti-CD137scFv consisting of the amino acid sequence of 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 by 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. A preferred length is 5 amino acids or more (the upper limit is not particularly limited, but is usually 30 amino acids or less, preferably 20 amino acids or less), and particularly preferably 10 amino acids. As the peptide linker, for example, a glycine-serine linker (GS linker) or a glycine-lysine-proline-glycine-serine linker (GKPGS linker) can be used. Examples of such linkers are as follows. Ser Gly-Ser Gly-Gly-Ser Ser-Gly-Gly Gly-Gly-Gly-Ser (Sequence ID 46) Ser-Gly-Gly-Gly (SEQ ID NO: 47) Gly-Gly-Gly-Gly-Ser (Sequence ID 48) Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 49) Gly-Gly-Gly-Gly-Gly-Ser (Sequence ID 50) Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 51) Gly-Gly-Gly-Gly-Gly-Gly-Ser (Sequence ID 52) Ser-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 53) Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (Sequence ID 54) (Gly-Gly-Gly-Gly-Ser)n (Ser-Gly-Gly-Gly-Gly)n Gly-Lys-Pro-Gly-Ser (Sequence ID 55) (Gly-Lys-Pro-Gly-Ser)n The above n represents an integer of 1 or more. In some embodiments, the above n is 1 to 10, 2 to 8, or 2 to 6. The length and sequence of the peptide linker can be appropriately selected by those skilled in the art depending on the purpose. In one embodiment, the linker used to link the anti-CLDN4 antibody or its antigen-binding fragment to the anti-CD137 antibody or its antigen-binding fragment is a GS linker or a GKPGS linker, and in one embodiment, it 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, but the anti-CLDN4-anti-CD137 bispecific antibody of the present invention can take various structures, such as the heavy chain amino-terminus of the anti-CLDN4 IgG antibody being linked to the carboxyl terminus of the anti-CD137scFv, the light chain amino-terminus of the anti-CLDN4 IgG antibody being linked to the carboxyl terminus of the anti-CD137scFv, the amino-terminus of the anti-CD137scFv being linked to the heavy chain carboxyl terminus of the anti-CLDN4 IgG antibody, or the amino-terminus of the anti-CD137scFv being linked to the light chain carboxyl terminus 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 anti-CD137scFv ligated to the heavy chain carboxy terminus of an anti-CLDN4 IgG antibody, or the amino terminus of anti-CD137scFv ligated to the light chain carboxy terminus 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 anti-CD137scFv ligated to the heavy chain carboxy terminus 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-CD137scFv, wherein the amino terminus of the anti-CD137scFv is linked to the heavy or light chain carboxyl terminus of the anti-CLDN4 IgG antibody via a linker, and the heavy chain variable region and light chain variable region of the anti-CD137scFv are any of (a) to (m) below: (a) A heavy chain variable region comprising CDR1 consisting of amino acid sequences 31 to 35 of SEQ ID NO: 10, CDR2 consisting of amino acid sequences 50 to 66 of SEQ ID NO: 10, and CDR3 consisting of amino acid sequences 99 to 107 of SEQ ID NO: 10, and a light chain variable region comprising CDR1 consisting of amino acid sequences 24 to 34 of SEQ ID NO: 12, CDR2 consisting of amino acid sequences 50 to 56 of SEQ ID NO: 12, and CDR3 consisting of amino acid sequences 89 to 98 of SEQ ID NO: 12; (b) A heavy chain variable region comprising CDR1 consisting of amino acid sequences 31 to 35 of SEQ ID NO: 14, CDR2 consisting of amino acid sequences 50 to 65 of SEQ ID NO: 14, and CDR3 consisting of amino acid sequences 98 to 107 of SEQ ID NO: 14, and a light chain variable region comprising CDR1 consisting of amino acid sequences 23 to 35 of SEQ ID NO: 16, CDR2 consisting of amino acid sequences 51 to 57 of SEQ ID NO: 16, and CDR3 consisting of amino acid sequences 90 to 100 of SEQ ID NO: 16; (c) A heavy chain variable region comprising CDR1 consisting of amino acid sequences 31 to 35 of SEQ ID NO: 18, CDR2 consisting of amino acid sequences 50 to 65 of SEQ ID NO: 18, and CDR3 consisting of amino acid sequences 98 to 107 of SEQ ID NO: 18, and a light chain variable region comprising CDR1 consisting of amino acid sequences 23 to 35 of SEQ ID NO: 20, CDR2 consisting of amino acid sequences 51 to 57 of SEQ ID NO: 20, and CDR3 consisting of amino acid sequences 90 to 100 of SEQ ID NO: 20; (d) A heavy chain variable region comprising CDR1 consisting of amino acid sequences from amino acid numbers 31 to 35 of SEQ ID NO: 22, CDR2 consisting of amino acid sequences from amino acid numbers 50 to 65 of SEQ ID NO: 22, and CDR3 consisting of amino acid sequences from amino acid numbers 98 to 110 of SEQ ID NO: 22, and a light chain variable region comprising CDR1 consisting of amino acid sequences from amino acid numbers 23 to 35 of SEQ ID NO: 24, CDR2 consisting of amino acid sequences from amino acid numbers 51 to 57 of SEQ ID NO: 24, and CDR3 consisting of amino acid sequences from amino acid numbers 90 to 100 of SEQ ID NO: 24; (e) The heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 118 of SEQ ID NO: 10 and the light chain variable region consisting of amino acid sequences from amino acid numbers 1 to 109 of SEQ ID NO: 12; (f) The heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 118 of SEQ ID NO: 14 and the light chain variable region consisting of amino acid sequences from amino acid numbers 1 to 111 of SEQ ID NO: 16; (g) The heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 118 of SEQ ID NO: 18 and the light chain variable region consisting of amino acid sequences from amino acid numbers 1 to 111 of SEQ ID NO: 20; (h) Heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 121 of SEQ ID NO: 22 and light chain variable region consisting of amino acid sequences from amino acid numbers 1 to 111 of SEQ ID NO: 24 (i) The heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 26 and the light chain variable region consisting of the amino acid sequence from amino acid numbers 134 to 242 of SEQ ID NO: 26; (j) Heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 118 of SEQ ID NO: 28 and light chain variable region consisting of amino acid sequences from amino acid numbers 134 to 244 of SEQ ID NO: 28; (k) Light chain variable region consisting of amino acid sequences from amino acid numbers 1 to 111 of SEQ ID NO: 30 and heavy chain variable region consisting of amino acid sequences from amino acid numbers 132 to 249 of SEQ ID NO: 30; (l) The heavy chain variable region consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 32 and the light chain variable region consisting of the amino acid sequence from amino acid numbers 134 to 244 of SEQ ID NO: 32; or (m) Heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 121 of SEQ ID NO: 34 and light chain variable region consisting of amino acid sequences 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-CD137scFv, wherein the amino terminus of the anti-CD137scFv is linked to the heavy or light chain carboxyl terminus of the anti-CLDN4 IgG antibody via a linker, and the anti-CD137scFv is any of the following (a) to (e): (a) Anti-CD137scFv consisting of the amino acid sequence from amino acid number 1 to 242 of SEQ ID NO: 26; (b) Anti-CD137scFv consisting of the amino acid sequence of amino acid numbers 1 to 244 of SEQ ID NO: 28; (c) Anti-CD137scFv consisting of the amino acid sequence from amino acid number 1 to 249 of SEQ ID NO: 30; (d) Anti-CD137scFv consisting of the amino acid sequence from amino acid number 1 to 244 of SEQ ID NO. 32; or (e) Anti-CD137scFv consisting of the amino acid sequence of 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-CD137scFv bispecific antibodies: (a) to (h) (a) The heavy chain of an anti-CLDN4 antibody including a heavy chain variable region comprising CDR1 consisting of amino acid sequences 31 to 35 of SEQ ID NO: 2, CDR2 consisting of amino acid sequences 50 to 66 of SEQ ID NO: 2, and CDR3 consisting of amino acid sequences 99 to 114 of SEQ ID NO: 2; the light chain of an anti-CLDN4 antibody including a light chain variable region comprising CDR1 consisting of amino acid sequences 24 to 35 of SEQ ID NO: 4, CDR2 consisting of amino acid sequences 51 to 57 of SEQ ID NO: 4, and CDR3 consisting of amino acid sequences 90 to 98 of SEQ ID NO: 4; and amino acid sequences 31 to 35 of SEQ ID NO: 10 A bispecific antibody comprising an anti-CD137scFv containing a CDR1 consisting of a sequence, a heavy chain variable region containing a CDR2 consisting of amino acid sequences from amino acid numbers 50 to 66 of SEQ ID NO: 10, and a CDR3 consisting of amino acid sequences from amino acid numbers 99 to 107 of SEQ ID NO: 10, and a light chain variable region containing a CDR1 consisting of amino acid sequences from amino acid numbers 24 to 34 of SEQ ID NO: 12, a CDR2 consisting of amino acid sequences from amino acid numbers 50 to 56 of SEQ ID NO: 12, and a CDR3 consisting of amino acid sequences from amino acid numbers 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) The heavy chain of an anti-CLDN4 antibody including a heavy chain variable region comprising CDR1 consisting of amino acid sequences 31 to 35 of SEQ ID NO: 2, CDR2 consisting of amino acid sequences 50 to 66 of SEQ ID NO: 2, and CDR3 consisting of amino acid sequences 99 to 114 of SEQ ID NO: 2; the light chain of an anti-CLDN4 antibody including a light chain variable region comprising CDR1 consisting of amino acid sequences 24 to 35 of SEQ ID NO: 4, CDR2 consisting of amino acid sequences 51 to 57 of SEQ ID NO: 4, and CDR3 consisting of amino acid sequences 90 to 98 of SEQ ID NO: 4; and amino acid sequences 31 to 35 of SEQ ID NO: 14 A bispecific antibody comprising an anti-CD137scFv containing a CDR1 consisting of a sequence, a heavy chain variable region containing a CDR2 consisting of amino acid sequences from amino acid numbers 50 to 65 of SEQ ID NO: 14, and a CDR3 consisting of amino acid sequences from amino acid numbers 98 to 107 of SEQ ID NO: 14, and a light chain variable region containing a CDR1 consisting of amino acid sequences from amino acid numbers 23 to 35 of SEQ ID NO: 16, a CDR2 consisting of amino acid sequences from amino acid numbers 51 to 57 of SEQ ID NO: 16, and a CDR3 consisting of amino acid sequences from amino acid numbers 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) The heavy chain of an anti-CLDN4 antibody including a heavy chain variable region comprising CDR1 consisting of amino acid sequences from amino acid numbers 31 to 35 of SEQ ID NO: 2, CDR2 consisting of amino acid sequences from amino acid numbers 50 to 66 of SEQ ID NO: 2, and CDR3 consisting of amino acid sequences from amino acid numbers 99 to 114 of SEQ ID NO: 2; the light chain of an anti-CLDN4 antibody including a light chain variable region comprising CDR1 consisting of amino acid sequences from amino acid numbers 24 to 35 of SEQ ID NO: 4, CDR2 consisting of amino acid sequences from amino acid numbers 51 to 57 of SEQ ID NO: 4, and CDR3 consisting of amino acid sequences from amino acid numbers 90 to 98 of SEQ ID NO: 4; and amino acid sequences from amino acid numbers 31 to 35 of SEQ ID NO: 18 A bispecific antibody comprising an anti-CD137scFv containing a heavy chain variable region including a CDR1 consisting of a sequence, a CDR2 consisting of amino acid sequences from amino acid numbers 50 to 65 of SEQ ID NO: 18, and a CDR3 consisting of amino acid sequences from amino acid numbers 98 to 107 of SEQ ID NO: 18, and a light chain variable region including a CDR1 consisting of amino acid sequences from amino acid numbers 23 to 35 of SEQ ID NO: 20, a CDR2 consisting of amino acid sequences from amino acid numbers 51 to 57 of SEQ ID NO: 20, and a CDR3 consisting of amino acid sequences from amino acid numbers 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) The heavy chain of an anti-CLDN4 antibody including a heavy chain variable region comprising CDR1 consisting of amino acid sequences from amino acid numbers 31 to 35 of SEQ ID NO: 2, CDR2 consisting of amino acid sequences from amino acid numbers 50 to 66 of SEQ ID NO: 2, and CDR3 consisting of amino acid sequences from amino acid numbers 99 to 114 of SEQ ID NO: 2; the light chain of an anti-CLDN4 antibody including a light chain variable region comprising CDR1 consisting of amino acid sequences from amino acid numbers 24 to 35 of SEQ ID NO: 4, CDR2 consisting of amino acid sequences from amino acid numbers 51 to 57 of SEQ ID NO: 4, and CDR3 consisting of amino acid sequences from amino acid numbers 90 to 98 of SEQ ID NO: 4; and amino acid sequences from amino acid numbers 31 to 35 of SEQ ID NO: 22 A bispecific antibody comprising an anti-CD137scFv containing a CDR1 consisting of a sequence, a heavy chain variable region including a CDR2 consisting of amino acid sequences from amino acid numbers 50 to 65 of SEQ ID NO: 22, and a CDR3 consisting of amino acid sequences from amino acid numbers 98 to 110 of SEQ ID NO: 22, and a light chain variable region including a CDR1 consisting of amino acid sequences from amino acid numbers 23 to 35 of SEQ ID NO: 24, a CDR2 consisting of amino acid sequences from amino acid numbers 51 to 57 of SEQ ID NO: 24, and a CDR3 consisting of amino acid sequences from amino acid numbers 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) The heavy chain of an anti-CLDN4 antibody containing a heavy chain variable region including CDR1 consisting of amino acid sequences 31 to 35 of SEQ ID NO: 6, CDR2 consisting of amino acid sequences 50 to 66 of SEQ ID NO: 6, and CDR3 consisting of amino acid sequences 99 to 112 of SEQ ID NO: 6; the light chain of an anti-CLDN4 antibody containing a light chain variable region including CDR1 consisting of amino acid sequences 24 to 35 of SEQ ID NO: 8, CDR2 consisting of amino acid sequences 51 to 57 of SEQ ID NO: 8, and CDR3 consisting of amino acid sequences 90 to 98 of SEQ ID NO: 8; and amino acid sequences 31 to 35 of SEQ ID NO: 10 A bispecific antibody comprising an anti-CD137scFv containing a CDR1 consisting of a sequence, a heavy chain variable region containing a CDR2 consisting of amino acid sequences from amino acid numbers 50 to 66 of SEQ ID NO: 10, and a CDR3 consisting of amino acid sequences from amino acid numbers 99 to 107 of SEQ ID NO: 10, and a light chain variable region containing a CDR1 consisting of amino acid sequences from amino acid numbers 24 to 34 of SEQ ID NO: 12, a CDR2 consisting of amino acid sequences from amino acid numbers 50 to 56 of SEQ ID NO: 12, and a CDR3 consisting of amino acid sequences from amino acid numbers 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) The heavy chain of an anti-CLDN4 antibody containing a heavy chain variable region including CDR1 consisting of amino acid sequences 31 to 35 of SEQ ID NO: 6, CDR2 consisting of amino acid sequences 50 to 66 of SEQ ID NO: 6, and CDR3 consisting of amino acid sequences 99 to 112 of SEQ ID NO: 6; the light chain of an anti-CLDN4 antibody containing a light chain variable region including CDR1 consisting of amino acid sequences 24 to 35 of SEQ ID NO: 8, CDR2 consisting of amino acid sequences 51 to 57 of SEQ ID NO: 8, and CDR3 consisting of amino acid sequences 90 to 98 of SEQ ID NO: 8; and amino acid sequences 31 to 35 of SEQ ID NO: 14 A bispecific antibody comprising an anti-CD137scFv containing a CDR1 consisting of a sequence, a heavy chain variable region containing a CDR2 consisting of amino acid sequences from amino acid numbers 50 to 65 of SEQ ID NO: 14, and a CDR3 consisting of amino acid sequences from amino acid numbers 98 to 107 of SEQ ID NO: 14, and a light chain variable region containing a CDR1 consisting of amino acid sequences from amino acid numbers 23 to 35 of SEQ ID NO: 16, a CDR2 consisting of amino acid sequences from amino acid numbers 51 to 57 of SEQ ID NO: 16, and a CDR3 consisting of amino acid sequences from amino acid numbers 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) The heavy chain of an anti-CLDN4 antibody containing a heavy chain variable region including CDR1 consisting of amino acid sequences 31 to 35 of SEQ ID NO: 6, CDR2 consisting of amino acid sequences 50 to 66 of SEQ ID NO: 6, and CDR3 consisting of amino acid sequences 99 to 112 of SEQ ID NO: 6; the light chain of an anti-CLDN4 antibody containing a light chain variable region including CDR1 consisting of amino acid sequences 24 to 35 of SEQ ID NO: 8, CDR2 consisting of amino acid sequences 51 to 57 of SEQ ID NO: 8, and CDR3 consisting of amino acid sequences 90 to 98 of SEQ ID NO: 8; and the amino acid sequence of SEQ ID NO: 18 A bispecific antibody comprising an anti-CD137scFv comprising a heavy chain variable region including a column-based CDR1, a CDR2 consisting of amino acid sequences from amino acid numbers 50 to 65 of SEQ ID NO: 18, and a CDR3 consisting of amino acid sequences from amino acid numbers 98 to 107 of SEQ ID NO: 18, and a light chain variable region including a CDR1 consisting of amino acid sequences from amino acid numbers 23 to 35 of SEQ ID NO: 20, a CDR2 consisting of amino acid sequences from amino acid numbers 51 to 57 of SEQ ID NO: 20, and a CDR3 consisting of amino acid sequences from amino acid numbers 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; or (h) The heavy chain of an anti-CLDN4 antibody including a heavy chain variable region comprising CDR1 consisting of amino acid sequences 31 to 35 of SEQ ID NO: 6, CDR2 consisting of amino acid sequences 50 to 66 of SEQ ID NO: 6, and CDR3 consisting of amino acid sequences 99 to 112 of SEQ ID NO: 6; the light chain of an anti-CLDN4 antibody including a light chain variable region comprising CDR1 consisting of amino acid sequences 24 to 35 of SEQ ID NO: 8, CDR2 consisting of amino acid sequences 51 to 57 of SEQ ID NO: 8, and CDR3 consisting of amino acid sequences 90 to 98 of SEQ ID NO: 8; and amino acid sequences 31 to 35 of SEQ ID NO: 22 A bispecific antibody comprising an anti-CD137scFv containing a heavy chain variable region including a CDR1 consisting of a sequence, 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 including 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, wherein the amino terminus of the anti-CD137scFv 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-CD137scFv bispecific antibodies: (a) A bispecific antibody comprising: (a) a heavy chain of an anti-CLDN4 antibody containing a heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 125 of SEQ ID NO: 2; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences from amino acid numbers 1 to 109 of SEQ ID NO: 4; and an anti-CD137scFv containing a heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 118 of SEQ ID NO: 26 and a light chain variable region consisting of amino acid sequences from amino acid numbers 134 to 242 of SEQ ID NO: 26, wherein the amino terminus of the anti-CD137scFv is linked to the carboxyl 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 containing a heavy chain variable region consisting of amino acid sequences 1 to 125 of SEQ ID NO: 2; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 4; and an anti-CD137scFv containing a heavy chain variable region consisting of amino acid sequences 1 to 118 of SEQ ID NO: 28 and a light chain variable region consisting of amino acid sequences 134 to 244 of SEQ ID NO: 28, wherein the amino terminus of the anti-CD137scFv is linked to the carboxyl 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 containing a heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 125 of SEQ ID NO: 2; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences from amino acid numbers 1 to 109 of SEQ ID NO: 4; and an anti-CD137scFv containing a light chain variable region consisting of amino acid sequences from amino acid numbers 1 to 111 of SEQ ID NO: 30 and a heavy chain variable region consisting of amino acid sequences from amino acid numbers 132 to 249 of SEQ ID NO: 30, wherein the amino terminus of the anti-CD137scFv is linked to the carboxyl 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 containing a heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 125 of SEQ ID NO: 2; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences from amino acid numbers 1 to 109 of SEQ ID NO: 4; and an anti-CD137scFv containing a heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 118 of SEQ ID NO: 32 and a light chain variable region consisting of amino acid sequences from amino acid numbers 134 to 244 of SEQ ID NO: 32, wherein the amino terminus of the anti-CD137scFv is linked to the carboxyl 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 containing a heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 125 of SEQ ID NO: 2; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences from amino acid numbers 1 to 109 of SEQ ID NO: 4; and an anti-CD137scFv containing a heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 121 of SEQ ID NO: 34 and a light chain variable region consisting of amino acid sequences from amino acid numbers 137 to 247 of SEQ ID NO: 34, 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 bispecific antibody comprising: a heavy chain of an anti-CLDN4 antibody containing a heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 123 of SEQ ID NO: 6; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences from amino acid numbers 1 to 109 of SEQ ID NO: 8; and an anti-CD137scFv containing a heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 118 of SEQ ID NO: 26 and a light chain variable region consisting of amino acid sequences from amino acid numbers 134 to 242 of SEQ ID NO: 26, 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 bispecific antibody comprising: a heavy chain of an anti-CLDN4 antibody containing a heavy chain variable region consisting of amino acid sequences 1 to 123 of SEQ ID NO: 6; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 8; and an anti-CD137scFv containing a heavy chain variable region consisting of amino acid sequences 1 to 118 of SEQ ID NO: 28 and a light chain variable region consisting of amino acid sequences 134 to 244 of SEQ ID NO: 28, wherein the amino terminus of the anti-CD137scFv is linked to the carboxyl 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 containing a heavy chain variable region consisting of amino acid sequences 1 to 123 of SEQ ID NO: 6; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 8; and an anti-CD137scFv containing a light chain variable region consisting of amino acid sequences 1 to 111 of SEQ ID NO: 30 and a heavy chain variable region consisting of amino acid sequences 132 to 249 of SEQ ID NO: 30, wherein the amino terminus of the anti-CD137scFv is linked to the carboxyl terminus of the heavy chain of the anti-CLDN4 antibody via a linker; (i) A bispecific antibody comprising: (i) a heavy chain of an anti-CLDN4 antibody containing a heavy chain variable region consisting of amino acid sequences 1 to 123 of SEQ ID NO: 6; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 8; and an anti-CD137scFv containing a heavy chain variable region consisting of amino acid sequences 1 to 118 of SEQ ID NO: 32 and a light chain variable region consisting of amino acid sequences 134 to 244 of SEQ ID NO: 32, wherein the amino terminus of the anti-CD137scFv is linked to the carboxyl 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 containing a heavy chain variable region consisting of amino acid sequences 1 to 123 of SEQ ID NO: 6; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 8; and an anti-CD137scFv containing a heavy chain variable region consisting of amino acid sequences 1 to 121 of SEQ ID NO: 34 and a light chain variable region consisting of amino acid sequences 137 to 247 of SEQ ID NO: 34, wherein the amino terminus of the anti-CD137scFv is linked to the heavy chain carboxy terminus of the anti-CLDN4 antibody via a linker; (k) A bispecific antibody comprising: a heavy chain of an anti-CLDN4 antibody containing a heavy chain variable region consisting of amino acid sequences 1 to 125 of SEQ ID NO: 2; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 4; and an anti-CD137scFv containing a heavy chain variable region consisting of amino acid sequences 1 to 118 of SEQ ID NO: 26 and a light chain variable region consisting of amino acid sequences 134 to 242 of SEQ ID NO: 26, wherein the amino terminus of the anti-CD137scFv is linked to the carboxyl 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 containing a heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 125 of SEQ ID NO: 2; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences from amino acid numbers 1 to 109 of SEQ ID NO: 4; and an anti-CD137scFv containing a heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 118 of SEQ ID NO: 28 and a light chain variable region consisting of amino acid sequences from amino acid numbers 134 to 244 of SEQ ID NO: 28, wherein the amino terminus of the anti-CD137scFv is linked to the carboxyl 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 containing a heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 125 of SEQ ID NO: 2; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences from amino acid numbers 1 to 109 of SEQ ID NO: 4; and an anti-CD137scFv containing a light chain variable region consisting of amino acid sequences from amino acid numbers 1 to 111 of SEQ ID NO: 30 and a heavy chain variable region consisting of amino acid sequences from amino acid numbers 132 to 249 of SEQ ID NO: 30, wherein the amino terminus of the anti-CD137scFv is linked to the carboxyl 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 containing a heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 125 of SEQ ID NO: 2; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences from amino acid numbers 1 to 109 of SEQ ID NO: 4; and an anti-CD137scFv containing a heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 118 of SEQ ID NO: 32 and a light chain variable region consisting of amino acid sequences from amino acid numbers 134 to 244 of SEQ ID NO: 32, wherein the amino terminus of the anti-CD137scFv is linked to the carboxyl 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 containing a heavy chain variable region consisting of amino acid sequences 1 to 125 of SEQ ID NO: 2; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 4; and an anti-CD137scFv containing a heavy chain variable region consisting of amino acid sequences 1 to 121 of SEQ ID NO: 34 and a light chain variable region consisting of amino acid sequences 137 to 247 of SEQ ID NO: 34, wherein the amino terminus of the anti-CD137scFv is linked to the carboxyl 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 containing a heavy chain variable region consisting of amino acid sequences 1 to 123 of SEQ ID NO: 6; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 8; and an anti-CD137scFv containing a heavy chain variable region consisting of amino acid sequences 1 to 118 of SEQ ID NO: 26 and a light chain variable region consisting of amino acid sequences 134 to 242 of SEQ ID NO: 26, wherein the amino terminus of the anti-CD137scFv is linked to the carboxyl 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 containing a heavy chain variable region consisting of amino acid sequences 1 to 123 of SEQ ID NO: 6; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 8; and an anti-CD137scFv containing a heavy chain variable region consisting of amino acid sequences 1 to 118 of SEQ ID NO: 28 and a light chain variable region consisting of amino acid sequences 134 to 244 of SEQ ID NO: 28, wherein the amino terminus of the anti-CD137scFv is linked to the carboxyl 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 containing a heavy chain variable region consisting of amino acid sequences 1 to 123 of SEQ ID NO: 6; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 8; and an anti-CD137scFv containing a light chain variable region consisting of amino acid sequences 1 to 111 of SEQ ID NO: 30 and a heavy chain variable region consisting of amino acid sequences 132 to 249 of SEQ ID NO: 30, wherein the amino terminus of the anti-CD137scFv is linked to the carboxyl terminus of the light chain of the anti-CLDN4 antibody via a linker; (s) A bispecific antibody comprising: (s) a heavy chain of an anti-CLDN4 antibody containing a heavy chain variable region consisting of amino acid sequences 1 to 123 of SEQ ID NO: 6; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 8; and an anti-CD137scFv containing a heavy chain variable region consisting of amino acid sequences 1 to 118 of SEQ ID NO: 32 and a light chain variable region consisting of amino acid sequences 134 to 244 of SEQ ID NO: 32, wherein the amino terminus of the anti-CD137scFv is linked to the carboxyl 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 containing a heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 123 of SEQ ID NO: 6; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences from amino acid numbers 1 to 109 of SEQ ID NO: 8; and an anti-CD137scFv containing a heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 121 of SEQ ID NO: 34 and a light chain variable region consisting of amino acid sequences from amino acid numbers 137 to 247 of SEQ ID NO: 34, wherein the amino terminus of the anti-CD137scFv is linked to the carboxyl 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 the anti-CLDN4-anti-CD137scFv bispecific antibody of (a) or (b) below: (a) A bispecific antibody comprising a polypeptide containing the heavy chain of an anti-CLDN4 antibody and anti-CD137scFv, consisting of the amino acid sequence from amino acid numbers 1 to 705 of SEQ ID NO: 36, and 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; or (b) A bispecific antibody comprising a heavy chain of an anti-CLDN4 antibody consisting of amino acid sequences from amino acid numbers 1 to 712 of SEQ ID NO: 38 and a polypeptide containing anti-CD137scFv, and a light chain of an anti-CLDN4 antibody consisting of amino acid sequences from amino acid numbers 1 to 215 of SEQ ID NO: 40.

[0051] In this specification, "post-translational modification" refers to modifications that occur in antibodies after translation when they are expressed in cells. Examples of post-translational modifications include pyroglutamylation, glycosylation, oxidation, deamidation, and glycation of glutamine or glutamic acid at the N-terminus of the heavy chain, as well as 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-translationally modified antibody is pyroglutamylation of the N-terminus of the heavy chain variable region and / or lysine deletion of the heavy chain C-terminus. It is known in the art that post-translationally modified antibodies by N-terminus pyroglutamylation or C-terminus lysine deletion do not affect antibody activity (Analytical Biochemistry, 2006, Vol.348, pp.24-39).

[0053] The anti-CLDN4-anti-CD137 bispecific antibody of the present invention binds to human CLDN4 and human CD137. Whether or not it binds to human CLDN4 and human CD137 can be confirmed using known methods for measuring binding activity. Examples of methods for measuring binding activity include ELISA and flow cytometry. ELISA or flow cytometry can be carried out using methods commonly used by those skilled in the art, for example, the methods described in Examples 3 or 5.

[0054] The anti-CLDN4-anti-CD137 bispecific antibody of the present invention can be readily prepared by a person skilled in the art using human CLDN4 and human CD137 as antigens and antibody production techniques known in the art, or it can be readily prepared by a person skilled in the art using methods known in the art based on sequence information of the heavy chain variable region and light chain variable region of the anti-CLDN4 antibody and anti-CD137 antibody disclosed herein.

[0055] The anti-CLDN4-anti-CD137 bispecific antibody of the present invention is not particularly limited, but can be produced, for example, by following the method described in <Method for Producing the Bispecific Antibody of the Present Invention> below.

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

[0057] In one embodiment, the polynucleotide of the bispecific antibody of the present invention is a polynucleotide comprising a base sequence that encodes the heavy chain variable region or light chain variable region of an anti-CLDN4 antibody, selected from the group consisting of (a) to (d) below: (a) A polynucleotide containing a nucleotide sequence that encodes the 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 containing a nucleotide sequence that encodes 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 containing a base sequence that encodes the light chain variable region of the anti-CLDN4 antibody, consisting of the amino acid sequence from amino acid number 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 (a) or (b) below: (a) 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 125 of SEQ ID NO: 2, and 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; or (b) 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, and 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: 8.

[0059] In one embodiment, the polynucleotide of the bispecific antibody of the present invention is a polynucleotide comprising a base sequence that encodes the heavy chain variable region or light chain variable region of an anti-CD137 antibody, selected from the group consisting of (a) to (r) below: (a) A polynucleotide containing a nucleotide sequence that encodes the 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 containing 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 109 of SEQ ID NO: 12; (c) A polynucleotide containing a base sequence that encodes the heavy chain variable region of the anti-CD137 antibody, consisting of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 14; (d) A polynucleotide containing 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: 16; (e) A polynucleotide containing a nucleotide sequence that encodes the heavy chain variable region of an anti-CD137 antibody, consisting of the amino acid sequence from amino acid number 1 to 118 of SEQ ID NO: 18; (f) A polynucleotide containing a base sequence that encodes 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. 20; (g) A polynucleotide containing a nucleotide sequence that encodes 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: 22; (h) A polynucleotide containing a base sequence that encodes the light chain variable region of the anti-CD137 antibody, consisting of amino acid sequences 1 to 111 of SEQ ID NO: 24; (i) A polynucleotide containing a nucleotide sequence encoding the heavy chain variable region of an anti-CD137 antibody, consisting of amino acid sequences from amino acid numbers 1 to 118 of SEQ ID NO: 26; (j) A polynucleotide containing a nucleotide sequence that encodes the light chain variable region of the anti-CD137 antibody, consisting of the amino acid sequence from amino acid numbers 134 to 242 of Sequence ID No. 26; (k) A polynucleotide containing a nucleotide sequence encoding the heavy chain variable region of the anti-CD137 antibody, consisting of the amino acid sequence from amino acid number 1 to 118 of SEQ ID NO: 28; (l) A polynucleotide containing a base sequence that encodes the light chain variable region of the anti-CD137 antibody, consisting of the amino acid sequence from amino acid numbers 134 to 244 of SEQ ID NO: 28; (m) A polynucleotide containing a base sequence encoding the light chain variable region of the anti-CD137 antibody, consisting of the amino acid sequence from amino acid number 1 to 111 of SEQ ID NO: 30; (n) A polynucleotide containing a nucleotide sequence that encodes the heavy chain variable region of the anti-CD137 antibody, consisting of the amino acid sequence from amino acid numbers 132 to 249 of SEQ ID NO: 30; (o) A polynucleotide containing a base sequence that encodes the heavy chain variable region of the anti-CD137 antibody, consisting of the amino acid sequence from amino acid number 1 to 118 of SEQ ID NO: 32; (p) A polynucleotide containing a base sequence that encodes the light chain variable region of the 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 base sequence that encodes the heavy chain variable region of an anti-CD137 antibody consisting of amino acid sequences 1 to 121 of SEQ ID NO: 34; or (r) A polynucleotide containing a base sequence that encodes the light chain variable region of the 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 the bispecific antibody of the present invention is a polynucleotide comprising a base sequence that encodes the heavy chain variable region and the light chain variable region of an anti-CD137 antibody, selected from the group consisting of (a) to (i) below: (a) A polynucleotide comprising a nucleotide sequence encoding the heavy chain variable region of an anti-CD137 antibody consisting of amino acid sequences 1 to 118 of SEQ ID NO: 10, and a polynucleotide comprising a nucleotide sequence encoding the light chain variable region of an anti-CD137 antibody consisting of amino acid sequences 1 to 109 of SEQ ID NO: 12; (b) A polynucleotide comprising a nucleotide sequence encoding the heavy chain variable region of an anti-CD137 antibody consisting of amino acid sequences 1 to 118 of SEQ ID NO: 14, and a polynucleotide comprising a nucleotide sequence encoding the light chain variable region of an anti-CD137 antibody consisting of amino acid sequences 1 to 111 of SEQ ID NO: 16; (c) A polynucleotide containing a nucleotide sequence encoding the heavy chain variable region of an anti-CD137 antibody consisting of amino acid sequences 1 to 118 of SEQ ID NO: 18, and a polynucleotide containing a nucleotide sequence encoding the light chain variable region of an anti-CD137 antibody consisting of amino acid sequences 1 to 111 of SEQ ID NO: 20; (d) A polynucleotide containing a nucleotide sequence encoding the heavy chain variable region of an anti-CD137 antibody consisting of amino acid sequences 1 to 121 of SEQ ID NO: 22, and a polynucleotide containing a nucleotide sequence encoding the light chain variable region of an anti-CD137 antibody consisting of amino acid sequences 1 to 111 of SEQ ID NO: 24; (e) A polynucleotide comprising a nucleotide sequence encoding the heavy chain variable region of an anti-CD137 antibody consisting of amino acid sequences from amino acid numbers 1 to 118 of SEQ ID NO: 26, and a polynucleotide comprising a nucleotide sequence encoding the light chain variable region of an anti-CD137 antibody consisting of amino acid sequences from amino acid numbers 134 to 242 of SEQ ID NO: 26; (f) A polynucleotide containing a nucleotide sequence encoding the heavy chain variable region of an anti-CD137 antibody consisting of amino acid sequences 1 to 118 of SEQ ID NO: 28, and a polynucleotide containing a nucleotide sequence encoding the light chain variable region of an anti-CD137 antibody consisting of amino acid sequences 134 to 244 of SEQ ID NO: 28; (g) A polynucleotide containing a nucleotide sequence encoding the light chain variable region of an anti-CD137 antibody consisting of amino acid sequences from amino acid numbers 1 to 111 of SEQ ID NO: 30, and a polynucleotide containing a nucleotide sequence encoding the heavy chain variable region of an anti-CD137 antibody consisting of amino acid sequences from amino acid numbers 132 to 249 of SEQ ID NO: 30; (h) A polynucleotide comprising a nucleotide sequence encoding the heavy chain variable region of an anti-CD137 antibody consisting of amino acid sequences 1 to 118 of SEQ ID NO: 32, and a polynucleotide comprising a nucleotide sequence encoding the light chain variable region of an anti-CD137 antibody consisting of amino acid sequences 134 to 244 of SEQ ID NO: 32; or (i) A polynucleotide comprising a nucleotide sequence encoding the heavy chain variable region of an anti-CD137 antibody consisting of amino acid sequences from amino acid numbers 1 to 121 of SEQ ID NO: 34, and a polynucleotide comprising a nucleotide sequence encoding the light chain variable region of an anti-CD137 antibody consisting of amino acid sequences 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 base sequence encoding anti-CD137scFv, selected from the group consisting of (a) to (e) below: (a) A polynucleotide containing a base sequence encoding anti-CD137scFv, consisting of the amino acid sequence from amino acid number 1 to 242 of SEQ ID NO: 26; (b) A polynucleotide containing a nucleotide sequence encoding anti-CD137scFv, consisting of the amino acid sequence from amino acid number 1 to 244 of SEQ ID NO: 28; (c) A polynucleotide containing a base sequence encoding anti-CD137scFv, consisting of amino acid sequences from amino acid number 1 to 249 of SEQ ID NO. 30; (d) A polynucleotide comprising a nucleotide sequence encoding anti-CD137scFv, consisting of the amino acid sequence from amino acid number 1 to 244 of SEQ ID NO: 32; or (e) A polynucleotide containing a base sequence encoding anti-CD137scFv, consisting of amino acid sequences from amino acid number 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 (a) to (e) below: (a) A polynucleotide comprising a nucleotide sequence encoding a polypeptide containing the heavy chain of an anti-CLDN4 antibody and anti-CD137scFv, consisting of the amino acid sequence from amino acid numbers 1 to 705 of SEQ ID NO: 36; (b) A polynucleotide comprising a nucleotide sequence encoding a polypeptide containing the heavy chain of an anti-CLDN4 antibody and anti-CD137scFv, consisting of the amino acid sequence from amino acid number 1 to 712 of SEQ ID NO. 38; (c) A polynucleotide containing the base sequence encoding the light chain of the anti-CLDN4 antibody, consisting of the amino acid sequence from amino acid number 1 to 215 of SEQ ID NO: 40; (d) A polynucleotide comprising a nucleotide sequence encoding a polypeptide containing the heavy chain of an anti-CLDN4 antibody consisting of amino acid sequences 1 to 705 of SEQ ID NO: 36 and anti-CD137scFv, and a polynucleotide comprising a nucleotide sequence encoding the light chain of an anti-CLDN4 antibody consisting of amino acid sequences 1 to 215 of SEQ ID NO: 40; or (e) A polynucleotide comprising a base sequence encoding the heavy chain of an anti-CLDN4 antibody consisting of amino acid sequences from amino acid numbers 1 to 712 of SEQ ID NO: 38 and a polypeptide containing anti-CD137scFv, and a polynucleotide comprising a base sequence encoding the light chain of an anti-CLDN4 antibody consisting of amino acid sequences from amino acid numbers 1 to 215 of SEQ ID NO: 40.

[0063] The polynucleotide of the bispecific antibody of the present invention can be readily prepared by those skilled in the art based on its base sequence using methods known in the art. For example, the polynucleotide of the bispecific antibody of the present invention can be synthesized using gene synthesis methods known in the art. Such gene synthesis methods may include various methods known to those skilled in the art, such as the antibody gene synthesis method described in International Publication No. 90 / 07861.

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

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

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

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

[0068] In one embodiment, the expression vector for the bispecific antibody of the present invention comprises a polynucleotide selected from (a) or (b) below: (a) A polynucleotide comprising a nucleotide sequence encoding a polypeptide containing the heavy chain of an anti-CLDN4 antibody consisting of amino acid sequences 1 to 705 of SEQ ID NO: 36 and anti-CD137scFv, and a polynucleotide comprising a nucleotide sequence encoding the light chain of an anti-CLDN4 antibody consisting of amino acid sequences 1 to 215 of SEQ ID NO: 40; or (b) A polynucleotide comprising a nucleotide sequence encoding the heavy chain of an anti-CLDN4 antibody consisting of amino acid sequences 1 to 712 of SEQ ID NO: 38 and a polypeptide containing anti-CD137scFv, and a polynucleotide comprising a nucleotide sequence encoding the light chain of an anti-CLDN4 antibody consisting of amino acid sequences 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 can produce 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 include the pcDNA series (Thermo Fisher Scientific), pALTER®-MAX (Promega), pHEK293 Ultra Expression Vector (Takara Bio Inc.), pEE6.4 or pEE12.4 (Lonza Biologics). Examples of viral vectors include lentiviruses, adenoviruses, retroviruses, and adeno-associated viruses. For example, when using a lentivirus to introduce the polynucleotide of the present invention into cells, the lentivirus could be the pLVSIN-CMV / EF1α vector (Takara Bio Inc.), the pLenti vector (Thermo Fisher Scientific Inc.), etc. In one embodiment, the vectors used for the expression vector of the bispecific antibody of the present invention are pcDNA3.4-TOPO® (Thermo Fisher Scientific) and pcDNA3.1 (Thermo Fisher Scientific).

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

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

[0072] <Host cell of the present invention> The present invention also provides host cells transformed with the polynucleotide of the bispecific antibody of the present invention or the expression vector of the bispecific antibody of the present invention (also referred to as "the polynucleotide, etc." of the present invention). The host cells of the present invention contain the polynucleotide, etc. of the present invention within the cell. The introduced polynucleotide, etc. of the present invention may or may not be incorporated into the genomic DNA of the host cell. The cells used as host cells may be cells that can be cultured in vitro or cells that are in vivo. If the host cells are cells that can be cultured in vitro, the host cells of the present invention can be produced by introducing the polynucleotide, etc. of the present invention into the cells in vitro. The method for transforming the host cells is not particularly limited, but for example, methods commonly used by those skilled in the art, such as the calcium phosphate method, the electroporation method, or the lipofection method, can be used. If the host cells are cells that are in vivo, the method for introducing the polynucleotide, etc. of the present invention into the host cells is not particularly limited, but nucleic acid delivery carriers (cationic carriers or non-cationic carriers (e.g., liposomes, lipid nanoparticles (LNPs), etc.), etc.) 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 by methods such as electroporation and express antibodies or polypeptides. Examples of cells that can be cultured in vitro include various cells such as conventional cells or artificially established cells that are commonly used in the art of the present invention (e.g., animal cells (e.g., CHO-K1 cells, ExpiCHO-S® cells, CHOK1SV cells, CHO-DG44 cells, HEK293 cells, Expi293F cells, NS0 cells), insect cells (e.g., Sf9), bacteria (e.g., Escherichia species), yeasts (e.g., Saccharomyces species, Pichia species), etc.). In one embodiment, the host cell of the present invention is an Expi293F cell, a CHO-K1 cell, or an ExpiCHO-S cell.

[0074] The selection of host cells transformed in vitro can be carried out by methods commonly used by those skilled in the art. Selection methods may include, for example, drug selection methods using drug selection marker genes and drugs such as tetracycline, ampicillin, neomycin, or hygromycin, as well as cell isolation methods such as ultradilution, single-cell sorting, or colony pickup.

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

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

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

[0078] In one embodiment, the host cell contains a polynucleotide selected from (a) or (b) below: (a) A polynucleotide comprising a nucleotide sequence encoding a polypeptide containing the heavy chain of an anti-CLDN4 antibody consisting of amino acid sequences 1 to 705 of SEQ ID NO: 36 and anti-CD137scFv, and a polynucleotide comprising a nucleotide sequence encoding the light chain of an anti-CLDN4 antibody consisting of amino acid sequences 1 to 215 of SEQ ID NO: 40; or (b) A polynucleotide comprising a nucleotide sequence encoding the heavy chain of an anti-CLDN4 antibody consisting of amino acid sequences 1 to 712 of SEQ ID NO: 38 and a polypeptide containing anti-CD137scFv, and a polynucleotide comprising a nucleotide sequence encoding the light chain of an anti-CLDN4 antibody consisting of amino acid sequences 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 polypeptides (a) to (j) below: (a) A polynucleotide comprising a base sequence encoding a polypeptide comprising the heavy chain of an anti-CLDN4 antibody including a heavy chain variable region consisting of amino acid sequences 1 to 125 of SEQ ID NO: 2, and an anti-CD137scFv comprising the heavy chain variable region of an anti-CD137 antibody consisting of amino acid sequences 1 to 118 of SEQ ID NO: 26 and the light chain variable region of an anti-CD137 antibody consisting of amino acid sequences 134 to 242 of SEQ ID NO: 26, wherein the amino terminal of the anti-CD137scFv is linked to the heavy chain carboxyl terminal of the anti-CLDN4 antibody via a linker; and a polynucleotide comprising a base sequence encoding the light chain of an anti-CLDN4 antibody including a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 4; (b) A polynucleotide comprising a base sequence encoding a polypeptide comprising the heavy chain of an anti-CLDN4 antibody comprising a heavy chain variable region consisting of amino acid sequences 1 to 125 of SEQ ID NO: 2, and an anti-CD137scFv comprising the heavy chain variable region of an anti-CD137 antibody consisting of amino acid sequences 1 to 118 of SEQ ID NO: 28 and the light chain variable region of an anti-CD137 antibody consisting of amino acid sequences 134 to 244 of SEQ ID NO: 28, wherein the amino terminal of the anti-CD137scFv is linked to the carboxyl terminal of the heavy chain of the anti-CLDN4 antibody via a linker, and a polynucleotide comprising a base sequence encoding the light chain of an anti-CLDN4 antibody comprising the light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 4; (c) A polynucleotide comprising a base sequence encoding a polypeptide comprising the heavy chain of an anti-CLDN4 antibody including a heavy chain variable region consisting of amino acid sequences 1 to 125 of SEQ ID NO: 2, and an anti-CD137scFv comprising the light chain variable region of an anti-CD137 antibody consisting of amino acid sequences 1 to 111 of SEQ ID NO: 30 and the heavy chain variable region of an anti-CD137 antibody consisting of amino acid sequences 132 to 249 of SEQ ID NO: 30, wherein the amino terminus of the anti-CD137scFv is linked to the heavy chain carboxy terminus of the anti-CLDN4 antibody via a linker, and a polynucleotide comprising a base sequence encoding the light chain of an anti-CLDN4 antibody including a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 4; (d) A polynucleotide comprising a base sequence encoding a polypeptide comprising the heavy chain of an anti-CLDN4 antibody including a heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 125 of SEQ ID NO: 2, and an anti-CD137scFv comprising the heavy chain variable region of an anti-CD137 antibody consisting of amino acid sequences from amino acid numbers 1 to 118 of SEQ ID NO: 32 and the light chain variable region of an anti-CD137 antibody consisting of amino acid sequences from amino acid numbers 134 to 244 of SEQ ID NO: 32, wherein the amino terminal of the anti-CD137scFv is linked to the heavy chain carboxyl terminal of the anti-CLDN4 antibody via a linker, and a polynucleotide comprising a base sequence encoding the light chain of an anti-CLDN4 antibody including a light chain variable region consisting of amino acid sequences from amino acid numbers 1 to 109 of SEQ ID NO: 4; (e) A polynucleotide comprising a base sequence encoding a polypeptide comprising the heavy chain of an anti-CLDN4 antibody including a heavy chain variable region consisting of amino acid sequences 1 to 125 of SEQ ID NO: 2, and an anti-CD137scFv comprising the heavy chain variable region of an anti-CD137 antibody consisting of amino acid sequences 1 to 121 of SEQ ID NO: 34 and the light chain variable region of an anti-CD137 antibody consisting of amino acid sequences 137 to 247 of SEQ ID NO: 34, wherein the amino terminal of the anti-CD137scFv is linked to the heavy chain carboxyl terminal of the anti-CLDN4 antibody via a linker, and a polynucleotide comprising a base sequence encoding the light chain of an anti-CLDN4 antibody including a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 4; (f) A polynucleotide comprising a base sequence encoding a polypeptide comprising the heavy chain of an anti-CLDN4 antibody including a heavy chain variable region consisting of amino acid sequences 1 to 123 of SEQ ID NO: 6, and an anti-CD137scFv comprising the heavy chain variable region of an anti-CD137 antibody consisting of amino acid sequences 1 to 118 of SEQ ID NO: 26 and the light chain variable region of an anti-CD137 antibody consisting of amino acid sequences 134 to 242 of SEQ ID NO: 26, wherein the amino terminal of the anti-CD137scFv is linked to the heavy chain carboxyl terminal of the anti-CLDN4 antibody via a linker, and a polynucleotide comprising a base sequence encoding the light chain of an anti-CLDN4 antibody including a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 8; (g) A polynucleotide comprising a base sequence encoding a polypeptide comprising the heavy chain of an anti-CLDN4 antibody including a heavy chain variable region consisting of amino acid sequences 1 to 123 of SEQ ID NO: 6, and an anti-CD137scFv comprising the heavy chain variable region of an anti-CD137 antibody consisting of amino acid sequences 1 to 118 of SEQ ID NO: 28 and the light chain variable region of an anti-CD137 antibody consisting of amino acid sequences 134 to 244 of SEQ ID NO: 28, wherein the amino terminal of the anti-CD137scFv is linked to the carboxyl terminal of the heavy chain of the anti-CLDN4 antibody via a linker, and a polynucleotide comprising a base sequence encoding the light chain of an anti-CLDN4 antibody including a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 8; (h) A polynucleotide comprising a base sequence encoding a polypeptide comprising the heavy chain of an anti-CLDN4 antibody including a heavy chain variable region consisting of amino acid sequences 1 to 123 of SEQ ID NO: 6, and an anti-CD137scFv comprising the light chain variable region of an anti-CD137 antibody consisting of amino acid sequences 1 to 111 of SEQ ID NO: 30 and the heavy chain variable region of an anti-CD137 antibody consisting of amino acid sequences 132 to 249 of SEQ ID NO: 30, wherein the amino terminus of the anti-CD137scFv is linked to the carboxyl terminus of the heavy chain of the anti-CLDN4 antibody via a linker, and a polynucleotide comprising a base sequence encoding the light chain of an anti-CLDN4 antibody including a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 8; (i) A polynucleotide comprising a base sequence encoding a polypeptide comprising the heavy chain of an anti-CLDN4 antibody comprising a heavy chain variable region consisting of amino acid sequences 1 to 123 of SEQ ID NO: 6, and an anti-CD137scFv comprising the heavy chain variable region of an anti-CD137 antibody consisting of amino acid sequences 1 to 118 of SEQ ID NO: 32 and the light chain variable region of an anti-CD137 antibody consisting of amino acid sequences 134 to 244 of SEQ ID NO: 32, wherein the amino terminal of the anti-CD137scFv is linked to the carboxyl terminal of the heavy chain of the anti-CLDN4 antibody via a linker, and a polynucleotide comprising a base sequence encoding the light chain of an anti-CLDN4 antibody comprising the light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 8; or (j) A polynucleotide comprising a base sequence encoding a polypeptide comprising the heavy chain of an anti-CLDN4 antibody comprising a heavy chain variable region consisting of amino acid sequences 1 to 123 of SEQ ID NO: 6, and an anti-CD137scFv comprising the heavy chain variable region of an anti-CD137 antibody consisting of amino acid sequences 1 to 121 of SEQ ID NO: 34 and the light chain variable region of an anti-CD137 antibody consisting of amino acid sequences 137 to 247 of SEQ ID NO: 34, wherein the amino terminal of the anti-CD137scFv is linked to the heavy chain carboxyl terminal of the anti-CLDN4 antibody via a linker, and a polynucleotide comprising a base sequence encoding the light chain of an anti-CLDN4 antibody comprising the light chain variable region consisting of amino acid sequences 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) described below: (a) A host cell comprising a polynucleotide containing a nucleotide sequence encoding a polypeptide including the heavy chain of an anti-CLDN4 antibody consisting of amino acid sequences 1 to 705 of SEQ ID NO: 36 and anti-CD137scFv, and a polynucleotide containing a nucleotide sequence encoding the light chain of an anti-CLDN4 antibody consisting of amino acid sequences 1 to 215 of SEQ ID NO: 40; or (b) A host cell comprising a polynucleotide comprising a nucleotide sequence encoding a polypeptide containing the heavy chain and anti-CD137scFv 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 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.

[0081] In one embodiment of the host cell of the present invention, the host cell is a host cell comprising a polynucleotide comprising a nucleotide sequence encoding a polypeptide containing 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 anti-CD137scFv, and 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.

[0082] <Method for producing the bispecific antibody of the present invention> The present invention also provides a method for producing the anti-CLDN4-anti-CD137 bispecific antibody of the present invention (also referred to as the "production method of the present invention"). The production method of the present invention may include a method for producing the polynucleotide of the bispecific antibody of the present invention, a method for producing an expression vector for the bispecific antibody of the present invention, and a method for producing the host cells of the present invention. Furthermore, the production method of the present invention may include a step of culturing the host cells described in <host cells of the present invention> above and expressing the antibody in the cells or the culture supernatant, a method for recovering, isolating and purifying the antibody, etc. However, the production method of the present invention is not limited to these methods, as long as the anti-CLDN4-anti-CD137 bispecific antibody of the present invention is produced.

[0083] In one embodiment, the production method of the present invention includes the step of culturing the following host cells (a) to (j): (a) A host cell comprising a polynucleotide comprising a base sequence encoding a polypeptide containing the heavy chain of an anti-CLDN4 antibody comprising a heavy chain variable region consisting of amino acid sequences 1 to 125 of SEQ ID NO: 2, and an anti-CD137scFv comprising the heavy chain variable region of an anti-CD137 antibody consisting of amino acid sequences 1 to 118 of SEQ ID NO: 26 and the light chain variable region of an anti-CD137 antibody consisting of amino acid sequences 134 to 242 of SEQ ID NO: 26, wherein the amino terminal of the anti-CD137scFv is linked to the carboxyl terminal of the heavy chain of the anti-CLDN4 antibody via a linker, and a polynucleotide comprising a base sequence encoding the light chain of an anti-CLDN4 antibody comprising the light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 4; (b) A host cell comprising a polynucleotide comprising a base sequence encoding a polypeptide containing the heavy chain of an anti-CLDN4 antibody comprising a heavy chain variable region consisting of amino acid sequences 1 to 125 of SEQ ID NO: 2, and an anti-CD137scFv comprising the heavy chain variable region of an anti-CD137 antibody consisting of amino acid sequences 1 to 118 of SEQ ID NO: 28 and the light chain variable region of an anti-CD137 antibody consisting of amino acid sequences 134 to 244 of SEQ ID NO: 28, wherein the amino terminal of the anti-CD137scFv is linked to the carboxyl terminal of the heavy chain of the anti-CLDN4 antibody via a linker, and a polynucleotide comprising a base sequence encoding the light chain of an anti-CLDN4 antibody comprising the light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 4; (c) A host cell comprising a polynucleotide comprising a heavy chain of an anti-CLDN4 antibody including a heavy chain variable region consisting of amino acid sequences 1 to 125 of SEQ ID NO: 2, and an anti-CD137scFv including a light chain variable region of an anti-CD137 antibody consisting of amino acid sequences 1 to 111 of SEQ ID NO: 30 and a heavy chain variable region of an anti-CD137 antibody consisting of amino acid sequences 132 to 249 of SEQ ID NO: 30, wherein the amino terminus of the anti-CD137scFv is linked to the carboxyl terminus of the heavy chain of the anti-CLDN4 antibody via a linker, and a polynucleotide comprising a polynucleotide comprising a light chain encoding the light chain of an anti-CLDN4 antibody including a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 4; (d) A host cell comprising a polynucleotide comprising a heavy chain of an anti-CLDN4 antibody including a heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 125 of SEQ ID NO: 2, and an anti-CD137scFv including a heavy chain variable region of an anti-CD137 antibody consisting of amino acid sequences from amino acid numbers 1 to 118 of SEQ ID NO: 32 and a light chain variable region of an anti-CD137 antibody consisting of amino acid sequences from amino acid numbers 134 to 244 of SEQ ID NO: 32, wherein the amino terminal of the anti-CD137scFv is linked to the carboxyl terminal of the heavy chain of the anti-CLDN4 antibody via a linker, and a polynucleotide comprising a polynucleotide comprising a light chain encoding the light chain of an anti-CLDN4 antibody including a light chain variable region consisting of amino acid sequences from amino acid numbers 1 to 109 of SEQ ID NO: 4; (e) A host cell comprising a polynucleotide comprising a heavy chain of an anti-CLDN4 antibody including a heavy chain variable region consisting of amino acid sequences 1 to 125 of SEQ ID NO: 2, and an anti-CD137scFv including a heavy chain variable region of an anti-CD137 antibody consisting of amino acid sequences 1 to 121 of SEQ ID NO: 34 and a light chain variable region of an anti-CD137 antibody consisting of amino acid sequences 137 to 247 of SEQ ID NO: 34, wherein the amino terminal of the anti-CD137scFv is linked to the carboxyl terminal of the heavy chain of the anti-CLDN4 antibody via a linker, and a polynucleotide comprising a polynucleotide comprising a light chain encoding the light chain of an anti-CLDN4 antibody including a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 4; (f) A host cell comprising a polynucleotide comprising a heavy chain of an anti-CLDN4 antibody including a heavy chain variable region consisting of amino acid sequences 1 to 123 of SEQ ID NO: 6, and an anti-CD137scFv including a heavy chain variable region of an anti-CD137 antibody consisting of amino acid sequences 1 to 118 of SEQ ID NO: 26 and a light chain variable region of an anti-CD137 antibody consisting of amino acid sequences 134 to 242 of SEQ ID NO: 26, wherein the amino terminal of the anti-CD137scFv is linked to the carboxyl terminal of the heavy chain of the anti-CLDN4 antibody via a linker, and a polynucleotide comprising a polynucleotide comprising a light chain encoding the light chain of an anti-CLDN4 antibody including a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 8; (g) A host cell comprising a polynucleotide containing a base sequence encoding a polypeptide comprising the heavy chain of an anti-CLDN4 antibody including a heavy chain variable region consisting of amino acid sequences 1 to 123 of SEQ ID NO: 6, and an anti-CD137scFv including the heavy chain variable region of an anti-CD137 antibody consisting of amino acid sequences 1 to 118 of SEQ ID NO: 28 and the light chain variable region of an anti-CD137 antibody consisting of amino acid sequences 134 to 244 of SEQ ID NO: 28, wherein the amino terminal of the anti-CD137scFv is linked to the carboxyl terminal of the heavy chain of the anti-CLDN4 antibody via a linker, and a polynucleotide containing a base sequence encoding the light chain of an anti-CLDN4 antibody including a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 8; (h) A host cell comprising a polynucleotide comprising a heavy chain of an anti-CLDN4 antibody containing a heavy chain variable region consisting of amino acid sequences 1 to 123 of SEQ ID NO: 6, and an anti-CD137scFv containing a light chain variable region of an anti-CD137 antibody consisting of amino acid sequences 1 to 111 of SEQ ID NO: 30 and an anti-CD137scFv containing a heavy chain variable region of an anti-CD137 antibody consisting of amino acid sequences 132 to 249 of SEQ ID NO: 30, wherein the amino terminus of the anti-CD137scFv is linked to the carboxyl terminus of the heavy chain of the anti-CLDN4 antibody via a linker, and a polynucleotide comprising a polynucleotide comprising a light chain encoding the light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 8; (i) A host cell comprising a polynucleotide comprising a base sequence encoding a polypeptide containing the heavy chain of an anti-CLDN4 antibody comprising a heavy chain variable region consisting of amino acid sequences 1 to 123 of SEQ ID NO: 6, and an anti-CD137scFv comprising the heavy chain variable region of an anti-CD137 antibody consisting of amino acid sequences 1 to 118 of SEQ ID NO: 32 and the light chain variable region of an anti-CD137 antibody consisting of amino acid sequences 134 to 244 of SEQ ID NO: 32, wherein the amino terminal of the anti-CD137scFv is linked to the carboxyl terminal of the heavy chain of the anti-CLDN4 antibody via a linker, and a polynucleotide comprising a base sequence encoding the light chain of an anti-CLDN4 antibody comprising the light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 8; or (j) A host cell comprising a polynucleotide comprising a base sequence encoding a polypeptide containing the heavy chain of an anti-CLDN4 antibody comprising a heavy chain variable region consisting of amino acid sequences 1 to 123 of SEQ ID NO: 6, and an anti-CD137scFv comprising the heavy chain variable region of an anti-CD137 antibody consisting of amino acid sequences 1 to 121 of SEQ ID NO: 34 and the light chain variable region of an anti-CD137 antibody consisting of amino acid sequences 137 to 247 of SEQ ID NO: 34, wherein the amino terminal of the anti-CD137scFv is linked to the carboxyl terminal of the heavy chain of the anti-CLDN4 antibody via a linker, and a polynucleotide comprising a base sequence encoding the light chain of an anti-CLDN4 antibody comprising the light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 8.

[0084] In one embodiment, the production method of the present invention includes the step of culturing host cells of the following (a) or (b): (a) Host cells containing a polynucleotide comprising a nucleotide sequence encoding a polypeptide including the heavy chain of an anti-CLDN4 antibody consisting of amino acid sequences 1 to 705 of SEQ ID NO: 36 and anti-CD137scFv, and a nucleotide sequence encoding the light chain of an anti-CLDN4 antibody consisting of amino acid sequences 1 to 215 of SEQ ID NO: 40; or (b) A host cell comprising a polynucleotide containing a nucleotide sequence encoding a polypeptide including the heavy chain and anti-CD137scFv 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 polynucleotide containing 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.

[0085] The host cells of the present invention can be cultured by known methods. Culture conditions, such as temperature, pH of the culture medium, and culture time, can be appropriately selected by those skilled in the art. When the host cells are animal cells, suitable culture media can be, for example, MEM medium containing about 5-20% fetal bovine serum (Science, 1959, Vol. 130, pp. 432-437), D-MEM medium (Virol., 1959, Vol. 8, pp. 396), RPMI-1640 medium (J. Am. Med. Assoc., 1967, Vol. 199, pp. 519), 199 medium (Exp. Biol. Med., 1950, Vol. 73, pp. 1-8), etc. The pH of the culture medium is, for example, about 6-8, and the culture is usually carried out at about 30-40°C for about 15-336 hours, with aeration and stirring as necessary. When the host cells are insect cells, a culture medium such as Grace's medium containing fetal bovine serum (Proc. Natl. Acad. Sci. USA., 1985, Vol. 82, p. 8404) can be used. The pH of the medium is, for example, about 5 to 8, and the culture is usually carried out at about 20 to 40°C for about 15 to 100 hours, with aeration and stirring as needed. When the host cells are E. coli or yeast, a liquid medium containing nutrients is suitable as the culture medium. 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. Examples of carbon sources include glucose, dextran, soluble starch, and sucrose, while examples of inorganic or organic nitrogen sources include ammonium salts, nitrates, amino acids, corn slush liquor, peptone, casein, meat extract, soybean meal, and potato extract. The culture medium may optionally contain other nutrients (e.g., 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 E. coli, the culture medium can be, for example, LB medium or M9 medium (Molecular Cloning, Cold Spring Harbor Laboratory, Vol. 3, A2.2). Culturing is usually carried out at about 14 to 43°C for about 3 to 24 hours, with aeration and stirring as needed.When the host cells are yeast, a culture medium such as Burkholder Minimal Medium (Proc. Natl. Acad. Sci. USA., 1980, Vol. 77, p. 4505) can be used. Culturing is usually carried out at approximately 20-35°C for approximately 14-144 hours, with aeration and stirring as needed. 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 may include, in addition to the step of culturing the host cells of the present invention and expressing the anti-CLDN4-anti-CD137 bispecific antibody, 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 utilizing solubility such as salting out and solvent precipitation, methods utilizing differences in molecular weight such as dialysis, ultrafiltration, and gel filtration, methods utilizing charge such as ion exchange chromatography and hydroxyapatite chromatography, methods utilizing specific affinity such as affinity chromatography, methods utilizing differences in hydrophobicity such as reversed-phase high-performance liquid chromatography, and methods utilizing differences in isoelectric point such as isoelectric focusing. In one embodiment, the antibody secreted into the culture supernatant can be purified by various chromatography methods, for example, column chromatography using a protein A column or a protein G column.

[0087] The anti-CLDN4-anti-CD137 bispecific antibody and the 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 Compositions, etc. of the Invention> The present invention also provides pharmaceutical compositions, etc. (also referred to as "pharmaceutical compositions of the present invention"), comprising the anti-CLDN4-anti-CD137 bispecific antibody of the present invention. Pharmaceutical compositions of the present invention include pharmaceutical compositions comprising the anti-CLDN4-anti-CD137 bispecific antibody of the present invention and pharmaceutically acceptable excipients. 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, for example, parenteral preparations such as injections and intravenous infusions, which can be administered by appropriate methods such as intravenous administration, subcutaneous administration, intraperitoneal administration, and intratumor administration. In formulation, excipients, carriers, additives, etc., can be used according to these dosage forms, within a pharmaceutically acceptable range.

[0089] The pharmaceutical compositions of the present invention may include post-translational modifications of the anti-CLDN4-anti-CD137 bispecific antibody of the present invention. For example, pharmaceutical compositions containing antibodies that have undergone either or both of the following: deletion of the C-terminal lysine or pyroglutamylation of the N-terminal region are also included in the present invention.

[0090] In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition containing any of the following (a) to (j) anti-CLDN4-anti-CD137 bispecific antibody of the present invention and / or a post-translational modification of said antibody: (a) an anti-CLDN4-anti-CD137 bispecific antibody and / or a post-translational modification of said antibody comprising: (a) a heavy chain of an anti-CLDN4 antibody containing a heavy chain variable region consisting of amino acid sequences 1 to 125 of SEQ ID NO: 2; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 4; and an anti-CD137scFv containing a heavy chain variable region consisting of amino acid sequences 1 to 118 of SEQ ID NO: 26 and a light chain variable region consisting of amino acid sequences 134 to 242 of SEQ ID NO: 26, 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) an anti-CLDN4-anti-CD137 bispecific antibody and / or a post-translational modification of said antibody comprising: a heavy chain of an anti-CLDN4 antibody containing a heavy chain variable region consisting of amino acid sequences 1 to 125 of SEQ ID NO: 2; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 4; and an anti-CD137scFv containing a heavy chain variable region consisting of amino acid sequences 1 to 118 of SEQ ID NO: 28 and a light chain variable region consisting of amino acid sequences 134 to 244 of SEQ ID NO: 28, wherein the amino terminus of the anti-CD137scFv is linked to the carboxyl 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-translational modification of said antibody comprising: a heavy chain of an anti-CLDN4 antibody containing a heavy chain variable region consisting of amino acid sequences 1 to 125 of SEQ ID NO: 2; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 4; and an anti-CD137scFv containing a light chain variable region consisting of amino acid sequences 1 to 111 of SEQ ID NO: 30 and a heavy chain variable region consisting of amino acid sequences 132 to 249 of SEQ ID NO: 30, 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) an anti-CLDN4-anti-CD137 bispecific antibody and / or a post-translational modification of said antibody comprising: a heavy chain of an anti-CLDN4 antibody containing a heavy chain variable region consisting of amino acid sequences 1 to 125 of SEQ ID NO: 2; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 4; and an anti-CD137scFv containing a heavy chain variable region consisting of amino acid sequences 1 to 118 of SEQ ID NO: 32 and a light chain variable region consisting of amino acid sequences 134 to 244 of SEQ ID NO: 32, 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) an anti-CLDN4-anti-CD137 bispecific antibody and / or a post-translational modification of said antibody comprising: a heavy chain of an anti-CLDN4 antibody containing a heavy chain variable region consisting of amino acid sequences 1 to 125 of SEQ ID NO: 2; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 4; and an anti-CD137scFv containing a heavy chain variable region consisting of amino acid sequences 1 to 121 of SEQ ID NO: 34 and a light chain variable region consisting of amino acid sequences 137 to 247 of SEQ ID NO: 34, 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) An anti-CLDN4-anti-CD137 bispecific antibody and / or a post-translational modification of said antibody comprising: a heavy chain of an anti-CLDN4 antibody containing a heavy chain variable region consisting of amino acid sequences 1 to 123 of SEQ ID NO: 6; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 8; and an anti-CD137scFv containing a heavy chain variable region consisting of amino acid sequences 1 to 118 of SEQ ID NO: 26 and a light chain variable region consisting of amino acid sequences 134 to 242 of SEQ ID NO: 26, 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) an anti-CLDN4-anti-CD137 bispecific antibody and / or a post-translational modification of said antibody comprising: a heavy chain of an anti-CLDN4 antibody containing a heavy chain variable region consisting of amino acid sequences 1 to 123 of SEQ ID NO: 6; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 8; and an anti-CD137scFv containing a heavy chain variable region consisting of amino acid sequences 1 to 118 of SEQ ID NO: 28 and a light chain variable region consisting of amino acid sequences 134 to 244 of SEQ ID NO: 28, wherein the amino terminus of the anti-CD137scFv is linked to the heavy chain carboxy terminus of the anti-CLDN4 antibody via a linker; (h) An anti-CLDN4-anti-CD137 bispecific antibody and / or a post-translational modification of said antibody comprising: a heavy chain of an anti-CLDN4 antibody containing a heavy chain variable region consisting of amino acid sequences 1 to 123 of SEQ ID NO: 6; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 8; and an anti-CD137scFv containing a light chain variable region consisting of amino acid sequences 1 to 111 of SEQ ID NO: 30 and a heavy chain variable region consisting of amino acid sequences 132 to 249 of SEQ ID NO: 30, wherein the amino terminus of the anti-CD137scFv is linked to the heavy chain carboxy terminus of the anti-CLDN4 antibody via a linker; (i) an anti-CLDN4-anti-CD137 bispecific antibody and / or a post-translational modification of said antibody comprising: (i) a heavy chain of an anti-CLDN4 antibody containing a heavy chain variable region consisting of amino acid sequences 1 to 123 of SEQ ID NO: 6; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 8; and an anti-CD137scFv containing a heavy chain variable region consisting of amino acid sequences 1 to 118 of SEQ ID NO: 32 and a light chain variable region consisting of amino acid sequences 134 to 244 of SEQ ID NO: 32, wherein the amino terminus of the anti-CD137scFv is linked to the carboxyl terminus of the heavy chain of the anti-CLDN4 antibody via a linker; or (j) An anti-CLDN4-anti-CD137 bispecific antibody and / or a post-translational modification of said antibody comprising: a heavy chain of an anti-CLDN4 antibody containing a heavy chain variable region consisting of amino acid sequences 1 to 123 of SEQ ID NO: 6; a light chain of an anti-CLDN4 antibody containing a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 8; and an anti-CD137scFv containing a heavy chain variable region consisting of amino acid sequences 1 to 121 of SEQ ID NO: 34 and a light chain variable region consisting of amino acid sequences 137 to 247 of SEQ ID NO: 34, wherein the amino terminus of the anti-CD137scFv is linked to the heavy chain carboxy terminus of the anti-CLDN4 antibody via a linker.

[0091] In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition containing the anti-CLDN4-anti-CD137 bispecific antibody of the present invention and / or a post-translational modification of said antibody as described below: (a) an anti-CLDN4-anti-CD137 bispecific antibody and / or a post-translational modification of said antibody, comprising a polypeptide containing 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 anti-CD137scFv, 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; or (b) An anti-CLDN4-anti-CD137 bispecific antibody and / or a post-translational modification of said antibody, comprising a polypeptide containing the heavy chain of an anti-CLDN4 antibody consisting of the amino acid sequence of amino acids 1 to 712 of SEQ ID NO: 38 and anti-CD137scFv, and a light chain of an anti-CLDN4 antibody 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 and / or post-translational modification of the antibody added in formulation of the present invention varies depending on the severity and age of the patient's symptoms, the dosage form of the formulation used, and the antibody's binding titer, but for example, amounts of approximately 0.001 mg / kg to 100 mg / kg can be used.

[0093] <Pharmaceutical applications 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 for the treatment of target cancers. The present invention also includes a method for treating cancer, comprising the step of administering a therapeutically effective amount of the anti-CLDN4-anti-CD137 bispecific antibody of the present invention to a target. Furthermore, the present invention includes the anti-CLDN4-anti-CD137 bispecific antibody of the present invention for use in the treatment of cancer. The present invention also includes the use of the anti-CLDN4-anti-CD137 bispecific antibody of the present invention in the manufacture of pharmaceutical compositions for the treatment of cancer. The cancers that can be treated according to the present invention are not particularly limited, but include, for example, various peritoneal disseminated cancers, gastric cancer, lung cancer, acute lymphoblastic leukemia, acute myeloid leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, B-cell lymphoma, multiple myeloma, T-cell lymphoma and other hematological cancers, myelodysplastic syndromes, adenocarcinoma, squamous cell carcinoma, adenosquamous cell carcinoma, anaplastic 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, head and neck cancer, Examples 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, pancreaticblastoma, pleuropulmonary blastoma, and retinoblastoma. In one embodiment, the cancers targeted by treatment according to 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, the cancers targeted by treatment according to the present invention are cancers in which CLDN4 is highly expressed compared to normal tissue. The cancers targeted by the treatment according to the present invention are preferably cancers in which CLDN4 is highly expressed compared to normal tissue, or cancers selected from the group consisting of colorectal cancer, rectal cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, bladder cancer, ovarian cancer, breast cancer, and prostate cancer.

[0094] <Anti-CLDN4 antibody of the present invention> The present invention also provides any of the following (a) to (c) anti-CLDN4 antibodies or antigen-binding fragments thereof: (a) An anti-CLDN4 antibody or its antigen-binding fragment comprising a heavy chain variable region including CDR1 consisting of amino acid sequences 31 to 35 of SEQ ID NO: 2, CDR2 consisting of amino acid sequences 50 to 66 of SEQ ID NO: 2, and CDR3 consisting of amino acid sequences 99 to 114 of SEQ ID NO: 2, and a light chain variable region including CDR1 consisting of amino acid sequences 24 to 35 of SEQ ID NO: 4, CDR2 consisting of amino acid sequences 51 to 57 of SEQ ID NO: 4, and CDR3 consisting of amino acid sequences 90 to 98 of SEQ ID NO: 4; (b) an anti-CLDN4 antibody or its antigen-binding fragment comprising a heavy chain variable region consisting of amino acid sequences 1 to 125 of SEQ ID NO: 2 and a light chain variable region consisting of amino acid sequences 1 to 109 of SEQ ID NO: 4; or (c) An anti-CLDN4 antibody or its antigen-binding fragment 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 its antigen-binding fragment of the present invention is an anti-CLDN4 antibody or its antigen-binding fragment comprising a heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 125 of SEQ ID NO: 2 and a light chain variable region consisting of amino acid sequences from amino acid numbers 1 to 109 of SEQ ID NO: 4, or an anti-CLDN4 antibody or its antigen-binding fragment comprising a heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 123 of SEQ ID NO: 6 and a light chain variable region consisting of amino acid sequences from amino acid numbers 1 to 109 of SEQ ID NO: 8. In one embodiment, the anti-CLDN4 antibody or its antigen-binding fragment of the present invention is a heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 125 of SEQ ID NO: 2. In one embodiment, the anti-CLDN4 antibody or its antigen-binding fragment of the present invention is an anti-CLDN4 antibody or its antigen-binding fragment comprising a heavy chain variable region consisting of amino acid sequences from amino acid numbers 1 to 123 of SEQ ID NO: 6 and a light chain variable region consisting of amino acid sequences from amino acid numbers 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 selected from any of the Igγ, Igμ, Igα, Igδ, or Igε constant regions. For example, Igγ can be selected from Igγ1, Igγ2, Igγ3, or Igγ4. In one embodiment, the heavy chain constant region is the Igγ1 constant region, for example, the 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 mutations, P331G, or P331S mutations to reduce ADCC and CDC. The light chain constant region of the anti-CLDN4 antibody of the present invention can be selected from any of the Igλ or Igκ constant regions. In one embodiment, the light chain constant region is the Igκ constant region, for example, the 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 containing a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 and a light chain containing 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 containing a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 6 and a light chain containing 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 containing a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 and a light chain containing 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 containing a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 6 and a light chain containing a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 8.

[0099] The present invention also provides an anti-CLDN4 antibody or its antigen-binding fragment (also referred to as "the complex of the present invention") to which carbohydrates, lipids, metals (including radioisotopes), organic compounds (including toxins, near-infrared fluorescent dyes, chelating agents), etc. (also referred to as "modifiers") are bound. In this specification, "modifier" refers to a non-peptide substance that is directly bound to the antibody or its antigen-binding fragment or via a linker, etc. The modifiers used in the complex of the present invention are not particularly limited and include, for example, 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 modifiers include Ac), organic compounds, toxins, near-infrared fluorescent dyes (e.g., IRDye®), and chelating agents. The modifier used in the complex may be directly bound to the anti-CLDN4 antibody or its antigen-binding fragment of the present invention, or it may be bound via any linker. In one embodiment, the complex of the present invention is an antibody-drug conjugate (ADC) of the anti-CLDN4 antibody or its antigen-binding fragment. The drug and linker used in the ADC may be selected from among 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 or its antigen-binding fragment on the cell surface. Such cells can be prepared by those skilled in the art using polynucleotides encoding the anti-CLDN4 antibody or its antigen-binding fragment. Various immune cells (T cells, NK cells, NKT cells, etc.) can be used to express the anti-CLDN4 antibody or its antigen-binding fragment.

[0101] In one embodiment, the anti-CLDN4 antibody or its antigen-binding fragment of the present invention, the complex of the present invention, and the antibody or antigen-binding fragment portion in cells expressing the anti-CLDN4 antibody or its antigen-binding fragment on the cell 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 lysine deletion at the C-terminus of the heavy chain.

[0102] The anti-CLDN4 antibody or its antigen-binding fragment of the present invention, the complex of the present invention, and cells expressing the anti-CLDN4 antibody or its antigen-binding fragment of the present invention on the 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 antibody or its antigen-binding fragment of the present invention disclosed herein, and information on the modifiers used in the complex of the present invention. The anti-CLDN4 antibody or its antigen-binding fragment of the present invention can be produced, for example, by the method described in the section "Method for Producing the Bispecific Antibody of the Present Invention."

[0103] The present invention also provides a pharmaceutical composition comprising the anti-CLDN4 antibody or its antigen-binding fragment, the complex of the present invention, and cells expressing the anti-CLDN4 antibody or its antigen-binding fragment on the cell surface (collectively referred to as "the anti-CLDN4 antibody, etc." in this section), 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 the step of administering a therapeutically effective amount of the anti-CLDN4 antibody, etc. of the present invention, 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 uses 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 of the above-mentioned <Pharmaceutical Composition, etc. of the Present Invention>. Cancers targeted for treatment by the pharmaceutical uses of the anti-CLDN4 antibody, etc. of the present invention include the cancers described above in the <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 a heavy chain variable region and a light chain variable region of any of the following (a) to (d): (a) A heavy chain variable region comprising CDR1 consisting of amino acid sequences 31 to 35 of SEQ ID NO: 10, CDR2 consisting of amino acid sequences 50 to 66 of SEQ ID NO: 10, and CDR3 consisting of amino acid sequences 99 to 107 of SEQ ID NO: 10, and a light chain variable region comprising CDR1 consisting of amino acid sequences 24 to 34 of SEQ ID NO: 12, CDR2 consisting of amino acid sequences 50 to 56 of SEQ ID NO: 12, and CDR3 consisting of amino acid sequences 89 to 98 of SEQ ID NO: 12; (b) A heavy chain variable region comprising CDR1 consisting of amino acid sequences 31 to 35 of SEQ ID NO: 14, CDR2 consisting of amino acid sequences 50 to 65 of SEQ ID NO: 14, and CDR3 consisting of amino acid sequences 98 to 107 of SEQ ID NO: 14, and a light chain variable region comprising CDR1 consisting of amino acid sequences 23 to 35 of SEQ ID NO: 16, CDR2 consisting of amino acid sequences 51 to 57 of SEQ ID NO: 16, and CDR3 consisting of amino acid sequences 90 to 100 of SEQ ID NO: 16; (c) A heavy chain variable region comprising CDR1 consisting of amino acid sequences 31 to 35 of SEQ ID NO: 18, CDR2 consisting of amino acid sequences 50 to 65 of SEQ ID NO: 18, and CDR3 consisting of amino acid sequences 98 to 107 of SEQ ID NO: 18, and a light chain variable region comprising CDR1 consisting of amino acid sequences 23 to 35 of SEQ ID NO: 20, CDR2 consisting of amino acid sequences 51 to 57 of SEQ ID NO: 20, and CDR3 consisting of amino acid sequences 90 to 100 of SEQ ID NO: 20; or (d) A heavy chain variable region comprising CDR1 consisting of amino acid sequences from amino acid numbers 31 to 35 of SEQ ID NO: 22, CDR2 consisting of amino acid sequences from amino acid numbers 50 to 65 of SEQ ID NO: 22, and CDR3 consisting of amino acid sequences from amino acid numbers 98 to 110 of SEQ ID NO: 22, and a light chain variable region comprising CDR1 consisting of amino acid sequences from amino acid numbers 23 to 35 of SEQ ID NO: 24, CDR2 consisting of amino acid sequences from amino acid numbers 51 to 57 of SEQ ID NO: 24, and CDR3 consisting of amino acid sequences from amino acid numbers 90 to 100 of SEQ ID NO: 24.

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

[0106] In one embodiment, the antigen-binding fragment of the anti-CD137 antibody of the present invention is scFv. In one embodiment, the antigen-binding fragment of the anti-CD137 antibody of the present invention is any of (a) to (e) below; (a) Anti-CD137scFv consisting of the amino acid sequence from amino acid number 1 to 242 of SEQ ID NO: 26; (b) Anti-CD137scFv consisting of the amino acid sequence of amino acid numbers 1 to 244 of SEQ ID NO: 28; (c) Anti-CD137scFv consisting of the amino acid sequence from amino acid number 1 to 249 of SEQ ID NO: 30; (d) Anti-CD137scFv consisting of the amino acid sequence from amino acid number 1 to 244 of SEQ ID NO. 32; or (e) Anti-CD137scFv consisting of the amino acid sequence of amino acid numbers 1 to 247 of SEQ ID NO: 34.

[0107] The anti-CD137 antibody or its antigen-binding fragment of the present invention can be prepared by those skilled in the art by referring to the descriptions herein, such as "<Anti-CLDN4-anti-CD137 bispecific antibody of the present invention>". The anti-CD137 antibody or its antigen-binding fragment of the present invention can be used, for example, in a bispecific antibody with an antibody against any TAA (anti-TAA antibody) used in the treatment of cancer. That is, the present invention also provides a bispecific antibody comprising the anti-CD137 antibody or its antigen-binding fragment of the present invention and an anti-TAA antibody or its antigen-binding fragment (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, but for example, 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 may be used. In one embodiment, the anti-TAA-anti-CD137 bispecific antibody of the present invention comprises an scFv of an anti-CD137 antibody, or in one embodiment, the anti-TAA-anti-CD137 bispecific antibody of the present invention comprises an scFv of an anti-CD137 antibody which is any of (a) to (e) below; (a) Anti-CD137scFv consisting of the amino acid sequence from amino acid number 1 to 242 of SEQ ID NO: 26; (b) Anti-CD137scFv consisting of the amino acid sequence of amino acid numbers 1 to 244 of SEQ ID NO: 28; (c) Anti-CD137scFv consisting of the amino acid sequence from amino acid number 1 to 249 of SEQ ID NO: 30; (d) Anti-CD137scFv consisting of the amino acid sequence from amino acid number 1 to 244 of SEQ ID NO. 32; or (e) Anti-CD137scFv consisting of the amino acid sequence of amino acid numbers 1 to 247 of SEQ ID NO: 34.

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

[0109] Specific examples are provided hereby for reference to further understand the present invention, but these are for illustrative purposes only and do not limit the invention. [Examples]

[0110] [Example 1: Obtaining antibodies that selectively bind to antigens expressed in peritoneal dissemination cancer cells] [1-1. Obtaining peritoneal dissemination cancer cells derived from patients] Peritoneal dissemination cancer cells were obtained from patients in accordance with the method described in the paper "Establishment of Peritoneal Metastatic Cancer (Gastric, Pancreatic, Ovarian Cancer, etc.) Cell Lines" by Fumiko Chiwaki and Hiromi Sasaki (in Hiromi Sasaki (ed.), "Practical Guide to Cancer Research Using Patient-Derived Cancer Models," Yodosha, 2019, pp. 28-37). Ascites fluid collected from the patient was centrifuged at room temperature at 430 × g for 3 minutes. After removing the supernatant, hemolysis buffer was added to the precipitate to lyse it. After centrifugation, the supernatant was removed, and the cells were washed with 50 mL of Dulbecco's PBS(-) (Nissui Pharmaceutical Co., Ltd., 05913, hereinafter referred to as "PBS(-)"). Then, the cells were collected by centrifugation at room temperature at 430 × g for 3 minutes. All cells from the collected ascites fluid were resuspended in RPMI-1640 (L-glutamine-containing) medium (Fujifilm Wako Pure Chemical Industries, Ltd., 189-02025) containing 10% FBS (Thermo Fisher Scientific, Inc., 10270-106) and ×1 Antibiotic-Antimycotic (Thermo Fisher Scientific, Inc., 15240062) (hereinafter, the medium after adding FBS etc. in Example 1 will be referred to as "RPMI-1640 medium"). Dilute the suspension and place 5 × 10 cells in a 100 mm collagen-coated dish (hereinafter referred to as "dish") (IWAKI Corporation, 4020-010). 6 ~1 × 10 7Seeds were sown in 10 mL increments and cultured in a 37°C, 5% CO2 incubator. Cells in ascites fluid include adherent cells and suspension cells. Adherent cells include not only cancer cells but also non-cancer cells (fibroblasts, peritoneal mesothelial cells, etc.). Since non-cancer cells detach more quickly than cancer cells, these cells were separated and removed from all cells in the ascites fluid. After removing non-cancer cells, the cancer cells proliferated to about 80% confluence of the dish area, and then half of the total cells were passed into a new dish. This process was repeated, and cells that were passed 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 from the culture dish were seeded into a new 100 mm dish and passed. Cells that were passed five or more times were designated as suspension cancer cells. When peritoneal disseminated cancer cells from a single patient proliferated containing both adherent and suspension cancer cells, these were designated as mixed cancer cells. In this specification, adherent cancer cells, suspension cancer cells, or mixed cancer cells isolated by the above method are collectively referred to as "peritoneal dissemination cancer cells." The six cells obtained (NSC-9C, NSC-15CF, NSC-16C, NSC-20C, NSC-22C, NSC-32C, (hereinafter also referred to as "six types of peritoneal dissemination cancer cells")) were used in subsequent studies.

[0111] [1-2: Production of anti-gastric cancer antigen antibody-producing hybridomas] Using the biomonoclonal antibody development technology "VelocImmune" (registered trademark VelocImmune antibody technology; Regeneron (US Patent No. 6596541)), velocimune mice were immunized with peritoneal dissemination cancer cells to obtain antibodies against cancer antigens expressed on anti-peritoneal dissemination cancer cells. Among the peritoneal dissemination cancer cells obtained in Example 1-1, three cells of NSC-9C, NSC-15CF, and NSC-16C or three cells of NSC-20C, NSC-22C, and NSC-32C were mixed, suspended in TiterMax (registered trademark) Gold ADJUVANT (Merck, T2684) or PBS(-), and a peritoneal dissemination cancer cell suspension was prepared. This suspension was administered to vero-simune mice several times for immunization. According to a conventional method, lymphocytes were collected from the lymph nodes of the immunized mice and hybridomas were prepared by cell fusion with mouse myeloma cells SP2 / 0 (ATCC, CRL-1581). Monoclonal colonies of hybridomas were isolated using an automatic picking device, and monoclonal hybridoma cells (hereinafter referred to as "clones") were obtained. The isolated clones were cultured in a 37 °C, 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 dissemination 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 "the antibody contained in the clone supernatant"). First, the binding between the antibody contained in the clone supernatant and the six types of peritoneal dissemination cancer cells obtained in Example 1-1 was measured by flow cytometry, and clones producing antibodies that strongly bind to peritoneal dissemination cancer cells were selected. BV421 Goat Anti-Mouse Ig (Becton, Dickinson and Company, 563846) was used for flow cytometry. In addition, in order to exclude clones that provide antibodies that bind to the cancer antigen EpCAM, the binding of the antibodies contained in the clone supernatants to human EpCAM-Myc-DDK-expressing CHO-K1 cells was measured. Human EpCAM-Myc-DDK-expressing CHO-K1 cells were prepared by transfecting CHO-K1 cells (ATCC, CCL-61) with EPCAM (Myc-DDK-tagged)-Human epithelial cell adhesion molecule (EPCAM) (ORIGENE, RC201989). The binding of the cells to the antibodies contained in the clone supernatants was measured by flow cytometry. BV421 Goat Anti-Mouse Ig was used for flow cytometry. Clones that provided supernatants that did not show binding activity to EpCAM-expressing cells were selected by excluding clones that bound to the cells. As a positive control, CD326 (EpCAM) Monoclonal Antibody (1B7) (eBioscience, 14-9326) was used. Furthermore, in order to select clones that selectively bind to peritoneal 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 the cultured human peritoneal mesothelial cells, and flow cytometry was used to measure the binding between the cultured human peritoneal mesothelial cells cultured in Mesothelial Cell Growth Medium (Zenbio, MSO-1) and the clones. BV421 Goat Anti-Mouse Ig was used for flow cytometry. Only clones that did not bind to the cultured human peritoneal mesothelial cells were selected. Through the above experiments, the 3D11 clone was obtained as a clone that provides an antibody that binds to 5 or more of the 6 types of peritoneal disseminated cancer cells and does not bind to human EpCAM and cultured peritoneal mesothelial cells.

[0113] (2) Antibody purification from hybridoma supernatant 3D11 clones were 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"). Antibody purification was performed according to standard procedures.

[0114] [1-4: Identification of antigen molecules recognized by 3D11] (1) Identification of candidate antigen 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 compared to 3D11 during the process of obtaining 3D11. Cell lysates of NSC-15CF were prepared. 3D11 and one of two control antibodies (1D10, 10B5) were added to the cell lysates, followed by the addition of Dynabeads Protein G (Life Technologies, 10003D), mixing, and washing. Proteins bound to Dynabeads Protein G were digested using Trypsin / LysC (Promega, V5072) to obtain a peptide mixture. The solution containing the peptide mixture was subjected to LC-MS / MS analysis using UltiMate3000RSLCnano (Thermo Fisher Scientific) and Orbitrap Fusion (Thermo Fisher Scientific). Comparative quantitative analysis and peptide / protein identification were performed on the obtained LC-MS / MS data using Progenesis QI for Proteomics (Waters) and Mascot (Matrix Science) software to identify the binding proteins. By comparing the binding proteins of 3D11 and the control antibody, CLDN4 was identified as a candidate antigen molecule for 3D11.

[0115] (2) Antigen determination for 3D11 To determine whether CLDN4, identified in (1), is the antigen of 3D11, binding experiments were performed on human CLDN4-Myc-DDK expressing CHO-K1 cells. Human CLDN4-Myc-DDK expressing CHO-K1 cells were created 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 it recognizes CLDN4 as an antigen.

[0116] [Example 2: Sequencing of 3D11 and production of human antibodies] The genes coding the heavy and light chains of 3D11 were cloned according to standard procedures, 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 velocimune mice are antibodies in which the variable regions of the endogenous immunoglobulin heavy and light chains are replaced with the corresponding human variable regions. In other words, antibodies obtained using velocimune technology are antibodies (also called "chimeric antibodies") that possess the variable region of a human antibody and the constant region of a mouse antibody.

[0117] [Example: Anti-CLDN4-anti-CD3 bispecific antibody used in the treatment of peritoneal cancer dissemination] Various types of drugs are known to treat TAAs, which are selectively expressed on 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 a groundbreaking method that can obtain cancer cell-selective cytotoxic activity at low antibody concentrations. Therefore, we investigated the production of bispecific T-cell-recruiting antibodies using 3D11.

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

[0119] (2) Fabrication of anti-CD3scFv The anti-CD3scFv moiety was constructed based on the sequences of the heavy chain and light chain variable regions of the mouse anti-CD3 antibody described in Japanese Patent No. 5686953. Humanization of anti-CD3scFv was performed according to the method described in the literature (Front Biosci., 2008, Vol. 13, pp. 1619-1633). Back mutations were introduced during this process. The three-dimensional structural information (PDB Code: 5FCS) was analyzed using the integrated computational chemistry system MOE provided by MOLSIS Inc. to determine the location of the back mutations within the framework region. The amino acid sequence of the humanized anti-CD3scFv is shown in SEQ ID NO: 41. The polynucleotide encoding the amino acid sequence described in SEQ ID NO: 41 was synthesized according to a conventional method and inserted into a pcDNA3.1(+) vector (Thermo Fisher Scientific, V79020).

[0120] (3) Preparation of anti-CLDN4(3D11)-anti-CD3scFv bispecific antibodies Using vectors encoding 3D11.1 and anti-CD3scFv, 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 prepared according to the method described in Example 7 of PCT / JP2021 / 41839. The prepared bispecific antibody is referred to as the anti-CLDN4(3D11)-anti-CD3scFv bispecific antibody.

[0121] (4) Evaluation of the efficacy of anti-CLDN4(3D11)-anti-CD3scFv 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 possess RTCC activity. Furthermore, the in vivo antitumor activity of the anti-CLDN4(3D11)-anti-CD3scFv bispecific antibody was evaluated according to the method described in Example 11 of PCT / JP2021 / 41839. The anti-CLDN4(3D11)-anti-CD3scFv bispecific antibody showed antitumor effects in an in vivo mouse model of gastric cancer peritoneal dissemination. These results suggest that the anti-CLDN4(3D11)-anti-CD3scFv bispecific antibody is effective in the treatment of cancer. Furthermore, to investigate the safety of the anti-CLDN4(3D11)-anti-CD3scFv bispecific antibody, a single dose was administered intravenously to cynomolgus monkeys. As a result, deaths and critical cases were observed in the monkeys administered the anti-CLDN4(3D11)-anti-CD3scFv bispecific antibody. In the deceased monkeys, blood IL-6 levels were significantly increased 1 and 6 hours after administration, and in the critically ill monkeys, blood IL-6 levels were significantly increased 1, 6, and 24 hours after administration. Based on these results, while the anti-CLDN4(3D11)-anti-CD3scFv bispecific antibody exhibits therapeutic effects in vitro and in vivo, concerns have arisen regarding serious side effects, which are thought to be due to an increase in cytokines. Therefore, it was determined that the anti-CLDN4(3D11)-anti-CD3scFv bispecific antibody is unsuitable as an antibody for use in the treatment of human cancer.

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

[0123] [3-1. Obtaining anti-CD137 antibodies] (1) Production of CD137-Fc fusion protein A polynucleotide encoding the extracellular region of human CD137 consisting of the amino acid sequence from amino acid number 1 to 186 of SEQ ID NO: 42 or the extracellular region of cynomolgus CD137 consisting of the amino acid sequence from amino acid number 1 to 186 of SEQ ID NO: 43 was inserted into the pFUSE-hIgG1-Fc1 vector (InvivoGen, pfuse-hg1fc1), respectively, to prepare expression vectors encoding fusion proteins of the extracellular region of CD137 and human Fc (hereinafter referred to as "human CD137-human Fc fusion protein" and "cynomolgus CD137-human Fc fusion protein", respectively). The prepared vectors were introduced 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 cultured. The proteins secreted into the culture supernatant were purified using MabSelect SuRe or HiTrap MabSelect SuRe (GE Healthcare, 11-0034-94) to obtain the human CD137-human Fc fusion protein and the cynomolgus CD137-human Fc fusion protein.

[0124] (2) Construction of a monkey CD137-His tag-3×FLAG tag fusion protein A vector was constructed by tandem-inserting polynucleotides encoding the extracellular region of monkey CD137 (amino acid sequence from amino acid 1 to 183 of SEQ ID NO: 43), the His tag of SEQ ID NO: 44, and the 3×FLAG tag of SEQ ID NO: 45 into a pcDNA3.4 TOPO vector. The constructed 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-3×FLAG tag fusion protein".

[0125] (3) Production and screening of anti-CD137 antibody-producing hybridoma clones Velosimune mice or AlivaMab mice (Ablexis, U.S. Patent No. 9346873) were immunized by administering 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 according to standard procedures, and hybridomas were created by cell fusion with mouse myeloma cells SP2 / 0. Single colonies of hybridomas were isolated using an automated picking device, and monoclonal hybridoma cells (hereinafter referred to as "clones") were obtained. Each clone was cultured for several days to obtain the culture supernatant. The binding of antibodies contained in the culture supernatant to CD137 was confirmed by the following method. Binding to CD137 was evaluated by ELISA. For evaluation, plates coated with either 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 (Southern Biotech, 1030-05) was used as the secondary antibody. Multiple clones that bind to both human CD137 and monkey CD137 were obtained using the above method.

[0126] (4) Obtain anti-CD137 antibody and confirm agonist 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 binding to CD137 and CD137 agonist activity. The binding of the anti-CD137 antibody to CD137 was confirmed using the ELISA method described in (3). The CD137 agonist activity of the anti-CD137 antibody was measured using the 4-1BB Bioassay Kit (Promega, JA2351) in the following manner: FcγRIIb CHO-K1 Cells (Promega, JA2251) were seeded in a 384-well plate (Greiner Bio-One, 781080) and cultured overnight at 37°C and 5% CO2. The culture supernatant was removed, and the purified antibodies of each clone and 4-1BB Effector Cells (Promega, JA2351) were added, and the cells were allowed to stand at 37°C and 5% CO2 for 6 hours. Bio-Glo Reagent (Promega, JA2351) was added, and chemiluminescence induced by 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 under conditions without FcγRIIb CHO-K1 Cells. Of the anti-CD137 antibodies evaluated, 3-34 derived from velocimune mice, and A2-32, A2-48, and A2-73 derived from AlivaMab mice, exhibited strong binding activity to human and monkey CD137, and showed CD137 agonist activity dependent on the presence of FcγRIIb CHO-K1 cells.

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

[0128] [3-2. Preparation of anti-CLDN4(3D11)-anti-CD137(3-34)scFv bispecific antibodies] Based on the sequences of the heavy and light chain variable regions of 3D11 and 3-34, a bispecific anti-CLDN4(3D11)-anti-CD137(3-34)scFv antibody (hereinafter also referred to as "3D11_3-34-j6_S") was designed. The constructed 3D11_3-34-j6_S has a GS linker linked to the heavy chain carboxyl terminus (also referred to as the "C-terminus") of a human IgG1κ-type anti-CLDN4(3D11) antibody, and the amino terminus of anti-CD137(3-34)scFv (SEQ ID NO: 26), which contains the heavy and light chain variable regions of the anti-CD137 antibody, is bound 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. Polynucleotides encoding the designed antibody were prepared and inserted into a pcDNA3.4 TOPO vector according to a standard procedure. The aforementioned expression vector was introduced into ExpiCHO-S cells, and 3D11_3-34-j6_S was purified from the culture supernatant. The prepared 3D11_3-34-j6_S was confirmed to have anti-CD137 binding activity and CD137 agonist activity in vitro, suggesting its potential efficacy as a cancer treatment drug.

[0129] [Example 4: Preparation of anti-CLDN4 (hKM3900)-anti-CD137scFv bispecific antibody] [4-1. Preparation of humanized anti-CLDN4 (hKM3900) antibody] The anti-CLDN4 antibody KM3900 has been reported to selectively bind to CLDN4 compared to other claudin family molecules such as CLDN6 (Patent Document 1). Therefore, based on this report, a humanized antibody of KM3900 was constructed. Specifically, based on the humanized amino acid sequence of the variable region of KM3900, a humanized antibody was designed from the sequences of the constant regions of human Igγ1 and human Igκ. The human Igγ1 constant region was modified by introducing amino acid mutations of L234A, L235A, and P331G, or L234A, L235A, and P331S. A humanized anti-CLDN4 (hKM3900) antibody was produced according to a standard method and used in subsequent studies. Antibodies with the L234A, L235A, and P331G amino acid mutations are referred to as "hKM3900," and antibodies with the L234A, L235A, and P331S amino acid mutations are referred to as "hKM3900_S." The amino acid sequence of the heavy chain variable region of hKM3900 is described in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region is described in SEQ ID NO: 8.

[0130] [4-2. Preparation of anti-CLDN4-anti-CD137scFv bispecific antibodies] Anti-CD137scFv was designed from the anti-CD137 antibodies (3-34, A2-32, A2-48, A2-73) obtained in Example 3, and bispecific antibodies of hKM3900 and anti-CD137scFv were produced (Table 2). The produced antibodies are collectively referred to as "anti-CLDN4(hKM3900)-anti-CD137scFv".

[0131] (1) Construction of a vector encoding an anti-CLDN4 (hKM3900)-anti-CD137scFv bispecific antibody. A bispecific anti-CLDN4 (hKM3900)-anti-CD137scFv antibody was designed based on the sequence of hKM3900 and the sequences of the heavy chain variable region and light chain variable region of the anti-CD137 antibody. The anti-CLDN4 (hKM3900)-anti-CD137scFv bispecific antibody has a GS linker linked to the heavy or light chain C-terminus of the IgG1-type anti-CLDN4 antibody (hKM3900), and the amino-terminus of anti-CD137scFv, including the heavy chain variable region and light chain variable region of the anti-CD137 antibody, is bound 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 on the designed antibodies is shown in Table 2. Polynucleotides encoding the designed antibodies were prepared and inserted into a pcDNA3.4 TOPO vector according to a standard procedure. The vectors created 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-CD137scFv bispecific antibody Using an anti-CLDN4(hKM3900)-anti-CD137scFv bispecific antibody expression vector, eleven types of anti-CLDN4(hKM3900)-anti-CD137scFv bispecific antibodies listed in Table 2 were produced. Specifically, the anti-CLDN4(hKM3900)-anti-CD137scFv bispecific antibody expression vector was 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 obtained culture supernatant by affinity purification using MabSelect SuRe. Furthermore, some of the anti-CLDN4(hKM3900)-anti-CD137scFv bispecific antibodies underwent additional purification 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). Hereafter, the anti-CLDN4(3D11)-anti-CD137(3-34)scFv bispecific antibodies and anti-CLDN4(hKM3900)-anti-CD137scFv bispecific antibodies prepared in Examples 3 and 4 may be collectively referred to as "anti-CLDN4-anti-CD137scFv bispecific antibodies."

[0134] [Example 5: Evaluation of binding activity and agonist activity of anti-CLDN4 (hKM3900)-anti-CD137scFv bispecific antibody] The binding activity of the anti-CLDN4(hKM3900)-anti-CD137scFv bispecific antibody to CLDN4 and CD137, as well as its CD137 agonist activity, were evaluated.

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

[0136] [Table 3]

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

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

[0139] [Table 4]

[0140] [5-4. Evaluation of CD137 agonist activity of anti-CLDN4-anti-CD137scFv bispecific antibody in the presence of CLDN4-expressing cancer cells] The specific CD137 agonist activity of the anti-CLDN4-anti-CD137scFv bispecific antibody, mediated through 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 placed in a 384-well plate (Greiner Bio-One, 781080) in a 1 × 10⁶ well. 4 The antibody was added to a concentration of IgG_L-3-34-j6 per well and incubated overnight at 37°C in 5% CO2. The medium was removed the following day. 12.5 μL / well of the test antibody, diluted in RPMI-1640 medium containing 1% FBS, was added to each well. The final antibody concentrations are shown in Figures 2-1 to 2-3. The test antibody used was 3D11_3-34-j6_S, an anti-CLDN4 (hKM3900)-anti-CD137scFv bispecific antibody as described in Table 2. hKM3900, ctrl IgG_L-3-34-j6 antibody, and urelumab (BMS, SEQ ID NOs. 3 and 6 as described in International Publication No. 2005 / 035584) were used as control antibodies. 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. 12.5 μL of this suspension was added to each well, and the plates were incubated at 37°C and 5% CO2 for 6 hours. 25 μL / well of Bio-Glo Reagent was added to the plates, 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-CD137scFv bispecific antibody showed 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, the same experiment as above was performed under NCI-H322-free conditions. As a result, CD137 agonist activity was not observed in the absence of CLDN4-expressing cancer cells (Figures 2-4 to 2-6). This result indicates that the anti-CLDN4-anti-CD137scFv bispecific antibody exerts CD137 agonist activity only in the coexistence of CLDN4-expressing cancer cells. On the other hand, while urelumab showed CD137 agonist activity in the presence of CLDN4-expressing cancer cells, its effect was weaker than that of the anti-CLDN4-anti-CD137 bispecific antibody. Furthermore, 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 agonist activity in tumor tissues that highly express CLDN4.

[0141] [Example 6: In vitro efficacy evaluation of anti-CLDN4-anti-CD137scFv bispecific antibody] We evaluated the interferon-γ production-promoting function and cancer cell-toxic activity of the anti-CLDN4-anti-CD137scFv bispecific antibody.

[0142] [6-1. Production and cryopreservation of expanded PanT cells] (1) Preparation of Expanded PanT Cells In Example 6, the mixture of RPMI-1640 (Sigma, R8758) 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), or HEPES (Thermo Fisher Scientific, 15630-080) at a final concentration of 1%. Anti-CD3 antibody (BioLegend, 317325), diluted to a final concentration of 1-3 μg / mL, was added to the dish, and the anti-CD3 antibody was immobilized. Using the PanT Cell Isolation Kit, human (Miltenyi Biotec, 130-096-535), PanT cells (including both CD4 T cells and CD8 T cells, hereinafter referred to as "PanT cells") were isolated from human peripheral blood mononuclear cells (LONZA, CC-2702) according to the manufacturer's recommended protocol. The isolated PanT cells were centrifuged, the supernatant was removed, and the cells were suspended in culture medium. The entire amount of PanT cells suspended in culture medium was seeded onto a dish immobilized with the aforementioned anti-CD3 antibody. Human IL-2 at a final concentration of 100-200 U / mL (PeproTech, 200-2) and anti-CD28 antibody at a final concentration of 1-4 μg / mL (BioLegend, 302923) 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 in dishes. Human IL-2 at a final concentration of 100-200 U / mL was then added, and the cells were cultured at 37°C in a 5% CO2 incubator. After 4 days, the entire volume of cells was harvested, centrifuged, and the supernatant was removed. The cells were then resuspended in a cell banker (Takara, CB011) or a ban banker (GS Lymphotek, CS-02-001), transferred to tubes, and frozen and stored at -80°C.The cells cryopreserved in this manner are referred to as "Expanded PanT cells" in this specification.

[0143] [6-2.60As6-Luc / GFP expression cell generation] The preparation of lentiviral solutions containing Luc / GFP was carried out using L293T cells (Thermo Fisher Scientific, K4975-00) according to standard procedures. For the introduction of the pCDH-CMV-GL3-EF1a-GFP-T2A-puro modified vector (donated by Associate Professor Ryo Takahashi, Department of Cell and Molecular Biology, Graduate School of Biomedical Sciences, Hiroshima University (PLoS One, 2015, Vol.10, e0123407)) into L293T cells, 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) were used. GFP-expressing lentiviruses grown in L293T cells were collected using a 45 μm Millex®-HV filter (Merck Millipore, SLHV033RS). The virus solution was dispensed into 1 mL cryovials (Nalgene, 5000-0020) and stored frozen at -80°C. According to the conventional method, 60As6-Luc / GFP-expressing lentivirus was infected into 60As6 cells (obtained from Dr. Gokichi Yanagihara of the Biomaterials Research Institute), which are CLDN4-expressing cancer cell lines, to produce 60As6-Luc / GFP-expressing cells. Polybrene (Santa Cruz, sc-134220) was used to increase the infection efficiency. As the medium, RPMI-1640 containing 10% FBS and 2 μg / mL Puromaycin (Thermo Fisher Scientific, A-11138-02) was used. In this specification, the established cells are referred to as "60As6-Luc / GFP cells". It was confirmed by flow cytometry that 60As6-Luc / GFP cells highly express CLDN4 endogenously. The constructed cells were used in subsequent experiments.

[0144] [6-3. Evaluation of the Function of Promoting Interferon-γ Production in the Co-culture System of Cancer Cells and T Cells with Anti-CLDN4 (3D11)-Anti-CD137scFv Bispecific Antibody] The function of promoting interferon-γ production of anti-CLDN4 (3D11)-anti-CD137scFv bispecific antibody in the co-culture system of 60As6-Luc / GFP cells and Expanded PanT cells was evaluated. 0.1 μg / 100 μL / well of anti-CD3 antibody was added to a flat-bottom 96-well plate (IWAKI, 3860-096) to immobilize the anti-CD3 antibody on the plate. 60As6-Luc / GFP cells were seeded on the plate at a density of 5×10 4 cells / 50 μL / well and cultured in a 37°C, 5% CO2 incubator. The next day, 2×10 5Expanded PanT cells were seeded at a concentration of 30 μL / well. The test antibody and isotype control were serially diluted in culture medium at a 5-fold common ratio starting from the highest concentration of 50,000 ng / mL, and 20 μL of each was added. Urelumab and anti-CLDN4(3D11)-anti-CD137scFv bispecific antibody (3D11_3-34-j6_S) were used as the test antibody, and IgG(lysozyme)-scFv(CD137) (in-house prepared) was used as the isotype control. After 6 days, the amount of interferon-γ produced in the supernatant was measured using the AlphaLISA interferon-γ measurement kit (Perkin Elmer, AL217C) according to the manufacturer's recommended protocol. Figure 3-1 shows the reaction curve between interferon-γ production and antibody concentration. The anti-CLDN4(3D11)-anti-CD137scFv bispecific antibody showed interferon-γ production promoting activity in a co-culture system of CLDN4-expressing cancer cell lines, specifically 60As6-Luc / GFP cells, and Expanded PanT cells. The interferon-γ production promoting activity of the anti-CLDN4(3D11)-anti-CD137scFv bispecific antibody was significantly stronger than that of urelumab.

[0145] [6-4. Evaluation of the interferon-γ production-promoting function of anti-CLDN4(hKM3900)-anti-CD137scFv 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-CD137scFv bispecific antibody in a co-culture system of 60As6-Luc / GFP cells and Expanded PanT cells was evaluated using the same method as in 6-3. The anti-CD3 antibody was immobilized in a 96-well plate at a concentration of 0.03 μg / 100 μL / well. 2 × 10⁶ 60As6-Luc / GFP cells were cultured. 4 Expanded PanT cells were seeded at 50 μL / well. 4Cells were seeded at 30 μL / well. Urelumab or anti-CLDN4 (hKM3900)-anti-CD137scFv bispecific antibodies (hKM3900_3-34-j6 or hKM3900_tA2-32LH), serially diluted from a maximum concentration of 5000 ng / mL at a 2-fold common ratio, were used as the test antibody. 20 μL each of the 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 reaction curve between interferon-γ production and antibody concentration. The anti-CLDN4 (hKM3900)-anti-CD137scFv bispecific antibody showed interferon-γ production promoting activity in a co-culture system of 60As6-Luc / GFP cells and Expanded PanT cells. The interferon-γ production promoting activity 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-CD137scFv bispecific antibody in T cell monoculture systems] The interferon-γ production-promoting function of an anti-CLDN4(3D11)-anti-CD137scFv bispecific antibody in a monoculture system of expanded PanT cells was evaluated. Anti-CD3 antibody was immobilized on a flat-bottomed 96-well plate by adding 0.1 μg / 100 μL / well of anti-CD3 antibody. 2 × 10⁶ cells were added to the immobilized plate. 5Expanded PanT cells were seeded at a rate of 80 μL / well. Urelumab and anti-CLDN4(3D11)-anti-CD3scFv bispecific antibody (3D11_3-34-j6_S) were used as the test antibodies, and IgG(lysozyme)-scFv(CD137) (in-house prepared) was used as the isotype control. The test antibody and isotype control were serially diluted in culture medium at a 5-fold common ratio starting from the maximum concentration of 50,000 ng / mL, and 20 μL of each was added. After 6 days, the amount of interferon-γ produced in the supernatant was measured using the AlphaLISA interferon-γ measurement kit in the same manner as in 6-3. Figure 3-3 shows the reaction curve between interferon-γ production and antibody concentration. The anti-CLDN4(3D11)-anti-CD137scFv bispecific antibody did not promote interferon-γ production in a T cell monoculture system, but urelumab did. This result means 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 absent.

[0147] [6-6. Evaluation of the interferon-γ production-promoting function of anti-CLDN4(hKM3900)-anti-CD137scFv bispecific antibody in T cell monoculture systems] The interferon-γ production-promoting function of the anti-CLDN4(hKM3900)-anti-CD137scFv bispecific antibody in a monoculture system of expanded PanT cells was evaluated using the same method as in 6-5. The anti-CD3 antibody was immobilized in a 96-well plate at a concentration of 0.03 μg / 100 μL / well. Expanded PanT cells were cultured in 8 × 10⁶ cells. 4Cells were seeded at 80 μL / well. Urelumab and anti-CLDN4 (hKM3900)-anti-CD137scFv bispecific antibodies (hKM3900_3-34-j6, hKM3900_tA2-32LH) were used as test antibodies. The test antibodies and isotype control (lysozyme antibody) were serially diluted at a 2-fold common ratio starting from a maximum concentration of 5000 ng / mL, and 20 μL of each was added. Figures 3-4 show the reaction curves between interferon-γ production and antibody concentration. The anti-CLDN4 (hKM3900)-anti-CD137scFv bispecific antibody did not show an interferon-γ production promoting function in a T cell monoculture system, but urelumab did show an interferon-γ production promoting function. The results from 6-5 and 6-6 indicate that in an environment without CLDN4-expressing cancer cell lines, the anti-CLDN4-anti-CD137scFv bispecific antibody does not show T cell activity enhancing effects, suggesting that the systemic immunostimulatory effects reported as a side effect of anti-CD137 antibodies may be reduced.

[0148] [6-7. Evaluation of cancer cytotoxic activity of anti-CLDN4(3D11)-anti-CD137scFv bispecific antibodies in a co-culture system of cancer cells and T cells] The cancer cytotoxic activity (inhibitory effect on cancer cell proliferation) of the anti-CLDN4(3D11)-anti-CD137scFv bispecific antibody was evaluated in a co-culture system of 60As6-Luc / GFP cells and expanded PanT cells. 60As6-Luc / GFP cells were cultured in 1 × 10⁶ well flat-bottom 96-well plates. 4 Expanded panT cells were seeded at 50 μL / well and cultured in a 37°C, 5% CO2 incubator. After 2 hours, 1 × 10⁶ cells of expanded panT cells were placed in the 96-well plate. 5Cells were seeded in 50 μL / well units. Urelumab and anti-CLDN4(3D11)-anti-CD137scFv bispecific antibodies were used as test antibodies. These antibodies were serially diluted in culture medium at a 10-fold common ratio starting from the highest concentration of 40,000 ng / mL, and 50 μL was added to each well. Furthermore, 50 μL of 40 ng / mL anti-CD3 antibody was added to each well, and the fluorescence (GFP) area of ​​each well was measured using an IncuCyte® ZOOM (Sartorius Corporation) in a 37°C, 5% CO2 incubator. The increase in fluorescence area from 0 hours after the start of measurement was used as an indicator of cell proliferation. Figure 4 shows the cancer cell cytotoxic effect (cancer cell proliferation rate) of the test antibodies. The vertical axis of Figure 4 shows the relative value of the increase in fluorescence area from 0 hours to 168 hours, with the well without the test antibody set to 100%. As shown in Figure 4, the anti-CLDN4(3D11)-anti-CD137scFv bispecific antibody exhibited cytotoxic activity in a co-culture 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 cancer cytotoxic activity of anti-CLDN4(hKM3900)-anti-CD137scFv bispecific antibody in a co-culture system of cancer cells and T cells] The cancer cell-killing activity of the anti-CLDN4 (hKM3900)-anti-CD137scFv 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-bottomed 96-well plate (Perkin Elmer, 6005680) and immobilized on the plate. After washing the plate twice with PBS(-), 2 × 10⁶ samples were taken. 4 60As6-Luc / GFP cells were seeded at 50 μL / well and cultured at 37°C in a 5% CO2 incubator. The following day, 4.0 × 10⁶ cells were placed in a 96-well plate. 4Expanded PanT cells were seeded at a concentration of 30 μL / well. Additionally, 20 μL each of the test antibody and isotype control were added, with concentrations ranging from 5000 ng / mL to 0.844 ng / mL. The test antibodies used were urelumab and anti-CLDN4 (hKM3900)-anti-CD137scFv bispecific antibodies (hKM3900_3-34-j6, hKM3900_tA2-32LH). Measurements were performed after 6 days. For some wells, 11 μL of 1% Triton-X100 (Sigma-Aldrich, T-9284) was added 30 minutes prior to measurement to kill the cells. Luciferase activity of 60As6-Luc / GFP cells in each well was measured using the ONE-Glo Luciferase Measurement Kit (Promega, E6120) according to the manufacturer's recommended protocol, using ARVO X-3 (Perkin Elmer). Cancer killing rates are expressed as relative values, with the average luciferase chemiluminescence of the antibody-free wells set to 0% and the average value of the wells with Triton-X100 added 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 co-culture system of 60As6-Luc / GFP cells and Expanded PanT cells, the addition of an anti-CLDN4 (hKM3900)-anti-CD137scFv bispecific antibody significantly increased the 60As6-Luc / GFP cell killing rate. On the other hand, no increase in cell toxicity was observed in the 60As6-Luc / GFP cell line with urelumab, an anti-CD137 specific antibody.

[0150] [Example 7: In vivo evaluation of anti-CLDN4-anti-CD137scFv bispecific antibody] [7-1. In vivo antitumor effects of anti-CLDN4-anti-CD137scFv bispecific antibodies] NCI-H322 cells were suspended in PBS(-) (WAKO Corporation, 045-29795) and 5.0 × 10⁶ cells were extracted. 6 A cell suspension at a concentration of 100 μL per cell was subcutaneously implanted into 7-week-old female mice (NOG, InVivoScience). Six days after implantation, the cell count was 3.4 × 10⁶. 6Human peripheral blood mononuclear cells (Lonza, CC-2702) were injected via tail vein at a rate of 200 μL per cell. Fifteen days after 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 distributed so that the tumor volume between groups was approximately equal (day 0). hKM3900_tA2-32LH was used as the test antibody. The test antibody and isotype control (anti-lysozyme antibody) were mixed in 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 (mm²] 3 )] = [Longest diameter of tumor (mm)] x [Shortest diameter of tumor (mm)] 2 × 0.5 As shown in Figure 6, a significant reduction in tumor volume was observed in the groups administered hKM3900_tA2-32LH at 0.1, 1, or 10 mg / kg. The vertical axis represents tumor volume, and the horizontal axis represents the number of days after the first dose. The solid and dashed lines show the change in the mean tumor volume for each group, and the error bars show the standard error of the mean. The significance probability P-value (*: P<0.05, **: P<0.01) was calculated by comparing the tumor volume on day 12 of the isotype control group and the hKM3900_tA2-32LH group using Dunnett's multiple comparison test. The number of cases on day 12 in the hKM3900_tA2-32LH 0.1 mg / kg group was 9.

[0151] [7-2. Safety study of anti-CLDN4(hKM3900)-anti-CD137scFv bispecific antibody in cynomolgus monkeys] To investigate the safety of anti-CLDN4(hKM3900)-anti-CD137scFv bispecific antibodies (hKM3900_3-34-j6 and hKM3900_tA2-32LH), a single dose of anti-CLDN4(hKM3900)-anti-CD137scFv bispecific antibodies was administered intravenously to cynomolgus monkeys. As a result, no particular abnormalities were observed in the serum cytokine concentrations of IL-6, TNF-α, etc., in the monkeys administered with anti-CLDN4(hKM3900)-anti-CD137scFv bispecific antibodies. Therefore, from a safety standpoint, anti-CLDN4(hKM3900)-anti-CD137scFv bispecific antibodies were judged to be suitable for use in the treatment of cancer.

[0152] [Example 8: Treatment of cancer with anti-CLDN4-anti-CD137 bispecific antibodies] [8-1. Confirmation of CLDN4 expression levels] To investigate the versatility of CLDN4 as a cancer treatment target, we examined CLDN4 RNA expression levels in cancer tissues using The Cancer Genome Atlas (TCGA). The results confirmed that CLDN4 expression was upregulated in cancer tissues such as colorectal cancer, bladder cancer, and lung cancer. This suggests that CLDN4 may be a therapeutic target not only for peritoneal dissemination cancer but also for other cancer types. Furthermore, we investigated CLDN4 RNA expression levels in normal tissues using The Genotype-Tissue Expression (GTEx). The results showed that CLDN4 was expressed in several normal tissues. However, CLDN4 expression levels in normal tissues were lower than in cancer tissues. Therefore, it is suggested that cancer drugs targeting CLDN4 may have a wide safety margin and a low risk of 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. Furthermore, the polynucleotide, expression vector, transformed host cells, and method for producing the antibody of the present invention are useful for producing the anti-CLDN4-anti-CD137 bispecific antibody. [Sequence Listing Free Text]

[0154] The numerical headings in the following sequence list <223> This section provides an explanation of "Artificial Sequence". Specifically, Sequence ID No. 1 in the sequence listing is the nucleotide sequence of the heavy chain variable region of 3D11, and Sequence ID No. 2 is the amino acid sequence of the heavy chain variable region of 3D11. Sequence ID No. 3 is the nucleotide sequence of the light chain variable region of 3D11, and Sequence ID No. 4 is the amino acid sequence of the light chain variable region of 3D11. Sequence ID No. 5 is the nucleotide sequence of the heavy chain variable region of hKM3900, and Sequence ID No. 6 is the amino acid sequence of the heavy chain variable region of hKM3900. Sequence ID No. 7 is the nucleotide sequence of the light chain variable region of hKM3900, and Sequence ID No. 8 is the amino acid sequence of the light chain variable region of hKM3900. Sequence ID No. 9 is the nucleotide sequence of the heavy chain variable region of 3-34, and Sequence ID No. 10 is the amino acid sequence of the heavy chain variable region of 3-34. Sequence ID No. 11 is the nucleotide sequence of the light chain variable region of 3-34, and Sequence ID No. 12 is the amino acid sequence of the light chain variable region of 3-34. Sequence ID 13 is the nucleotide sequence of the heavy chain variable region of A2-32, and Sequence ID 14 is the amino acid sequence of the heavy chain variable region of A2-32. Sequence ID 15 is the nucleotide sequence of the light chain variable region of A2-32, and Sequence ID 16 is the amino acid sequence of the light chain variable region of A2-32. Sequence ID 17 is the nucleotide sequence of the heavy chain variable region of A2-48, and Sequence ID 18 is the amino acid sequence of the heavy chain variable region of A2-48. Sequence ID 19 is the nucleotide sequence of the light chain variable region of A2-48, and Sequence ID 20 is the amino acid sequence of the light chain variable region of A2-48. Sequence ID 21 is the nucleotide sequence of the heavy chain variable region of A2-73, and Sequence ID 22 is the amino acid sequence of the heavy chain variable region of A2-73. Sequence ID 23 is the nucleotide sequence of the light chain variable region of A2-73, and Sequence ID 24 is the amino acid sequence of the light chain variable region of A2-73. Sequence ID 25 is the nucleotide sequence of 3-34-j6 scFv, and Sequence ID 26 is the amino acid sequence of 3-34-j6 scFv. Sequence ID 27 is the nucleotide sequence of tA2-32 scFv, and Sequence ID 28 is the amino acid sequence of tA2-32 scFv. Sequence ID 29 is the nucleotide sequence of tA2-32LH scFv, and Sequence ID 30 is the amino acid sequence of tA2-32LH scFv. Sequence ID 31 is the nucleotide sequence of tA2-48 scFv, and Sequence ID 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 the 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. Sequence ID 44 is the amino acid sequence of the His-tagged protein. Sequence ID 45 is the amino acid sequence of the 3×FLAG tag. Sequence IDs 46-54 are the amino acid sequences of the GS linker. Sequence ID 55 is the amino acid sequence of the GKPGS linker.

Claims

1. An anti-CD137 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region of any one of the following anti-CD137 antibodies (a) to (d): (a) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence of amino acid numbers 31 to 35 of SEQ ID NO: 10, a CDR2 consisting of the amino acid sequence of amino acid numbers 50 to 66 of SEQ ID NO: 10, and a CDR3 consisting of the amino acid sequence of 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 of amino acid numbers 24 to 34 of SEQ ID NO: 12, a CDR2 consisting of the amino acid sequence of amino acid numbers 50 to 56 of SEQ ID NO: 12, and a CDR3 consisting of the amino acid sequence of 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 of amino acid numbers 31 to 35 of SEQ ID NO: 14, a CDR2 consisting of the amino acid sequence of amino acid numbers 50 to 65 of SEQ ID NO: 14, and a CDR3 consisting of the amino acid sequence of 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 of amino acid numbers 23 to 35 of SEQ ID NO: 16, a CDR2 consisting of the amino acid sequence of amino acid numbers 51 to 57 of SEQ ID NO: 16, and a CDR3 consisting of the amino acid sequence of 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 of amino acid numbers 31 to 35 of SEQ ID NO: 18, a CDR2 consisting of the amino acid sequence of amino acid numbers 50 to 65 of SEQ ID NO: 18, and a CDR3 consisting of the amino acid sequence of 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 of amino acid numbers 23 to 35 of SEQ ID NO: 20, a CDR2 consisting of the amino acid sequence of amino acid numbers 51 to 57 of SEQ ID NO: 20, and a CDR3 consisting of the amino acid sequence of 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 of amino acid numbers 31 to 35 of SEQ ID NO:22, a CDR2 consisting of the amino acid sequence of amino acid numbers 50 to 65 of SEQ ID NO:22, and a CDR3 consisting of the amino acid sequence of 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 of amino acid numbers 23 to 35 of SEQ ID NO:24, a CDR2 consisting of the amino acid sequence of amino acid numbers 51 to 57 of SEQ ID NO:24, and a CDR3 consisting of the amino acid sequence of 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 number 1 to amino acid number 118 of SEQ ID NO: 10 and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to amino acid number 109 of SEQ ID NO: 12; (b) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to amino acid number 118 of SEQ ID NO: 14 and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to amino acid number 111 of SEQ ID NO: 16; (c) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 118 of SEQ ID NO: 18 and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 111 of SEQ ID NO: 20; (d) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 121 of SEQ ID NO: 22 and a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 111 of SEQ ID NO: 24; (e) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to amino acid number 118 of SEQ ID NO: 26 and a light chain variable region consisting of the amino acid sequence from amino acid number 134 to amino acid number 242 of SEQ ID NO: 26; (f) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to amino acid number 118 of SEQ ID NO: 28 and a light chain variable region consisting of the amino acid sequence from amino acid number 134 to amino acid number 244 of SEQ ID NO: 28; (g) a light chain variable region consisting of the amino acid sequence from amino acid number 1 to amino acid number 111 of SEQ ID NO: 30, and a heavy chain variable region consisting of the amino acid sequence from amino acid number 132 to amino acid number 249 of SEQ ID NO: 30; (h) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 118 of SEQ ID NO: 32 and a light chain variable region consisting of the amino acid sequence from amino acid number 134 to 244 of SEQ ID NO: 32; or (i) a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to amino acid number 121 of SEQ ID NO: 34, and a light chain variable region consisting of the amino acid sequence from amino acid number 137 to amino acid number 247 of SEQ ID NO:

34.

3. An anti-CD137 antibody as described in claim 1, which is an IgG antibody consisting of a heavy chain including a heavy chain variable region of the anti-CD137 antibody and a light chain including a light chain variable region.

4. An antigen-binding fragment described in 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 having any one of the following sequences (a) to (e): (a) an anti-CD137 scFv consisting of the amino acid sequence from amino acid number 1 to amino acid number 242 of SEQ ID NO: 26; (b) an anti-CD137 scFv consisting of the amino acid sequence from amino acid number 1 to amino acid number 244 of SEQ ID NO: 28; (c) an anti-CD137 scFv consisting of the amino acid sequence from amino acid number 1 to amino acid number 249 of SEQ ID NO: 30; (d) an anti-CD137 scFv consisting of the amino acid sequence from amino acid number 1 to amino acid number 244 of SEQ ID NO: 32; or (e) An anti-CD137 scFv consisting of the amino acid sequence from amino acid number 1 to amino acid number 247 of SEQ ID NO:

34.

6. An anti-CD137 antibody or antigen-binding fragment thereof described in 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 its antigen-binding fragment described in 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 described in claim 5.

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

10. A polynucleotide selected from the group consisting of the following (a) to (w), for use in producing an anti-TAA-anti-CD137 bispecific antibody: (a) a polynucleotide comprising a base sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid number 1 to amino acid number 118 of SEQ ID NO: 10; (b) a polynucleotide comprising a base sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid number 1 to amino acid number 109 of SEQ ID NO: 12; (c) a polynucleotide comprising a base sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid number 1 to amino acid number 118 of SEQ ID NO: 14; (d) a polynucleotide comprising a base sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid number 1 to amino acid number 111 of SEQ ID NO: 16; (e) a polynucleotide comprising a base sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid number 1 to amino acid number 118 of SEQ ID NO: 18; (f) a polynucleotide comprising a base sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid number 1 to amino acid number 111 of SEQ ID NO: 20; (g) a polynucleotide comprising a base sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid number 1 to amino acid number 121 of SEQ ID NO: 22; (h) a polynucleotide comprising a base sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid number 1 to amino acid number 111 of SEQ ID NO: 24; (i) a polynucleotide comprising a base sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid number 1 to amino acid number 118 of SEQ ID NO: 26; (j) a polynucleotide comprising a base 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 base sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid number 1 to amino acid number 118 of SEQ ID NO: 28; (l) a polynucleotide comprising a base 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 base sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid number 1 to amino acid number 111 of SEQ ID NO: 30; (n) a polynucleotide comprising a base 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 base sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid number 1 to amino acid number 118 of SEQ ID NO: 32; (p) a polynucleotide comprising a base 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 base sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid number 1 to amino acid number 121 of SEQ ID NO: 34; (r) a polynucleotide comprising a base 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; (s) a polynucleotide comprising a base sequence encoding an anti-CD137 scFv consisting of the amino acid sequence from amino acid number 1 to amino acid number 242 of SEQ ID NO: 26; (T) a polynucleotide comprising a base sequence encoding an anti-CD137 scFv consisting of the amino acid sequence from amino acid number 1 to amino acid number 244 of SEQ ID NO: 28; (u) a polynucleotide comprising a base sequence encoding an anti-CD137 scFv consisting of the amino acid sequence from amino acid number 1 to amino acid number 249 of SEQ ID NO: 30; (v) a polynucleotide comprising a base sequence encoding an anti-CD137 scFv consisting of the amino acid sequence from amino acid number 1 to amino acid number 244 of SEQ ID NO: 32; or (w) a polynucleotide comprising a base sequence encoding an anti-CD137 scFv consisting of the amino acid sequence from amino acid number 1 to amino acid number 247 of SEQ ID NO:

34.

11. An expression vector comprising the polynucleotide described in claim 10.

12. A host cell transformed with the expression vector described in claim 11.

13. A host cell comprising a polynucleotide selected from the group consisting of (a) to (w) below: (a) a polynucleotide comprising a base sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid number 1 to amino acid number 118 of SEQ ID NO: 10; (b) a polynucleotide comprising a base sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid number 1 to amino acid number 109 of SEQ ID NO: 12; (c) a polynucleotide comprising a base sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid number 1 to amino acid number 118 of SEQ ID NO: 14; (d) a polynucleotide comprising a base sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid number 1 to amino acid number 111 of SEQ ID NO: 16; (e) a polynucleotide comprising a base sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid number 1 to amino acid number 118 of SEQ ID NO: 18; (f) a polynucleotide comprising a base sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid number 1 to amino acid number 111 of SEQ ID NO: 20; (g) a polynucleotide comprising a base sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid number 1 to amino acid number 121 of SEQ ID NO: 22; (h) 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 1 to 111 of SEQ ID NO:

24. (i) a polynucleotide comprising a base sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid number 1 to amino acid number 118 of SEQ ID NO: 26; (j) a polynucleotide comprising a base 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 base sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid number 1 to amino acid number 118 of SEQ ID NO: 28; (l) a polynucleotide comprising a base 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 base sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid number 1 to amino acid number 111 of SEQ ID NO: 30; (n) a polynucleotide comprising a base 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 base sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid number 1 to amino acid number 118 of SEQ ID NO: 32; (p) a polynucleotide comprising a base 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 base sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence from amino acid number 1 to amino acid number 121 of SEQ ID NO: 34; (r) a polynucleotide comprising a base 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; (s) a polynucleotide comprising a base sequence encoding an anti-CD137 scFv consisting of the amino acid sequence from amino acid number 1 to amino acid number 242 of SEQ ID NO: 26; (T) a polynucleotide comprising a base sequence encoding an anti-CD137 scFv consisting of the amino acid sequence from amino acid number 1 to amino acid number 244 of SEQ ID NO: 28; (u) a polynucleotide comprising a base sequence encoding an anti-CD137 scFv consisting of the amino acid sequence from amino acid number 1 to amino acid number 249 of SEQ ID NO: 30; (v) a polynucleotide comprising a base sequence encoding an anti-CD137 scFv consisting of the amino acid sequence from amino acid number 1 to amino acid number 244 of SEQ ID NO: 32; or (w) a polynucleotide comprising a base sequence encoding an anti-CD137 scFv consisting of the amino acid sequence from amino acid number 1 to amino acid number 247 of SEQ ID NO:

34.

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

15. A pharmaceutical composition comprising an anti-CD137 antibody or antigen-binding fragment thereof described in any one of claims 1 to 5 and a pharma- ceutically acceptable excipient.

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

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

18. A pharmaceutical composition comprising the anti-TAA-anti-CD137 bispecific antibody of claim 7 and a pharma- ceutically acceptable excipient.

19. The pharmaceutical composition of claim 18 for use in the treatment of cancer.

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