Bispecific Anti-CLDN4 / Anti-CD137 antibody

Abstract

FIELD: biotechnology; medicine.SUBSTANCE: bispecific anti-CLDN4 / anti-CD137 antibody suitable for treating cancer. A bispecific anti-CLDN4 / anti-CD137 antibody, obtained using an anti-CLDN4 antibody and an anti-CD137 antibody, has agonistic activity against CD137, stimulates the production of interferon-γ T cells and exhibits cytotoxic activity against tumor cells expressing CLDN4 on their cell surface.EFFECT: bispecific anti-CLDN4 / anti-CD137 antibody of the present invention is proposed as a new agent for the treatment of human cancer.30 cl, 6 dwg, 4 tbl, 8 ex

Description

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

[0002] Claudin-4 (CLDN4) is a tetraspanin membrane protein belonging to the claudin family. It is expressed in epithelial cells and endothelial cells and plays an important role as a core molecule located in tight junctions. CLDN4 is highly expressed in tumor tissues of colorectal cancer, bladder cancer, ovarian cancer, etc., and it has been suggested that anti-CLDN4 antibodies may be applicable for the treatment or diagnosis of cancer (Patent Document 1 and Non-Patent Document 1). Furthermore, it has been reported that the use of both anti-CLDN4 antibodies and anti-epidermal growth factor receptor (EGFR) antibodies has an antitumor effect in an animal model (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 is reported to be expressed on the cell surface of immune cells such as T cells, B cells, natural killer (NK) cells, dendritic cells, eosinophils, and mast cells. Specifically, CD137 on T cells binds to the CD137 ligand on antigen-presenting cells and is known to be involved in T cell activation and survival as a costimulatory molecule (Non-patent Document 3). An agonistic anti-CD137 antibody exerts an antitumor effect by activating immune cells in the tumor microenvironment in an animal model (Non-patent Document 4). Urelumab, an agonistic anti-CD137 antibody, has shown therapeutic effect in clinical trials but is reported to cause liver toxicity as an adverse reaction (Non-patent Document 5).

[0004] As an innovative method for achieving selective cytotoxic activity against tumor cells with a low antibody concentration, a bispecific T cell recruiting antibody with various antibody formats has been reported. The bispecific T cell recruiting antibody is a bispecific antibody comprising an antibody against a tumor-associated antigen (TAA) expressed on the surface of tumor cells and an antibody that binds to a T cell, and studies on the effect of these antibodies in T cell immunotherapy are beginning (Non-Patent Document 6). Anti-CD3 antibody is often used as the T cell binding antibody, and bispecific T cell recruiting antibodies to various TAAs are currently being studied and developed.

[0005] In addition, bispecific T cell recruiting antibodies against CD137 and TAA have been seriously studied in recent years. Research is underway on bispecific anti-GPC3 / anti-CD137 antibodies, bispecific anti-HER2 / anti-CD137 antibodies, bispecific anti-PDL1 / anti-CD137 antibodies, bispecific anti-FAP / anti-CD137 antibodies, and the like that recognize TAA 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, and Non-Patent Documents 7 to 9). However, bispecific anti-CLDN4 / anti-CD137 antibodies have not been reported so far. Reference ListPatent Documents

[0006] Patent Document 1: WO 2008 / 114733Patent Document 2: WO 2015 / 156268Patent Document 3: WO 2016 / 177802Non-patent documents

[0007] Non-patent Document 1: Cancer Science, 2009: 100(9): pp. 1623-1630Non-patent Document 2: Oncotarget, 2018: 9(100): p. 37367-37378Non-patent Document 3: Cancer Science, 2020: 111(5): p. 1461-1467Non-patent Document 4: Cancer Immunology Immunotherapy, 2012: 61(5): p. 1721-1733Non-patent Document 5: Clinical Cancer Research, 2017: 23(8): p. 1929-1936Non-patent document L 6: mAbs, 2017: 9(2): p.182-212Non-patent document 7: Clinical Cancer Research, 2019: 25(19): p. 5878-5889Non-patent document 8: Clinical Cancer Research, 2020: 26(15): p. 4154-4167Non-patent document 9: Journal for Immunotherapy of Cancer, 2020; 8(2): e000238Summary of the inventionTechnical problem

[0008] The aim of the present invention is to provide a bispecific anti-CLDN4 / anti-CD137 antibody suitable for the treatment of cancer.Solution to the technical problem

[0009] The present inventors searched for an antibody that selectively binds to an antigen expressed in a peritoneal metastatic tumor cell to obtain 3D11, which is an anti-CLDN4 antibody (Examples 1 and 2). It was found that a bispecific T cell recruiting antibody using 3D11 and an anti-CD3 antibody was not suitable for the treatment of a cancer patient (Reference Example). Therefore, 3D11 and KM3900, which is a known anti-CLDN4 antibody, were used to attempt to obtain a bispecific T cell recruiting antibody with an anti-CD137 antibody (Examples 3 and 4). The resulting bispecific anti-CLDN4 / anti-CD137 antibody, which binds to CLDN4 and CD137 (Example 5), stimulated interferon-γ production by T cells in vitro and exhibited cytotoxic activity against tumor cells expressing CLDN4 on their cell surface (Example 6).Furthermore, it was confirmed that the obtained bispecific anti-CLDN4 / anti-CD137 antibody exhibited antitumor activity in a mouse model in vivo (Example 7-1) and could be safely used in cynomolgus macaques (Examples 7-2). It was suggested that the bispecific anti-CLBN4 / anti-CD137 antibody of the present invention is suitable for the treatment of cancer.

[0010] In particular, the present invention relates to the following items [1] to

[31] .1. A bispecific anti-CLDN4 / anti-CD137 antibody comprising 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, wherein the heavy chain variable region and the light chain variable region of the anti-CLDN4 antibody are any of the following (a) or (b): (a) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence at amino acid positions 31-35 of SEQ ID NO: 2, a CDR2 consisting of the amino acid sequence at amino acid positions 50-66 of SEQ ID NO: 2, and a CDR3 consisting of the amino acid sequence at amino acid positions 99-114 of SEQ ID NO: 2, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence at amino acid positions positions 24-35 of SEQ ID NO: 4, CDR2,consisting of the amino acid sequence at amino acid positions 51-57 of SEQ ID NO: 4, and CDR3 consisting of the amino acid sequence at amino acid positions 90-98 of SEQ ID NO: 4; or(b) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence at amino acid positions 31-35 of SEQ ID NO: 6, a CDR2 consisting of the amino acid sequence at amino acid positions 50-66 of SEQ ID NO: 6, and a CDR3 consisting of the amino acid sequence at amino acid positions 99-112 of SEQ ID NO: 6, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence at amino acid positions 24-35 of SEQ ID NO: 8, a CDR2 consisting of the amino acid sequence at amino acid positions 51-57 of SEQ ID NO: 8, and a CDR3 consisting of the amino acid sequence at amino acid positions 90-98 of SEQ ID NO: 8. 2. The bispecific antibody of claim 1,wherein the heavy chain variable region and the light chain variable region of the anti-CLDN4 antibody are any of the following (a) or (b): (a) a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-125 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 4; or (b) a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-123 of SEQ ID NO: 6 and a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 8.3. The bispecific antibody of any one of claims 1 and 2, comprising an IgG antibody (IgG anti-CLDN4 antibody) consisting of a heavy chain containing the variable region of the heavy chain and a light chain containing the variable region of the light chain of the anti-CLDN4 antibody.4. The bispecific antibody according to claim 3,comprising a LALA mutation (L234A and L235A, wherein the position of the mutation is an amino acid position in the constant region of human Igγ1 according to the EU numbering system) in the Fc region of an IgG anti-CLDN4 antibody. 5. The bispecific anti-CLDN4 / anti-CD137 antibody of claim 3, comprising a P331G mutation in the Fc region of an IgG anti-CLDN4 antibody (wherein the position of the mutation is an amino acid position in the constant region of human Igγ1 according to the EU numbering system). 6. The bispecific antibody of claim 3, comprising a LALA mutation and a P331G mutation in the Fc region of an IgG anti-CLDN4 antibody. 7. The bispecific antibody of any one of claims 1-6, wherein the heavy chain variable region and the light chain variable region of the anti-CD137 antibody are any of the following (a)-(d): (a) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence at amino acid positions 31-35 of SEQ ID NO: 10, a CDR2,consisting of the amino acid sequence at amino acid positions 50-66 of SEQ ID NO: 10, and a CDR3 consisting of the amino acid sequence at amino acid positions 99-107 of SEQ ID NO: 10, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence at amino acid positions 24-34 of SEQ ID NO: 12, a CDR2 consisting of the amino acid sequence at amino acid positions 50-56 of SEQ ID NO: 12, and a CDR3 consisting of the amino acid sequence at amino acid positions 89-98 of SEQ ID NO: 12;(b) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence at amino acid positions 31-35 of SEQ ID NO: 14, a CDR2 consisting of the amino acid sequence at amino acid positions 50-65 of SEQ ID NO: 14, and a CDR3 consisting from the amino acid sequence at amino acid positions 98-107 of SEQ ID NO: 14, and a light chain variable region containing CDR1,consisting of the amino acid sequence at amino acid positions 23-35 of SEQ ID NO: 16, a CDR2 consisting of the amino acid sequence at amino acid positions 51-57 of SEQ ID NO: 16, and a CDR3 consisting of the amino acid sequence at amino acid positions 90-100 of SEQ ID NO: 16;(c) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence at amino acid positions 31-35 of SEQ ID NO: 18, a CDR2 consisting of the amino acid sequence at amino acid positions 50-65 of SEQ ID NO: 18, and a CDR3 consisting of the amino acid sequence at amino acid positions 98-107 of SEQ ID NO: 18, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence at amino acid positions 23-35 of SEQ ID NO: 20, a CDR2 consisting from the amino acid sequence at amino acid positions 51-57 of SEQ ID NO: 20, and CDR3,consisting of the amino acid sequence at amino acid positions 90-100 of SEQ ID NO: 20; or (d) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence at amino acid positions 31-35 of SEQ ID NO: 22, a CDR2 consisting of the amino acid sequence at amino acid positions 50-65 of SEQ ID NO: 22, and a CDR3 consisting of the amino acid sequence at amino acid positions 98-110 of SEQ ID NO: 22, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence at amino acid positions 23-35 of SEQ ID NO: 24, a CDR2 consisting of the amino acid sequence at amino acid positions 51-57 of SEQ ID NO: 24, and a CDR3 consisting of the amino acid sequence at amino acid positions 90-100 of SEQ ID NO: 24.8. A bispecific antibody according to any one of claims 1 to 7,wherein the heavy chain variable region and the light chain variable region of the anti-CD137 antibody are any one of the following (a) to (i): (a) a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 10 and a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 12; (b) a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 14 and a light chain variable region consisting of the amino acid sequence at amino acid positions 1-111 of SEQ ID NO: 16; (c) a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 18 and a light chain variable region consisting of amino acid sequence at amino acid positions 1-111 of SEQ ID NO: 20;(d) a heavy chain variable region,consisting of the amino acid sequence at amino acid positions 1-121 of SEQ ID NO: 22, and a light chain variable region consisting of the amino acid sequence at amino acid positions 1-111 of SEQ ID NO: 24;(e) a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 26, and a light chain variable region consisting of the amino acid sequence at amino acid positions 134-242 of SEQ ID NO: 26;(f) a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 28, and a light chain variable region consisting of the amino acid sequence at amino acid positions 134-244 of SEQ ID NO: 28;(g) a light chain variable region consisting of the amino acid sequence at amino acid positions positions 1-111 of SEQ ID NO: 30, and a heavy chain variable region,consisting of the amino acid sequence at amino acid positions 132-249 of SEQ ID NO: 30;(h) a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 32 and a light chain variable region consisting of the amino acid sequence at amino acid positions 134-244 of SEQ ID NO: 32; or(i) a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-121 of SEQ ID NO: 34 and a light chain variable region consisting of the amino acid sequence at amino acid positions 137-247 of SEQ ID NO: 34.9. The bispecific antibody of any one of claims 7 and 8, comprising an anti-CD137 single chain variable fragment (anti-CD137 scFv) comprising a heavy chain variable region and a light chain variable region of an anti-CD137 antibody.10. The bispecific antibody of claim 9, wherein the anti-CD137 scFv is any one of the following (a) to (e): (a) an anti-CD137 scFv,consisting of the amino acid sequence at amino acid positions 1-242 of SEQ ID NO: 26;(b) an anti-CD137 scFv consisting of the amino acid sequence at amino acid positions 1-244 of SEQ ID NO: 28;(c) an anti-CD137 scFv consisting of the amino acid sequence at amino acid positions 1-249 of SEQ ID NO: 30;(d) an anti-CD137 scFv consisting of the amino acid sequence at amino acid positions 1-244 of SEQ ID NO: 32; or(e) an anti-CD137 scFv consisting of the amino acid sequence at amino acid positions 1-247 of SEQ ID NO: 34. 11. The bispecific antibody of any one of claims 9 and 10, comprising an IgG anti-CLDN4 antibody and an anti-CD137 scFv, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain or light chain of an IgG anti-CLDN4 antibody via a linker. 12. The bispecific anti-CLDN4 / anti-CD137 antibody described in any of the following (a) to (j): (a) a bispecific antibody comprising the heavy chain of an anti-CLDN4 antibody,comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-125 of SEQ ID NO: 2, and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 4, and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 26, and a light chain variable region consisting of the amino acid sequence at amino acid positions 134-242 of SEQ ID NO: 26, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker;(b) a bispecific antibody comprising a heavy chain of an anti-CLDN4 antibody comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-125 of SEQ ID NO: 2, and a light chain of an anti-CLDN4 antibody,comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 4, and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 28 and a light chain variable region consisting of the amino acid sequence at amino acid positions 134-244 of SEQ ID NO: 28, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of an anti-CLDN4 antibody via a linker;(c) a bispecific antibody comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-125 of SEQ ID NO: 2 and an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 4, and an anti-CD137 scFv comprising the light chain variable region,consisting of the amino acid sequence at amino acid positions 1-111 of SEQ ID NO: 30, and a heavy chain variable region consisting of the amino acid sequence at amino acid positions 132-249 of SEQ ID NO: 30, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of an anti-CLDN4 antibody via a linker; (d) a bispecific antibody comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-125 of SEQ ID NO: 2, and an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 4, and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 32, and a light chain variable region,consisting of the amino acid sequence at amino acid positions 134-244 of SEQ ID NO: 32, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of an anti-CLDN4 antibody via a linker; (e) a bispecific antibody comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-125 of SEQ ID NO: 2, and an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 4, and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-121 of SEQ ID NO: 34, and a light chain variable region consisting of the amino acid sequence at amino acid positions 137-247 of SEQ ID NO: 34,wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of an anti-CLDN4 antibody via a linker; (f) a bispecific antibody comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-123 of SEQ ID NO: 6 and an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 8 and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 26 and a light chain variable region consisting of the amino acid sequence at amino acid positions 134-242 of SEQ ID NO: 26, wherein the amino terminus an anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of an anti-CLDN4 antibody via a linker; (g) a bispecific antibody comprising the heavy chain of an anti-CLDN4 antibody,comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-123 of SEQ ID NO: 6, and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 8, and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 28, and a light chain variable region consisting of the amino acid sequence at amino acid positions 134-244 of SEQ ID NO: 28, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker;(h) a bispecific antibody comprising a heavy chain of an anti-CLDN4 antibody comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-123 of SEQ ID NO: 6, and a light chain of an anti-CLDN4 antibody,comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 8, and an anti-CD137 scFv comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-111 of SEQ ID NO: 30 and a heavy chain variable region consisting of the amino acid sequence at amino acid positions 132-249 of SEQ ID NO: 30, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of an anti-CLDN4 antibody via a linker;(i) a bispecific antibody comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-123 of SEQ ID NO: 6 and an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 8, and an anti-CD137 scFv comprising a heavy chain variable region,consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 32, and a light chain variable region consisting of the amino acid sequence at amino acid positions 134-244 of SEQ ID NO: 32, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker; or(j) a bispecific antibody comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-123 of SEQ ID NO: 6 and an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 8 and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-121 of SEQ ID NO: 34 and a light chain variable region,consisting of the amino acid sequence at amino acid positions 137-247 of SEQ ID NO: 34, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker. 13. The bispecific anti-CLDN4 / anti-CD137 antibody of any one of claims 11 and 12, wherein the linker is a GS linker. 14. The bispecific anti-CLDN4 / anti-CD137 antibody of claim 13, wherein the GS linker is a linker consisting of the amino acid sequence of SEQ ID NO: 54. 15. A bispecific anti-CLDN4 / anti-CD137 antibody as described in any one of (a) or (b): (a) a bispecific antibody comprising a polypeptide consisting of the amino acid sequence at amino acid positions 1-705 of SEQ ID NO: 36, comprising an anti-CLDN4 antibody heavy chain and an anti-CD137 scFv, and an anti-CLDN4 antibody light chain consisting of the amino acid sequence at amino acid positions 1-215 of SEQ ID NO: 40; or (b) a bispecific antibody comprising a polypeptide,consisting of the amino acid sequence at amino acid positions 1-712 of SEQ ID NO: 38, comprising a heavy chain of an anti-CLDN4 antibody and an anti-CD137 scFv, and a light chain of an anti-CLDN4 antibody consisting of the amino acid sequence at amino acid positions 1-215 of SEQ ID NO: 40. 16. The bispecific anti-CLDN4 / anti-CD137 antibody of any one of claims 1-15, which is post-translationally modified. 17. The bispecific anti-CLDN4 / anti-CD137 antibody of claim 16, wherein the post-translational modification is pyroglutamylation at the N-terminus of the heavy chain variable region and / or deletion of lysine at the C-terminus of the heavy chain. A polynucleotide having a nucleotide sequence encoding a heavy chain variable region or a light chain variable region of an anti-CLDN4 antibody, which is selected from the group consisting of the following (a)-(d): (a) a polynucleotide having a nucleotide sequence encoding a heavy chain variable region of an anti-CLDN4 antibody,consisting of an amino acid sequence at amino acid positions 1-125 of SEQ ID NO: 2;(b) a polynucleotide having a nucleotide sequence encoding a light chain variable region of an anti-CLDN4 antibody consisting of an amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 4;(c) a polynucleotide having a nucleotide sequence encoding a heavy chain variable region of an anti-CLDN4 antibody consisting of an amino acid sequence at amino acid positions 1-123 of SEQ ID NO: 6; or(d) a polynucleotide having a nucleotide sequence encoding a light chain variable region of an anti-CLDN4 antibody consisting of an amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 8.19. A polynucleotide having a nucleotide sequence encoding the heavy chain variable region or the light chain variable region of an anti-CD137 antibody, which is selected from the group,consisting of the following (a)-(r): (a) a polynucleotide having a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 10; (b) a polynucleotide having a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 12; (c) a polynucleotide having a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 14; (d) a polynucleotide having a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence at amino acid positions positions 1-111 of SEQ ID NO: 16;(e) a polynucleotide having a nucleotide sequence,encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 18;(f) a polynucleotide having a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence at amino acid positions 1-111 of SEQ ID NO: 20;(g) a polynucleotide having a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence at amino acid positions 1-121 of SEQ ID NO: 22;(h) a polynucleotide having a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence at amino acid positions 1-111 of SEQ ID NO: 24;(i) a polynucleotide having a nucleotide sequence encoding the variable region of the heavy chain of an anti-CD137 antibody,consisting of an amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 26;(j) a polynucleotide having a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of an amino acid sequence at amino acid positions 134-242 of SEQ ID NO: 26;(k) a polynucleotide having a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of an amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 28;(l) a polynucleotide having a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of an amino acid sequence at amino acid positions 134-244 of SEQ ID NO: 28;(m) a polynucleotide having a nucleotide sequence encoding a light chain variable region anti-CD137 antibodies,consisting of an amino acid sequence at amino acid positions 1-111 of SEQ ID NO: 30;(n) a polynucleotide having a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of an amino acid sequence at amino acid positions 132-249 of SEQ ID NO: 30;(o) a polynucleotide having a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of an amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 32;(p) a polynucleotide having a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of an amino acid sequence at amino acid positions 134-244 of SEQ ID NO: 32;(q) a polynucleotide having a nucleotide sequence encoding a heavy chain variable region anti-CD137 antibodies,consisting of the amino acid sequence at amino acid positions 1-121 of SEQ ID NO: 34; or (r) a polynucleotide having a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence at amino acid positions 137-247 of SEQ ID NO: 34.20. A polynucleotide having a nucleotide sequence encoding an anti-CD137 scFv that is selected from the group consisting of the following (a)-(e): (a) a polynucleotide having a nucleotide sequence encoding an anti-CD137 scFv consisting of the amino acid sequence at amino acid positions 1-242 of SEQ ID NO: 26; (b) a polynucleotide having a nucleotide sequence encoding an anti-CD137 scFv consisting of the amino acid sequence at amino acid positions 1-244 of SEQ ID NO: 28; (c) a polynucleotide having a nucleotide sequence encoding an anti-CD137 scFv,consisting of an amino acid sequence at amino acid positions 1-249 of SEQ ID NO: 30;(d) a polynucleotide having a nucleotide sequence encoding an anti-CD137 scFv consisting of an amino acid sequence at amino acid positions 1-244 of SEQ ID NO: 32; or(e) a polynucleotide having a nucleotide sequence encoding an anti-CD137 scFv consisting of an amino acid sequence at amino acid positions 1-247 of SEQ ID NO: 34.21. A polynucleotide for use in the production of a bispecific anti-CLDN4 / anti-CD137 antibody according to claim 15, which is selected from the group consisting of the following (a)-(e): (a) a polynucleotide having a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence at amino acid positions 1-705 of SEQ ID NO: 36, comprising the heavy chain of an anti-CLDN4 antibody and an anti-CD137 scFv; (b) a polynucleotide having a nucleotide sequence encoding a polypeptide,consisting of the amino acid sequence at amino acid positions 1-712 of SEQ ID NO: 38, comprising a heavy chain of an anti-CLDN4 antibody and an anti-CD137 scFv;(c) a polynucleotide having a nucleotide sequence encoding a light chain of an anti-CLDN4 antibody consisting of the amino acid sequence at amino acid positions 1-215 of SEQ ID NO: 40;(d) a polynucleotide having a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence at amino acid positions 1-705 of SEQ ID NO: 36, comprising the heavy chain of an anti-CLDN4 antibody and an anti-CD137 scFv, and a polynucleotide having a nucleotide sequence encoding a light chain of an anti-CLDN4 antibody consisting of the amino acid sequence at amino acid positions 1-215 of SEQ ID NO: 40; or(e) a polynucleotide having a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence at amino acid positions 1-712 of SEQ ID NO: 38,comprising a heavy chain of an anti-CLDN4 antibody and an anti-CD137 scFv, and a polynucleotide having a nucleotide sequence encoding a light chain of an anti-CLDN4 antibody consisting of the amino acid sequence at amino acid positions 1-215 of SEQ ID NO: 40.22. An expression vector comprising the polynucleotide of any one of claims 18-21.23. A host cell transformed with the expression vector of claim 22.24. A host cell comprising a polynucleotide selected from the group consisting of (a)-(dd):(a) a polynucleotide having a nucleotide sequence encoding a heavy chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence at amino acid positions 1-125 of SEQ ID NO: 2;(b) a polynucleotide having a nucleotide sequence encoding a light chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 4;(c) a polynucleotide,having a nucleotide sequence encoding a heavy chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence at amino acid positions 1-123 of SEQ ID NO: 6;(d) a polynucleotide having a nucleotide sequence encoding a light chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 8;(e) a polynucleotide having a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 10;(f) a polynucleotide having a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 12;(g) a polynucleotide having a nucleotide sequence encoding the variable region of the heavy chain of an anti-CD137 antibody,consisting of an amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 14;(h) a polynucleotide having a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of an amino acid sequence at amino acid positions 1-111 of SEQ ID NO: 16;(i) a polynucleotide having a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of an amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 18;(j) a polynucleotide having a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of an amino acid sequence at amino acid positions 1-111 of SEQ ID NO: 20;(k) a polynucleotide having a nucleotide sequence encoding a heavy chain variable region anti-CD137 antibodies,consisting of an amino acid sequence at amino acid positions 1-121 of SEQ ID NO: 22;(l) a polynucleotide having a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of an amino acid sequence at amino acid positions 1-111 of SEQ ID NO: 24;(m) a polynucleotide having a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of an amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 26;(n) a polynucleotide having a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of an amino acid sequence at amino acid positions 134-242 of SEQ ID NO: 26;(o) a polynucleotide having a nucleotide sequence encoding a heavy chain variable region anti-CD137 antibodies,consisting of an amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 28;(p) a polynucleotide having a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of an amino acid sequence at amino acid positions 134-244 of SEQ ID NO: 28;(q) a polynucleotide having a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of an amino acid sequence at amino acid positions 1-111 of SEQ ID NO: 30;(r) a polynucleotide having a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of an amino acid sequence at amino acid positions 132-249 of SEQ ID NO: 30;(s) a polynucleotide having a nucleotide sequence encoding a heavy chain variable region anti-CD137 antibodies,consisting of an amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 32;(t) a polynucleotide having a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of an amino acid sequence at amino acid positions 134-244 of SEQ ID NO: 32;(u) a polynucleotide having a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of an amino acid sequence at amino acid positions 1-121 of SEQ ID NO: 34;(v) a polynucleotide having a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of an amino acid sequence at amino acid positions 137-247 of SEQ ID NO: 34;(w) a polynucleotide having a nucleotide sequence encoding an anti-CD137 scFv consisting of the amino acid sequence at amino acid positions 1-242 of SEQ ID NO: 26;(x) polynucleotide,having a nucleotide sequence encoding an anti-CD137 scFv consisting of the amino acid sequence at amino acid positions 1-244 of SEQ ID NO: 28; (y) a polynucleotide having a nucleotide sequence encoding an anti-CD137 scFv consisting of the amino acid sequence at amino acid positions 1-249 of SEQ ID NO: 30; (z) a polynucleotide having a nucleotide sequence encoding an anti-CD137 scFv consisting of the amino acid sequence at amino acid positions 1-244 of SEQ ID NO: 32; (aa) a polynucleotide having a nucleotide sequence encoding an anti-CD137 scFv consisting of the amino acid sequence at amino acid positions 1-247 of SEQ ID NO: 34; (bb) a polynucleotide having a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence at amino acid positions 1-705 SEQ ID NO: 36, comprising the heavy chain of an anti-CLDN4 antibody and an anti-CD137 scFv;(cc) polynucleotide,having a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence at amino acid positions 1-712 of SEQ ID NO: 38, comprising a heavy chain of an anti-CLDN4 antibody and an anti-CD137 scFv; or (dd) a polynucleotide having a nucleotide sequence encoding a light chain of an anti-CLDN4 antibody consisting of the amino acid sequence at amino acid positions 1-215 of SEQ ID NO: 40.25. A host cell comprising a polynucleotide according to any one of the following paragraphs (a) or (b): (a) a polynucleotide having a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence at amino acid positions 1-705 of SEQ ID NO: 36, comprising the heavy chain of an anti-CLDN4 antibody and an anti-CD137 scFv, and a polynucleotide having a nucleotide sequence encoding a light chain of an anti-CLDN4 antibody consisting of the amino acid sequence at amino acid positions 1-215 of SEQ ID NO: 40; or (b) a polynucleotide,having a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence at amino acid positions 1-712 of SEQ ID NO: 38, comprising a heavy chain of an anti-CLDN4 antibody and an anti-CD137 scFv, and a polynucleotide having a nucleotide sequence encoding a light chain of an anti-CLDN4 antibody consisting of the amino acid sequence at amino acid positions 1-215 of SEQ ID NO: 40. 26. A method for producing a bispecific anti-CLDN4 / anti-CD137 antibody, comprising the step of culturing a host cell according to any one of claims 23-25. 27. A pharmaceutical composition comprising the bispecific anti-CLDN4 / anti-CD137 antibody according to any one of claims 1-17 and a pharmaceutically acceptable excipient. 28. The bispecific anti-CLDN4 / anti-CD137 antibody according to any one of claims 1-17 for use in the treatment of cancer.29. The pharmaceutical composition of claim 27 for use in the treatment of cancer.30. A method for treating cancer,comprising the step of administering a therapeutically effective amount of the bispecific anti-CLDN4 / anti-CD137 antibody of any one of claims 1 to 17 to a subject. 31. The use of the bispecific anti-CLDN4 / anti-CD137 antibody of any one of claims 1 to 17 in the manufacture of a pharmaceutical composition for use in the treatment of cancer. Advantageous effects of the invention,

[0011] The bispecific anti-CLDN4 / anti-CD137 antibody of the present invention binds to both CLDN4, which is a tumor antigen, and CD137 expressed on the cell surface of an immune cell such as a T cell, to activate immune cells around the tumor cell and thereby enhance the killing activity of the tumor cell. The bispecific anti-CLDN4 / anti-CD137 antibody of the present invention or a pharmaceutical composition containing the antibody can be used for the treatment of various types of cancer. Brief Description of the Figures

[0012] Fig. 1-1 shows the binding activity of the test antibodies to CD137, measured and estimated using ELISA. The ordinate axis of the graph indicates the value obtained by subtracting the optical density at 570 nm from the optical density at 450 nm, and the abscissa axis indicates the concentration of the test antibody. Each symbol indicates the mean absorbance value obtained by adding each test antibody, and the vertical error line indicates the standard deviation. Fig. 1-2 shows the binding activity of the test antibodies to CD137, measured and estimated using ELISA. The ordinate axis of the graph indicates the value obtained by subtracting the optical density at 570 nm from the optical density at 450 nm, and the abscissa axis indicates the concentration of the test antibody. Each of the symbols indicates the mean absorbance value obtained by adding each of the test antibodies. In Fig.1-3 show the binding activity of the test antibodies to CD137, measured and estimated using ELISA. The ordinate axis of the graphs indicates the value obtained by subtracting the optical density at 570 nm from the optical density at 450 nm, and the abscissa axis indicates the concentration of the test antibody. Each of the symbols indicates the average optical density value obtained upon the addition of each of the test antibodies. Figs. 1-4 show the binding activity of the test antibodies to CD137, measured and estimated using ELISA. The ordinate axis of the graphs indicates the value obtained by subtracting the optical density at 570 nm from the optical density at 450 nm, and the abscissa axis indicates the concentration of the test antibody. Each of the symbols indicates the average optical density value obtained upon the addition of each of the test antibodies. Fig.2-1 shows the agonistic activity of the test antibodies for CD137 obtained by co-culturing tumor cells expressing CLDN4 (NCI-H322 cells) and effector 4-1BB cells. The ordinate axis of the graphs indicates the relative luminescence value, and the abscissa axis indicates the concentration of the test antibody. Each symbol indicates the mean value of the relative luminescence value obtained upon the addition of each of the test antibodies, and the vertical error bar indicates the standard deviation. Fig. 2-2 shows the agonistic activity of the test antibodies for CD137 obtained by co-culturing tumor cells expressing CLDN4 (NCI-H322 cells) and effector 4-1BB cells. The ordinate axis of the graphs indicates the relative luminescence value, and the abscissa axis indicates the concentration of the test antibody. Each symbol indicates the mean value of the relative luminescence obtained upon addition of each of the antibodies tested.Figures 2-3 show the agonist activity of the test antibodies for CD137 obtained in co-culture of tumor cells expressing CLDN4 (NCI-H322 cells) and 4-1BB effector cells. The relative luminescence value is indicated on the ordinate axis of the graphs, and the concentration of the test antibody is indicated on the abscissa axis. Each symbol indicates the mean value of the relative luminescence value obtained upon the addition of each of the test antibodies, and the vertical error bar indicates the standard deviation. Figures 2-4 show the agonist activity of the test antibodies for CD137 obtained in a monoculture of 4-1BB effector cells. The relative luminescence value is indicated on the ordinate axis of the graphs, and the concentration of the test antibody is indicated on the abscissa axis. Each symbol indicates the mean value of the relative luminescence obtained upon addition of each of the tested antibodies, and the vertical error bar indicates the standard deviation.Fig. 2-5 shows the agonist activity for CD137 of the test antibodies, obtained on a monoculture of 4-1BB effector cells. The ordinate axis of the graphs indicates the relative luminescence value, and the abscissa axis indicates the concentration of the test antibody. Each of the symbols indicates the average value of the relative luminescence value obtained upon the addition of each of the test antibodies. Fig. 2-6 shows the agonist activity for CD137 of the test antibodies, obtained on a monoculture of 4-1BB effector cells. The ordinate axis of the graphs indicates the relative luminescence value, and the abscissa axis indicates the concentration of the test antibody. Each of the symbols indicates the average value of the relative luminescence value obtained upon the addition of each of the test antibodies, and the vertical error line indicates the standard deviation. Fig.Figure 3-1 shows the stimulatory function for interferon-γ production obtained by adding each test antibody to the coculture system of 60As6-Luc / GFP cells and expanded pan-T cells. The ordinate axis shows the amount of interferon-γ produced (pg / ml) obtained 6 days after the addition of the antibody, and the abscissa axis shows the antibody concentration. Each symbol indicates the average amount of interferon-γ produced at each antibody concentration. Figure 3-2 shows the stimulatory function for interferon-γ production obtained by adding each test antibody to the coculture system of 60As6-Luc / GFP cells and expanded pan-T cells. The ordinate axis of the graphs shows the amount of interferon-γ produced (pg / ml), obtained 4 days after the addition of the antibody, and the abscissa axis shows the concentration of the antibody.Each symbol indicates the mean amount of interferon-γ produced at each antibody concentration, and the vertical error bar indicates the standard deviation. Figure 3-3 shows the amount of interferon-γ produced by adding each test antibody to the expanded pan-T cell monoculture system. The ordinate axis of the graphs indicates the amount of interferon-γ produced (pg / ml) obtained 6 days after adding the antibody, and the abscissa axis indicates the antibody concentration. Each symbol indicates the mean amount of interferon-γ produced at each antibody concentration. Figure 3-4 shows the amount of interferon-γ produced by adding each test antibody to the expanded pan-T cell monoculture system. The ordinate axis of the graphs shows the amount of interferon-γ produced (pg / ml), obtained 5 days after the addition of the antibody, and the abscissa axis shows the concentration of the antibody.Each symbol indicates the mean amount of interferon-γ produced at each antibody concentration, and the vertical error bar indicates the standard deviation. Figure 4 shows the tumor cell cytotoxic activity (tumor cell growth inhibitory activity) of each test antibody in a coculture system of 60As6-Luc / GFP cells and expanded pan-T cells. The ordinate axis of the graphs indicates the cell growth rate obtained under the assumption that the fluorescence area increasing from 0 h to 168 h after the start of the measurement in the well to which the test antibody was not added corresponds to 100%. The abscissa axis indicates the antibody concentration. Each symbol indicates the mean tumor cell growth rate for each antibody. 5 shows the cytotoxic activity for tumor cells obtained by adding each tested antibody to the co-culture system of 60As6-Luc / GFP cells and expanded pan T cells.The ordinate axis of the graph shows the tumor cell death rate (%) obtained under the assumption that in the measured chemiluminescence values ​​of 60As6-Luc / GFP cells analyzed 6 days after the addition of the antibody, the average value obtained without the addition of the antibody corresponds to a death rate of 0%, and the average value obtained with the addition of Triton-X100 corresponds to a death rate of 100%. The abscissa axis shows the antibody concentration. Each symbol indicates the average cancer cell death rate at each antibody concentration, and the vertical error line indicates the standard deviation. Fig. 6 shows the change in tumor volume in mice transplanted with PBMCs subcutaneously bearing NCI-H322 tumor cells. The abscissa axis of the graph shows the day after the first administration, the ordinate axis shows the average tumor volume, and the vertical error line indicates the standard deviation. Description of embodiments.

[0013] Next, the present invention will be described in detail. Definitions In the present invention, the terms have the meanings commonly used in the art by those skilled in the art unless otherwise specifically defined.

[0014] An antibody (or immunoglobulin) is a glycoprotein whose basic structure is a four-chain, bilaterally symmetrical Y-shaped structure, comprising two heavy chains of the same sequence and two light chains of the same sequence. There are five classes of antibodies: IgG, IgM, IgA, IgD, and IgE. The basic structure of the antibody molecule is common to these classes: two heavy chains with a molecular weight of 50,000 to 70,000 and two light chains with a molecular weight of 20,000 to 30,000 are linked by a disulfide bond and a non-covalent bond to form an antibody molecule having a Y-shaped four-chain structure with a molecular weight of 150,000 to 190,000. The heavy chain is a polypeptide chain typically containing about 440 amino acids, has a structure specific to each class, and is designated Igγ, Igμ, Igα, Igδ, and Igε, respectively, as IgG, IgM, IgA, IgD, or IgE.IgG has subclasses IgG1, IgG2, IgG3, and IgG4, and the heavy chains corresponding to these subclasses are designated Igγ1, Igγ2, Igγ3, and Igγ4. The light chain is a polypeptide chain typically containing approximately 220 amino acids, and it is known to have two types: the λ type and the κ type, which are designated Igλ and Igκ, respectively. Each of the two types of light chains can be paired with either type of heavy chain.

[0015] In an antibody molecule, the heavy chain has four intrachain disulfide bonds (five in Igμ and Igε), the light chain has two intrachain disulfide bonds, and thus one loop is formed by 100-110 amino acid residues. The three-dimensional structures of these loops are the same and are referred to as a structural unit or domain. In both the heavy chain and the light chain, the domain located at the N-terminus is referred to as the variable region and has an amino acid sequence that varies even among antibodies obtained from the same class (or subclass) of the same animal species and is known to be involved in the formation of specific antibody-antigen bonds. The domain located on the side downstream of the variable region at the C-terminus has an amino acid sequence that is essentially constant in each class or subclass and is referred to as the constant region.The heavy chain, from the N-terminus to the C-terminus, has a heavy-chain variable region (VH) and a heavy-chain constant region (CH). CH is further divided at the N-terminus into three domains: the CH1 domain, the CH2 domain, and the CH3 domain. The light chain, from the N-terminus to the C-terminus, has a light-chain variable region (VL) and a light-chain constant region (CL).

[0016] The amino acid sequence of the three complementarity-determining regions (CDRs) present in VH and VL varies greatly and contributes to the variability of the variable regions. Each CDR is a region consisting of 5-10 amino acid residues, which is located in the order CDR1, CDR2, and CDR3 at the N-terminus of each heavy chain and light chain and comes into contact with the antigen to form the antibody binding site. It is known that the CDRs of the heavy chain contribute more to antigen binding than the CDRs of the light chain, and that CDR3 makes the largest contribution among CDR1-CDR3. On the other hand, the region without the CDRs of the variable region, referred to as the framework region (FR), consists of FR1-FR4 and shows relatively little variation in amino acid sequence.

[0017] When an antibody is treated with papain, a protease, three antibody fragments are produced. The two fragments located at the N-terminal end are referred to as the Fab region (antigen-binding fragment, fragment binding antigen). The Fab region refers to the region consisting of the VH of the heavy chain, part of the CH1 domain and hinge region, and the light chain (VL and CL), and binds to the antigen via the VH and VL (antigen-binding site) included in the Fab region. In addition, the fragment located at the C-terminal end is referred to as the Fc region (fragment crystallizable, fragment crystallizable).

[0018] Within the context of the present invention, an “IgG antibody” refers to an antibody having a Y-shaped structure comprising two Fab regions and an Fc region. In one embodiment, the two Fab regions of an IgG antibody comprise the same Fab region sequences. In one embodiment, the two Fab regions of an IgG antibody comprise different Fab region sequences.

[0019] In the context of the present invention, the terms "antigen-binding fragment" and "antibody fragment" can be used interchangeably and refer to a molecule that comprises at least one polypeptide chain comprising a heavy chain variable region and a light chain variable region of an immunoglobulin molecule and having antigen-binding activity. Representative examples of the antigen-binding fragment include a single-chain variable fragment (scFv), a Fab fragment, a Fab' fragment, and an F(ab')2 fragment. Here, "scFv" refers to a monovalent antigen-binding fragment comprising VH and VL linked via a linker. A Fab fragment is a monovalent antigen-binding fragment comprising a fragment consisting of a light chain, VH and CH1 domain of a heavy chain, and a portion of a hinge region.The Fab' fragment is a monovalent antigen-binding fragment containing a fragment consisting of the light chain, VH, and CH1 domain of the heavy chain, and a portion of the hinge region. This portion of the hinge region contains a cysteine ​​residue contained in the disulfide bond between the heavy chains. The F(ab')2 fragment is a bivalent antigen-binding fragment containing Fab' fragments linked by a disulfide bond. The term "monovalent" means that it contains one antigen-binding site, and the term "bivalent" means that it contains two antigen-binding sites.

[0020] In the context of the present invention, a "bispecific antibody" refers to an antibody molecule comprising two antibodies or antigen-binding fragments that specifically bind to different antigens and have binding activity against the corresponding antigens. As bispecific antibodies, bispecific antibodies having different structures are known to those skilled in the art (Non-Patent Document 6). In the context of the present invention, the term "antibody" encompasses full-length antibodies (various immunoglobulins, in particular IgG antibodies), antigen-binding fragments, and bispecific antibodies of any structure, unless specifically limited by context.

[0021] In the context of the present invention, the IgG type anti-CLDN4 antibody is referred to as an "IgG anti-CLDN4 antibody".

[0022] In the context of the present invention, a “human antibody” refers to an antibody having an amino acid sequence of a human immunoglobulin. In the context of the present invention, a “humanized antibody” refers to an antibody in which part, most, or all of the amino acid residues, except for the CDRs, are substituted with amino acid residues derived from a human immunoglobulin molecule. The method for humanization is not particularly limited, and for obtaining a humanized antibody, reference can be made, for example, to U.S. Patent No. 5,225,539, U.S. Patent No. 6,180,370, and the like.

[0023] The number of amino acid residues of the antibody used herein may be indicated by the Kabat numbering system or the EU numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., 1991, NIH Publication No. 91-3242)) in accordance with the numbering system.

[0024] Within the context of the present invention, the term “link” or “linked” means that a plurality of components (such as a Fab region and an Fc region) are connected directly or through an intermediary (such as a peptide linker). Here, the term “peptide linker” means at least one amino acid sequence that can be introduced by a genetic engineering method used to link a plurality of 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 the context of the present invention, the term “identity” means the identity value obtained using EMBOSS Needle (Nucleic Acids Res., 2015, Vol.43, p.W580-W584) with default parameters. The parameters are as follows: Gap Opening Penalty=10 Gap Extension Penalty=0.5 Matrix=EBLOSUM62 Gap Ending Penalty=false

[0026] Bispecific anti-CLDN4 / anti-CD137 antibody of the present inventionThe present invention relates to a bispecific antibody binding to CLDN4 and CD137 (also referred to herein as an "anti-CLDN4 / anti-CD137 bispecific antibody"). The bispecific anti-CLDN4 / anti-CD137 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 bispecific anti-CLDN4 / anti-CD137 antibody of the present invention includes a bispecific antibody comprising an IgG anti-CLDN4 antibody and an anti-CD137 scFv (also referred to herein as an "anti-CD137 scFv"). In one embodiment, the bispecific anti-CLDN4 / anti-CD137 antibody of the present invention is a bispecific antibody comprising an IgG anti-CLDN4 antibody and an anti-CD137 scFv.

[0027] In the context of the present invention, an “anti-CLDN4 antibody” is an antibody capable of binding to human CLDN4, and an “anti-CD137 antibody” is an antibody capable of binding to human CD137. Using a known method for assessing binding activity, the extent to which an antibody binds to human CLDN4 or human CD137 can be determined. Examples of the method for measuring binding activity include a method such as enzyme-linked immunosorbent assay (ELISA) or flow cytometry. ELISA or flow cytometry can be performed by a method commonly used by those skilled in the art, and the method described in Examples 3 or 5, for example, can be used.

[0028] The bispecific anti-CLDN4 / anti-CD137 antibody of the present invention may have any structure as long as it binds to CLDN4 and CD137, and examples thereof include a bispecific antibody having the structure described in Non-Patent Document 6. In one embodiment, the bispecific anti-CLDN4 / anti-CD137 antibody of the present invention may be a conjugate in which a Fab region of an anti-CLDN4 antibody and a Fab region of an anti-CD137 antibody are linked to each other, a conjugate of an IgG anti-CLDN4 antibody and an anti-CD137 antibody of the IgG type (also referred to herein as an "IgG anti-CD137 antibody"), a conjugate of an IgG anti-CLDN4 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 IgG anti-CD137 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 bispecific anti-CLDN4 / anti-CD137 antibody of the present invention comprises a full-length anti-CLDN4 antibody and a full-length anti-CD137 antibody. In one embodiment, the bispecific anti-CLDN4 / anti-CD137 antibody of the present invention comprises an IgG anti-CLDN4 antibody and an IgG anti-CD137 antibody. The two antibodies can be linked to each other via a linker. In one embodiment, the bispecific anti-CLDN4 / anti-CD137 antibody of the present invention comprises a full-length anti-CLDN4 antibody and an antigen-binding fragment of an anti-CD137 antibody. The binding site of the two antibodies is not particularly limited, and the antibodies are linked directly or via a linker. In one embodiment, the bispecific anti-CLDN4 / anti-CD137 antibody of the present invention comprises an IgG anti-CLDN4 antibody and an anti-CD137 scFv (referred to herein as “anti-CD137 scFv”).In one embodiment, the bispecific anti-CLDN4 / anti-CD137 antibody of the present invention comprises an IgG anti-CLDN4 antibody and an anti-CD137 scFv, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody. In one embodiment, the bispecific anti-CLDN4 / anti-CD137 antibody of the present invention comprises an IgG anti-CLDN4 antibody and an anti-CD137 scFv, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the light chain of the anti-CLDN4 antibody. In one embodiment, the bispecific anti-CLDN4 / anti-CD137 antibody of the present invention comprises an IgG anti-CLDN4 antibody and an anti-CD137 scFv, wherein the carboxy terminus of the anti-CD137 scFv is linked to the amino terminus of the heavy chain of the anti-CLDN4 antibody. In one embodiment, the bispecific anti-CLDN4 / anti-CD137 antibody of the present invention comprises an IgG anti-CLDN4 antibody and an anti-CD137 scFv, wherein the carboxy terminus of the anti-CD137 scFv is linked to the amino terminus of the light chain of the anti-CLDN4 antibody.In one embodiment, the bispecific anti-CLDN4 / anti-CD137 antibody of the present invention comprises an antigen-binding fragment of an anti-CLDN4 antibody and an antigen-binding fragment of an anti-CD137 antibody. In one embodiment, the bispecific anti-CLDN4 / anti-CD137 antibody of the present invention comprises an anti-CLDN4 antibody and an anti-CD137 scFv. In one embodiment, the bispecific anti-CLDN4 / anti-CD137 antibody of the present invention comprises an anti-CD137 scFv.

[0030] In the bispecific anti-CLDN4 / anti-CD137 antibody of the present invention, the heavy chain variable regions and the light chain variable regions of the anti-CLDN4 antibody and the anti-CD137 antibody may be derived from a human antibody or may be derived from a humanized antibody, or a combination thereof. In one embodiment, the bispecific anti-CLBN4 / anti-CD137 antibody of the present invention is an antibody comprising a human antibody, a humanized antibody, or a combination thereof.

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

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

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

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

[0035] When the bispecific anti-CLDN4 / anti-CD137 antibody of the present invention comprises an Fc region, the Fc region of the bispecific antibody may comprise a mutation that results in a decrease in antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). L234A is a substitution of leucine to alanine at amino acid position 234 in the constant region of human Igγ1. L235A is a substitution of leucine to alanine at amino acid position 235 in the constant region of human Igγ1. The amino acid mutations L234A and L235A in the constant region of human Igγ1 are referred to as the “LALA mutation”. Here, positions 234 and 235 are amino acid positions in the constant region of human Igγ1 according to the EU numbering system. This mutation is known to result in decreased ADCC and CDC antibodies (Mol. Immunol., 1992, Vol. 29, p. 633-639; and J. Immunol., 2000, Vol. 164, p. 4178-4184).P331G or P331S is a substitution of proline for glycine or serine at amino acid position 331 in the constant region of human Igγ1. In this case, position 331 is the amino acid position in the constant region of human Igγ1 according to the EU numbering system. This mutation is known to reduce CDC antibodies (J. Immunol., 2000, Vol. 164(8), pp. 4178-4184).

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

[0037] The present inventors have created a novel anti-CD137 antibody or a binding fragment thereof (scFv) in the process of creating the bispecific anti-CLDN4 / anti-CD137 antibody of the present invention. The present inventors have identified a bispecific anti-CLDN4 / anti-CD137 antibody that does not transduce the CD137 signal when bound to CD137 alone, but transduces the CD137 signal only when simultaneously bound to CD137 and CLDN4. The antibody does not exhibit agonist activity unless it binds to CLDN4. On the other hand, binding of the bispecific anti-CLDN4 / anti-CD137 antibody to CLDN4 and CD137 results in CD137 signal transduction and T cell activation. This activity is believed to attenuate hepatotoxicity, etc. observed in clinical trials of urelumab and is therefore expected to be a preferred profile as an anti-CD137 antibody used in a bispecific antibody.

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

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

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

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

[0042] The type and length of the linker used to link the heavy chain variable region and the light chain variable region of the anti-CD137 antibody in the anti-CD137 scFv are not particularly limited and can be appropriately selected by those skilled in the art. A peptide linker can be used as the linker. The preferred length is 5 amino acids or more (the upper limit is not particularly limited, but is usually 30 amino acids or less, and preferably 20 amino acids or less), and is particularly preferably 15 amino acids. As the 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 include the following: In the above examples, n is an integer equal to 1 or greater. In one aspect, n is from 1 to 10, from 2 to 8, or from 2 to 6. The length and sequence of the linker can be appropriately selected by those skilled in the art depending on the purpose. In one embodiment, the linker used in the anti-CD137 scFv is a GS linker. In one embodiment, the linker used in the anti-CD137 scFv is a GS (Gly-Gly-Gly-Gly-Ser)n linker, and n is either 3 or 4.

[0043] In one embodiment, the anti-CLDN4 / anti-CD137 bispecific antibody of the present invention comprises an anti-CD137 scEv described in any one of the following (a) to (e): (a) an anti-CD137 scEv consisting of the amino acid sequence at amino acid positions 1-242 of SEQ ID NO: 26; (b) an anti-CD137 scEv consisting of the amino acid sequence at amino acid positions 1-244 of SEQ ID NO: 28; (c) an anti-CD137 scEv consisting of the amino acid sequence at amino acid positions 1-249 of SEQ ID NO: 30; (d) an anti-CD137 scFv consisting of the amino acid sequence at amino acid positions 1-244 of SEQ ID NO: 32; or(e) an anti-CD137 scFv 7 consisting of the amino acid sequence at amino acid positions 1-247 of SEQ ID NO: 34.

[0044] In the bispecific anti-CLDN4 / anti-CD137 antibody of the present invention, the anti-CLDN4 antibody or its antigen-binding fragment can be linked to the anti-CD137 antibody or its antigen-binding fragment via a linker. The type and length of the linker for linking the anti-CLDN4 antibody or its antigen-binding fragment to 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 can be used as the linker. The preferred length is 5 or more amino acids (wherein the upper limit is not particularly limited, but is usually 30 or less amino acids, and preferably 20 or less amino acids), and is 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 include the following:. In the above examples, n is an integer equal to 1 or more. In one aspect, n is from 1 to 10, from 2 to 8, or from 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 as a linker for linking the anti-CD137 antibody or antigen-binding fragment thereof to the anti-CD137 antibody or antigen-binding fragment thereof is a GS linker or a GKPGS linker, and in another embodiment, it is a linker having the amino acid sequence of SEQ ID NO: 54.

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

[0046] In one embodiment, the anti-CLBN4 / anti-CD137 bispecific antibody of the present invention is a CLDN4 / anti-CD137 bispecific antibody comprising an IgG anti-CLDN4 antibody and an anti-CD137 scFv, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain or the light chain of the IgG anti-CLDN4 antibody through a linker, and the heavy chain variable region and the light chain variable region of the anti-CD137 scEv are described in any one of the following (a) to (m): (a) a heavy chain variable region comprising CBR1 consisting of the amino acid sequence at amino acid positions 31 to 35 of SEQ IB NO: 10, CBR2 consisting of the amino acid sequence at amino acid positions 50 to 66 of SEQ IB NO: 10, and CBR3 consisting of the amino acid sequence at amino acid positions 99-107 of SEQ IB NO: 10, and a light chain variable region comprising CBR1 consisting of the amino acid sequence at amino acid positions 24-34 of SEQ IB NO: 12, CBR2,consisting of the amino acid sequence at amino acid positions 50-56 of SEQ ID NO: 12, and a CDR3 consisting of the amino acid sequence at amino acid positions 89-98 of SEQ ID NO: 12;(b) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence at amino acid positions 31-35 of SEQ ID NO: 14, a CDR2 consisting of the amino acid sequence at amino acid positions 50-65 of SEQ ID NO: 14, and a CDR3 consisting of the amino acid sequence at amino acid positions 98-107 of SEQ ID NO: 14, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence at amino acid positions 23-35 of SEQ ID NO: 16, a CDR2 consisting of the amino acid sequence at amino acid positions 51-57 of SEQ ID NO: 16, and a CDR3 consisting from the amino acid sequence at amino acid positions 90-100 of SEQ ID NO: 16;(c) a heavy chain variable region comprising CDR1,consisting of the amino acid sequence at amino acid positions 31-35 of SEQ ID NO: 18, a CDR2 consisting of the amino acid sequence at amino acid positions 50-65 of SEQ ID NO: 18, and a CDR3 consisting of the amino acid sequence at amino acid positions 98-107 of SEQ ID NO: 18, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence at amino acid positions 23-35 of SEQ ID NO: 20, a CDR2 consisting of the amino acid sequence at amino acid positions 51-57 of SEQ ID NO: 20, and a CDR3 consisting of the amino acid sequence at amino acid positions 90-100 of SEQ ID NO: 20;(d) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence at amino acid positions 31-35 of SEQ ID NO: 22, a CDR2 consisting from the amino acid sequence at amino acid positions 50-65 of SEQ ID NO: 22, and CDR3,consisting of the amino acid sequence at amino acid positions 98-110 of SEQ ID NO: 22, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence at amino acid positions 23-35 of SEQ ID NO: 24, a CDR2 consisting of the amino acid sequence at amino acid positions 51-57 of SEQ ID NO: 24, and a CDR3 consisting of the amino acid sequence at amino acid positions 90-100 of SEQ ID NO: 24;(e) a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 10, and a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 12;(f) a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 14, and the variable region of the light chain,consisting of the amino acid sequence at amino acid positions 1-111 of SEQ ID NO: 16;(g) a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 18, and a light chain variable region consisting of the amino acid sequence at amino acid positions 1-111 of SEQ ID NO: 20;(h) a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-121 of SEQ ID NO: 22, and a light chain variable region consisting of the amino acid sequence at amino acid positions 1-111 of SEQ ID NO: 24;(i) a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 26, and a light chain variable region consisting of the amino acid sequence at amino acid positions 134-242 SEQ ID NO: 26;(j) heavy chain variable region,consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 28, and a light chain variable region consisting of the amino acid sequence at amino acid positions 134-244 of SEQ ID NO: 28;(k) a light chain variable region consisting of the amino acid sequence at amino acid positions 1-111 of SEQ ID NO: 30, and a heavy chain variable region consisting of the amino acid sequence at amino acid positions 132-249 of SEQ ID NO: 30;(l) a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 32, and a light chain variable region consisting of the amino acid sequence at amino acid positions 134-244 of SEQ ID NO: 32; or (m) a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-121 of SEQ ID NO: 34, and a light chain variable region,consisting of the amino acid sequence at amino acid positions 137-247 of SEQ ID NO: 34.,

[0047] In one embodiment, the anti-CLDN4 / anti-CD137 bispecific antibody of the present invention is a anti-CLDN4 / anti-CD137 bispecific antibody comprising an IgG anti-CLDN4 antibody and an anti-CD137 scFv, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain or the light chain of the IgG anti-CLDN4 antibody via a linker, and the anti-CD137 scFv is described in any one of the following (a) to (e): (a) an anti-CD137 scFv consisting of the amino acid sequence at amino acid positions 1 to 242 of SEQ ID NO: 26; (b) an anti-CD137 scFv consisting of the amino acid sequence at amino acid positions 1 to 244 of SEQ ID NO: 28; (c) an anti-CD137 scFv consisting of the amino acid sequences at amino acid positions 1-249 of SEQ ID NO: 30;(d) an anti-CD137 scFv consisting of the amino acid sequence at amino acid positions 1-244 of SEQ ID NO: 32; or(e) an anti-CD137 scFv consisting of the amino acid sequence at amino acid positions 1-247 of SEQ ID NO: 34.

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

[0049] In one embodiment, the bispecific anti-CLDN4 / anti-CD137 antibody of the present invention is the bispecific anti-CLDN4 / anti-CD137 scFv described in any one of the following (a) to (j): (a) a bispecific antibody comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1 to 125 of SEQ ID NO: 2, and an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1 to 109 of SEQ ID NO: 4, and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1 to 118 of SEQ ID NO: 26, and a variable region a light chain consisting of the amino acid sequence at amino acid positions 134-242 of SEQ ID NO: 26,wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of an anti-CLDN4 antibody via a linker; (b) a bispecific antibody comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-125 of SEQ ID NO: 2 and an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 4 and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 28 and a light chain variable region consisting of the amino acid sequence at amino acid positions 134-244 of SEQ ID NO: 28, wherein the amino terminus an anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of an anti-CLDN4 antibody via a linker; (c) a bispecific antibody comprising the heavy chain of an anti-CLDN4 antibody,comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-125 of SEQ ID NO: 2, and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 4, and an anti-CD137 scFv comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-111 of SEQ ID NO: 30, and a heavy chain variable region consisting of the amino acid sequence at amino acid positions 132-249 of SEQ ID NO: 30, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker;(d) a bispecific antibody comprising a heavy chain of an anti-CLDN4 antibody comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-125 of SEQ ID NO: 2, and a light chain of an anti-CLDN4 antibody,comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 4, and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 32 and a light chain variable region consisting of the amino acid sequence at amino acid positions 134-244 of SEQ ID NO: 32, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of an anti-CLDN4 antibody via a linker; (e) a bispecific antibody comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-125 of SEQ ID NO: 2 and an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 4, and an anti-CD137 scFv comprising a heavy chain variable region,consisting of the amino acid sequence at amino acid positions 1-121 of SEQ ID NO: 34, and a light chain variable region consisting of the amino acid sequence at amino acid positions 137-247 of SEQ ID NO: 34, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of an anti-CLDN4 antibody via a linker; (f) a bispecific antibody comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-123 of SEQ ID NO: 6, and an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 8, and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 26, and a light chain variable region,consisting of the amino acid sequence at amino acid positions 134-242 of SEQ ID NO: 26, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of an anti-CLDN4 antibody via a linker; (g) a bispecific antibody comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-123 of SEQ ID NO: 6, and an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 8, and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 28, and a light chain variable region consisting of the amino acid sequence at amino acid positions 134-244 of SEQ ID NO: 28,wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of an anti-CLDN4 antibody via a linker; (h) a bispecific antibody comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-123 of SEQ ID NO: 6 and an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 8 and an anti-CD137 scFv comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-111 of SEQ ID NO: 30 and a heavy chain variable region consisting of the amino acid sequence at amino acid positions 132-249 of SEQ ID NO: 30, wherein the amino terminus an anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of an anti-CLDN4 antibody via a linker;(i) a bispecific antibody comprising the heavy chain of an anti-CLDN4 antibody,comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-123 of SEQ ID NO: 6, and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 8, and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 32, and a light chain variable region consisting of the amino acid sequence at amino acid positions 134-244 of SEQ ID NO: 32, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker;(j) a bispecific antibody comprising a heavy chain of an anti-CLDN4 antibody comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-123 of SEQ ID NO: 6, and a light chain of an anti-CLDN4 antibody,comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 8, and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-121 of SEQ ID NO: 34 and a light chain variable region consisting of the amino acid sequence at amino acid positions 137-247 of SEQ ID NO: 34, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of an anti-CLDN4 antibody via a linker; (k) a bispecific antibody comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-125 of SEQ ID NO: 2 and an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 4, and an anti-CD137 scFv comprising a heavy chain variable region,consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 26, and a light chain variable region consisting of the amino acid sequence at amino acid positions 134-242 of SEQ ID NO: 26, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of an anti-CLDN4 antibody via a linker; (l) a bispecific antibody comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-125 of SEQ ID NO: 2, and an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 4, and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 28, and a light chain variable region,consisting of the amino acid sequence at amino acid positions 134-244 of SEQ ID NO: 28, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of an anti-CLDN4 antibody via a linker; (m) a bispecific antibody comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-125 of SEQ ID NO: 2 and an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 4, and an anti-CD137 scFv comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-111 of SEQ ID NO: 30 and a heavy chain variable region consisting of the amino acid sequence at amino acid positions 132-249 of SEQ ID NO: 30,wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of an anti-CLDN4 antibody via a linker; (n) a bispecific antibody comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-125 of SEQ ID NO: 2 and an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 4 and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 32 and a light chain variable region consisting of the amino acid sequence at amino acid positions 134-244 of SEQ ID NO: 32, wherein the amino terminus an anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of an anti-CLDN4 antibody via a linker;(o) a bispecific antibody comprising the heavy chain of an anti-CLDN4 antibody,comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-125 of SEQ ID NO: 2, and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 4, and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-121 of SEQ ID NO: 34, and a light chain variable region consisting of the amino acid sequence at amino acid positions 137-247 of SEQ ID NO: 34, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker; (p) a bispecific antibody comprising a heavy chain of an anti-CLDN4 antibody comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-123 of SEQ ID NO: 6, and a light chain of an anti-CLDN4 antibody,comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 8, and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 26 and a light chain variable region consisting of the amino acid sequence at amino acid positions 134-242 of SEQ ID NO: 26, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of an anti-CLDN4 antibody via a linker via a linker; (q) a bispecific antibody comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-123 of SEQ ID NO: 6 and an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 8, and an anti-CD137 scFv,comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 28 and a light chain variable region consisting of the amino acid sequence at amino acid positions 134-244 of SEQ ID NO: 28, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of an anti-CLDN4 antibody via a linker; (r) a bispecific antibody comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-123 of SEQ ID NO: 6 and an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 8, and an anti-CD137 scFv comprising a variable region a light chain consisting of the amino acid sequence at amino acid positions 1-111 of SEQ ID NO: 30, and a heavy chain variable region,consisting of the amino acid sequence at amino acid positions 132-249 of SEQ ID NO: 30, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of an anti-CLDN4 antibody via a linker; (s) a bispecific antibody comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-123 of SEQ ID NO: 6, and an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 8, and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 32, and a light chain variable region consisting of the amino acid sequence at amino acid positions 134-244 of SEQ ID NO: 32,wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker; or (t) a bispecific antibody comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-123 of SEQ ID NO: 6 and an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 8 and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-121 of SEQ ID NO: 34 and a light chain variable region consisting of the amino acid sequence at amino acid positions 137-247 of SEQ ID NO: 34, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the anti-CLDN4 antibody heavy chain via a linker.

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

[0051] In the context of the present invention, the term “post-translational modification” refers to the fact that an antibody expressed in a cell is modified after translation. Examples of post-translational modification include modifications such as pyroglutamylation, glycosylation, oxidation, deamidation, or glycation of glutamine or glutamic acid at the N-terminus of the heavy chain, and removal of lysine by hydrolysis of lysine at the C-terminus of the heavy chain by carboxypeptidase. Such post-translational modification is known to occur in various antibodies (J. Pharm. Sci., 2008, Vol. 97, p. 2426-2447).

[0052] In one embodiment, the bispecific anti-CLDN4 / anti-CD137 antibody of the present invention may be post-translationally modified. In one embodiment, the post-translational modification is pyroglutamylation at the N-terminus of the heavy chain variable region and / or deletion of lysine at the C-terminus of the heavy chain. It is known in the art that post-translational modification by pyroglutamylation at the N-terminus or deletion of lysine at the C-terminus does not affect the activity of the antibody (Analytical Biochemistry, 2006, Vol. 348, p. 24-39).

[0053] The bispecific anti-CLDN4 / anti-CD137 antibody of the present invention binds to human CLDN4 and human CD137. Using a known method for evaluating binding activity, the extent to which the antibody binds or does not bind to human CLDN4 and human CD137 can be tested. Examples of the method for evaluating binding activity include ELISA and flow cytometry. ELISA or flow cytometry can be performed by a method commonly used by those skilled in the art, and a method such as that described in Examples 3 or 5 can be used.

[0054] The bispecific anti-CLDN4 / anti-CD137 antibody of the present invention can be easily produced by persons skilled in the art using human CLDN4 and human CD137 as antigens, using a method for producing antibodies known in the art, or can be easily produced by persons skilled in the art based on the sequence information and the like of the heavy chain variable regions and light chain variable regions of the anti-CLDN4 antibody and the anti-CD137 antibody described herein, using a method known in the art.

[0055] The bispecific anti-CLDN4 / anti-CD137 antibody of the present invention can be produced, for example, according to the method described in the section “Producing Method of the Bispecific Antibody of the Invention” described below, although the production method is not particularly limited to this method.

[0056] Polynucleotide of the bispecific antibody of the present inventionThe present invention also relates to a polynucleotide useful for producing the bispecific anti-CLDN4 / anti-CD137 antibody of the present invention (also referred to as the "polynucleotide 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 having a nucleotide sequence encoding a heavy chain variable region or a light chain variable region of an anti-CLDN4 antibody, which is selected from the group consisting of the following (a) to (d): (a) a polynucleotide having a nucleotide sequence encoding a heavy chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence at amino acid positions 1-125 of SEQ ID NO: 2; (b) a polynucleotide having a nucleotide sequence encoding a light chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 4;(c) a polynucleotide having a nucleotide sequence encoding a heavy chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence at amino acid positions 1-123 of SEQ ID NO: 6; or (d) a polynucleotide having a nucleotide sequence encoding a light chain variable region of an anti-CLDN4 antibody consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 8.

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

[0059] In one embodiment, the polynucleotide of the bispecific antibody of the present invention is a polynucleotide having a nucleotide sequence encoding a heavy chain variable region or a light chain variable region of an anti-CLDN4 antibody that is selected from the group consisting of (a) to (r) of the following: (a) a polynucleotide having a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 10; (b) a polynucleotide having a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 12;(c) a polynucleotide having a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 14;(d) a polynucleotide having a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence at amino acid positions 1-111 of SEQ ID NO: 16;(e) a polynucleotide having a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 18;(f) a polynucleotide having a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence at amino acid positions 1-111 of SEQ ID NO: 20;(g) a polynucleotide having a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence at amino acid positions 1-121 of SEQ ID NO: 22;(h) a polynucleotide having a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence at amino acid positions 1-111 of SEQ ID NO: 24;(i) a polynucleotide having a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 26;(j) a polynucleotide having a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence at amino acid positions 134-242 of SEQ ID NO: 26;(k) a polynucleotide having a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 28; (l) a polynucleotide having a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence at amino acid positions 134-244 of SEQ ID NO: 28; (m) a polynucleotide having a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence at amino acid positions 1-111 of SEQ ID NO: 30; (n) a polynucleotide having a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence at amino acid positions 132-249 SEQ ID NO: 30;(o) a polynucleotide having a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 32;(p) a polynucleotide having a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence at amino acid positions 134-244 of SEQ ID NO: 32;(q) a polynucleotide having a nucleotide sequence encoding a heavy chain variable region of an anti-CD137 antibody consisting of the amino acid sequence at amino acid positions 1-121 of SEQ ID NO: 34; or(r) a polynucleotide having a nucleotide sequence encoding a light chain variable region of an anti-CD137 antibody consisting of the amino acid sequence at amino acid positions 137-247 of SEQ ID NO: 34.;

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

[0061] In one embodiment, the polynucleotide of the bispecific antibody of the present invention is a polynucleotide having a nucleotide sequence encoding an anti-CD137 scFv that is selected from the group consisting of the following (a) to (e): (a) a polynucleotide having a nucleotide sequence encoding an anti-CD137 scFv consisting of the amino acid sequence at amino acid positions 1-242 of SEQ ID NO: 26; (b) a polynucleotide having a nucleotide sequence encoding an anti-CD137 scFv consisting of the amino acid sequence at amino acid positions 1-244 of SEQ ID NO: 28; (c) a polynucleotide having a nucleotide sequence encoding an anti-CD137 scFv consisting of the amino acid sequence at amino acid positions 1-249 of SEQ ID NO: 30;(d) a polynucleotide having a nucleotide sequence encoding an anti-CD137 scFv consisting of the amino acid sequence at amino acid positions 1-244 of SEQ ID NO: 32; or(e) a polynucleotide having a nucleotide sequence encoding an anti-CD137 scFv consisting of the amino acid sequence at amino acid positions 1-247 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): (a) a polynucleotide having a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence at amino acid positions 1-705 of SEQ ID NO: 36, comprising a heavy chain of an anti-CLDN4 antibody and an anti-CD137 scFv; (b) a polynucleotide having a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence at amino acid positions 1-712 of SEQ ID NO: 38, comprising a heavy chain of an anti-CLDN4 antibody and an anti-CD137 scFv; (c) a polynucleotide having a nucleotide sequence encoding a light chain of an anti-CLDN4 antibody consisting of the amino acid sequence at amino acid positions 1-215 of SEQ ID NO: 40;(d) a polynucleotide having a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence at amino acid positions 1-705 of SEQ ID NO: 36, comprising the heavy chain of an anti-CLDN4 antibody and an anti-CD137 scFv, and a polynucleotide having a nucleotide sequence encoding the light chain of an anti-CLDN4 antibody consisting of the amino acid sequence at amino acid positions 1-215 of SEQ ID NO: 40; or(e) a polynucleotide having a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence at amino acid positions 1-712 of SEQ ID NO: 38, containing the heavy chain of an anti-CLDN4 antibody and an anti-CD137 scFv, and a polynucleotide having a nucleotide sequence encoding the light chain of an anti-CLDN4 antibody, consisting of the amino acid sequence at amino acid positions 1-215 of SEQ ID NO: 40.;

[0063] The bispecific antibody polynucleotide of the present invention can be easily obtained by those skilled in the art based on the nucleotide sequence using a method known in the art. For example, the bispecific antibody polynucleotide of the present invention can be synthesized using a gene synthesis method known in the art. Various methods known to those skilled in the art can be used as such a gene synthesis method, such as the antibody gene synthesis method described in WO90 / 07861.

[0064] Expression vector of the bispecific antibody of the inventionThe present invention also relates to an expression vector containing a polynucleotide of the bispecific antibody of the present invention (also referred to as the "expression vector of the bispecific antibody of the present invention"). These polynucleotides may be respectively contained in different vectors, or a number of polynucleotides may be contained in a single vector.

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

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

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

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

[0069] The expression vector of the bispecific antibody of the present invention is not particularly limited, provided that it can produce the polynucleotide of the present invention in various host cells such as a eukaryotic cell (e.g., an animal cell, an insect cell, a plant cell, or yeast) and / or a prokaryotic cell (such as E. coli). Examples of such an expression vector include a plasmid vector and a viral vector. As the plasmid vector, for example, pcDNA series vectors (Thermo Fisher Scientific), pALTER (R)-MAX (Promega Corporation), pHEK293 Ultra Expression Vector (Takara Bio Inc.), pEE 6.4 or pEE 12.4 (Lonza Biologies), or the like can be used. As the viral vector, for example, a lentivirus, adenovirus, retrovirus, or adeno-associated virus-based vector can be used.For example, when a lentivirus is used to introduce the polynucleotide of the present invention into a cell, the pLVSIN-CMV / EF1α vector (Takara Bio Inc.), the pLenti vector (Thermo Fisher Scientific), or the like can be used as the lentivirus. In one embodiment, the vector used as the expression vector of the bispecific antibody of the present invention is pcDNA 3.4-TOPO (R) (Thermo Fisher Scientific) or pcDNA 3.1 (Thermo Fisher Scientific).

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

[0071] When an animal cell, an insect cell, or yeast is used as the host cell, the expression vector for the bispecific antibody of the present invention may comprise a start codon and a stop codon. In this case, an enhancer sequence, 5' and 3' untranslated regions of the gene encoding the antibody of the present invention or its heavy or light chain, a secretory signal sequence, a splice junction, a polyadenylation site, a replication unit (replicon), and the like may be contained. When E. coli is used as the host cell, the expression vector of the present invention may comprise a start codon, a stop codon, a terminator region, and a replication unit (replicon).The expression vector of the present invention may comprise a drug resistance selection marker gene commonly used depending on the purpose (e.g., a tetracycline resistance gene, an ampicillin resistance gene, a kanamycin resistance gene, a neomycin resistance gene, or a dihydrofolate reductase gene).

[0072] Host Cell of the InventionThe present invention also relates to a host cell transformed with a polynucleotide of the bispecific antibody of the present invention or an expression vector of the bispecific antibody of the present invention (also referred to as "a polynucleotide or the like of the present invention") (also referred to as "a host cell of the present invention"). The host cell of the present invention contains a polynucleotide or the like of the present invention. The introduced polynucleotide or the like of the present invention may or may not integrate into the genomic DNA of the host cell. The cell used as the host cell may be either a cell that can be cultured in vitro or an in vivo cell.When the host cell is a cell that can be cultured in vitro, the host cell of the present invention can be obtained by introducing a polynucleotide or the like of the present invention into the cell in vitro. The method for transforming the host cell is not particularly limited, and, for example, a method commonly used by those skilled in the art, such as a calcium phosphate method, an electroporation-based method, or a lipofection-based method, can be used. When the host cell is an in vivo cell, the method for introducing a polynucleotide or the like of the present invention into the host cell is not particularly limited, and a nucleic acid delivery carrier (a cationic carrier or a non-cationic carrier (for example, including, but not limited to, a liposome and a lipid nanoparticle (LNP)) and the like) can be used.

[0073] A cell that can be cultured in vitro is not particularly limited, as long as it can be transformed with the expression vector used or by a method such as electroporation to express an antibody or a polypeptide. Examples of cells that can be cultured in vitro include various cells, including ordinary cells commonly used in the art and artificially generated cells (e.g., animal cells (such as CHO-K1 cell, ExpiCHO-S(R) cell and CHOK1SV cell, CHO-DG44 cell, HEK293 cell, Expi293F cell and NS0 cell), insect cells (such as Sf9), bacteria (e.g., belonging to the genus Escherichia) and yeast (e.g., cells belonging to the genus Saccharomyces and the genus Pichia)). In one embodiment, the host cell of the present invention is an Expi293F cell, a CHO-K1 cell, or an ExpiCHO-S cell.

[0074] In vitro selection of the transformed host cell can be carried out by a method commonly used by those skilled in the art. The selection method can be, for example, a drug resistance selection method using a drug resistance selection marker gene such as tetracycline, ampicillin, neomycin, or hygromycin, or a cell isolation method such as the limiting dilution method, single cell sorting method, or colony picking method.

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

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

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

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

[0079] In one embodiment of the host cell of the present invention, the host cell comprises the polynucleotides described in the following (a) to (j): (a) a polynucleotide having a nucleotide sequence encoding a polypeptide comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1 to 125 of SEQ ID NO: 2, and an anti-CD137 scFv comprising an anti-CD137 antibody heavy chain variable region consisting of the amino acid sequence at amino acid positions 1 to 118 of SEQ ID NO: 26, and an anti-CD137 antibody light chain variable region consisting of the amino acid sequence at amino acid positions 134 to 242 of SEQ ID NO: 26, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus heavy chain of anti-CLDN4 antibody via a linker;and a polynucleotide having a nucleotide sequence encoding an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 4;(b) a polynucleotide having a nucleotide sequence encoding a polypeptide comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-125 of SEQ ID NO: 2, and an anti-CD137 scFv comprising an anti-CD137 antibody heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 28, and an anti-CD137 antibody light chain variable region consisting of the amino acid sequence at amino acid positions 134-244 of SEQ ID NO: 28, wherein the amino terminus anti-CD137 scFv is linked to the carboxyl terminus of the heavy chain of the anti-CLDN4 antibody via a linker;and a polynucleotide having a nucleotide sequence encoding an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 4;(c) a polynucleotide having a nucleotide sequence encoding a polypeptide comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-125 of SEQ ID NO: 2, and an anti-CD137 scFv comprising an anti-CD137 antibody light chain variable region consisting of the amino acid sequence at amino acid positions 1-111 of SEQ ID NO: 30, and an anti-CD137 antibody heavy chain variable region consisting of the amino acid sequence at amino acid positions 132-249 of SEQ ID NO: 30, wherein the amino terminus anti-CD137 scFv is linked to the carboxyl terminus of the heavy chain of the anti-CLDN4 antibody via a linker;and a polynucleotide having a nucleotide sequence encoding an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 4;(d) a polynucleotide having a nucleotide sequence encoding a polypeptide comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-125 of SEQ ID NO: 2, and an anti-CD137 scFv comprising an anti-CD137 antibody heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 32, and an anti-CD137 antibody light chain variable region consisting of the amino acid sequence at amino acid positions 134-244 of SEQ ID NO: 32, wherein the amino terminus anti-CD137 scFv is linked to the carboxyl terminus of the heavy chain of the anti-CLDN4 antibody via a linker;and a polynucleotide having a nucleotide sequence encoding an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 4;(e) a polynucleotide having a nucleotide sequence encoding a polypeptide that comprises an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-125 of SEQ ID NO: 2, and an anti-CD137 scFv comprising an anti-CD137 antibody heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-121 of SEQ ID NO: 34, and an anti-CD137 antibody light chain variable region consisting of the amino acid sequence at amino acid positions 137-247 of SEQ ID NO: 34, wherein the amino terminus anti-CD137 scFv is linked to the carboxyl terminus of the heavy chain of the anti-CLDN4 antibody via a linker;and a polynucleotide having a nucleotide sequence encoding an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 4;(f) a polynucleotide having a nucleotide sequence encoding a polypeptide comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-123 of SEQ ID NO: 6, and an anti-CD137 scFV comprising an anti-CD137 antibody heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 26, and an anti-CD137 antibody light chain variable region consisting of the amino acid sequence at amino acid positions 134-242 of SEQ ID NO: 26, wherein the amino terminus anti-CD137 scFv is linked to the carboxyl terminus of the heavy chain of the anti-CLDN4 antibody via a linker;and a polynucleotide having a nucleotide sequence encoding an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 8; (g) a polynucleotide having a nucleotide sequence encoding a polypeptide comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-123 of SEQ ID NO: 6, and an anti-CD137 scFv comprising an anti-CD137 antibody heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 28, and an anti-CD137 antibody light chain variable region consisting of the amino acid sequence at amino acid positions 134-244 of SEQ ID NO: 28, wherein the amino terminus anti-CD137 scFv is linked to the carboxyl terminus of the heavy chain of the anti-CLDN4 antibody via a linker;and a polynucleotide having a nucleotide sequence encoding an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 8;(h) a polynucleotide having a nucleotide sequence encoding a polypeptide comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-123 of SEQ ID NO: 6, and an anti-CD137 scFv comprising an anti-CD137 antibody light chain variable region consisting of the amino acid sequence at amino acid positions 1-111 of SEQ ID NO: 30, and an anti-CD137 antibody heavy chain variable region consisting of the amino acid sequence at amino acid positions 132-249 of SEQ ID NO: 30, where the amino terminus anti-CD137 scFv is linked to the carboxyl terminus of the heavy chain of the anti-CLDN4 antibody via a linker;and a polynucleotide having a nucleotide sequence encoding an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 8;(i) a polynucleotide having a nucleotide sequence encoding a polypeptide comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-123 of SEQ ID NO: 6, and an anti-CD137 scFV comprising an anti-CD137 antibody heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 32, and an anti-CD137 antibody light chain variable region consisting of the amino acid sequence at amino acid positions 134-244 of SEQ ID NO: 32, wherein the amino terminus anti-CD137 scFv is linked to the carboxyl terminus of the heavy chain of the anti-CLDN4 antibody via a linker;and a polynucleotide having a nucleotide sequence encoding a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of an amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 8; or(j) a polynucleotide having a nucleotide sequence encoding a polypeptide comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-123 of SEQ ID NO: 6, and an anti-CD137 scFv comprising an anti-CD137 antibody heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-121 of SEQ ID NO: 34, and an anti-CD137 antibody light chain variable region consisting of the amino acid sequence at amino acid positions 137-247 of SEQ ID NO: 34, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the anti-CLDN4 antibody heavy chain via a linker;and a polynucleotide having a nucleotide sequence encoding a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of an amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 8.;

[0080] In one embodiment of the host cell of the present invention, the host cell is a host cell described in the following (a) or (b): (a) a host cell comprising a polynucleotide having a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence at amino acid positions 1-705 of SEQ ID NO: 36, comprising a heavy chain of an anti-CLDN4 antibody and an anti-CD137 scFv, and a polynucleotide having a nucleotide sequence encoding a light chain of an anti-CLDN4 antibody consisting of the amino acid sequence at amino acid positions 1-215 of SEQ ID NO: 40;or(b) a host cell comprising a polynucleotide having a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence at amino acid positions 1-712 of SEQ ID NO: 38, comprising the heavy chain of an anti-CLDN4 antibody and an anti-CD137 scFv, a polynucleotide having a nucleotide sequence encoding the light chain of an anti-CLDN4 antibody consisting of the amino acid sequence at amino acid positions 1-215 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 having a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence at amino acid positions 1-712 of SEQ ID NO: 38, comprising the heavy chain of an anti-CLDN4 antibody and an anti-CD137 scFv, and a polynucleotide having a nucleotide sequence encoding a light chain of an anti-CLDN4 antibody consisting of the amino acid sequence at amino acid positions 1-215 of SEQ ID NO: 40.

[0082] Method for Producing a Bispecific Antibody of the InventionThe present invention also relates to a method for producing the bispecific anti-CLDN4 / anti-CD137 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 a 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 a host cell of the present invention, as described above. Furthermore, the production method of the present invention may include a step of culturing the host cell described above in the "Host Cell of the Invention" section to express the antibody in the cell or culture supernatant, a method for collecting, isolating, and purifying the antibody, and the like.However, the production method of the present invention is not limited to these methods, as long as the bispecific anti-CLDN4 / anti-CD137 antibody of the present invention is produced.

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

[0084] In one embodiment, the production method of the present invention includes a step of culturing the host cell described in the following (a) or (b): (a) a host cell comprising a polynucleotide having a nucleotide sequence encoding a polypeptide consisting of an amino acid sequence at amino acid positions 1-705 of SEQ ID NO: 36, comprising a heavy chain of an anti-CLDN4 antibody and an anti-CD137 scFv, and a polynucleotide having a nucleotide sequence encoding a light chain of an anti-CD137 antibody consisting of an amino acid sequence at amino acid positions 1-215 of SEQ ID NO: 40;or(b) a host cell comprising a polynucleotide having a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence at amino acid positions 1-712 of SEQ ID NO: 38, comprising the heavy chain of an anti-CLDN4 antibody and an anti-CD137 scFv, and a polynucleotide having a nucleotide sequence encoding a light chain of an anti-CLDN4 antibody consisting of the amino acid sequence at amino acid positions 1-215 of SEQ ID NO: 40.

[0085] The host cell of the present invention can be cultured by a known method. Culture conditions such as temperature, pH of the medium, and culture time can be appropriately selected by those skilled in the art. When the host cell is, for example, an animal cell, MEM medium (Science, 1959, Vol. 130, pp. 432-437) containing about 5% to 20% fetal bovine serum, D-MEM medium (Virol., 1959, Vol. 8, p. 396), RPMI-1640 medium (J. Am. Med. Assoc., 1967, Vol. 199, p. 519), 199 medium (Exp. Biol. Med., 1950, Vol. 73, pp. 1-8) or the like can be used. The pH of the medium is, for example, about 6 to 8, and the culture is usually carried out at a temperature of about 30°C to 40°C for about 15 to 336 hours with aeration or stirring, if necessary. When the host cell is an insect cell, the medium can be, for example, Grace's medium (Proc. Natl. Acad. Sci. USA., 1985, Vol. 82, p.8404) containing fetal bovine serum or the like. The pH of the medium is, for example, about 5 to 8, and the culturing is usually carried out at a temperature of about 20°C to 40°C for about 15 to 100 hours with aeration or stirring, if necessary. When the host cell is, for example, E. coli or yeast, a liquid medium containing a nutrient source is suitably used as the medium. The nutrient medium contains, for example, a carbon source, an inorganic nitrogen source, or an organic nitrogen source necessary for the growth of the transformed host cell. Examples of the carbon source include glucose, dextran, soluble starch, and sucrose, examples of the inorganic nitrogen source or the organic nitrogen source include ammonium salts, nitric acid salts, amino acids, corn steep liquor, peptone, casein, meat extract, soybean meal, and potato extract.If necessary, other nutrients (e.g., inorganic salts (such as calcium chloride, sodium dihydrogen phosphate, and magnesium chloride) or vitamins), an antibiotic (such as tetracycline, neomycin, ampicillin, or kanamycin), or the like may be contained. The pH of the medium is, for example, about 5 to 8. When the host cell is E. coli, the medium can be, for example, LB medium, M9 medium (Molecular Cloning, Cold Spring Harbor Laboratory, Vol. 3, A2.2), or the like. Cultivation is usually carried out at a temperature of 14°C to 43°C for about 3 to 24 hours with aeration or stirring if necessary. When the host cell is yeast, the medium can be, for example, Burkholder's minimal medium (Proc. Natl. Acad. Sci. USA., 1980, Vol. 77, p. 4505) or the like. Cultivation is usually carried out at a temperature of 20°C to 35°C for approximately 14-144 hours with aeration or stirring, if necessary.By such culturing, the bispecific anti-CLBN4 / anti-CD137 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 cell of the present invention to express the bispecific anti-CLBN4 / anti-CD137 antibody, a step of collecting, isolating or purifying the anti-CLBN4 / anti-CD137 antibody from the host cell. Examples of the isolation or purification method include a solubility-based method such as a salting-out method or a solvent precipitation method, a molecular weight-based method such as dialysis, ultrafiltration, and gel filtration, a charge-based method such as ion-exchange chromatography or hydroxyapatite chromatography, a specific affinity-based method such as affinity chromatography, a hydrophobicity-based method such as reversed-phase high-performance liquid chromatography, and an isoelectric point-based method such as isoelectric focusing.In one embodiment, the antibody secreted into the culture supernatant can be purified by various chromatographies, such as column chromatography using a protein A column or a protein G column.

[0087] The bispecific anti-CLDN4 / anti-CD137 antibody of the present invention includes a bispecific anti-CLDN4 / anti-CD137 antibody and a bispecific anti-CLDN4 / anti-CD137 scFv obtained by the production method of the present invention.

[0088] Pharmaceutical Composition or the Like According to the InventionThe present invention also relates to a pharmaceutical composition or the like containing the bispecific anti-CLDN4 / anti-CD137 antibody of the present invention (also referred to as the "pharmaceutical composition of the present invention"). The pharmaceutical composition of the present invention includes a pharmaceutical composition containing the bispecific anti-CLDN4 / anti-CD137 antibody of the present invention and a pharmaceutically acceptable excipient. The pharmaceutical composition of the present invention can be prepared by a commonly used method with an excipient commonly used in the art, namely, a pharmaceutical excipient, a pharmaceutical carrier, and the like.Examples of the dosage form of such a pharmaceutical composition include parenteral formulations such as injections and drops, and administration can be carried out by a suitable route such as intravenous, subcutaneous, intraperitoneal, or intratumoral administration. The composition can use an excipient, carrier, additive, etc. suitable for the dosage form within a pharmaceutically acceptable range.

[0089] The pharmaceutical composition of the present invention may comprise a post-translationally modified product of the bispecific anti-CLDN4 / anti-CD137 antibody of the present invention. For example, the present invention may encompass a pharmaceutical composition comprising an antibody or the like comprising either a lysine deletion at the C-terminus or pyroglutamylation at the N-terminus.

[0090] In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition comprising the bispecific anti-CLDN4 / anti-CD137 antibody of the present invention and / or a post-translationally modified antibody product described in any one of the following (a) to (j): (a) a bispecific anti-CLDN4 / anti-CD137 antibody and / or a post-translationally modified antibody product comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1 to 125 of SEQ ID NO: 2 and an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1 to 109 of SEQ ID NO: 4 and an anti-CD137 scFv comprising a heavy chain variable region consisting of amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 26, and a light chain variable region,consisting of the amino acid sequence at amino acid positions 134-242 of SEQ ID NO: 26, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of an anti-CLDN4 antibody via a linker; (b) a bispecific anti-CLDN4 antibody and / or a post-translationally modified antibody product comprising a heavy chain of an anti-CLDN4 antibody comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-125 of SEQ ID NO: 2, and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 4, and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 28, and a light chain variable region consisting of the amino acid sequence at amino acid positions 134-244 of SEQ ID NO: 28,wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of an anti-CLDN4 antibody via a linker; (c) a bispecific anti-CLDN4 / anti-CD137 antibody and / or a post-translationally modified antibody product comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-125 of SEQ ID NO: 2 and an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 4 and an anti-CD137 scFv comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-111 of SEQ ID NO: 30 and a heavy chain variable region consisting of the amino acid sequence at amino acid positions 132-249 SEQ ID NO: 30,wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker; (d) a bispecific anti-CLDN4 / anti-CD137 antibody and / or a post-translationally modified antibody product comprising a heavy chain of an anti-CLDN4 antibody comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-125 of SEQ ID NO: 2, and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 4, and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 32, and a light chain variable region consisting of the amino acid sequence at amino acid positions 134-244 SEQ ID NO: 32,wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker; (e) a bispecific anti-CLDN4 / anti-CD137 antibody and / or a post-translationally modified antibody product comprising a heavy chain of an anti-CLDN4 antibody comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-125 of SEQ ID NO: 2, and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 4, and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-121 of SEQ ID NO: 34, and a light chain variable region consisting of the amino acid sequence at amino acid positions 137-247 SEQ ID NO: 34,wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of an anti-CLDN4 antibody via a linker; (f) a bispecific anti-CLDN4 / anti-CD137 antibody and / or a post-translationally modified antibody product comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-123 of SEQ ID NO: 6 and an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 8 and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 26 and a light chain variable region consisting of the amino acid sequence at amino acid positions 134-242 SEQ ID NO: 26,wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of an anti-CLDN4 antibody via a linker; (g) a bispecific anti-CLDN4 / anti-CD137 antibody and / or a post-translationally modified antibody product comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-123 of SEQ ID NO: 6 and an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 8 and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 28 and a light chain variable region consisting of the amino acid sequence at amino acid positions 134-244 SEQ ID NO: 28,wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of an anti-CLDN4 antibody via a linker; (h) a bispecific anti-CLDN4 / anti-CD137 antibody and / or a post-translationally modified antibody product comprising a heavy chain of an anti-CLDN4 antibody comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-123 of SEQ ID NO: 6, and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 8, and an anti-CD137 scFv comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-111 of SEQ ID NO: 30, and a heavy chain variable region consisting of the amino acid sequence at amino acid positions 132-249 SEQ ID NO: 30,wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of an anti-CLDN4 antibody via a linker; (i) a bispecific anti-CLDN4 / anti-CD137 antibody and / or a post-translationally modified antibody product comprising a heavy chain of an anti-CLDN4 antibody comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-123 of SEQ ID NO: 6, and a light chain of an anti-CLDN4 antibody comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 8, and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 32, and a light chain variable region consisting of the amino acid sequence at amino acid positions 134-244 SEQ ID NO: 32,wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain of the anti-CLDN4 antibody via a linker; or(j) a bispecific anti-CLDN4 / anti-CD137 antibody and / or post-translationally modified antibody product comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-123 of SEQ ID NO: 6 and an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 8 and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-121 of SEQ ID NO: 34 and a light chain variable region consisting of the amino acid sequence at amino acid positions 137-247 of SEQ ID NO: 34, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxyl terminus of the heavy chain of the anti-CLDN4 antibody via a linker.

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

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

[0093] Pharmaceutical use of the bispecific anti-CLDN4 / anti-CD137 antibody of the inventionThe bispecific anti-CLDN4 / anti-CD137 antibody of the present invention and a pharmaceutical composition containing the same can be used to treat cancer in a subject. Furthermore, the present invention includes a method for treating cancer, comprising the step of administering to the subject a therapeutically effective amount of the bispecific anti-CLDN4 / anti-CD137 antibody of the present invention. Furthermore, the present invention covers the bispecific anti-CLDN4 / anti-CD137 antibody of the present invention for use in the treatment of cancer. Furthermore, the present invention includes the use of the bispecific anti-CLDN4 / anti-CD137 antibody of the present invention in the manufacture of a pharmaceutical composition for the treatment of cancer. The cancer to be treated by the present invention is not particularly limited, and examples thereof include various metastatic peritoneal cancers, gastric cancer,lung cancer, blood cancer such as acute lymphoblastic leukemia, acute myelogenous leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, multiple myeloma, and T-cell lymphoma, solid cancers such as myelodysplastic syndrome, adenocarcinoma, squamous cell carcinoma, adenosquamous cell carcinoma, undifferentiated carcinoma, large cell carcinoma, non-small cell lung cancer, small cell lung cancer, mesothelioma, skin cancer, cutaneous T-cell lymphoma, breast cancer, prostate cancer, bladder cancer, vaginal cancer, cervical cancer, head and neck cancer, uterine cancer, cervical cancer, liver cancer, gallbladder cancer, bile duct cancer, kidney cancer, pancreatic cancer, colon cancer, colorectal cancer, rectal cancer, cancer small intestine cancer, stomach cancer, esophageal cancer, testicular cancer, ovarian cancer and brain tumor, cancer of bone tissue, cartilage tissue, adipose tissue, muscle tissue,vascular tissue and hematopoietic tissue, sarcomas such as chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma and soft tissue sarcoma, and blastomas such as glioblastoma, glioblastoma multiforme, hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma and retinoblastoma. In one embodiment, the cancer treated according to the present invention is colorectal cancer, non-small cell lung cancer, small cell lung cancer, bladder cancer, ovarian cancer, breast cancer or prostate cancer. In one embodiment, the cancer to be treated according to the present invention is a cancer in which CLDN4 is expressed at a high level compared to normal tissue. The cancer to be treated according to the present invention is preferably a cancer in which CLDN4 is expressed at a high level compared to normal tissue,or a cancer selected from the group consisting of colorectal cancer, rectal cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, bladder cancer, ovarian cancer, breast cancer, and prostate cancer.

[0094] Anti-CLDN4 antibody of the inventionThe present invention also relates to an anti-CLDN4 antibody or antigen-binding fragment thereof described in any one of the following (a) to (c): (a) an anti-CLDN4 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence at amino acid positions 31 to 35 of SEQ ID NO: 2, a CDR2 consisting of the amino acid sequence at amino acid positions 50 to 66 of SEQ ID NO: 2, and a CDR3 consisting of the amino acid sequence at amino acid positions 99 to 114 of SEQ ID NO: 2, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence at amino acid positions 24 to 35 of SEQ ID NO: 4, a CDR2 consisting of the amino acid sequence at amino acid positions 51 to 57 of SEQ ID NO: 4, and CDR3 consisting of the amino acid sequence at amino acid positions 90-98 of SEQ ID NO: 4;(b) an anti-CLDN4 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-125 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 4; or (c) an anti-CLDN4 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-123 of SEQ ID NO: 6 and a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 8.

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

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

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

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

[0099] The present invention also relates to an anti-CLDN4 antibody of the present invention or an antigen-binding fragment thereof to which a carbohydrate, a lipid, a metal (including a radioisotope), an organic compound (including a toxin, a near-infrared fluorescent dye and a chelating agent) or the like (also referred to as a “modifier”) is bound (also referred to as a “complex of the present invention”). In the context of the present invention, the term “modifier” refers to a non-peptide substance bound to the antibody or an antigen-binding fragment thereof directly or through a linker and the like. The modifier for use in the complex of the present invention is not particularly limited, and examples thereof include polyethylene glycol, sugar chains, phospholipids, radioactive isotopes (e.g., zirconium-89 ( 89 Zr), yttrium-90 ( 90 Y), indium-111 ( 111 In), astatine-211 ( 211 At) and actinium-225 ( 225Ac)), organic compounds, toxins, near-infrared fluorescent dyes (e.g., IRDye(R)), chelating agents. The modifier for use in the complex may be directly linked to the anti-CLDN4 antibody of the present invention or an antigen-binding fragment thereof or may be linked thereto through any linker. In one embodiment, the complex of the present invention is an antibody-drug conjugate (ADC) of an anti-CLDN4 antibody or an antigen-binding fragment thereof. The chemical drug and linker for use in the ADC may be selected from drugs and linkers commonly used by those skilled in the art.

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

[0101] In one embodiment, the anti-CLDN4 antibody of the present invention or an antigen-binding fragment thereof, the complex of the present invention and a portion of the antibody or antigen-binding fragment in a cell expressing the anti-CLDN4 antibody of the present invention or an antigen-binding fragment thereof on the cell surface may be post-translationally modified. In one embodiment, the post-translational modification is pyroglutamylation at the N-terminus of the heavy chain variable region and / or deletion of lysine at the C-terminus of the heavy chain.

[0102] The anti-CLDN4 antibody of the present invention or its antigen-binding fragment, the complex of the present invention, and the cell expressing the anti-CLDN4 antibody of the present invention or its antigen-binding fragment on its cell surface can be obtained by those skilled in the art based on the VH and VL sequence information of the anti-CLDN4 antibody of the present invention or its antigen-binding fragment and the information on the modifier for use in the complex of the present invention disclosed herein, using a method known in the art. The anti-CLDN4 antibody of the present invention or its antigen-binding fragment can be obtained, for example, according to the method described in the section "Method for Producing a Bispecific Antibody of the Present Invention", although it is not particularly limited thereto.

[0103] The present invention also relates to a pharmaceutical composition comprising the anti-CLDN4 antibody of the present invention or an antigen-binding fragment thereof, the complex of the present invention, and a cell expressing the anti-CLDN4 antibody of the present invention or an antigen-binding fragment thereof on its cell surface (hereinafter collectively referred to as the "anti-CLDN4 antibody etc. of the present invention" in this section), and a pharmaceutically acceptable excipient. The pharmaceutical composition can be used for the treatment of cancer. The present invention further relates to a method for treating cancer, comprising the step of administering a therapeutically effective amount of the anti-CLDN4 antibody etc. of the present invention to a subject, the anti-CLDN4 antibody etc. of the present invention for use in the treatment of cancer, and for use in the production of a pharmaceutical composition for the treatment of cancer of the anti-CLDN4 antibody etc. of the present invention.Pharmaceutical use of the anti-CLDN4 antibody and the like according to the present invention can be carried out by those skilled in the art, as described in the section "Pharmaceutical Composition or the Like According to the Invention" described above. Examples of cancers to be treated by pharmaceutical use of the anti-CLDN4 antibody and the like according to the present invention include the cancers described above in the section "Pharmaceutical Composition or the Like According to the Invention."

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

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

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

[0107] The anti-CD137 antibody of the present invention or an antigen-binding fragment thereof can be produced by those skilled in the art with reference to the section "Bispecific anti-CD137 / anti-CD137 Antibody of the Invention" and the like as described herein. The anti-CD137 antibody of the present invention or an antigen-binding fragment thereof can be used in a bispecific antibody with an antibody to any TAA (anti-TAA antibody), for example, used in the treatment of cancer. In other words, the present invention also relates to a bispecific antibody comprising the anti-CD137 antibody of the present invention or an antigen-binding fragment thereof and an anti-TAA antibody or an antigen-binding fragment thereof (also referred to as the "bispecific anti-TAA / anti-CD137 antibody of the present invention").TAA is not particularly limited if it is expressed on the cell surface of the tumor, and 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 can be used, for example.In one embodiment, the bispecific anti-TAA / anti-CD137 antibody of the present invention comprises an anti-CD137 antibody scFv, and in another embodiment, the bispecific anti-TAA / anti-CD137 antibody of the present invention comprises an anti-CD137 antibody scFv described in any one of the following (a) to (e): (a) an anti-CD137 scFv consisting of the amino acid sequence at amino acid positions 1-242 of SEQ ID NO: 26; (b) an anti-CD137 scFv consisting of the amino acid sequence at amino acid positions 1-244 of SEQ ID NO: 28; (c) an anti-CD137 scFv consisting of the amino acid sequence at amino acid positions 1-249 of SEQ ID NO: 30; (d) an anti-CD137 scFv consisting of the amino acid sequence at amino acid positions 1-244 of SEQ ID NO: 32; or(e) an anti-CD137 scFv consisting of the amino acid sequence at amino acid positions 1-247 of SEQ ID NO: 34.

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

[0109] Specific examples will now be provided, which should be referred to for a further understanding of the present invention, and it should be noted that these examples are merely illustrative and do not limit the present invention. Examples

[0110] Examples 1: Production of an antibody that selectively binds to an antigen expressed in a peritoneal metastatic tumor cell1-1. Preparation of patient-derived peritoneal metastatic tumor cellsPeritoneal metastatic tumor cells from the patient were obtained according to the method described in the literature by Fumiko Chiwaki and Hiroki Sasaki “Fukumaku Teni Gan (I Sui, Ranso Gan Nado) Saibo Kabu No Juritsu (in Japanese) (Establishment of Peritoneal Metastasis Cancer (such as Gastric, Pancreatic abd Ovarian Cancer) Cell Lines” (Practical Guide for Cancer Research using Patient-Derived Experimental Model, edited by Hiroki Sasaki, Yodosha Company, Ltd., 2019, p. 28-37).Ascites fluid collected from the patient was centrifuged at room temperature and 430× g for 3 min, the supernatant was removed, and hemolysis buffer was added to the pellet to induce hemolysis. After centrifugation The supernatant was removed and Dulbecco's PBS (-) (Nissui Pharmaceutical Co.) was added., Ltd., 05913; hereinafter referred to as “PBS(-)”) in an amount of 50 ml to wash the cells. Thereafter, the resulting cells were collected by centrifugation at room temperature and 430× g for 3 min. The whole cells in the collected ascites fluid were resuspended in RPMI-1640 medium (containing L-glutamine) (FUJIFILM Wako Pure Chemical Corporation, 189-02025) containing 10% FBS (Thermo Fisher Scientific, 10270-106) and ×1 antibiotic-antimycotic mixture (Thermo Fisher Scientific, 15240062) (where the medium after adding FBS etc. in Example 1 is hereinafter referred to as “RPMI-1640 medium”). The suspension was diluted, and the cells were seeded at a number of 5×10. 6 up to 1×10 7cells / 10 ml in each case per 100 mm diameter collagen-coated dish (hereinafter referred to as “dish”) (IWAKI, 4020-010) for culture at 37°C in a 5% CO2 incubator. There are adherent cells and floating cells in the ascites fluid, where the adherent cells include not only tumor cells but also non-tumor cells (such as fibroblasts and peritoneal mesothelial cells). Non-tumor cells have the ability to detach in a shorter time than tumor cells, and therefore, these cells were removed by separating them from whole cells in the ascites fluid. After removing the non-tumor cells, tumor cells were cultured to approximately 80% confluency on the dish area, and then the passaging process of 1 / 2 whole cells was repeated on a new dish, and the cells that were passaged 5 times or more were considered adherent tumor cells.For floating cells, 5 ml of culture supernatant and 5 ml of RPMI-1640 medium were plated onto a new 100 mm dish from the culture dish used for cell passaging, and cells that had been passaged 5 or more times were considered floating tumor cells. When peritoneal metastatic tumor cells obtained from a single patient grew with both adherent tumor cells and floating tumor cells, they were considered mixed tumor cells. For the purposes of the present invention, adherent tumor cells, floating tumor cells, or mixed tumor cells isolated by the above-mentioned method are generally referred to as "peritoneal metastatic tumor cells." The obtained six cells (NSC-9C, NSC-15CF, NSC-16C, NSC-20C, NSC-22C, and NSC-32C (hereinafter also referred to as “six types of peritoneal metastatic tumor cells”)) were used in the subsequent study.

[0111] 1-2: Preparation of a hybridoma producing an antibody against a gastric cancer antigenPeritoneal metastatic tumor cells were immunized into Veloclmmune mice obtained using human monoclonal antibody development technology (Veloclmmune® antibody technology; Regeneron (U.S. Patent No. 6,596,541)) to produce antibodies against a tumor antigen expressed on anti-peritoneal metastatic cells. From the peritoneal metastatic tumor cells obtained in Example 1-1, three types of NSC-9C, NSC-15CF, and NSC-16C cells or three types of NSC-20C, NSC-22C, and NSC-32C cells were mixed, and the resulting cells were suspended in Titermax(R) gold adjuvant (Merck, T2684) or PBS(-) to prepare a suspension peritoneal metastatic tumor cells. Veloclmmune mice were immunized with this suspension several times.Lymphocytes were collected from the lymph nodes of immunized mice and subjected to cell fusion with SP2 / 0 murine myeloma cells (ATCC, CRL-1581) to produce hybridomas using the standard method. Individual hybridoma colonies were isolated using an automated hybridoma cell harvester for monoclonal antibody-producing cells (hereinafter referred to as "clones"). The isolated clone was cultured at 37°C in an 8% CO2 incubator, and after approximately 14 days, the supernatant was collected in a 96-well plate for the next experiment.

[0112] 1-3: Selection of an Antibody Selectively Binding to a Peritoneal Metastatic Tumor Cell (1) Testing Binding to a Peritoneal Metastatic Tumor Cell and a Cell Expressing EpCAMK The cell clone supernatant obtained in Example 1-2 contains an antibody (hereinafter referred to as "the antibody contained in the clone supernatant"). First, the binding of the antibody contained in the clone supernatant to six types of peritoneal metastatic tumor cells obtained in Example 1-1 was evaluated using flow cytometry to select a clone producing an antibody that strongly binds to the peritoneal metastatic tumor cells. Goat anti-mouse Ig BV421 (Becton, Dickinson and Company, 563846) was used for flow cytometry.In addition, the binding of the antibody contained in the clone supernatant to CHO-K1 cells expressing human EpCAM-Myc-DDK was assessed to exclude clones that mediate antibody binding to EpCAM, a tumor antigen. CHO-K1 cells expressing human EpCAM-Myc-DDK were obtained 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 antibody contained in the clone supernatant to the cells was assessed by flow cytometry. Goat anti-mouse Ig BV421 was used for flow cytometry. Clones binding to EpCAM-expressing cells were excluded from clone selection, thereby providing a supernatant that did not exhibit cell binding activity. The CD326 (EpCAM) monoclonal antibody (1B7) (eBioscience, 14-9326) was used as a positive control.In addition, clones that bind to cultured human peritoneal mesothelial cells were excluded from the selection of clones to obtain those that selectively bind to peritoneal metastatic tumor cells. Human mesothelial cells (Zenbio, MES-F, lot MESM050311A) (hereinafter also referred to as "cultured human peritoneal mesothelial cells") were used as cultured human peritoneal mesothelial cells, and the binding between cultured peritoneal mesothelial cells in mesothelial cell growth medium (Zenbio, MSO-1) and their clone was assessed by flow cytometry. Goat anti-mouse Ig BV421 was used for flow cytometry. Only clones that did not bind to cultured human peritoneal mesothelial cells were selected.Through the above experiment, clone 3D11 was obtained as a clone that provided antibody binding to 5 or more of 6 peritoneal metastatic tumor cells but not to human EpCAM and cultured peritoneal mesothelial cells.

[0113] (2) Isolation of Antibody from Hybridoma SupernatantClone 3D11 was cultured in CD hybridoma medium (Thermo Eisner Scientific, 11279023). From the resulting culture supernatants, the 3D11 antibody (hereinafter referred to as “3D11”) was purified using MabSelect SuRe (GE Healthcare, 17-5438-02). The antibody was purified in the usual manner.

[0114] 1-4: Identification of the Antigen Molecule Recognized by 3D11(1) Identification of the Candidate Antigen Molecule by LC-MS / MS AnalysisA candidate antigen molecule for 3D11 was identified. 1D10 and 10B5 were used as the control antibodies in this experiment. These antibodies showed a different binding pattern to peritoneal metastatic tumor cells than 3D11 during the preparation of 3D11. NSC-15CF cell homogenate was prepared. 3D11 and one of the two control antibodies (1D10, 10B5) were added to the cell homogenate, and then Bynabeads Protein G magnetic particles (Life Technologies, 10003D) were added, followed by mixing and washing. Bynabeads Protein G-bound protein was cleaved using trypsin / LysC (Promega Corporation, V5072) to yield a peptide mixture.A solution containing the peptide mixture was analyzed by LC-MS / MS using an UltiMate 3000 RSLCnano (Thermo Eisher Scientific) and Orbitrap Fusion (Thermo Eisher Scientific). The resulting LC-MS / MS data were subjected to comparative quantification and peptide / protein identification using Progenic QI for Proteomics (Waters) and Mascot (Matrix Science) software to identify binding proteins. 3D11-binding proteins and control antibodies were compared, and CLDN4 was identified as a candidate antigen molecule for 3D11.

[0115] (2) Determination of 3D11 AntigenA binding experiment with CHO-K1 cells expressing human CLDN4-Myc-DDK was performed to determine whether CLDN4, thus identified in (1), is an antigen for 3D11. CHO-K1 cells expressing human CLDN4-Myc-DDK were prepared by transfecting CHO-K1 cells with CLDN4 (Myc-DDK-tagged)-human claudin 4 (CLDN4) (ORIGENE, RC200490). As a result, it was confirmed that 3D11 binds to human CHO-K1 cells expressing CLDN4-Myc-DDK and thus recognizes CLDN4 as an antigen.

[0116] Examples 2: Determination of 3D11 sequence and production of human antibody

[0116] The gene encoding the heavy chain and light chain of 3D11 was cloned using a conventional method to determine the nucleotide sequence and amino acid sequence of 3D11. The amino acid sequence of the variable region of the heavy chain of 3D11 is shown in SEQ ID NO: 2, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO: 4. The antibody produced by Veloclmmune mice is an antibody in which the endogenous variable regions of the immunoglobulin heavy chain and light chain are replaced by the corresponding human variable regions. In other words, the antibody produced using the Veloclmmune method is an antibody having a variable region of a human antibody and a constant region of a mouse antibody (also called a “chimeric antibody”).

[0117] Reference Example: Bispecific Anti-CLDN4 / Anti-CD3 Antibody for Use in the Treatment of Peritoneal Cancer Metastasis Various types of drugs, such as antibodies, antibody-drug conjugates (ADCs), and chimeric antigen receptor T cell (CAR-T) therapy, are known as therapeutic agents for TAAs selectively expressed on tumor cells. Among them, bispecific T cell recruiting antibodies (T cell recruiting antibodies) are known as an innovative method that can achieve selective cytotoxic activity against tumor cells with a low antibody concentration. Therefore, a bispecific T cell recruiting antibody was investigated using 3D11.

[0118] (1) Production of humanized anti-CLDN4 antibodyA bispecific human anti-CLDN4 / anti-CD3 scFv antibody was produced as follows. A portion of the human anti-CLDN4 antibody was produced by linking the constant region of the human IgG1 heavy chain to the variable region of the 3D11 heavy chain and the constant region of the human light chain to the variable region of the light chain in a conventional manner. In addition, the LALA mutation (L234 and L235A) in which the amino acids at amino acid positions 238 and 239 (EU numbering system: 234 and 235) of the heavy chain are each substituted for a leucine (L) by an alanine (A), a knob-in-hole mutation in which the amino acids at amino acid positions 370, 372, and 411 (EU numbering system: 366, 368, and 407) are respectively substituted from threonine (T) to serine (S), L to A, and tyrosine (Y) to valine (V), and a mutation in which the amino acid at amino acid position 301 (EU numbering system: 297) is substituted from asparagine (N) to A.The resulting humanized anti-CLDN4 antibody is designated 3D11.1. The polynucleotide encoding the amino acid sequence of 3D11.1 was synthesized using a conventional method and inserted into the pcDNA3.4 TOPO vector (Thermo Fisher Scientific).

[0119] (2) Production of anti-CD3 scFvA portion of the anti-CD3 scFv was produced based on the sequences of the heavy chain variable region and light chain variable region of the mouse anti-CD3 antibody described in Japanese Patent No. 5686953. Humanization of the anti-CD3 scFv was carried out according to the method described in the literature (Front Biosci., 2008, Vol. 13, pp. 1619-1633). In this method, a back mutation was introduced. The three-dimensional structure information (PDB code: 5FCS) was analyzed using the integrated computational chemistry system MOE provided by MOLSIS Inc. to determine the positions for introducing a back mutation in the framework region. The amino acid sequence of the humanized anti-CD3 scFv is shown in SEQ ID NO: 41. The polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 41 was synthesized in a routine manner and inserted into the pcDNA3.1 (+) vector (Thermo Fisher Scientific, V79020).

[0120] (3) Production of anti-CLDN4 (3D11) / anti-CD3 scFv bispecific antibodyA vector encoding 3D11.1 and anti-CD3 scFv was used to produce a bispecific antibody consisting of an Fab region of an anti-CLDN4 antibody and an scFv region and an Fc region of an anti-CD3 antibody, according to the method described in Example 7 of PCT / JP 2021 / 41839. The bispecific antibody thus obtained was named "anti-CLDN4 (3D11) / anti-CD3 scFv bispecific antibody."

[0121] (4) Evaluation of the therapeutic effect of the anti-CLDN4(3D11) / anti-CD3 scFv bispecific antibodyThe in vitro redirected T cell cytotoxicity (RTCC) activity of the anti-CLDN4(3D11) / anti-CD3 scFv bispecific antibody was evaluated according to the method described in Example 9 of PCT / JP 2021 / 41839. The anti-CLDN4(3D11) / anti-CD3 scFv bispecific antibody was confirmed to have RTCC activity. In addition, the in vivo antitumor activity of the anti-CLDN4(3D11) / anti-CD3 scFv bispecific antibody was evaluated according to the method described in Example 11 of PCT / JP 2021 / 41839. The anti-CLDN4(3D11) / anti-CD3 scFv bispecific antibody exhibited an antitumor effect in a mouse model of peritoneal metastases from gastric cancer in vivo. These data suggest that the anti-CLBN4(3D11) / anti-CD3 scFv bispecific antibody is effective for cancer treatment.In addition, to investigate the safety of the anti-CLDN4(3D11) / anti-CD3 scFv bispecific antibody, the anti-CLDN4(3D11) / anti-CD3 scFv bispecific antibody was administered intravenously to cynomolgus macaques at a single dose. As a result, death and atonal state occurred in the monkeys administered the anti-CLDN4(3D11) / anti-CD3 scFv bispecific antibody. Blood IL-6 levels were markedly increased in the dead monkeys at 1 h and 6 h after administration, as well as in the monkeys in atonal state at 1 h, 6 h, and 24 h after administration. Based on these results, although the anti-CLDN4(3D11) / anti-CD3 scFv bispecific antibody exerted therapeutic effects in vitro and in vivo, concerns were raised about a serious adverse reaction, presumably due to increased cytokine levels. Thus, it was concluded that the anti-CLDN4(3D11) / anti-CD3 scFv bispecific antibody is not considered as an antibody for the treatment of human cancer.

[0122] Example 3: Production of anti-CLDN4(3D11) / anti-CD3 scFv bispecific antibodyA bispecific antibody with anti-CD137 scEv (hereinafter referred to as “anti-CLDN4(3D11) / anti-CD137 scEv bispecific antibody”) was produced using 3D11 for research.

[0123] 3-1. Production of anti-CD137 antibody (1) Production of CD137-Fc fusion proteinPolynucleotides encoding the human CD137 extracellular region consisting of the amino acid sequence at amino acid positions 1 to 186 of SEQ ID NO: 42 or the monkey CD137 extracellular region consisting of the amino acid sequence at amino acid positions 1 to 186 of SEQ ID NO: 43, respectively, were inserted into pFUSE-hIgG1-Fc1 vectors (InvivoGen, pfuse-hglfcl) to obtain expression vectors encoding fusion proteins of the extracellular regions of CD137 and human Fc (hereinafter, respectively referred to as the “human CD137-human Fc fusion protein” and the “monkey CD137-human Fc fusion protein”).Each of the vectors obtained in this way was introduced into Expi293F cells (Thermo Fisher Scientific, A14527) or ExpiCHO-S cells (Thermo Fisher Scientific, A29133) using the Expi293 Expression Kit (Thermo Fisher Scientific, A14635) or the ExpiCHO Expression Kit (Thermo Fisher Scientific, A29133) for subsequent culturing. Proteins secreted into the culture supernatants 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 monkey CD137-human Fc fusion protein.

[0124] (2) Preparation of monkey CD137-His-tag-3×FLAG-tag fusion proteinPolynucleotides encoding the extracellular region of monkey CD137 consisting of the amino acid sequence at amino acid positions 1-183 of SEQ ID NO: 43, His-tag of SEQ ID NO: 44, and 3×FLAG-tag of SEQ ID NO: 45 were inserted sequentially into the pcdna3.4 TOPO vector to obtain a vector. The thus obtained vector was introduced into ExpiCHO-S cells using the ExpiEectamine CHO Transfection Kit (Thermo Eisher Scientific, A29129) for culture. The protein secreted into the culture supernatant was purified using HisTrap excel (GE Healthcare Bioscience, 17-3712-06). The fusion protein obtained in this way is referred to as "monkey CD137-His-tag-3×FLAG-tag fusion protein".

[0125] (3) Preparation and screening of hybridoma clone producing anti-CD137 antibody Veloclmmune mice or AlivaMab mice (Ablexis, US Patent No. 9346873) were immunized with human CD137-human Fc fusion protein or human CD137-His tag protein (R&D Systems, 9220-4B) and immunoadjuvants several times. Lymphocytes were collected from the lymph nodes of the immunized mice by a routine method and subjected to cell fusion with SP2 / 0 murine myeloma cells to obtain hybridomas. Single colonies of hybridomas were isolated using an automated device for collecting hybridoma cells producing monoclonal antibodies (hereinafter referred to as “clone”). Each clone was cultured for several days to obtain a culture supernatant. The binding of antibodies to CD137 contained in the culture supernatant was analyzed using the following method. Binding to CD137 was assessed using ELISA.The assay utilized a plate coated with any of the following: human CD137-His-tag, human CD137-human Fc fusion protein, monkey CD137-human Fc fusion protein and human Fc protein, and goat anti-mouse IgG. Human ads-HRP (SouthernBiotech, 1030-05) was used as the secondary antibody. This method yielded a series of clones binding to both human and monkey CD137.

[0126] (4) Preparation of anti-CD137 antibody and testing of agonist activityThe clones obtained in Example (3) were cultured in Hybridoma-SFM medium (Thermo Fisher Scientific, 12045-076), and anti-CD137 antibody was purified from the resulting culture supernatant using MabSelect SuRe. The purified anti-CD137 antibodies were tested for binding to CD137 and agonist activity for CD137. Binding of anti-CD137 antibodies to CD137 was tested using the ELISA described in Example (3). Evaluation of the agonist activity for CD137 by anti-CD137 antibodies was performed using the Bioassy 4-1BB Bioassay Kit (Promega Corporation, JA2351) as follows. FcγRIIb CHO-K1 cells (Promega Corporation, JA2251) were seeded in a 384-well plate (Greiner Bio-One, 781080) for cultivation at 37°C and 5% CO2 overnight.The culture supernatant was removed, purified antibodies from each clone described above and 4-1BB effector cells (Promega Corporation, JA2351) were added to it, and the resulting mixture was incubated at 37°C and 5% CO2 for 6 h. Bio-Glo reagent (Promega Corporation, JA2351) was added to the resulting mixture to measure chemiluminescence by induction of luciferase expression in response to CD137 agonist activity. A similar experiment as described above was performed in the absence of FcγRIIb CHO-K1 cells to evaluate the agonist activity for CD137 in the absence of FcγRIIb CHO-K1 cells. Among the anti-CD137 antibodies evaluated in this way, the 3-34 antibody obtained from Veloclmmune mice and the A2-32, A2-48, and A2-73 antibodies obtained from AlivaMab mice had high binding activity to human and monkey CD137 and exhibited agonist activity for CD137 dependent on the presence of FcγRIIb CHO-K1 cells.

[0127] (5) Identification of the gene sequence of anti-CD137 antibody From each of the clones providing anti-CD137 antibodies (3-34, A2-32, A2-48, and A2-73), cell lysate was prepared using a routine method, cDNA was synthesized, and the nucleotide sequence of the antibody was determined. The SEQ ID NO of the nucleotide sequence thus identified is shown in Table 1.

[0128] 3-2. Preparation of anti-CLDN4(3D11) / anti-CD137 bispecific antibody scFv (3-34)The anti-CLDN4(3D11) / anti-CD137 bispecific antibody scEv (3-34) (hereinafter also referred to as “3D11_3-34-j6_S”) was developed based on the heavy chain variable region and light chain variable region sequences of 3D11 and 3-34. In the resulting antibody 3D11_3-34-j6_S, the GS linker is linked to the carboxy terminus of the heavy chain (also referred to as the "C-terminus") of the human IgG1κ anti-CLDN4 antibody (3D11), and the amino terminus of the anti-CD137 scFv (3-34) (SEQ ID NO: 26), which contains the heavy chain variable region and the light chain variable region of the anti-CD137 antibody, is linked to the C-terminus of the GS linker. In addition, amino acid mutations of L234A, L235A, and P331S were introduced into the constant region of human IgG1 3D11. Polynucleotides encoding the constructed antibody were obtained and inserted into pcDNA3.4 TOPO vectors in the usual manner.Expression vectors were genetically introduced into ExpiCHO-S cells, and 3D11_3-34-j6_S was isolated from the culture supernatant. The resulting antibody, 3D11_3-34-j6_S, was confirmed to possess anti-CD137-binding activity and CD137 agonist activity in vitro, suggesting its potential as an antitumor therapeutic.

[0129] Examples 4: Production of anti-CLDN4 (hKM3900) / anti-CD137 scFv[4-1] bispecific antibody Production of humanized anti-CLDN4 antibody (hKM3900) It was reported that KM3900, which is an anti-CLDN4 antibody, selectively binds to CLDN4 compared with other claudin family molecules such as CLDN6 (PTL 1). Therefore, humanized antibody KM3900 was produced based on this report. Specifically, based on the humanized amino acid sequence of the variable region of KM3900, a humanized antibody was constructed from the sequences of the constant region of human Igγ1 and the constant region of human Igκ. The amino acid mutations L234A, L235A, and P331G or the amino acid mutations L234A, L235A, and P331S were introduced into the constant region of human Igγ1. Humanized anti-CLDN4 antibody (hKM3900) was routinely produced for use in the following study.An antibody containing the amino acid mutations L234A, L235A, and P331G is designated "hKM3900," and an antibody containing the amino acid mutations L234A, L235A, and P331S is designated "hKM3900_S." The amino acid sequence of the variable region of the heavy chain of hKM3900 is shown in SEQ ID NO: 6, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO: 8.

[0130] 4-2. Production of bispecific anti-CLDN4 / anti-CD137 scFvAnti-CD137 scFv was constructed based on the anti-CD137 antibodies obtained in Example 3 (3-34, A2-32, A2-48, and A2-73) to produce bispecific antibodies hKM3900 and anti-CD137 scFv (Table 2). The antibody thus obtained is hereinafter referred to as “anti-CLDN4(hKM3900) / anti-CD137 scFv”.

[0131] (1) Construction of a vector encoding an anti-CLDN4(hKM3900) / anti-CD137 scFv bispecific antibodyThe anti-CLDN4(hKM3900) / anti-CD137 scFv bispecific antibody was constructed based on the hKM3900 sequence and the heavy chain variable region and light chain variable region sequences of an anti-CD137 antibody. In the anti-CLDN4(hKM3900) / anti-CD137 scFv bispecific antibody, the GS linker is linked to the C-terminus of the heavy chain or light chain of anti-CLDN4 (hKM3900) IgG1, and the amino terminus of anti-CD137 scFv containing the heavy chain variable region and the light chain variable region of the anti-CD137 antibody is linked to the C-terminus of the GS linker. In addition, amino acid mutations of L234A, L235A, and P331G or P331S were introduced into the constant region of human IgG1 hKM3900. Information on the engineered antibodies is presented in Table 2. Each polynucleotide encoding the engineered antibodies was prepared and inserted into the pcDNA3.4 TOPO vector in a routine manner.The vector obtained in this way is hereinafter referred to as “anti-CLDN4 (hKM3900) / anti-CD137 scFv bispecific antibody expression vector”.

[0132]

[0133] (2) Production of anti-CLDN4 (hKM3900) / anti-CD137 scFv bispecific antibodyThe anti-CLDN4 (hKM3900) / anti-CD137 scFv bispecific antibody expression vectors were used to produce eleven anti-CLDN4 (hKM3900) / anti-CD137 scFv bispecific antibodies, as shown in Table 2. Specifically, the anti-CLDN4 (hKM3900) / anti-CD137 scFv bispecific antibody expression vectors were introduced into ExpiCHO-S cells using the ExpiFeetamine CHO Transfection Kit to induce the secretion of anti-CLDN4 / anti-CD137 scFv bispecific antibodies into the culture supernatant. From the resulting culture supernatant, the bispecific anti-CLDN4 (hKM3900) / anti-CD137 scFv antibodies were purified by affinity purification using MabSelect SuRe.In addition, some of the anti-CLDN4 (hKM3900) / anti-CD137 scFv bispecific antibodies were further purified by size exclusion chromatography using HiLoad 26 / 600 superdex 200 pg (GE Healthcare, 28-9893-36) or Superdex 200 Enhance 10 / 300 GL (GE Healthcare, 28-9909-44). Hereinafter, the anti-CLDN4 (3D11) / anti-CD137 scFv (3-34) bispecific antibody and the anti-CLDN4 (hKM3900) / anti-CD137 scFv bispecific antibody obtained in Examples 3 and 4 are collectively referred to as the “anti-CLDN4 / anti-CD137 scFv bispecific antibody” in some cases.

[0134] Example 5: Evaluation of the binding activity and agonistic activity of the bispecific anti-CLDN4 (hKM3900) / anti-CD137 scFv antibodyThe bispecific anti-CLDN4 (hKM3900) / anti-CD137 scFv antibody was evaluated for its binding activity to CLDN4 and CD137, and its agonistic activity to CD137.

[0135] 5-1. Evaluation of the Binding Activity of Anti-CLDN4(hKM3900) / Anti-CD137 scFv Bispecific Antibody Using Flow CytometryThe binding activity of the anti-CLDN4(hKM3900) / anti-CD137 scFv bispecific antibody to CLDN4 was evaluated by flow cytometry. The CLDN4 gene derived from CLDN4 (Myc-DDK-tagged)-human claudin 4 was inserted into the mammalian expression vector pCMV6-AC-GFP (ORIGENE, PS100010) to generate the human CLDN4 expression vector. Human CLDN4 expression vectors were introduced into CHO-K1 cells to generate CHO-K1 cells expressing human CLDN4. hKM3900 and two bispecific anti-CLDN4 (hKM3900) / anti-CD137 scFv antibodies (hKM3900_tA2-32LH and hKM3900_3-34-j6) were used as the test antibodies.For each test antibody, serial dilutions were prepared using staining buffer (FBS) (BD Bioscience, 554-656) from a maximum concentration of 100 μg / mL to 5.08 ng / mL with a typical ratio of 3, and CHO-K1 expressing human CLDN4 were stained with a diluted solution of each test antibody. Allophycocyanin (APC) fragment of F(ab')2 goat anti-human IgG AffiniPure, specific for the Fcγ fragment (Jackson ImmunoResearch, 109-136-170), was used as the secondary antibody. Fluorescence intensity was measured using a FACS Canto II (BD Bioscience). The measurement results were analyzed using GraphPad Prism software to calculate the apparent dissociation constant (KD) between human CLDN4 and the anti-CLDN4(hKM3900) / anti-CD137 scFv bispecific antibody. The apparent KD values ​​obtained in this way are presented in Table 3.Two bispecific anti-CLDN4 (hKM3900) / anti-CD137 scFv antibodies exhibited the same binding activity to human CLDN4 as the humanized anti-CLDN4 antibody (hKM3900). Other bispecific anti-CLDN4 (hKM3900) / anti-CD137 scFv antibodies are expected to exhibit equivalent binding activity to CLDN4.

[0136]

[0137] 5-2. Evaluation of the binding activity of anti-CLDN4(hKM3900) / anti-CD137 scFv bispecific antibody to CD137 using ELISAThe binding activity of anti-CLDN4(hKM3900) / anti-CD137 scFv bispecific antibody to CD137 was determined using ELISA. ELISA was performed according to the usual method. 20 ng / 20 μl / well of human CD137-His-tag protein (R&D Systems, 9220-4B) was immobilized in a 384-well plate (Thermo Fisher Scientific, 464718). Eleven anti-CLDN4 (hKM3900) / anti-CD137 scFv bispecific antibodies and the ctrl IgG_L-3-34-j6 antibody shown in Table 2 were used as the test antibodies. The ctrl IgG L-3-34-J6 antibody is an antibody in which anti-CD137 scFv (3-34-j6) is linked to the carboxyl terminus of the light chain of an isotype control antibody. The test antibodies were serially diluted with TBS-T (Nippon Gene Co., Ltd., 310-07375) containing 5% blocking reagent (Nacalai Tesque, Inc., 03953-95) and added at 20 μl / well.The antibody had a final concentration as shown in Fig. 1-1-1-4. Goat anti-human IgG Fc, Multi-Species SPads-HRP (SouthernBiotech, 2014-05) was used as the secondary antibody. hKM3900 was used as a control antibody. An Infinite M200 PRO reader (TECAN) was used to measure the optical density at 450 nm and 570 nm. The results are shown in Fig. 1-1-1-4. All anti-CLDN4(hKM3900) / anti-CD137 scFv bispecific antibodies bound to human CD137-His-tag protein in a concentration-dependent manner. Based on the obtained data, it was found that the binding position of anti-CD137 scFv does not affect the binding activity for human CD137.

[0138] 5-3. Evaluation of the binding activity of anti-CLDN4(hKM3900) / anti-CD137 scFv bispecific antibody to CD137 by surface plasmon resonance. The binding activity of anti-CLDN4(hKM3900) / anti-CD137 scFv bispecific antibody to CD137 was determined by surface plasmon resonance (SPR). Biacore T200 (GE Healthcare Japan) was used for the SPR analysis. The ligand obtained by fixation of two anti-CLDN4(hKM3900) / anti-CD137 scFv bispecific antibodies (hKM3900-3-34-j6, hKM3900_tA2-32LH) with protein A of the S series sensor chip (GE Healthcare, 29-1275-56) was used. Serial dilutions of the human CD137-His-tag fusion protein or the monkey CD137-His-tag-3×FLAG-tag fusion protein obtained in Example 3-1 (2) using HBS-EP+ were used as the analyte, from a maximum concentration of 200 nM to 0.0977 nM with a typical ratio of 2.The analytes were added to the channel having the bound ligand at a rate of 50 μl / min for 2 min, then HBS-EP+ was added to them at a rate of 50 μl / min for 10 min, and thus, the binding and dissociation of the anti-CLDN4 (hKM3900) / anti-CD137 scFv bispecific antibody to and from CD137 took place. The results obtained in this experiment were used to calculate the association rate constant (ka), the dissociation rate constant (kd), and the dissociation constant (KD) between human CD137 and the anti-CLDN4 (hKM3900) / anti-CD137 scFv bispecific antibody using data analysis software (BIA Evaluation). The ka, kd, and KD values ​​are shown in Table 4. As a result, it was found that the anti-CLDN4 (hKM3900) / anti-CD137 scFv bispecific antibody exhibited binding activity equivalent to that of anti-human CD137 and anti-monkey CD137 antibodies.

[0139]

[0140] 5-4. Evaluation of CD137 agonist activity of anti-CLDN4 / anti-CD137 scFv bispecific antibody in coexistence with CLDN4-expressing tumor cells. The specific CD137 agonist activity of anti-CLDN4 / anti-CD137 scFv bispecific antibody on CLDN4-expressing tumor cells was evaluated using the Bioassay 4-1BB assay 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 the suspension. CLDN4 was expressed in NCI-H322. The NCI-H322 suspension was added to a 384-well plate (Greiner Bio-One, 781080) at a density of 1×10 4cells / well and the resulting cells were cultured at 37°C and 5% CO2 overnight. The following day, the medium was removed. The test antibody diluted in RPMI-1640 medium containing 1% FBS was added at 12.5 μl / well. The antibody has a final concentration as shown in Fig. 2-1 - Fig. 2-3. The test antibody was 3D11_3-34-j6_S and the bispecific anti-CLDN4(hKM3900) / anti-CD137 scFv antibody listed in Table 2. The control antibody was hKM3900, the control antibody IgG_L-3-34-j6, and urelumab (BMS, SEQ ID NO: 3 and SEQ ID NO: 6, described in International Patent Publication WO 2005 / 035584). 4-1BB effector cells supplied in the Bioassy 4-1BB assay kits were suspended in 7 ml RPMI-1640 containing 1% FBS per vial, the resulting suspension was added at 12.5 μl to each well, and the resulting suspension was added at 12.5 µl into each well and incubated at 37°C and 5% CO2 for 6 h.Bio-Glo reagent was added to the plate at 25 μl / well, and chemiluminescence was measured using EnVision 2103 (Perkin Elmer). The results are shown in Fig. 2-1-2-3. The anti-CLDN4 / anti-CD137 scFv bispecific antibody exhibited strong agonistic activity for CD137 on 4-1BB effector cells when co-existed with CLDN4-expressing tumor cells. A similar experiment as described above was performed under NCI-H322-free conditions to evaluate the agonistic activity for CD137 in the absence of CLDN4-expressing tumor cells. As a result, the antibody did not exhibit agonistic activity for CD137 in the absence of CLDN4-expressing tumor cells (Fig. 2-4 - Fig. 2-6). Based on this result, it was shown that the bispecific anti-CLDN4 / anti-CD137 scFv antibody exhibits agonistic activity for CD137 only when co-existing with CLDN4-expressing tumor cells.On the other hand, urelumab exhibited CD137 agonist activity in the presence of CLDN4-expressing tumor cells, but its effect was weak compared to that of the bispecific anti-CLDN4 / anti-CD137 antibody. Furthermore, urelumab exhibited CD137 agonist activity even in the absence of CLDN4-expressing tumor cells. Based on these results, it is expected that the bispecific anti-CLDN4 / anti-CD137 antibody will exhibit less toxicity than urelumab and will exhibit specific and potent agonist activity against CD137 in tumor tissue in which CLDN4 is highly expressed.

[0141] Example 6: In vitro evaluation of the effect of the bispecific anti-CLDN4 / anti-CD137 scFv antibodyThe stimulating function for the production of interferon-γ and the cytotoxic activity against tumor cells of the bispecific anti-CLDN4 / anti-CD137 scFv antibody were evaluated.

[0142] 6-1. Preparation and cryopreservation of expanded pan T cells(1) Preparation of expanded pan T cellIn Example 6, a mixture obtained by adding FBS (Cytiva, SH30084.03) at a final concentration of 10% and penicillin-streptomycin mixture (Thermo Fisher Scientific, 15070-063) at a final concentration of 1% to RPMI-1640 (Sigma, R8758)) is referred to as “culture medium”. Each compound selected from the essential amino acids MEM (Merck, M7145), sodium pyruvate (Merck, S8636), GlutaMAX I (Thermo Fisher Scientific, 35050-061) and HEPES (Thermo Fisher Scientific, 15630-080) could be added to the culture medium at a final concentration of 1%. Anti-CD3 antibody (BioLegend, 317325), diluted to a final concentration of 1 to 3 μg / ml, was added to the plate and the anti-CD3 antibody was immobilized.The Human Pan-T Cell Isolation Kit (MilteNY Biotec, 130-096-535) was used to isolate pan-T cells (including both CD4T and CD8T cells; hereinafter referred to as "pan-T cells") from human peripheral blood mononuclear cells (LONZA, CC-2702) according to the manufacturer's recommended protocol. The isolated pan-T cells were centrifuged, the supernatant was removed, and the resulting material was suspended in culture medium. Whole pan-T cells suspended in culture medium were plated onto a plate immobilized with anti-CD3 antibody. In addition, human IL-2 (PeproTech, 200-2) at a final concentration of 100 to 200 U / ml and anti-CD28 (BioLegend, 302923) at a final concentration of 1 to 4 μg / ml were added, and the resulting mixture was cultured at 37°C in an incubator with 5% CO2. After 3 days, the resulting cells were collected, suspended in culture medium, and plated.Human IL-2 was also added to the dishes at a final concentration of 100 to 200 U / ml, and the resulting mixture was cultured at 37°C in an incubator with 5% CO2. After 4 days, whole cells were collected for centrifugation, the supernatant was removed, the resulting cells were resuspended in a Cell Banker (Takara, CB011) or Bambanker (GC LYMPHOTEC Inc., CS-02-001), and the resulting cells were divided into flasks for subsequent cryopreservation at -80°C. Cells cryopreserved in this manner are referred to herein as "expanded pan-T cells."

[0143] 6-2. Preparation of 60As6-Luc / GFP-expressing cellsPreparation of Luc / GFP-containing lentivirus solution was performed using L293T cells (Thermo Fisher Scientific, K4975-00) according to the usual protocol. Opti-MEM medium with reduced serum, GlutaMAX supplement (Thermo Fisher Scientific, 51985-034), MISSION (R) lentivirus packaging mix (SIGMA, SEP001), and Lipofectamine LTX (Thermo Fisher Scientific, 15338100) were used to insert the pCDH-CMV-GL3-EF1a-GFP-T2A-puro vector (kindly provided by Associate Professor Ryo Takahashi of Hiroshima University, Graduate School of Biomedical and Health Sciences, Department of Cell and Molecular Biology (PLoS One, 2015, Vol. 10, e0123407)) into L293T cells. To collect GFP-expressing lentivirus cultured in L293T cells, a Millex (R)-HV filter with a 45-μm pore size (Merck Millipore, SLHV033RS) was used. The virus solution was placed into 1-ml cryovials (Nalgene, 5000-0020) for cryopreservation at -80°C.A tumor cell line expressing CLDN4, 60As6 cells (obtained from Dr. Kazuyoshi Yanagihara of the Institute of Biomaterials), was infected with a lentivirus expressing 60As6-Luc / GFP using a conventional method for obtaining 60As6-Luc / GFP-expressing cells. Polybrene (SantaCruz, sc-134220) was used to enhance the infection efficiency. RPMI-1640 containing 10% FBS and 2 μg / mL puromycin (Thermo Fisher Scientific, A-11138-02) was used as the medium. The cells obtained in this way are referred to here as “60As6-Luc / GFP cells.” Flow cytometry confirmed that CLDN4 was inherently highly expressed in 60As6-Luc / GFP cells. The cells constructed in this way were used in subsequent experiments.

[0144] 6-3. Evaluation of the stimulatory function of the anti-CLDN4 (3D11) / anti-CD137 scFv bispecific antibody on interferon-γ production in the coculture system of tumor cells and T cells. The stimulatory function of the anti-CLDN4 (3D11) / anti-CD137 scFv bispecific antibody on interferon-γ production was evaluated in the coculture system of 60As6-Luc / GFP cells and expanded pan-T cells. Anti-CD3 antibody at a concentration of 0.1 μg / 100 μl / well was added to a flat-bottomed 96-well plate (IWAKI, 3860-096) to immobilize the anti-CD3 antibody on the plate. 60As6-Luc / GFP cells were seeded in a plate at a density of 5×10 4 cells / 50 μl / well for cultivation at 37°C in an incubator with 5% CO2. The next day, expanded pan-T cells were seeded in a plate at a concentration of 2×10 5cells / 30 μl / well. The test antibodies and isotype control were serially diluted with culture medium from a maximum concentration of 50,000 ng / ml at a total ratio of 5 and added in an amount of 20 μl each. Urelumab and the bispecific antibody anti-CLDN4(3D11) / anti-CD137 scFv (3D11_3-34-j6_S) were used as the test antibody, and IgG (lysozyme)-scFv (CD137) (prepared by the inventors) 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 detection kit (Perkin Elmer, AL217C) according to the manufacturer's recommended protocol. In Fig. 3-1 shows the reaction curve of the amount of interferon-γ produced depending on the antibody concentration.The anti-CLDN4(3D11) / anti-CD137 scFv bispecific antibody exhibited stimulating activity against interferon-γ production in a coculture system of a CLDN4-expressing tumor cell line, 60As6-Luc / GFP cells, and expanded pan-T cells. The stimulating effect of anti-CLDN4(3D11) / anti-CD137 scFv bispecific antibodies on interferon-γ production was significantly stronger than that of urelumab.

[0145] 6-4. Evaluation of the Stimulating Function for Interferon-γ Production of the Anti-CLDN4(hKM3900) / Anti-CD137 scFv Bispecific Antibody in the Tumor Cell-T Cell Co-culture SystemThe stimulating function for interferon-γ production of the anti-CLDN4(hKM3900) / anti-CD137 scFv bispecific antibody in the co-culture system of 60As6-Luc / GFP cells and expanded pan-T cells was evaluated by the same method as described in Example 6-3. The anti-CD3 antibody was immobilized in a 96-well plate at a concentration of 0.03 μg / 100 μl / well. 60As6-Luc / GFP cells were seeded at 2×10 4 cells / 50 µl / well. Expanded pan-T cells were seeded at 8×10 4cells / 30 μl / well. Serially diluted urelumab from a maximum concentration of 5000 ng / ml with a standard ratio of 2 or a bispecific antibody anti-CLDN4 (hKM3900) / anti-CD137 scFv (hKM3900_3-34-j6 or hKM3900_tA2-32LH) were used as the test antibody. Each of the test antibodies and the isotype control (anti-lysozyme antibody) were added in an amount of 20 μl each. After 4 days, the amount of interferon-γ produced in the supernatant was measured. Fig. 3-2 shows the reaction curve of the amount of interferon-γ produced depending on the antibody concentration. The bispecific anti-CLDN4 (hKM3900) / anti-CD137 scFv antibody stimulated interferon-γ production in a coculture system of 60As6-Luc / GFP cells and expanded pan-T cells. The stimulatory effect of these bispecific antibodies on interferon-γ production was significantly stronger than that of urelumab.

[0146] 6-5. Evaluation of the stimulatory function for interferon-γ production of anti-CLDN4(3D11) / anti-CD137 scFv bispecific antibody in the T cell monoculture system. The stimulatory function for interferon-γ production of anti-CLDN4(3D11) / anti-CD137 scFv bispecific antibody was evaluated in the expanded pan-T cell monoculture system. Anti-CD3 antibody was added to a flat-bottomed 96-well plate at a dose of 0.1 μg / 100 μl / well to immobilize the anti-CD3 antibody on the plate. Expanded pan-T cells were seeded on the immobilized plate at a dose of 2 × 10 5cells / 80 μl / well. Urelumab and the bispecific antibody anti-CLDN4(3D11) / anti-CD3 scFv (3D11_3-34-j6_S) were used as the test antibody, and IgG(lysozyme)-scFv (CD137) (produced by the inventors) was used as the isotype control. The test antibodies and the isotype control were serially diluted with the culture medium from the maximum concentration of 50,000 ng / ml with the usual ratio of 5 and added in an amount of 20 μl each. After 6 days, the amount of interferon-γ produced in the supernatant was measured using the AlphaLISA interferon-γ detection kit in a similar manner to that described in Example 6-3. Fig. 3-3 shows the reaction curve of the amount of interferon-γ produced depending on the antibody concentration. The bispecific antibody anti-CLDN4(3D11) / anti-CD137 scFv did not exhibit stimulatory function on interferon-γ production in the T cell monoculture system, but urelumab exhibited stimulatory activity on interferon-γ production.The obtained result indicates that the bispecific anti-CLDN4(3D11) / anti-CD137 scFv antibody does not exhibit T cell enhancing activity in an environment lacking a CLDN4-expressing tumor cell line.

[0147] 6-6. Evaluation of the stimulatory function of anti-CLDN4(hKM3900) / anti-CD137 scFv bispecific antibody on interferon-γ production in the T cell monoculture systemThe stimulatory function of anti-CLDN4(hKM3900) / anti-CD137 scFv bispecific antibody on interferon-γ production in the expanded pan T cell monoculture system was evaluated by the same method as in Example 6-5. The anti-CD3 antibody was immobilized in a 96-well plate at a concentration of 0.03 μg / 100 μL / well. The expanded pan T cells were seeded at 8×10 4cells / 80 μl / well. Urelumab and the bispecific antibody anti-CLDN4 (hKM3900) / anti-CD137 scEv (hKM3900_3-34-j6 or hKM3900_tA2-32LH) were used as test antibodies. The test antibodies and the isotype control (anti-lysozyme antibody) were serially diluted from a maximum concentration of 5000 ng / ml with a standard ratio of 2 and added in an amount of 20 μl each. Figures 3-4 show the reaction curve of interferon-γ production depending on the antibody concentration. The bispecific antibody anti-CLDN4(hKM3900) / anti-CD137 scEv did not exhibit a stimulatory function on interferon-γ production in the T cell monoculture system, but urelumab exhibited a stimulatory function on interferon-γ production.The results described in Examples 6-5 and 6-6 indicate that the anti-CLDN4 / anti-CD137 scEv bispecific antibody does not exhibit T cell enhancing activity in an environment lacking CLDN4-expressing tumor cells and suggest a reduction in the systemic immunostimulatory activity reported as an adverse reaction of the anti-CD137 antibody.

[0148] 6-7. Evaluation of cytotoxic activity against tumor cells of the bispecific anti-CLDN4(3D11) / anti-CD137 scFv antibody in a co-culture system of tumor cells and T cells. The anti-tumor cytotoxic activity (tumor cell growth inhibitory activity) of the bispecific anti-CLDN4(3D11) / anti-CD137 scFv antibody was evaluated in a co-culture system of 60As6-Luc / GFP cells and expanded pan-T cells. 60As6-Luc / GFP cells were seeded in a flat-bottomed 96-well plate at a rate of 1×10 4cells / 50 μl / each well for cultivation at 37°C in an incubator with 5% CO2. After 2 h, expanded pan-T cells were seeded into the resulting 96-well plate at a rate of 1×10 5cells / 50 μl / each well. Urelumab and the bispecific anti-CLDN4(3D11) / anti-CD137 scFv antibody were used as the test antibodies. These antibodies were serially diluted with the culture medium from a maximum concentration of 40,000 ng / ml at a typical ratio of 10 and added in an amount of 50 μl each. In addition, 40 ng / ml anti-CD3 antibodies were added in an amount of 50 μl each, and the fluorescence area (GFP) in each well was measured using IncuCyte (R) ZOOM (Sartorius) at 37°C in an incubator with 5% CO2. The fluorescence area increasing from 0 h after the start of the measurement was used as an indicator of cell growth. In Fig. Figure 4 shows the cytotoxic activity of the test antibody against tumor cells (tumor cell growth rate). The ordinate in Figure 4 indicates the relative value obtained under the assumption that, as the fluorescence area increases from 0 h to 168 h, the well to which the test antibody was not added corresponds to 100%.As shown in Fig. 4, the anti-CLDN4(3D11) / anti-CD137 scFv bispecific antibody exhibited cytotoxic activity in a coculture system of 60As6-Luc / GFP cells and expanded pan-T cells. The cytotoxic activity of this bispecific antibody was significantly higher than that of urelumab.

[0149] 6-8. Evaluation of cytotoxic activity of anti-CLDN4(hKM3900) / anti-CD137 scFv bispecific antibody against tumor cells in a co-culture system of tumor cells and T cells. The killing activity of anti-CLDN4(hKM3900) / anti-CD137 scFv bispecific antibody against tumor cells was evaluated in a co-culture system of 60As6-Luc / GFP cells and expanded pan-T cells. Anti-CD3 antibody was added to a flat-bottomed 96-well plate (Perkin Elmer, 6005680) at a concentration of 1 ng / 100 μl / each well for immobilization in the plate. The plate was washed twice with PBS (-), and 60As6-Luc / GFP cells were seeded into it at a concentration of 2×10 4cells / 50 μl / well and the resulting cells were cultured at 37°C in an incubator with 5% CO2. The next day, expanded pan-T cells were seeded in a 96-well plate at a rate of 4.0 × 10 4cells / 30 μl / well. In addition, the test antibody and isotype control were added at a concentration of 5000 ng / ml to 0.844 ng / ml in an amount of 20 μl each. Urelumab and the bispecific antibody anti-CLDN4 (hKM3900) / anti-CD137 scFv (hKM3900_3-34-j6 or hKM3900_tA2-32LH) were used as the test antibody. Measurements were performed after 6 days. In some wells, 1% Triton-X100 (Sigma-Aldrich, T-9284) was added in an amount of 11 μl per well for cell killing 30 min before the measurement. The ONE-Glo Luciferase Assay Kit (Promega Corporation, E6120) was used according to the manufacturer's recommended protocol to measure the luciferase activity of 60As6-Luc / GFP cells in each well using ARVO X-3 (Perkin Elmer).The tumor cell death rate is shown as a relative value, assuming that the average luciferase chemiluminescence value in the well without the antibody is 0% and the average value obtained in the well with Triton-X100 is 100%. An anti-lysozyme antibody (prepared by the authors) was used as an isotype control. Fig. 5 shows the 60As6-Luc / GFP cell death curve. In a coculture system of 60As6-Luc / GFP cells and expanded pan-T cells, the addition of the anti-CLDN4 (hKM3900) / anti-CD137 scFv bispecific antibody significantly increased the death rate of 60As6-Luc / GFP cells. On the other hand, urelumab, a specific anti-CD137 antibody, did not increase the cell death rate of the 60As6-Luc / GFP cell line.

[0150] Example 7: In vivo evaluation of anti-CLDN4 / anti-CD137 bispecific antibody scFv7-1. In vivo antitumor effect of anti-CLDN4 / anti-CD137 bispecific antibody scFvNCI-H322 cells were suspended in PBS (-) (WAKO, 045-29795) and the cell suspension was 5.0×10 6 cells / 100 μl were inoculated subcutaneously into 7-week-old female mice (NOG, In-Vivo Science Inc.). Six days after inoculation, 3.4×10 6cells / 200 μl human peripheral blood mononuclear cells (Lonza, CC-2702). Fifteen days after human peripheral blood mononuclear cell transplantation, mice were divided into groups to begin administration of the test antibody. Each group consisted of 10 mice, and the mice were distributed so that tumor volumes between groups were almost equivalent to each other (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 (mm 3 ) = largest tumor diameter (mm) × smallest tumor diameter (mm) 2× 0.5As shown in Fig. 6, significant tumor volume reduction effect was observed in the groups administered with 0.1, 1, or 10 mg / kg hKM3900_tA2-32LH. The ordinate axis indicates tumor volume, and the abscissa axis indicates the day after the first administration. The solid and dotted lines indicate the shift in the mean tumor volume in each group, and the vertical error line indicates the standard error of the mean. Significance values ​​P (*: P<0.05 and **: P<0.01) were calculated by comparing the tumor volume on day 12 in the isotype control administration group and the hKM3900_tA2-32LH administration group using Dunnett's multiple comparison test. The number of cases in the 0.1 mg / kg hKM3900_tA2-32LH administration group on day 12 was 9.

[0151] 7-2. Safety Testing of Anti-CLDN4(hKM3900) / Anti-CD137 scFv Bispecific Antibody in Cynomolgus Macaques To test the safety of the anti-CLDN4(hKM3900) / anti-CD137 scFv bispecific antibodies (hKM3900_3-34-j6 and hKM3900_tA2-32LH), each of the anti-CLDN4(hKM3900) / anti-CD137 scFv bispecific antibodies was administered intravenously at a single dose to cynomolgus macaques. As a result, no specific abnormalities in the levels of cytokines such as IL-6 and TNF-α were observed in the serum of monkeys administered the anti-CLDN4(hKM3900) / anti-CD137 scFv bispecific antibodies. Thus, it was concluded that the anti-CLDN4 (hKM3900) / anti-CD137 scFv bispecific antibodies are considered as antibodies for cancer treatment in terms of safety.

[0152] Examples 8: Cancer Treatment Using Bispecific Anti-CLDN4 / Anti-CD137 Antibody8-1. Determination of CLDN4 Expression LevelTo clarify the applicability of CLDN4 as a therapeutic target for cancer, the RNA expression level of CLDN4 in tumor tissues was examined in The Cancer Genome Atlas (TCGA). As a result, it was confirmed that CLDN4 expression was increased in tumor tissues of colorectal cancer, bladder cancer, lung cancer, etc., therefore, it was suggested that CLDN4 is a possible therapeutic target not only for metastatic peritoneal cancer but also for other types of cancer. In addition, the RNA expression level of CLDN4 in normal tissues was examined using genotype tissue expression (GTEx). As a result, CLDN4 was found to be expressed in several normal tissues. However, the level of CLDN4 expression was lower in normal tissues than in tumor tissues.Thus, it was suggested that an antitumor agent therapeutically targeting CLDN4 is a possible therapeutic agent with a large guaranteed safety margin and a low probability of adverse reactions. Industrial applicability.

[0153] It is expected that the bispecific anti-CLDN4 / anti-CD137 antibody of the present invention will be useful for the treatment of cancer. In addition, the polynucleotide, expression vector, transformed host cell, and method for producing the antibody of the present invention are useful for producing the bispecific anti-CLDN4 / anti-CD137 antibody.Sequence listing free text

[0154] ​​In the digital title <223> The following sequence listing describes an "artificial sequence." Specifically, SEQ ID NO: 1 in the sequence listing shows the nucleotide sequence of the variable region of the heavy chain of 3D11, and SEQ ID NO: 2 shows the amino acid sequence of the variable region of the heavy chain of 3D11. SEQ ID NO: 3 shows the nucleotide sequence of the variable region of the light chain of 3D11, and SEQ ID NO: 4 shows the amino acid sequence of the variable region of the light chain of 3D11. SEQ ID NO: 5 shows the nucleotide sequence of the variable region of the heavy chain of hKM3900, and SEQ ID NO: 6 shows the amino acid sequence of the variable region of the heavy chain of hKM3900. SEQ ID NO: 7 shows the nucleotide sequence of the variable region of the light chain of hKM3900, and SEQ ID NO: 8 shows the amino acid sequence of the variable region of the light chain of hKM3900. SEQ ID NO: 9 shows the nucleotideheavy chain variable region sequence 3-34, and SEQ ID NO: 10 shows the amino acid sequence of heavy chain variable region 3-34. SEQ ID NO: 11 shows the nucleotide sequence of light chain variable region 3-34, and SEQ ID NO: 12 shows the amino acid sequence of light chain variable region 3-34. SEQ ID NO: 13 shows the nucleotide sequence of heavy chain variable region A2-32, and SEQ ID NO: 14 shows the amino acid sequence of heavy chain variable region A2-32. SEQ ID NO: 15 shows the nucleotide sequence of light chain variable region A2-32, and SEQ ID NO: 16 shows the amino acid sequence of light chain variable region A2-32. SEQ ID NO: 17 shows the nucleotide sequence of the variable region of the heavy chain of A2-48, and SEQ ID NO: 18 shows the amino acid sequence of the variable region of the heavy chain of A2-48. SEQ ID NO: 19 shows the nucleotidethe sequence of the variable region of the light chain A2-48, and SEQ ID NO: 20 shows the amino acid sequence of the variable region of the light chain A2-48. SEQ ID NO: 21 shows the nucleotide sequence of the variable region of the heavy chain A2-73, and SEQ ID NO: 22 shows the amino acid sequence of the variable region of the heavy chain A2-73. SEQ ID NO: 23 shows the nucleotide sequence of the variable region of the light chain A2-73, and SEQ ID NO: 24 shows the amino acid sequence of the variable region of the light chain A2-73. SEQ ID NO: 25 shows the nucleotide sequence of 3-34-j6 scFv, and SEQ ID NO: 26 shows the amino acid sequence of 3-34-j6 scFv. SEQ ID NO: 27 shows the nucleotide sequence of tA2-32 scFv, and SEQ ID NO: 28 shows the amino acid sequence of tA2-32 scFv. SEQ ID NO: 29 shows the nucleotide sequence of tA2-32LH scFv, and SEQ ID NO: 30 shows the amino acid sequence of tA2-32LH scFv. SEQID NO: 31 shows the nucleotide sequence of tA2-48 scFv, and SEQ ID NO: 32 shows the amino acid sequence of tA2-48 scFv. SEQ ID NO: 33 shows the nucleotide sequence of tA2-73 scFv, and SEQ ID NO: 34 shows the amino acid sequence of tA2-73 scFv. SEQ ID NO: 35 shows the nucleotide sequence of the heavy chain of hKM3900_3-34-j6, and SEQ ID NO: 36 shows the amino acid sequence of the heavy chain of hKM3900_3-34-J6. SEQ ID NO: 37 shows the nucleotide sequence of the heavy chain of hKM3900_tA2-32LH, and SEQ ID NO: 38 shows the amino acid sequence of the heavy chain of hKM3900_tA2-32LH. SEQ ID NO: 39 shows the nucleotide sequence of the hKM3900 light chain, and SEQ ID NO: 40 shows the amino acid sequence of the hKM3900 light chain. SEQ ID NO: 41 shows the amino acid sequence of the human anti-CD3 scFv-Fc fusion protein. SEQ ID NO: 42 shows the amino acid sequence of human CD137. SEQ ID NO: 43 showsthe amino acid sequence of monkey CD137. SEQ ID NO: 44 sets forth the amino acid sequence of the His-tag protein. SEQ ID NO: 45 sets forth the amino acid sequence of the 3×FLAG-tag. SEQ ID NOs: 46-54 represent the amino acid sequences of the GS linkers. SEQ ID NO: 55 sets forth the amino acid sequence of the GKPGS linker. --->Sequence Listing <110> Astellas Pharma Inc.NATIONAL CANCER CENTER <120> BISPECIFIC ANTI-CLDN4 / ANTI-CD137 ANTIBODY <130> FA1535-21350 <150> JP2021-72429 <151> 2021-04-22 <160> 55 <170> PatentIn version 3.5 <210> 1 <211> 375 <212> DNA <213> Artificial sequence <220> <223> 3D11 VH <220> <221> CDS <222> (1)..(375) <400> 1cag gtt cag ttg gga cag tct ggc ccc gaa gtg aag aaa cct ggc gcc 48Gln Val Gln Leu Gly Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Ala1 5 10 15tct gtg aag gtg tcc tgc aag gct tcc ggc tac tcc ttt atc tcc tac 96Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Ile Ser Tyr20 25 30ggc atc tcc tgg gtc cga cag gct cctgga caa ggc ttg gaa tgg atg 144Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met35 40 45ggc tgg tcc tcc gtg tac aac ggc aac acc aac tac gtg cag aaa ttc 192Gly Trp Ser Ser Val Tyr Asn Gly Asn Thr Asn Tyr Val Gln Lys Phe50 55 60cag ggc aga gtg acc atg acc acc gac acc tct gcc tcc acc gcc tac 240Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Ala Ser Thr Ala Tyr65 70 75 80atg gaa ctg cgg tcc ctg aga tct gat gac gcc gcc gtg tac tac tgc 288Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Ala Ala Val Tyr Tyr Cys85 90 95gcc aga gac tct tac tcc tcc ggc tgg tct tac tac tac tcc ggc atg 336Ala Arg Asp Ser Tyr Ser Ser Gly Trp Ser Tyr Tyr Tyr Ser Gly Met100 105 110gat gtg tgg ggc cag ggc aca aca gtg acc gtg tcc tcc 375Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser115 120 125 <210> 2 <211> 125 <212> PROTEIN <213> Artificial sequence <220> <223> Synthetic construction <400> 2Gln Val Gln Leu Gly Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly TyrSer Phe Ile Ser Tyr20 25 30Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met35 40 45Gly Trp Ser Ser Val Tyr Asn Gly Asn Thr Asn Tyr Val Gln Lys Phe50 55 60Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Ala Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Ala Ala Val Tyr Tyr Cys85 90 95Ala Arg Asp Ser Tyr Ser Ser Gly Trp Ser Tyr Tyr Tyr Ser Gly Met100 105 110Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser115 120 125<210> 3<211> 327<212> ДНК<213> Искусственная последовательность<220><223> 3D11 VL<220><221> CDS<222> (1)..(327)<400> 3gag att gtg ctg acc cag tct cct ggc aca ctg tct ttg agc cct ggc 48Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15gag aga gct acc ctg tcc tgt aga gcc tct cag tcc gtg tcc tcc tct 96Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser20 25 30tac ctg gcc tgg tat cag cag aag cct gga cag gct ccc cgg ctg ttg 144Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu35 40 45atc tac ggc gct tcttct aga gcc aca ggc atc cct gac cgg ttc tcc 192Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser50 55 60gga tct ggc tct ggc acc gat ttc acc ctg acc atc aac cgg ctg gaa 240Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Arg Leu Glu65 70 75 80ccc gag gat ttc gcc gtg tac tac tgc cag cag tac ggc tcc tct cct 288Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro85 90 95tac tct ttt ggc cag ggc acc aag ctg gaa atc ​​aag cgg 327Tyr Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg100 105 <210> 4 <211> 109 <212> PROTEIN <213> Artificial sequence <220> <223> Synthetic construction <400> 4Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala ValTyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro85 90 95Tyr Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg100 105<210> 5<211> 369<212> ДНК<213> Искусственная последовательность<220><223> hKM3900 VH<220><221> CDS<222> (1)..(369)<400> 5gaa gtg cag ctg gtt cag tct ggc gcc gaa gtg aag aaa cct ggc gcc 48Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15tcc gtg aag atc tcc tgc aag gct tct ggc tac acc ttc acc gac tac 96Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr20 25 30tac atg aac tgg gtc cga cag gct cct gga cag gga ctt gag tgg atg 144Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met35 40 45gga gat gtg gtg ccc aac aac ggc gtg ccc acc tac aac cag aaa ttc 192Gly Asp Val Val Pro Asn Asn Gly Val Pro Thr Tyr Asn Gln Lys Phe50 55 60aag ggc aga gtg acc atc acc gcc gac aag tct acc tcc acc gcc tac 240Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80atg gaa ctg cgg agc ctg aga tct gag gac acc gcc gtg tac tac tgc 288Met Glu Leu ArgSer Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys85 90 95gcc aga cct cac tac tac tac gcc ggc aga tct ggc gcc atg gat tat 336Ala Arg Pro His Tyr Tyr Tyr Ala Gly Arg Ser Gly Ala Met Asp Tyr100 105 110tgg gga cag ggc acc ctg gtc acc gtg tcc tct 369Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser115 120 <210> 6 <211> 123 <212> PROTEIN <213> Artificial sequence <220> <223> Synthetic construction <400> 6Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr20 25 30Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met35 40 45Gly Asp Val Val Pro Asn Asn Gly Val Pro Thr Tyr Asn Gln Lys Phe50 55 60Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys85 90 95Ala Arg Pro His Tyr Tyr Tyr Ala Gly Arg Ser Gly Ala Met Asp Tyr100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser115 120 <210> 7 <211> 327 <212> DNA <213> Artificialпоследовательность<220><223> hKM3900 VL<220><221> CDS<222> (1)..(327)<400> 7gac atc cag ctg acc cag tct cca tcc tct ctg tct gcc tct gtg ggc 48Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15gac aga gtg aca att acc tgc acc gcc tcc tcc acc gtg tcc tct acc 96Asp Arg Val Thr Ile Thr Cys Thr Ala Ser Ser Thr Val Ser Ser Thr20 25 30tac ctg cac tgg tat cag cag aag ccc ggc aag gct ccc aag ctg ctg 144Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu35 40 45atc tac tcc acc tcc aat ctg gcc tct ggc gtg ccc tct aga ttc tcc 192Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser50 55 60gga tct ggc tct gga acc gac tat acc ctg aca atc tcc agc ctg cag 240Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln65 70 75 80cct gag gac ttc gcc acc tac tac tgc cac cag tac cac aga tcc cca 288Pro Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Tyr His Arg Ser Pro85 90 95cct acc ttt ggc cag ggc acc aag ctg gaa atc aag cgt 327Pro Thr Phe Gly Gln Gly Thr Lys Leu GluIle Lys Arg100 105 <210> 8 <211> 109 <212> PROTEIN <213> Artificial sequence <220> <223> Synthetic construction <400> 8Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Thr Ala Ser Ser Thr Val Ser Ser Thr20 25 30Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu35 40 45Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser50 55 60Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln65 70 75 80Pro Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Tyr His Arg Ser Pro85 90 95Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg100 105 <210> 9 <211> 354 <212> DNA <213> Artificial sequence <220> <223> 3-34 VH <220> <221> CDS <222> (1)..(354) <400> 9cag gtc cag ctg cag cag tct gag gct gag ctg gtg agg cct ggg gcc 48Gln Val Gln Leu Gln Gln Ser Glu Ala Glu Leu Val Arg Pro Gly Ala1 5 10 15tca gtg aag att tcc tgc aag gct ttt ggc tac acc ttc aca aac cat 96Ser Val Lys Ile Ser Cys Lys Ala Phe Gly Tyr Thr Phe Thr AsnHis20 25 30cat ata aac tgg gtg aag cag agg cct gga cag ggc ctg gac tgg att 144His Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Asp Trp Ile35 40 45gga tat att aat cct tat aat gat tat act acc ttc aac cgg aag ttc 192Gly Tyr Ile Asn Pro Tyr Asn Asp Tyr Thr Thr Phe Asn Arg Lys Phe50 55 60aag gac aag gcc aca ttg act gta cac aaa tcc tcc agc aca gcc tat 240Lys Asp Lys Ala Thr Leu Thr Val His Lys Ser Ser Ser Thr Ala Tyr65 70 75 80atg gag ctt agc ggc ctg aca tct gac gac tct gca gtc tat tac tgt 288Met Glu Leu Ser Gly Leu Thr Ser Asp Asp Ser Ala Val Tyr Tyr Cys85 90 95gca aga acc tgg ggt cac tgg tat ttc gat ctc tgg ggc cgt ggc acc 336Ala Arg Thr Trp Gly His Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr100 105 110gtg gtc act gtc tcc tca 354Val Val Thr Val Ser Ser115 <210> 10 <211> 118 <212> PROTEIN <213> Artificial sequence <220> <223> Synthetic construction <400> 10Gln Val Gln Leu Gln Gln Ser Glu Ala Glu Leu Val Arg Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Phe Gly Tyr Thr Phe ThrAsn His20 25 30His Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Asp Trp Ile35 40 45Gly Tyr Ile Asn Pro Tyr Asn Asp Tyr Thr Thr Phe Asn Arg Lys Phe50 55 60Lys Asp Lys Ala Thr Leu Thr Val His Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Gly Leu Thr Ser Asp Asp Ser Ala Val Tyr Tyr Cys85 90 95Ala Arg Thr Trp Gly His Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr100 105 110Val Val Thr Val Ser Ser115<210> 11<211> 327<212> ДНК<213> Искусственная последовательность<220><223> 3-34 VL<220><221> CDS<222> (1)..(327)<400> 11gaa ata gtg atg acg cag tct cca gcc acc ctg tct gtg tct cca ggg 48Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly1 5 10 15gaa aga gcc acc ctc tcc tgt agg gcc agt cag agt gtt agc agc aac 96Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn20 25 30tta gcc tgg tac cag cag aaa cct ggc cag gct ccc agg ctc ctc atc 144Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile35 40 45tat gat tca tcc acc agg gcc act ggt atc cca gcc agg ttc agt ggc192Tyr Asp Ser Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly50 55 60agt ggg tct ggg aca gac ttc act ctc acc atc agc agc ctg cag tct 240Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser65 70 75 80gaa gat ttt gca gtt tat tac tgt cag cag tat aat aac tgg cct ccg 288Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Pro Pro85 90 95tgg acg ttc ggc caa ggg acc aag gtg gaa atc ​​aaa cgg 327Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg100 105 <210> 12 <211> 109 <212> PROTEIN <213> Artificial sequence <220> <223> Synthetic construction <400> 12Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile35 40 45Tyr Asp Ser Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Pro8590 95Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg100 105<210> 13<211> 354<212> ДНК<213> Искусственная последовательность<220><223> A2-32 VH<220><221> CDS<222> (1)..(354)<400> 13cag gtg cag ctg cag gaa tcg ggc cca gga ctg gtg aag cct tcg gag 48Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15acc ctg tcc ctc acc tgc act gtc tct ggc ggc tcc atc agt ggt tat 96Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Gly Tyr20 25 30tac tgg agc tgg atc cgg cag ccc cca ggg aag ggc ctg gag tgg att 144Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile35 40 45gga tat atc tat tac agt gga ttc acc aac tac aac ccc tcc ctc aag 192Gly Tyr Ile Tyr Tyr Ser Gly Phe Thr Asn Tyr Asn Pro Ser Leu Lys50 55 60agt cga gtc acc ata tca gta gac acg tcc aag aac cag ttc tcc ctg 240Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75 80aaa ctg agc tct gtg acc gct gcg gac acg gcc gtg tat tat tgt acg 288Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr TyrCys Thr85 90 95aga gat cac tcc cac tac tac tac atg gac gtc tgg ggc caa ggg acc 336Arg Asp His Ser His Tyr Tyr Tyr Met Asp Val Trp Gly Gln Gly Thr100 105 110acg gtc acc gtc tcc tca 354Thr Val Thr Val Ser Ser115 <210> 14 <211> 118 <212> PROTEIN <213> Artificial sequence <220> <223> Synthetic construction <400> 14Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Gly Tyr20 25 30Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile35 40 45Gly Tyr Ile Tyr Tyr Ser Gly Phe Thr Asn Tyr Asn Pro Ser Leu Lys50 55 60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Thr85 90 95Arg Asp His Ser His Tyr Tyr Tyr Met Asp Val Trp Gly Gln Gly Thr100 105 110Thr Val Thr Val Ser Ser115 <210> 15 <211> 333 <212> DNA <213> Artificial sequence <220> <223> A2-32 VL <220> <221> CDS <222> (1)..(333) <400> 15cag tct gtg ctg act cag ccaccc tca gcg tct ggg acc ccc ggg cag 48Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5 10 15agg gtc acc atc tct tgt tct gga agc agc tcc aac atc gga agt aat 96Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn20 25 30act gta aac tgg tac cag cag ttc cca gga acg gcc ccc aaa ctc ctc 144Thr Val Asn Trp Tyr Gln Gln Phe Pro Gly Thr Ala Pro Lys Leu Leu35 40 45atc tat agt aat aat cag cgg ccc tca ggg gtc cct gac cga ttc tct 192Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser50 55 60ggc tcc aag tct ggc acc tca gcc tcc ctg gcc atc agt ggg ctc cag 240Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln65 70 75 80tct gag gat gag gct gat tat tac tgt gca gga tgg gat gac agc ctg 288Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Gly Trp Asp Asp Ser Leu85 90 95aat ggt ccg gta ttc ggc gga ggg acc aag ctg acc gtc cta ggt 333Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly100 105 110<210> 16<211> 111<212> БЕЛОК<213> Искусственнаяsubsequence <220> <223> Synthetic construction <400> 16Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn20 25 30Thr Val Asn Trp Tyr Gln Gln Phe Pro Gly Thr Ala Pro Lys Leu Leu35 40 45Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg...

Claims

1. A bispecific anti-CLDN4 / anti-CD137 antibody comprising 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, (a) wherein the heavy chain variable region and the light chain variable region of the anti-CLDN4 antibody comprise a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence at amino acid positions 31-35 of SEQ ID NO: 2, a CDR2 consisting of the amino acid sequence at amino acid positions 50-66 of SEQ ID NO: 2, and a CDR3 consisting of the amino acid sequence at amino acid positions 99-114 of SEQ ID NO: 2, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence at amino acid positions 24-35 of SEQ ID NO: 4, a CDR2 consisting of the amino acid sequence at amino acid positions 51-57 of SEQ ID NO: 4, and a CDR3 consisting of the amino acid sequence at amino acid positions 90-98 of SEQ ID NO: 4; and wherein the heavy chain variable region and the light chain variable region of the anti-CD137 antibody comprise: a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence at amino acid positions 31-35 of SEQ ID NO: 10, a CDR2 consisting of the amino acid sequence at amino acid positions 50-66 of SEQ ID NO: 10, and a CDR3 consisting of the amino acid sequence at amino acid positions 99-107 of SEQ ID NO: 10, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence at amino acid positions 24-34 of SEQ ID NO: 12, a CDR2 consisting of the amino acid sequence at amino acid positions 50-56 of SEQ ID NO: 12, and a CDR3 consisting of the amino acid sequence at amino acid positions 89-98 of SEQ ID NO: 12; or (b) wherein the heavy chain variable region and the light chain variable region of the anti-CLDN4 antibody comprise a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence at amino acid positions 31-35 of SEQ ID NO: 6, a CDR2 consisting of the amino acid sequence at amino acid positions 50-66 of SEQ ID NO: 6, and a CDR3 consisting of the amino acid sequence at amino acid positions 99-112 of SEQ ID NO: 6, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence at amino acid positions 24-35 of SEQ ID NO: 8, a CDR2 consisting of the amino acid sequence at amino acid positions 51-57 of SEQ ID NO: 8, and a CDR3 consisting of the amino acid sequence at amino acid positions 90-98 of SEQ ID NO: 8; and wherein the heavy chain variable region and the light chain variable region of the anti-CD137 antibody comprise one of the following variants: (i) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence at amino acid positions 31-35 of SEQ ID NO: 10, a CDR2 consisting of the amino acid sequence at amino acid positions 50-66 of SEQ ID NO: 10, and a CDR3 consisting of the amino acid sequence at amino acid positions 99-107 of SEQ ID NO: 10, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence at amino acid positions 24-34 of SEQ ID NO: 12, a CDR2 consisting of the amino acid sequence at amino acid positions 50-56 of SEQ ID NO: 12, and a CDR3 consisting of the amino acid sequence at amino acid positions 89-98 of SEQ ID NO: 12; (ii) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence at amino acid positions 31-35 of SEQ ID NO: 14, a CDR2 consisting of the amino acid sequence at amino acid positions 50-65 of SEQ ID NO: 14, and a CDR3 consisting of the amino acid sequence at amino acid positions 98-107 of SEQ ID NO: 14, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence at amino acid positions 23-35 of SEQ ID NO: 16, a CDR2 consisting of the amino acid sequence at amino acid positions 51-57 of SEQ ID NO: 16, and a CDR3 consisting of the amino acid sequence at amino acid positions 90-100 of SEQ ID NO: 16; (iii) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence at amino acid positions 31-35 of SEQ ID NO: 18, a CDR2 consisting of the amino acid sequence at amino acid positions 50-65 of SEQ ID NO: 18, and a CDR3 consisting of the amino acid sequence at amino acid positions 98-107 of SEQ ID NO: 18, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence at amino acid positions 23-35 of SEQ ID NO: 20, a CDR2 consisting of the amino acid sequence at amino acid positions 51-57 of SEQ ID NO: 20, and a CDR3 consisting of the amino acid sequence at amino acid positions 90-100 of SEQ ID NO: 20; or (iv) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence at amino acid positions 31-35 of SEQ ID NO: 22, a CDR2 consisting of the amino acid sequence at amino acid positions 50-65 of SEQ ID NO: 22, and a CDR3 consisting of the amino acid sequence at amino acid positions 98-110 of SEQ ID NO: 22, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence at amino acid positions 23-35 of SEQ ID NO: 24, a CDR2 consisting of the amino acid sequence at amino acid positions 51-57 of SEQ ID NO: 24, and a CDR3 consisting of the amino acid sequence at amino acid positions 90-100 of SEQ ID NO:

24.

2. The bispecific antibody of claim 1, wherein the heavy chain variable region and the light chain variable region of the anti-CLDN4 antibody are any of the following (a) or (b): (a) a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-125 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 4; or (b) a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-123 of SEQ ID NO: 6 and a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO:

8.

3. A bispecific antibody according to any one of claims 1 and 2, comprising an IgG antibody consisting of a heavy chain containing a variable region of a heavy chain and a light chain containing a variable region of a light chain of an anti-CLDN4 antibody (IgG anti-CLDN4 antibody).

4. The bispecific antibody of claim 3, comprising a LALA mutation (L234A and L235A, wherein the position of the mutation is an amino acid position in the constant region of human Igγ1 according to the EU numbering system) in the Fc region of an IgG anti-CLDN4 antibody.

5. The bispecific antibody of claim 3, comprising a P331G mutation in the Fc region of an IgG anti-CLDN4 antibody (wherein the position of the mutation is an amino acid position in the constant region of human Igγ1 according to the EU numbering system).

6. The bispecific antibody according to claim 3, comprising a LALA mutation and a P331G mutation in the Fc region of an IgG anti-CLDN4 antibody.

7. The bispecific antibody of any one of claims 1 to 6, wherein the heavy chain variable region and the light chain variable region of the anti-CD137 antibody are any of the following (a) to (i): (a) a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 10 and a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 12; (b) a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 14 and a light chain variable region consisting of the amino acid sequence at amino acid positions 1-111 of SEQ ID NO: 16; (c) a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 18 and a light chain variable region consisting of the amino acid sequence at amino acid positions 1-111 of SEQ ID NO: 20; (d) a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-121 of SEQ ID NO: 22 and a light chain variable region consisting of the amino acid sequence at amino acid positions 1-111 of SEQ ID NO: 24; (e) a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 26 and a light chain variable region consisting of the amino acid sequence at amino acid positions 134-242 of SEQ ID NO: 26; (f) a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 28 and a light chain variable region consisting of the amino acid sequence at amino acid positions 134-244 of SEQ ID NO: 28; (g) a light chain variable region consisting of the amino acid sequence at amino acid positions 1-111 of SEQ ID NO: 30 and a heavy chain variable region consisting of the amino acid sequence at amino acid positions 132-249 of SEQ ID NO: 30; (h) a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 32 and a light chain variable region consisting of the amino acid sequence at amino acid positions 134-244 of SEQ ID NO: 32; or (i) a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-121 of SEQ ID NO: 34, and a light chain variable region consisting of the amino acid sequence at amino acid positions 137-247 of SEQ ID NO:

34.

8. A bispecific antibody according to any one of claims 6 and 7, comprising an anti-CD137 single-chain variable fragment (anti-CD137 scFv) comprising the variable region of the heavy chain and the variable region of the light chain of an anti-CD137 antibody.

9. The bispecific antibody of claim 8, wherein the anti-CD137 scFv is any of the following (a)-(e): (a) an anti-CD137 scFv consisting of the amino acid sequence at amino acid positions 1-242 of SEQ ID NO: 26; (b) an anti-CD137 scFv consisting of the amino acid sequence at amino acid positions 1-244 of SEQ ID NO: 28; (c) an anti-CD137 scFv consisting of the amino acid sequence at amino acid positions 1-249 of SEQ ID NO: 30; (d) an anti-CD137 scFv consisting of the amino acid sequence at amino acid positions 1-244 of SEQ ID NO: 32; or (e) an anti-CD137 scFv consisting of the amino acid sequence at amino acid positions 1-247 of SEQ ID NO:

34.

10. A bispecific antibody according to any one of claims 8 and 9, comprising an IgG anti-CLDN4 antibody and an anti-CD137 scFv, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the heavy chain or light chain of the IgG anti-CLDN4 antibody via a linker.

11. The bispecific antibody of any one of claims 1 to 8, wherein the bispecific antibody is any of those described in any of the following points (a) to (j): (a) a bispecific antibody comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-125 of SEQ ID NO: 2 and an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 4 and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 26 and a light chain variable region consisting of the amino acid sequence at amino acid positions 134-242 of SEQ ID NO: 26, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the anti-CLDN4 antibody heavy chain via a linker; (b) a bispecific antibody comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-125 of SEQ ID NO: 2 and an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 4 and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 28 and a light chain variable region consisting of the amino acid sequence at amino acid positions 134-244 of SEQ ID NO: 28, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the anti-CLDN4 antibody heavy chain via a linker; (c) a bispecific antibody comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-125 of SEQ ID NO: 2 and an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 4 and an anti-CD137 scFv comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-111 of SEQ ID NO: 30 and a heavy chain variable region consisting of the amino acid sequence at amino acid positions 132-249 of SEQ ID NO: 30, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the anti-CLDN4 antibody heavy chain via a linker; (d) a bispecific antibody comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-125 of SEQ ID NO: 2 and an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 4 and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 32 and a light chain variable region consisting of the amino acid sequence at amino acid positions 134-244 of SEQ ID NO: 32, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the anti-CLDN4 antibody heavy chain via a linker; (e) a bispecific antibody comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-125 of SEQ ID NO: 2 and an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 4 and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-121 of SEQ ID NO: 34 and a light chain variable region consisting of the amino acid sequence at amino acid positions 137-247 of SEQ ID NO: 34, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the anti-CLDN4 antibody heavy chain via a linker; (f) a bispecific antibody comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-123 of SEQ ID NO: 6 and an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 8 and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 26 and a light chain variable region consisting of the amino acid sequence at amino acid positions 134-242 of SEQ ID NO: 26, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the anti-CLDN4 antibody heavy chain via a linker; (g) a bispecific antibody comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-123 of SEQ ID NO: 6 and an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 8 and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 28 and a light chain variable region consisting of the amino acid sequence at amino acid positions 134-244 of SEQ ID NO: 28, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the anti-CLDN4 antibody heavy chain via a linker; (h) a bispecific antibody comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-123 of SEQ ID NO: 6 and an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 8 and an anti-CD137 scFv comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-111 of SEQ ID NO: 30 and a heavy chain variable region consisting of the amino acid sequence at amino acid positions 132-249 of SEQ ID NO: 30, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the anti-CLDN4 antibody heavy chain via a linker; (i) a bispecific antibody comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-123 of SEQ ID NO: 6 and an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 8 and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-118 of SEQ ID NO: 32 and a light chain variable region consisting of the amino acid sequence at amino acid positions 134-244 of SEQ ID NO: 32, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the anti-CLDN4 antibody heavy chain via a linker; or (j) a bispecific antibody comprising an anti-CLDN4 antibody heavy chain comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-123 of SEQ ID NO: 6 and an anti-CLDN4 antibody light chain comprising a light chain variable region consisting of the amino acid sequence at amino acid positions 1-109 of SEQ ID NO: 8 and an anti-CD137 scFv comprising a heavy chain variable region consisting of the amino acid sequence at amino acid positions 1-121 of SEQ ID NO: 34 and a light chain variable region consisting of the amino acid sequence at amino acid positions 137-247 of SEQ ID NO: 34, wherein the amino terminus of the anti-CD137 scFv is linked to the carboxy terminus of the anti-CLDN4 antibody heavy chain via a linker.

12. The bispecific anti-CLDN4 / anti-CD137 antibody of any one of claims 10 and 11, wherein the linker is a GS linker.

13. The bispecific anti-CLDN4 / anti-CD137 antibody of claim 12, wherein the GS linker is a linker consisting of the amino acid sequence of SEQ ID NO:

54.

14. A bispecific anti-CLDN4 / anti-CD137 antibody as described in any of the following (a) or (b): (a) a bispecific antibody comprising a polypeptide consisting of the amino acid sequence at amino acid positions 1-705 of SEQ ID NO: 36, comprising the heavy chain of an anti-CLDN4 antibody and an anti-CD137 scFv, and a light chain of an anti-CLDN4 antibody consisting of the amino acid sequence at amino acid positions 1-215 of SEQ ID NO: 40; or (b) a bispecific antibody comprising a polypeptide consisting of an amino acid sequence at amino acid positions 1-712 of SEQ ID NO: 38, comprising a heavy chain of an anti-CLDN4 antibody and an anti-CD137 scFv, and a light chain of an anti-CLDN4 antibody consisting of an amino acid sequence at amino acid positions 1-215 of SEQ ID NO:

40.

15. The bispecific anti-CLDN4 / anti-CD137 antibody of any one of claims 1-14, which is post-translationally modified.

16. The bispecific anti-CLDN4 / anti-CD137 antibody of claim 15, wherein the post-translational modification is pyroglutamylation at the N-terminus of the heavy chain variable region and / or a lysine deletion at the C-terminus of the heavy chain.

17. A set of polynucleotides for producing an anti-CLDN4 antibody in the bispecific anti-CLDN4 / anti-CD137 antibody according to claim 2, comprising a first polynucleotide encoding the heavy chain of this antibody to CLDN4, and a second polynucleotide encoding the light chain of this antibody to CLDN4.

18. A set of polynucleotides for producing an anti-CD137 antibody in the bispecific anti-CLDN4 / anti-CD137 antibody according to claim 7, comprising a first polynucleotide encoding the heavy chain of this antibody to CD137, and a second polynucleotide encoding the light chain of this antibody to CD137.

19. The set of polynucleotides according to claim 18, having a nucleotide sequence encoding an anti-CD137 scFv, which is selected from the group consisting of the following items (a)-(e): (a) a polynucleotide having a nucleotide sequence encoding an anti-CD137 scFv consisting of the amino acid sequence at amino acid positions 1-242 of SEQ ID NO: 26; (b) a polynucleotide having a nucleotide sequence encoding an anti-CD137 scFv consisting of the amino acid sequence at amino acid positions 1-244 of SEQ ID NO: 28; (c) a polynucleotide having a nucleotide sequence encoding an anti-CD137 scFv consisting of the amino acid sequence at amino acid positions 1-249 of SEQ ID NO: 30; (d) a polynucleotide having a nucleotide sequence encoding an anti-CD137 scFv consisting of the amino acid sequence at amino acid positions 1-244 of SEQ ID NO: 32; or (e) a polynucleotide having a nucleotide sequence encoding an anti-CD137 scFv consisting of the amino acid sequence at amino acid positions 1-247 of SEQ ID NO:

34.

20. A set of polynucleotides for producing a bispecific anti-CLDN4 / anti-CD137 antibody according to claim 14, comprising a first polynucleotide encoding the heavy chain of this antibody to CLDN4, a second polynucleotide encoding the light chain of this antibody to CLDN4, a third polynucleotide encoding the heavy chain of this antibody to CD137, and a fourth polynucleotide encoding the light chain of this antibody to CD137.

21. An expression vector containing a set of polynucleotides according to claims 17 and 18 or a set of polynucleotides according to claim 20.

22. A host cell for producing a bispecific anti-CLDN4 / anti-CD137 antibody, wherein the host cell is transformed with the expression vector according to claim 21.

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

40.

24. The host cell of claim 22 or 23, comprising a polynucleotide according to any of the following points (a) or (b): (a) a polynucleotide having a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence at amino acid positions 1-705 of SEQ ID NO: 36, comprising a heavy chain of an anti-CLDN4 antibody and an anti-CD137 scFv, and a polynucleotide having a nucleotide sequence encoding a light chain of an anti-CLDN4 antibody consisting of the amino acid sequence at amino acid positions 1-215 of SEQ ID NO: 40; or (b) a polynucleotide having a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence at amino acid positions 1-712 of SEQ ID NO: 38, containing the heavy chain of an anti-CLDN4 antibody and an anti-CD137 scFv, and a polynucleotide having a nucleotide sequence encoding the light chain of an anti-CLDN4 antibody, consisting of the amino acid sequence at amino acid positions 1-215 of SEQ ID NO:

40.

25. A method for producing a bispecific anti-CLDN4 / anti-CD137 antibody, comprising the step of culturing a host cell according to any one of claims 22-24.

26. A pharmaceutical composition for treating cancer associated with CLDN4 expression, comprising a bispecific anti-CLDN4 / anti-CD137 antibody according to any one of claims 1-16 and a pharmaceutically acceptable excipient.

27. A bispecific anti-CLDN4 / anti-CD137 antibody according to any one of claims 1-16 for use in the treatment of cancer associated with CLDN4 expression.

28. The pharmaceutical composition of claim 26 for use in the treatment of cancer associated with CLDN4 expression.

29. A method for treating cancer associated with CLDN4 expression, comprising the step of administering a therapeutically effective amount of a bispecific anti-CLDN4 / anti-CD137 antibody according to any one of claims 1-16 to a subject.

30. Use of a bispecific anti-CLDN4 / anti-CD137 antibody according to any one of claims 1-16 in the manufacture of a pharmaceutical composition for use in the treatment of cancer associated with CLDN4 expression.

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