Cytotoxic inducing therapeutic agent to be used in treating cancer

Abstract

To provide an anticancer agent, a combination therapy, and a pharmaceutical composition to be used in the combination therapy in which a T-cell can be efficiently and specifically mobilized to a target cancer cell, especially a CLDN6 expression cell such as a cancer cell, and cancer is treated through cytotoxic activity of the T-cell to the target cancer tissue containing the CLDN6 expression cell.SOLUTION: The present invention provides an anticancer agent including multispecific antigen-binding molecules which targets CLDN6, a combination therapy of the anticancer agent and at least one of other anticancer agents, and a pharmaceutical composition to be used in the combination therapy.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to an anti-cancer agent comprising a multispecific antigen-binding molecule that targets claudin-6, and a combination therapy with at least one other anti-cancer agent. [Background technology]

[0002] The claudin family is a family of cell membrane proteins with a molecular weight of approximately 23 kDa that have four transmembrane domains and form tight junctions. The claudin family contains 24 members in humans and mice, and each member of the claudin family is known to exhibit a very unique expression pattern depending on the epithelial cell type (Non-Patent Documents 1 to 4). In epithelial cell sheets, a mechanism operates to prevent substances from leaking (diffusing) into the intercellular space, and the intercellular adhesion system known as tight junctions has been shown to actually play a central role as a "barrier" in this mechanism to prevent leakage.

[0003] The tight junction molecule claudin 6 (CLDN6) is a member of the claudin family of proteins and is not transcriptionally expressed in normal biological tissues (Non-Patent Documents 5 and 6). However, its expression is elevated in several types of cancer, including ovarian cancer, NSCLC, and gastric cancer (Non-Patent Documents 7 to 9). Regarding anti-CLDN6 antibodies, monospecific antibodies against CLDN6 have been reported to have ADCC activity or internalization activity against CLDN6-positive cancer cell lines (Patent Documents 1 to 5). To date, a T cell-redirecting bispecific antibody targeting CLDN6, designated 6PHU3, has been created using a bispecific sc(Fv)2 format with anti-CD3 / anti-CLDN6 specificity (Patent Documents 6 to 7). In preclinical evaluation, 6PHU3 has been reported to exhibit potent killing of cancer cells in vitro and in vivo (Non-Patent Document 10). [Prior art documents] [Chartered documents]

[0004]

Patent Document 1

Patent document 2

Patent document 3

Patent document 4

Patent document 5

Patent document 6

Patent document 7

Non-licensed literature

[0005] [Non-licensed document 1] Furuse and Tsukita, TRENDS in Cell Biology 2006, 16: 181 [Non-licensed document 2] Wilcox, et al., Cell 2001, 104: 165 [Non-licensed document 3] Rahner, et al., GASTROENTEROLOGY 2001, 120: 411

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

[0006] An object of the present disclosure is to provide an anticancer agent containing as an active ingredient a multispecific antigen-binding molecule that can efficiently and specifically recruit T cells to target cancer cells, particularly CLDN6-expressing cells such as cancer cells, and can treat cancer through the cytotoxic activity of T cells against target cancer tissues containing CLDN6-expressing cells. Another object of the present invention is to provide a combination therapy using the multispecific antigen-binding molecule and other drugs. [Means for solving the problem]

[0007] The present inventors have discovered a multispecific antigen-binding molecule that can bind to both CD3 and CD137 (4-1BB) and contains a first antigen-binding moiety that binds to either CD3 or CD137 (i.e., dual-binding to CD3 and CD137, but not simultaneously), and a second antigen-binding moiety that can bind to molecules specifically expressed in cancer tissues, particularly claudin 6 (CLDN6). The present inventors have demonstrated that the multispecific antigen-binding molecule of the present invention damages cancer cells, including CLDN6-expressing cancer cells. The present invention provides anticancer agents containing the multispecific antigen-binding molecule as an active ingredient, combination therapies using the multispecific antigen-binding molecule and at least one other anticancer agent, and pharmaceutical compositions comprising a combination of the multispecific antigen-binding molecule and an anticancer agent.

[0008] More specifically, the present disclosure provides: (A-1) An anticancer agent comprising the following multispecific antigen-binding molecule as an active ingredient: A multispecific antigen-binding molecule comprising: (i) a first antigen-binding portion capable of binding to CD3 and CD137, the first antigen-binding portion binding to either CD3 or CD137; and (ii) a second antigen-binding portion capable of binding to claudin 6 (CLDN6). (A-2) An anticancer agent comprising, as an active ingredient, a multispecific antigen-binding molecule according to any one of (1) to (6) below: (1) A multispecific antigen-binding molecule comprising: a first antibody variable region comprising a complementarity-determining region (CDR) 1 of SEQ ID NO: 11, a CDR 2 of SEQ ID NO: 17, and a CDR 3 of SEQ ID NO: 23; a second antibody variable region comprising a CDR 1 of SEQ ID NO: 32, a CDR 2 of SEQ ID NO: 36, and a CDR 3 of SEQ ID NO: 40; a third antibody variable region comprising a complementarity-determining region (CDR) 1 of SEQ ID NO: 7, a CDR 2 of SEQ ID NO: 13, and a CDR 3 of SEQ ID NO: 19; and a fourth antibody variable region comprising a CDR 1 of SEQ ID NO: 29, a CDR 2 of SEQ ID NO: 33, and a CDR 3 of SEQ ID NO: 37; (2) A multispecific antigen-binding molecule comprising: a first antibody variable region comprising a complementarity-determining region (CDR) 1 of SEQ ID NO: 9, a CDR 2 of SEQ ID NO: 15, and a CDR 3 of SEQ ID NO: 21; a second antibody variable region comprising a CDR 1 of SEQ ID NO: 31, a CDR 2 of SEQ ID NO: 35, and a CDR 3 of SEQ ID NO: 39; a third antibody variable region comprising a complementarity-determining region (CDR) 1 of SEQ ID NO: 8, a CDR 2 of SEQ ID NO: 14, and a CDR 3 of SEQ ID NO: 20; and a fourth antibody variable region comprising a CDR 1 of SEQ ID NO: 30, a CDR 2 of SEQ ID NO: 34, and a CDR 3 of SEQ ID NO: 38; (3) A multispecific antigen-binding molecule comprising: a first antibody variable region comprising a complementarity-determining region (CDR) 1 of SEQ ID NO: 10, a CDR 2 of SEQ ID NO: 16, and a CDR 3 of SEQ ID NO: 22; a second antibody variable region comprising a CDR 1 of SEQ ID NO: 31, a CDR 2 of SEQ ID NO: 35, and a CDR 3 of SEQ ID NO: 39; a third antibody variable region comprising a complementarity-determining region (CDR) 1 of SEQ ID NO: 8, a CDR 2 of SEQ ID NO: 14, and a CDR 3 of SEQ ID NO: 20; and a fourth antibody variable region comprising a CDR 1 of SEQ ID NO: 30, a CDR 2 of SEQ ID NO: 34, and a CDR 3 of SEQ ID NO: 38; (4) A multispecific antigen-binding molecule comprising: a first antibody variable region comprising a complementarity-determining region (CDR) 1 of SEQ ID NO: 12, a CDR 2 of SEQ ID NO: 18, and a CDR 3 of SEQ ID NO: 24; a second antibody variable region comprising a CDR 1 of SEQ ID NO: 32, a CDR 2 of SEQ ID NO: 36, and a CDR 3 of SEQ ID NO: 40; a third antibody variable region comprising a complementarity-determining region (CDR) 1 of SEQ ID NO: 7, a CDR 2 of SEQ ID NO: 13, and a CDR 3 of SEQ ID NO: 19; and a fourth antibody variable region comprising a CDR 1 of SEQ ID NO: 29, a CDR 2 of SEQ ID NO: 33, and a CDR 3 of SEQ ID NO: 37; (5) A multispecific antigen-binding molecule comprising: a first antibody variable region comprising a complementarity-determining region (CDR) 1 of SEQ ID NO: 11, a CDR 2 of SEQ ID NO: 17, and a CDR 3 of SEQ ID NO: 23; a second antibody variable region comprising a CDR 1 of SEQ ID NO: 32, a CDR 2 of SEQ ID NO: 36, and a CDR 3 of SEQ ID NO: 40; a third antibody variable region comprising a complementarity-determining region (CDR) 1 of SEQ ID NO: 29, a CDR 2 of SEQ ID NO: 33, and a CDR 3 of SEQ ID NO: 37; and a fourth antibody variable region comprising a CDR 1 of SEQ ID NO: 7, a CDR 2 of SEQ ID NO: 13, and a CDR 3 of SEQ ID NO: 19; (6) A multispecific antigen-binding molecule comprising: a first antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 12, CDR 2 of SEQ ID NO: 18, and CDR 3 of SEQ ID NO: 24; a second antibody variable region comprising CDR 1 of SEQ ID NO: 32, CDR 2 of SEQ ID NO: 36, and CDR 3 of SEQ ID NO: 40; a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 29, CDR 2 of SEQ ID NO: 33, and CDR 3 of SEQ ID NO: 37; and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 7, CDR 2 of SEQ ID NO: 13, and CDR 3 of SEQ ID NO: 19. (A-3) The anticancer agent of (A-2), wherein at least one selected from the group consisting of the first antibody variable region, the second antibody variable region, the third antibody variable region, and the fourth antibody variable region comprises a human antibody framework or a humanized antibody framework. (A-4) An anticancer agent comprising, as an active ingredient, a multispecific antigen-binding molecule according to any one of the following (I) to (VI): (I) A multispecific antigen-binding molecule comprising: a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 5; a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 28; a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 1; and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 25; (II) a multispecific antigen-binding molecule comprising: a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 3; a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 27; a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 2; and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 26; (III) A multispecific antigen-binding molecule comprising: a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 4; a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 27; a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 2; and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 26; (IV) a multispecific antigen-binding molecule comprising: a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 6; a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 28; a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 1; and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 25; (V) a multispecific antigen-binding molecule comprising: a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 5; a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 28; a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 25; and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 1; (VI) A multispecific antigen-binding molecule comprising: a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 6; a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 28; a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 25; and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 1. (A-5) Any one of the anticancer agents (A-2) to (A-4), wherein the first antibody variable region and the second antibody variable region are capable of binding to CD3 and CD137 and constitute a first antigen-binding moiety that binds to either CD3 or CD137, and the third antibody variable region and the fourth antibody variable region constitute a second antigen-binding moiety that can bind to CLDN6. (A-6) An anticancer agent according to any one of (A-2) to (A-4), wherein the first antibody variable region and the second antibody variable region constitute a first antigen-binding moiety that binds to CD3, and the third antibody variable region and the fourth antibody variable region constitute a second antigen-binding moiety that can bind to CLDN6. (A-7) An anticancer agent according to any one of (A-2) to (A-4), wherein the first antibody variable region and the second antibody variable region constitute a first antigen-binding portion that binds to CD137, and the third antibody variable region and the fourth antibody variable region constitute a second antigen-binding portion that can bind to CLDN6. (A-8)(i) a first antigen-binding portion that binds to CD3; and (ii) a second antigen-binding moiety that binds to claudin 6 (CLDN6); An anticancer agent comprising, as an active ingredient, a multispecific antigen-binding molecule comprising: The first antigen-binding portion is selected from the following (a1) to (a4): (a1) a first antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 9, CDR 2 of SEQ ID NO: 15, and CDR 3 of SEQ ID NO: 21, and a second antibody variable region comprising CDR 1 of SEQ ID NO: 31, CDR 2 of SEQ ID NO: 35, and CDR 3 of SEQ ID NO: 39; (a2) a first antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 10, CDR 2 of SEQ ID NO: 16, and CDR 3 of SEQ ID NO: 22, and a second antibody variable region comprising CDR 1 of SEQ ID NO: 31, CDR 2 of SEQ ID NO: 35, and CDR 3 of SEQ ID NO: 39; (a3) a first antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 11, CDR 2 of SEQ ID NO: 17, and CDR 3 of SEQ ID NO: 23, and a second antibody variable region comprising CDR 1 of SEQ ID NO: 32, CDR 2 of SEQ ID NO: 36, and CDR 3 of SEQ ID NO: 40; (a4) a first antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 12, CDR 2 of SEQ ID NO: 18, and CDR 3 of SEQ ID NO: 24, and a second antibody variable region comprising CDR 1 of SEQ ID NO: 32, CDR 2 of SEQ ID NO: 36, and CDR 3 of SEQ ID NO: 40; An anticancer drug containing one of the following: (A-9) The second antigen-binding moiety is selected from the following (b1) to (b3): (b1) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 8, CDR 2 of SEQ ID NO: 14, and CDR 3 of SEQ ID NO: 20, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 30, CDR 2 of SEQ ID NO: 34, and CDR 3 of SEQ ID NO: 38; (b2) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 7, CDR 2 of SEQ ID NO: 13, and CDR 3 of SEQ ID NO: 19, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 29, CDR 2 of SEQ ID NO: 33, and CDR 3 of SEQ ID NO: 37; (b3) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 29, CDR 2 of SEQ ID NO: 33, and CDR 3 of SEQ ID NO: 37, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 7, CDR 2 of SEQ ID NO: 13, and CDR 3 of SEQ ID NO: 19. An anticancer agent (A-8) comprising any one of the following: (A-10)(i) a first antigen-binding portion that binds to CD3; and (ii) a second antigen-binding moiety that binds to claudin 6 (CLDN6); An anticancer agent comprising, as an active ingredient, a multispecific antigen-binding molecule comprising: The second antigen-binding moiety is selected from the following (b1) to (b3): (b1) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 8, CDR 2 of SEQ ID NO: 14, and CDR 3 of SEQ ID NO: 20, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 30, CDR 2 of SEQ ID NO: 34, and CDR 3 of SEQ ID NO: 38; (b2) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 7, CDR 2 of SEQ ID NO: 13, and CDR 3 of SEQ ID NO: 19, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 29, CDR 2 of SEQ ID NO: 33, and CDR 3 of SEQ ID NO: 37; (b3) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 29, CDR 2 of SEQ ID NO: 33, and CDR 3 of SEQ ID NO: 37, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 7, CDR 2 of SEQ ID NO: 13, and CDR 3 of SEQ ID NO: 19. An anticancer drug containing one of the following: (A-11) The anticancer agent of any one of (A-8) to (A-10), wherein at least one selected from the group consisting of the first antibody variable region, the second antibody variable region, the third antibody variable region, and the fourth antibody variable region comprises a human antibody framework or a humanized antibody framework. (A-12) (i) a first antigen-binding portion that binds to CD3; and (ii) a second antigen-binding moiety that binds to claudin 6 (CLDN6); An anticancer agent comprising, as an active ingredient, a multispecific antigen-binding molecule comprising: The first antigen-binding portion is selected from the following (c1) to (c4): (c1) a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 3, and a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 27; (c2) a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 4, and a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 27; (c3) a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 5, and a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 28; (c4) a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 6, and a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 28 An anticancer drug containing one of the following: (A-13) The second antigen-binding portion is selected from the group consisting of the following (d1) to (d3): (d1) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 2, and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 26; (d2) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 1, and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 25; (d3) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 25, and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 1 An anticancer agent according to (A-12), comprising any one of the following: (A-14)(i) a first antigen-binding portion that binds to CD3; and (ii) a second antigen-binding moiety that binds to claudin 6 (CLDN6); An anticancer agent comprising, as an active ingredient, a multispecific antigen-binding molecule comprising: The second antigen-binding portion is selected from the following (d1) to (d3): (d1) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 2, and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 26; (d2) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 1, and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 25; (d3) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 25, and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 1 An anticancer drug containing one of the following: (A-15)(i) a first antigen-binding moiety that binds to CD137; and (ii) a second antigen-binding moiety that binds to claudin 6 (CLDN6); An anticancer agent comprising, as an active ingredient, a multispecific antigen-binding molecule comprising: The first antigen-binding portion is selected from the following (a1) to (a4): (a1) a first antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 9, CDR 2 of SEQ ID NO: 15, and CDR 3 of SEQ ID NO: 21, and a second antibody variable region comprising CDR 1 of SEQ ID NO: 31, CDR 2 of SEQ ID NO: 35, and CDR 3 of SEQ ID NO: 39; (a2) a first antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 10, CDR 2 of SEQ ID NO: 16, and CDR 3 of SEQ ID NO: 22, and a second antibody variable region comprising CDR 1 of SEQ ID NO: 31, CDR 2 of SEQ ID NO: 35, and CDR 3 of SEQ ID NO: 39; (a3) a first antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 11, CDR 2 of SEQ ID NO: 17, and CDR 3 of SEQ ID NO: 23, and a second antibody variable region comprising CDR 1 of SEQ ID NO: 32, CDR 2 of SEQ ID NO: 36, and CDR 3 of SEQ ID NO: 40; (a4) a first antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 12, CDR 2 of SEQ ID NO: 18, and CDR 3 of SEQ ID NO: 24, and a second antibody variable region comprising CDR 1 of SEQ ID NO: 32, CDR 2 of SEQ ID NO: 36, and CDR 3 of SEQ ID NO: 40; An anticancer drug containing one of the following: (A-16) The second antigen-binding moiety is one of the following (b1) to (b3): (b1) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 8, CDR 2 of SEQ ID NO: 14, and CDR 3 of SEQ ID NO: 20, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 30, CDR 2 of SEQ ID NO: 34, and CDR 3 of SEQ ID NO: 38; (b2) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 7, CDR 2 of SEQ ID NO: 13, and CDR 3 of SEQ ID NO: 19, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 29, CDR 2 of SEQ ID NO: 33, and CDR 3 of SEQ ID NO: 37; (b3) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 29, CDR 2 of SEQ ID NO: 33, and CDR 3 of SEQ ID NO: 37, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 7, CDR 2 of SEQ ID NO: 13, and CDR 3 of SEQ ID NO: 19. An anticancer agent according to (A-15), comprising any one of the following: (A-17)(i) a first antigen-binding moiety that binds to CD137; and (ii) a second antigen-binding moiety that binds to claudin 6 (CLDN6); An anticancer agent comprising, as an active ingredient, a multispecific antigen-binding molecule comprising: The second antigen-binding moiety is selected from the following (b1) to (b3): (b1) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 8, CDR 2 of SEQ ID NO: 14, and CDR 3 of SEQ ID NO: 20, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 30, CDR 2 of SEQ ID NO: 34, and CDR 3 of SEQ ID NO: 38; (b2) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 7, CDR 2 of SEQ ID NO: 13, and CDR 3 of SEQ ID NO: 19, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 29, CDR 2 of SEQ ID NO: 33, and CDR 3 of SEQ ID NO: 37; (b3) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 29, CDR 2 of SEQ ID NO: 33, and CDR 3 of SEQ ID NO: 37, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 7, CDR 2 of SEQ ID NO: 13, and CDR 3 of SEQ ID NO: 19. An anticancer drug containing one of the following: (A-18) The anticancer agent of any one of (A-15) to (A-17), wherein at least one selected from the group consisting of the first antibody variable region, the second antibody variable region, the third antibody variable region, and the fourth antibody variable region comprises a human antibody framework or a humanized antibody framework. (A-19)(i) a first antigen-binding moiety that binds to CD137; and (ii) a second antigen-binding moiety that binds to claudin 6 (CLDN6); An anticancer agent comprising, as an active ingredient, a multispecific antigen-binding molecule comprising: The first antigen-binding portion is selected from the following (c1) to (c4): (c1) a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 3, and a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 27; (c2) a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 4, and a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 27; (c3) a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 5, and a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 28; (c4) a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 6, and a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 28 An anticancer drug containing one of the following: (A-20) The second antigen-binding portion is selected from the group consisting of the following (d1) to (d3): (d1) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 2, and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 26; (d2) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 1, and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 25; (d3) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 25, and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 1 An anticancer agent according to (A-19), comprising any one of the following: (A-21)(i) a first antigen-binding moiety that binds to CD137; and (ii) a second antigen-binding moiety that binds to claudin 6 (CLDN6); An anticancer agent comprising, as an active ingredient, a multispecific antigen-binding molecule comprising: The second antigen-binding portion is selected from the following (d1) to (d3): (d1) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 2, and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 26; (d2) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 1, and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 25; (d3) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 25, and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 1 An anticancer drug containing one of the following: (A-22) (c1)~(c4) below: (c1) a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 3, and a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 27; (c2) a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 4, and a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 27; (c3) a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 5, and a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 28; (c4) a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 6, and a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 28 An anticancer agent comprising, as an active ingredient, a multispecific antigen-binding molecule comprising any one of the following: (A-23) (d1)~(d3) below: (d1) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 2, and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 26; (d2) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 1, and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 25; (d3) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 25, and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 1 The anticancer agent of (A-22), further comprising any one of the following: (A-24) (d1)~(d3) below: (d1) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 2, and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 26; (d2) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 1, and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 25; (d3) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 25, and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 1 An anticancer agent comprising, as an active ingredient, a multispecific antigen-binding molecule comprising any one of the following: (A-25) (a1) to (a4) below: (a1) a first antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 9, CDR 2 of SEQ ID NO: 15, and CDR 3 of SEQ ID NO: 21, and a second antibody variable region comprising CDR 1 of SEQ ID NO: 31, CDR 2 of SEQ ID NO: 35, and CDR 3 of SEQ ID NO: 39; (a2) a first antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 10, CDR 2 of SEQ ID NO: 16, and CDR 3 of SEQ ID NO: 22, and a second antibody variable region comprising CDR 1 of SEQ ID NO: 31, CDR 2 of SEQ ID NO: 35, and CDR 3 of SEQ ID NO: 39; (a3) a first antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 11, CDR 2 of SEQ ID NO: 17, and CDR 3 of SEQ ID NO: 23, and a second antibody variable region comprising CDR 1 of SEQ ID NO: 32, CDR 2 of SEQ ID NO: 36, and CDR 3 of SEQ ID NO: 40; (a4) a first antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 12, CDR 2 of SEQ ID NO: 18, and CDR 3 of SEQ ID NO: 24, and a second antibody variable region comprising CDR 1 of SEQ ID NO: 32, CDR 2 of SEQ ID NO: 36, and CDR 3 of SEQ ID NO: 40; An anticancer agent comprising, as an active ingredient, a multispecific antigen-binding molecule comprising any one of the following: (A-26) (b1) to (b3) below: (b1) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 8, CDR 2 of SEQ ID NO: 14, and CDR 3 of SEQ ID NO: 20, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 30, CDR 2 of SEQ ID NO: 34, and CDR 3 of SEQ ID NO: 38; (b2) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 7, CDR 2 of SEQ ID NO: 13, and CDR 3 of SEQ ID NO: 19, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 29, CDR 2 of SEQ ID NO: 33, and CDR 3 of SEQ ID NO: 37; (b3) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 29, CDR 2 of SEQ ID NO: 33, and CDR 3 of SEQ ID NO: 37, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 7, CDR 2 of SEQ ID NO: 13, and CDR 3 of SEQ ID NO: 19. The anticancer agent of (A-25), further comprising any one of the following: (A-27) (b1) to (b3) below: (b1) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 8, CDR 2 of SEQ ID NO: 14, and CDR 3 of SEQ ID NO: 20, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 30, CDR 2 of SEQ ID NO: 34, and CDR 3 of SEQ ID NO: 38; (b2) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 7, CDR 2 of SEQ ID NO: 13, and CDR 3 of SEQ ID NO: 19, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 29, CDR 2 of SEQ ID NO: 33, and CDR 3 of SEQ ID NO: 37; (b3) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 29, CDR 2 of SEQ ID NO: 33, and CDR 3 of SEQ ID NO: 37, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 7, CDR 2 of SEQ ID NO: 13, and CDR 3 of SEQ ID NO: 19. An anticancer agent comprising, as an active ingredient, a multispecific antigen-binding molecule comprising any one of the following: (A-28) The anticancer agent of any one of (A-1) to (A-27), wherein the multispecific antigen-binding molecule further comprises (iii) an Fc domain that exhibits reduced binding affinity to a human Fcγ receptor compared to a native human IgG1 Fc domain. (A-29) The anticancer agent of (A-28), wherein the Fc domain is composed of a first Fc region subunit and a second Fc region subunit. (A-30) The Fc domain is (e1) or (e2) below: (e1) a first Fc region subunit comprising a Cys at position 349, a Ser at position 366, an Ala at position 368, and a Val at position 407, and a second Fc region subunit comprising a Cys at position 354 and a Trp at position 366; (e2) a first Fc region subunit containing Glu at position 439 and a second Fc region subunit containing Lys at position 356 wherein the amino acid positions are numbered according to the EU index. (A-31) The first and / or second Fc region subunit is (f1) or (f2) below: (f1) Ala at position 234 and Ala at position 235; (f2) Ala at 234th place, Ala at 235th place, and Ala at 297th place wherein the amino acid positions are numbered according to the EU index. (A-32) The anticancer agent according to any one of (A-29) to (A-31), wherein the Fc domain further exhibits stronger FcRn-binding affinity to human FcRn compared to a native human IgG1 Fc domain. (A-33) The anticancer agent of (A-32), wherein the first and / or second Fc region subunits contain Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440, and the amino acid positions are numbered according to the EU index. (A-34) A first antibody variable region of a first antigen-binding portion is fused to a first heavy chain constant region, a second antibody variable region of the first antigen-binding portion is fused to a first light chain constant region, a third antibody variable region of the second antigen-binding portion is fused to a second heavy chain constant region, and a fourth antibody variable region of the second antigen-binding portion is fused to a second light chain constant region; The constant region is selected from the following (g1) to (g7): (g1) a first heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 74, a first light chain constant region comprising the amino acid sequence of SEQ ID NO: 87, a second heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 73, and a second light chain constant region comprising the amino acid sequence of SEQ ID NO: 88; (g2) a first heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 74, a first light chain constant region comprising the amino acid sequence of SEQ ID NO: 85, a second heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 81, and a second light chain constant region comprising the amino acid sequence of SEQ ID NO: 86; (g3) a first heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 79, a first light chain constant region comprising the amino acid sequence of SEQ ID NO: 72, a second heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 80, and a second light chain constant region comprising the amino acid sequence of SEQ ID NO: 89; (g4) a first heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 83, a first light chain constant region comprising the amino acid sequence of SEQ ID NO: 87, a second heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 82, and a second light chain constant region comprising the amino acid sequence of SEQ ID NO: 88; (g5) a first heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 83, a first light chain constant region comprising the amino acid sequence of SEQ ID NO: 85, a second heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 84, and a second light chain constant region comprising the amino acid sequence of SEQ ID NO: 86; (g6) a first heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 77, a first light chain constant region comprising the amino acid sequence of SEQ ID NO: 72, a second heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 78, and a second light chain constant region comprising the amino acid sequence of SEQ ID NO: 89; (g7) A first heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 75, a first light chain constant region comprising the amino acid sequence of SEQ ID NO: 72, a second heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 76, and a second light chain constant region comprising the amino acid sequence of SEQ ID NO: 89. Any one of the anticancer agents (A-1) to (A-27). (A-35) (h01)~(h18) below: (h01) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 42 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 51, and a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 56 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 69; (h02) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 41 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 50, and a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 54 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 68; (h03) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 41 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 50, and a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 55 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 68; (h04) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 42 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 51, and a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 57 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 69; (h05) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 44 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 52, and a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 60 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 70; (h06) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 44 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 52, and a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 61 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 70; (h07) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 45 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 50, and a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 62 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 68; (h08) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 45 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 50, and a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 63 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 68; (h09) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 46 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 51, and a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 64 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 69; (h10) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 46 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 51, and a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 65 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 69; (h11) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 47 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 52, and a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 66 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 70; (h12) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 47 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 52, and a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 67 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 70; (h13) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 48 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 53, and a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 56 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 71; (h14) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 48 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 53, and a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 57 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 71; (h15) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 49 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 53, and a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 64 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 71; (h16) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 49 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 53, and a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 65 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 71; (h17) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 43 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 52, and a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 58 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 70; and (h18) A heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 43 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 52, and a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 59 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 70. A multispecific antigen-binding molecule comprising a combination of four polypeptide chains, each of which is one of: An anti-cancer drug containing as an active ingredient. (A-36) (i) the antibody variable region contained in chain 3 and the antibody variable region contained in chain 4 form a first antigen-binding portion that can bind to CD3 and CD137 and binds to either CD3 or CD137; (ii) the antibody variable region contained in chain 1 and the antibody variable region contained in chain 2 form a second antigen-binding moiety capable of binding to claudin 6 (CLDN6); (iii) the antibody Fc region subunit contained in chain 1 and the antibody Fc region subunit contained in chain 3 form an Fc domain; (A-35) Anticancer drug. (A-37) (i) the antibody variable region contained in chain 3 and the antibody variable region contained in chain 4 form a first antigen-binding moiety that binds to CD3; (ii) the antibody variable region contained in chain 1 and the antibody variable region contained in chain 2 are forming a second antigen-binding portion that binds to claudin 6 (CLDN6); (iii) the antibody Fc region subunit contained in chain 1 and the antibody Fc region subunit contained in chain 3 form an Fc domain; (A-35) Anticancer drug. (A-38) (i) the antibody variable region contained in chain 3 and the antibody variable region contained in chain 4 form a first antigen-binding moiety that binds to CD137; (ii) the antibody variable region contained in chain 1 and the antibody variable region contained in chain 2 form a second antigen-binding moiety that binds to claudin 6 (CLDN6); (iii) the antibody Fc region subunit contained in chain 1 and the antibody Fc region subunit contained in chain 3 form an Fc domain; (A-35) Anticancer drug. (A-39) An anticancer agent comprising, as an active ingredient, a multispecific antigen-binding molecule that binds to epitopes that overlap and / or compete with the epitopes on CLDN6 and CD3 / CD137 to which any of the multispecific antigen-binding molecules of (A-5) to (A-7) binds. (A-40) Any one of the anticancer agents (A-1) to (A-39), wherein the target cancer is a CLDN6-positive cancer. (A-41) Any one of the anticancer agents (A-1) to (A-40), wherein the target cancer is at least one cancer selected from the group consisting of ovarian cancer, non-small cell lung cancer, gastric cancer, liver cancer, endometrial cancer, germ cell tumor, colorectal cancer, bladder cancer, and atypical teratoid rhabdoid tumor. (A-42) An anticancer agent selected from any one of (A-1) to (A-41), wherein the target cancer is cancer that has metastasized to the peritoneum. (A-43) An anticancer agent according to any one of (A-1) to (A-42), wherein the target cancer is peritoneal disseminated cancer. (A-44) An anticancer agent selected from any one of (A-1) to (A-43) for treating a patient having cancer that is refractory to treatment with the at least one other anticancer agent or an anticancer agent different from the at least one other anticancer agent. (A-45) Any one of the anticancer agents (A-40) to (A-44), wherein the CLDN6-positive cancer is a cancer that has been previously treated with another anticancer agent. (A-46) Any one of the anticancer agents (A-40) to (A-45), wherein the CLDN6-positive cancer is a cancer for which the desired effect has not been achieved by treatment with the administration of another anticancer agent alone. (A-47) The anticancer agent according to any one of (A-1) to (A-46), further comprising a pharmaceutically acceptable carrier. (A-48) The anticancer agent of any one of (A-1) to (A-47), wherein the multispecific antigen-binding molecule induces cytotoxicity. (A-49) The anticancer agent according to (A-48), wherein the cytotoxicity is T cell-dependent cytotoxicity.

[0009] Additionally, the present disclosure provides: (B-1) A pharmaceutical composition for use in combination with at least one other anticancer agent, comprising the following multispecific antigen-binding molecule as an active ingredient: A multispecific antigen-binding molecule comprising: (i) a first antigen-binding portion capable of binding to CD3 and CD137, the first antigen-binding portion binding to either CD3 or CD137; and (ii) a second antigen-binding portion capable of binding to claudin 6 (CLDN6). (B-2) A pharmaceutical composition comprising, as an active ingredient, the multispecific antigen-binding molecule according to any one of (A-2) to (A-39), for use in combination with at least one other anticancer agent. (B-3) The pharmaceutical composition according to (B-1) or (B-2), wherein the target cancer is a CLDN6-positive cancer. (B-4) Any one of the pharmaceutical compositions (B-1) to (B-3), wherein the target cancer is any cancer selected from the group consisting of ovarian cancer, non-small cell lung cancer, gastric cancer, liver cancer, endometrial cancer, germ cell tumor, colorectal cancer, bladder cancer, and atypical teratoid rhabdoid tumor. (B-5) The pharmaceutical composition according to any one of (B-1) to (B-4), wherein the target cancer is cancer that has metastasized to the peritoneum. (B-6) Any one of the pharmaceutical compositions (B-1) to (B-5) wherein the target cancer is peritoneal disseminated cancer. (B-7) Any one of the pharmaceutical compositions (B-1) to (B-6) for treating a patient having cancer that is refractory to treatment with the at least one other anticancer agent or an anticancer agent different from the at least one other anticancer agent. (B-8) Any one of the pharmaceutical compositions (B-1) to (B-7), wherein the target cancer is a cancer that has been treated with the at least one other anticancer agent or an anticancer agent different from the at least one other anticancer agent. (B-9) Any one of the pharmaceutical compositions (B-1) to (B-8), wherein the target cancer is a cancer for which the desired effect has not been achieved by treatment with the administration of the at least one other anticancer agent alone. (B-10) The pharmaceutical composition of any one of (B-1) to (B-9), wherein the multispecific antigen-binding molecule and the at least one other anticancer agent are administered separately or sequentially. (B-11) The pharmaceutical composition of any one of (B-1) to (B-10), wherein the multispecific antigen-binding molecule is administered before, simultaneously with, and / or after administration of the at least one other anticancer agent. (B-12) The pharmaceutical composition of any one of (B-1) to (B-11), wherein the multispecific antigen-binding molecule is administered to a patient with cancer in which CLDN6 expression has increased due to administration of the at least one other anticancer agent. (B-13) The pharmaceutical composition of any one of (B-1) to (B-12), wherein the multispecific antigen-binding molecule is administered to a patient with cancer in which TGFβ expression has increased due to administration of the at least one other anticancer agent. (B-14) The pharmaceutical composition of any one of (B-1) to (B-13), wherein the multispecific antigen-binding molecule is administered to a patient with cancer in which TGFβ1 expression has increased due to administration of the at least one other anticancer agent. (B-15) The pharmaceutical composition of any one of (B-1) to (B-14), wherein the antitumor effect of the multispecific antigen-binding molecule is enhanced by administration of the at least one other anticancer agent. (B-16) The pharmaceutical composition of any one of (B-1) to (B-15), wherein the at least one other anticancer agent is at least one selected from the group consisting of a chemotherapeutic agent, an immune checkpoint inhibitor, and a PARP inhibitor. (B-17) The pharmaceutical composition of any one of (B-1) to (B-16), wherein the at least one other anticancer agent is an agent that enhances the expression of CLDN6 in cells. (B-18) The pharmaceutical composition according to any one of (B-1) to (B-17), wherein the at least one other anticancer agent is a drug having a TGFβ inducing effect. (B-19) Any one of the pharmaceutical compositions of (B-16) to (B-18), wherein the at least one other anticancer agent is a platinum agent, an alkaloid, or an antimetabolite. (B-20) The pharmaceutical composition of any one of (B-16) to (B-19), wherein the at least one other anticancer agent is a platinum preparation. (B-21) The pharmaceutical composition of any one of (B-16) to (B-20), wherein the at least one other anticancer agent is an alkaloid. (B-22) The pharmaceutical composition of any one of (B-16) to (B-19), wherein the at least one other anticancer agent is a topoisomerase inhibitor. (B-23) The pharmaceutical composition of any one of (B-16) to (B-19), wherein the at least one other anticancer agent is an antimetabolite. (B-24) The pharmaceutical composition of any one of (B-16) to (B-19), wherein the at least one other anticancer agent is carboplatin or cisplatin. (B-25) The pharmaceutical composition of any one of (B-16) to (B-19), wherein the at least one other anticancer agent is irinotecan. (B-26) The pharmaceutical composition of any one of (B-16) to (B-19), wherein the at least one other anticancer drug is gemcitabine. (B-27) The pharmaceutical composition of (B-16), wherein the at least one other anticancer agent is an anti-PD-L1 antibody. (B-28) The pharmaceutical composition of (B-16), wherein the at least one other anticancer drug is olaparib. (B-29) A cytotoxicity inducer, a cell proliferation suppressor, a cell proliferation inhibitor, an immune response activator, a cancer therapeutic agent, or a cancer preventive agent, comprising any one of the pharmaceutical compositions (B-1) to (B-28). (B2-1) A pharmaceutical composition for use in combination with at least one TGFβ-inducing therapy, comprising the following multispecific antigen-binding molecule as an active ingredient: A multispecific antigen-binding molecule comprising: (i) a first antigen-binding portion capable of binding to CD3 and CD137, the first antigen-binding portion binding to either CD3 or CD137; and (ii) a second antigen-binding portion capable of binding to claudin 6 (CLDN6). (B2-2) The pharmaceutical composition according to (B2-1), wherein the treatment for inducing TGFβ is administration of an agent that induces TGFβ. (B2-3) The pharmaceutical composition according to (B2-1) or (B2-2), wherein the treatment for inducing TGFβ is administration of an agent that induces TGFβ1. (B2-4) The pharmaceutical composition according to any one of (B2-1) to (B2-3), wherein the target cancer is a CLDN6-positive cancer. (B2-5) Any one of the pharmaceutical compositions (B2-1) to (B2-4), wherein the target cancer is any cancer selected from the group consisting of ovarian cancer, non-small cell lung cancer, gastric cancer, liver cancer, endometrial cancer, germ cell tumor, colorectal cancer, bladder cancer, and atypical teratoid rhabdoid tumor. (B2-6) The pharmaceutical composition according to any one of (B2-1) to (B2-5), wherein the target cancer is cancer that has metastasized to the peritoneum. (B2-7) Any one of the pharmaceutical compositions (B2-1) to (B2-6), wherein the target cancer is peritoneal disseminated cancer. (B2-8) The pharmaceutical composition according to any one of (B2-1) to (B2-7) for treating a patient with cancer refractory to treatment with a therapy that induces TGFβ. (B2-9) Any one of the pharmaceutical compositions (B2-1) to (B2-8), wherein the target cancer is a cancer that has previously been treated with a therapy that induces TGFβ, or a cancer for which the desired effect has not been achieved by treatment with a therapy that induces TGFβ. (B2-10) Any one of the pharmaceutical compositions (B2-1) to (B2-9), wherein the pharmaceutical composition comprising the multispecific antigen-binding molecule is administered separately from or consecutively with the treatment that induces TGFβ. (B2-11) The pharmaceutical composition of any one of (B2-2) to (B2-10), wherein the multispecific antigen-binding molecule is administered before, simultaneously with, and / or after the TGFβ-inducing treatment. (B2-12) The pharmaceutical composition of any one of (B2-1) to (B2-11), wherein the multispecific antigen-binding molecule is administered to a patient with cancer in which CLDN6 expression has been increased by a treatment that induces TGFβ. (B2-13) The pharmaceutical composition of any one of (B2-1) to (B2-12), wherein the multispecific antigen-binding molecule is administered to a patient with cancer in which TGFβ expression has been increased by a treatment that induces TGFβ. (B2-14) The pharmaceutical composition of any one of (B2-1) to (B2-13), wherein the multispecific antigen-binding molecule is administered to a patient with cancer in which TGFβ1 expression has been increased by a treatment that induces TGFβ. (B2-15) The pharmaceutical composition according to any one of (B2-1) to (B2-14), wherein the antitumor effect of the multispecific antigen-binding molecule is enhanced by treatment that induces TGFβ. (B2-16) A cytotoxicity inducer, a cell proliferation suppressor, a cell proliferation inhibitor, an immune response activator, a cancer therapeutic agent, or a cancer preventive agent, comprising the pharmaceutical composition of any one of (B2-1) to (B2-15). (B3-1) A pharmaceutical composition for use in combination with at least one CLDN6 expression inducer, comprising the following multispecific antigen-binding molecule as an active ingredient: A multispecific antigen-binding molecule comprising: (i) a first antigen-binding portion capable of binding to CD3 and CD137, the first antigen-binding portion binding to either CD3 or CD137; and (ii) a second antigen-binding portion capable of binding to claudin 6 (CLDN6). (B3-2) The pharmaceutical composition according to (B3-1), wherein the target cancer is a CLDN6-positive cancer. (B3-3) The pharmaceutical composition of (B3-1) or (B3-2), wherein the target cancer is any cancer selected from the group consisting of ovarian cancer, non-small cell lung cancer, gastric cancer, liver cancer, endometrial cancer, germ cell tumor, colorectal cancer, bladder cancer, and atypical teratoid rhabdoid tumor. (B3-4) The pharmaceutical composition according to any one of (B3-1) to (B3-3), wherein the target cancer is cancer that has metastasized to the peritoneum. (B3-5) Any one of the pharmaceutical compositions (B3-1) to (B3-4), wherein the target cancer is peritoneal disseminated cancer. (B3-6) The pharmaceutical composition according to any one of (B3-1) to (B3-5) for treating a patient having cancer refractory to treatment with the CLDN6 expression inducer. (B3-7) Any one of the pharmaceutical compositions (B3-1) to (B3-6), wherein the target cancer is a cancer that has previously been treated with the CLDN6 expression inducer, or a cancer for which the desired effect has not been achieved by treatment with the CLDN6 expression inducer. (B3-8) Any one of the pharmaceutical compositions (B3-1) to (B3-7), wherein the pharmaceutical composition comprising the multispecific antigen-binding molecule is administered separately or consecutively with the CLDN6 expression inducer. (B3-9) The pharmaceutical composition of any one of (B3-1) to (B3-8), wherein the multispecific antigen-binding molecule is administered before, simultaneously with, and / or after administration of the CLDN6 expression inducer. (B3-10) The pharmaceutical composition of any one of (B3-1) to (B3-9), wherein the multispecific antigen-binding molecule is administered to a patient with cancer in which CLDN6 expression has been increased by administration of the CLDN6 expression inducer. (B3-11) The pharmaceutical composition of any one of (B3-1) to (B3-10), wherein the multispecific antigen-binding molecule is administered to a patient with cancer in which TGFβ expression has been increased by administration of the CLDN6 expression inducer. (B3-12) The pharmaceutical composition of any one of (B3-1) to (B3-11), wherein the multispecific antigen-binding molecule is administered to a patient with cancer in which TGFβ1 expression has been increased by administration of the CLDN6 expression inducer. (B3-13) The pharmaceutical composition of any one of (B3-1) to (B3-12), wherein the antitumor effect of the multispecific antigen-binding molecule is enhanced by administration of the CLDN6 expression inducer. (B3-14) A cytotoxicity inducer, a cell proliferation suppressor, a cell proliferation inhibitor, an immune response activator, a cancer therapeutic agent, or a cancer preventive agent, comprising the pharmaceutical composition of any one of (B3-1) to (B3-13).

[0010] Additionally, the present disclosure provides: (C-1) A multispecific antigen-binding molecule comprising (i) a first antigen-binding moiety capable of binding to both CD3 and CD137 and capable of binding to either CD3 or CD137, and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6), wherein the first antigen-binding moiety is an antigen-binding moiety capable of binding only to CD3, and has a higher cytotoxic activity than a multispecific antigen-binding molecule comprising: An anti-cancer drug containing as an active ingredient. (C-2) A multispecific antigen-binding molecule comprising (i) a first antigen-binding moiety capable of binding to both CD3 and CD137 and binding to either CD3 or CD137, and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6), wherein the first antigen-binding moiety is an antigen-binding moiety capable of binding only to CD3, and the multispecific antigen-binding molecule has reduced toxicity compared to a case where the first antigen-binding moiety is an antigen-binding moiety capable of binding only to CD3. An anti-cancer drug containing as an active ingredient. (C-3) A multispecific antigen-binding molecule comprising (1) a first antigen-binding portion capable of binding to a T cell receptor complex, and (2) a second antigen-binding portion capable of binding to CLDN6, which has a T cell cytotoxic activity equivalent to or greater than that of a multispecific antibody (CS3348) comprising an antigen-binding portion capable of binding to a T cell receptor complex, the antigen-binding portion comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 194 and a light chain comprising the amino acid sequence of SEQ ID NO: 192, and an antigen-binding portion capable of binding to CLDN6, the antigen-binding portion comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 193 and a light chain comprising the amino acid sequence of SEQ ID NO: 195. An anti-cancer drug containing as an active ingredient. (C-4) The anticancer agent of any one of (C-1) to (C-3), wherein the multispecific antigen-binding molecule further comprises (3) an Fc domain that exhibits reduced binding affinity to a human Fcγ receptor compared to a native human IgG1 Fc domain. (C-5) The anticancer agent of (C-1), (C-3), or (C-4), further characterized in that the multispecific antigen-binding molecule has lower toxicity compared to the multispecific antibody (CS3348). (C-6) Any one of the anticancer agents (C-1) to (C-5), wherein the first antigen-binding portion comprises a combination of antibody variable regions selected from the following (a1) or (a2), or a combination of antibody variable regions functionally equivalent thereto: (a1) a first antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 11, CDR 2 of SEQ ID NO: 17, and CDR 3 of SEQ ID NO: 23; and a second antibody variable region comprising CDR 1 of SEQ ID NO: 32, CDR 2 of SEQ ID NO: 36, and CDR 3 of SEQ ID NO: 40; (a2) A first antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 10, CDR 2 of SEQ ID NO: 16, and CDR 3 of SEQ ID NO: 22; and a second antibody variable region comprising CDR 1 of SEQ ID NO: 31, CDR 2 of SEQ ID NO: 35, and CDR 3 of SEQ ID NO: 39. (C-7) Any one of the anticancer agents (C-1) to (C-6), wherein the second antigen-binding portion comprises a combination of antibody variable regions selected from the following (b1) or (b2), or a combination of antibody variable regions functionally equivalent thereto: (b1) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 7, CDR 2 of SEQ ID NO: 13, and CDR 3 of SEQ ID NO: 19; and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 29, CDR 2 of SEQ ID NO: 33, and CDR 3 of SEQ ID NO: 37; (b2) a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 8, CDR 2 of SEQ ID NO: 14, and CDR 3 of SEQ ID NO: 20; and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 30, CDR 2 of SEQ ID NO: 34, and CDR 3 of SEQ ID NO: 38. (C-8) Any one of the anticancer agents (C-1) to (C-7), wherein the first antigen-binding portion comprises a combination of antibody variable regions selected from the following (c1) or (c2), or a combination of antibody variable regions functionally equivalent thereto: (c1) a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 5, and a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 28; (c2) A first antibody variable region comprising the amino acid sequence of SEQ ID NO: 4, and a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 27. (C-9) Any one of the anticancer agents (C-1) to (C-8), wherein the second antigen-binding portion comprises a combination of antibody variable regions selected from the following (d1) or (d2), or a combination of antibody variable regions functionally equivalent thereto: (d1) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 1, and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 25; (d2) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 2, and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 26. (C-10) The anticancer agent according to any one of (C-4) to (C-9), wherein the Fc domain is composed of a first Fc region subunit and a second Fc region subunit. (C-11) The Fc domain is (e1) or (e2) below: (e1) a first Fc region subunit comprising a Cys at position 349, a Ser at position 366, an Ala at position 368, and a Val at position 407, and a second Fc region subunit comprising a Cys at position 354 and a Trp at position 366; (e2) a first Fc region subunit containing Glu at position 439 and a second Fc region subunit containing Lys at position 356 wherein the amino acid positions are numbered according to the EU index. (C-12) The first and / or second Fc region subunit is (f1) or (f2) below: (f1) Ala at position 234 and Ala at position 235; (f2) Ala at 234th place, Ala at 235th place, and Ala at 297th place wherein the amino acid positions are numbered according to the EU index. (C-13) The anticancer agent according to any one of (C-4) to (C-12), wherein the Fc domain further exhibits stronger FcRn-binding affinity to human FcRn compared to a native human IgG1 Fc domain. (C-14) The anticancer agent according to any one of (C-1) to (C-13), further comprising a pharmaceutically acceptable carrier.

[0011] Additionally, the present disclosure provides: (D-1) A pharmaceutical composition comprising at least one other anticancer agent as an active ingredient, for use in combination with a multispecific antigen-binding molecule comprising (i) a first antigen-binding moiety capable of binding to CD3 and CD137 and binding to either CD3 or CD137, and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6). (D-2) The pharmaceutical composition of (D-1), wherein the multispecific antigen-binding molecule is the multispecific antigen-binding molecule of any of (A-2) to (A-39). (D-3) The pharmaceutical composition of any one of (D-1) and (D-2), wherein the target cancer is a CLDN6-positive cancer. (D-4) Any one of the pharmaceutical compositions (D-1) to (D-3), wherein the target cancer is any cancer selected from the group consisting of ovarian cancer, non-small cell lung cancer, gastric cancer, liver cancer, endometrial cancer, germ cell tumor, colorectal cancer, bladder cancer, and atypical teratoid rhabdoid tumor. (D-5) The pharmaceutical composition according to any one of (D-1) to (D-4), wherein the target cancer is cancer that has metastasized to the peritoneum. (D-6) Any one of the pharmaceutical compositions (D-1) to (D-5), wherein the target cancer is peritoneal disseminated cancer. (D-7) Any one of the pharmaceutical compositions (D-1) to (D-6) for treating a patient having cancer that is refractory to treatment with the at least one other anticancer agent or an anticancer agent different from the at least one other anticancer agent. (D-8) Any one of the pharmaceutical compositions (D-1) to (D-7), wherein the target cancer is a cancer that has been treated with the at least one other anticancer agent or an anticancer agent different from the at least one other anticancer agent. (D-9) Any one of the pharmaceutical compositions (D-1) to (D-8), wherein the target cancer is a cancer for which the desired effect has not been achieved by treatment with the administration of the at least one other anticancer agent alone. (D-10) The pharmaceutical composition of any one of (D-1) to (D-9), wherein the at least one other anticancer agent is administered separately or sequentially with the multispecific antigen-binding molecule. (D-11) The pharmaceutical composition of any one of (D-1) to (D-10), wherein the at least one other anticancer agent is administered before, simultaneously with, and / or after the multispecific antigen-binding molecule. (D-12) The pharmaceutical composition of any one of (D-1) to (D-11), wherein the administration of the multispecific antigen-binding molecule enhances the antitumor effect of the at least one other anticancer agent. (D-13) The pharmaceutical composition of any one of (D-1) to (D-12), wherein the at least one other anticancer agent is at least one selected from the group consisting of a chemotherapeutic agent, an immune checkpoint inhibitor, and a PARP inhibitor. (D-14) The pharmaceutical composition of any one of (D-1) to (D-13), wherein the at least one other anticancer agent is an agent that enhances the expression of CLDN6 in cells. (D-15) The pharmaceutical composition according to any one of (D-1) to (D-14), wherein the at least one other anticancer agent is an agent that induces the expression of TGFβ in cells. (D-16) The pharmaceutical composition of any one of (D-1) to (D-15), wherein the at least one other anticancer agent is an agent that induces the expression of TGFβ1 in cells. (D-17) The pharmaceutical composition of any one of (D-13) to (D-16), wherein the at least one other anticancer agent is a platinum agent, an alkaloid, or an antimetabolite. (D-18) The pharmaceutical composition of any one of (D-13) to (D-17), wherein the at least one other anticancer agent is a platinum preparation. (D-19) The pharmaceutical composition according to any one of (D-13) to (D-17), wherein the at least one other anticancer agent is a plant alkaloid. (D-20) The pharmaceutical composition according to any one of (D-13) to (D-17), wherein the at least one other anticancer agent is a topoisomerase inhibitor. (D-21) The pharmaceutical composition according to any one of (D-13) to (D-17), wherein the at least one other anticancer agent is an antimetabolite. (D-22) The pharmaceutical composition of any one of (D-13) to (D-17), wherein the at least one other anticancer agent is carboplatin or cisplatin. (D-23) The pharmaceutical composition of any one of (D-13) to (D-17), wherein the at least one other anticancer agent is irinotecan. (D-24) The pharmaceutical composition according to any one of (D-13) to (D-17), wherein the at least one other anticancer agent is gemcitabine. (D-25) The pharmaceutical composition of (D-13), wherein the at least one other anticancer agent is an anti-PD-L1 antibody. (D-26) The pharmaceutical composition of (D-13), wherein the at least one other anticancer drug is olaparib. (D-27) A cytotoxicity inducer, a cell proliferation suppressor, a cell proliferation inhibitor, an immune response activator, a cancer therapeutic agent, or a cancer preventive agent, comprising any one of the pharmaceutical compositions (D-1) to (D-26).

[0012] Additionally, the present disclosure provides: (E-1) A pharmaceutical composition for treating or preventing cancer, comprising the following multispecific antigen-binding molecule in combination with at least one other anticancer agent: A multispecific antigen-binding molecule comprising: (i) a first antigen-binding portion capable of binding to CD3 and CD137, the first antigen-binding portion binding to either CD3 or CD137; and (ii) a second antigen-binding portion capable of binding to claudin 6 (CLDN6). (E-2) The pharmaceutical composition of (E-1), wherein the multispecific antigen-binding molecule is the multispecific antigen-binding molecule of any of (A-2) to (A-39). (E-3) The pharmaceutical composition of any one of (E-1) or (E-2), wherein the cancer is a CLDN6-positive cancer. (E-4) Any one of the pharmaceutical compositions (E-1) to (E-3), wherein the cancer is any cancer selected from the group consisting of ovarian cancer, non-small cell lung cancer, gastric cancer, liver cancer, endometrial cancer, germ cell tumor, colorectal cancer, bladder cancer, and atypical teratoid rhabdoid tumor. (E-5) The pharmaceutical composition of any one of (E-1) to (E-4), wherein the cancer is cancer that has metastasized to the peritoneum. (E-6) The pharmaceutical composition according to any one of (E-1) to (E-5), wherein the cancer is a peritoneal disseminated cancer. (E-7) Any one of the pharmaceutical compositions (E-1) to (E-6) for treating a patient having cancer that is refractory to treatment with the at least one other anticancer agent or an anticancer agent different from the at least one other anticancer agent. (E-8) Any one of the pharmaceutical compositions (E-1) to (E-7), wherein the cancer is a cancer that has previously been treated with the at least one other anticancer agent or an anticancer agent different from the at least one other anticancer agent. (E-9) The pharmaceutical composition of any one of (E-1) to (E-8), wherein the cancer is a cancer for which the desired effect has not been achieved by treatment with the administration of the at least one other anticancer agent alone. (E-10) The pharmaceutical composition of any one of (E-1) to (E-9), wherein the antitumor effect of the multispecific antigen-binding molecule is enhanced by administration of the at least one other anticancer agent. (E-11) The pharmaceutical composition of any one of (E-1) to (E-9), wherein the administration of the multispecific antigen-binding molecule enhances the antitumor effect of the at least one other anticancer agent. (E-12) The pharmaceutical composition according to any one of (E-1) to (E-11), wherein the pharmaceutical composition is a combination drug. (E-13) The pharmaceutical composition of any one of (E-1) to (E-12), wherein the multispecific antigen-binding molecule and the at least one other anticancer agent are administered separately or sequentially. (E-14) The pharmaceutical composition of (E-13), wherein the multispecific antigen-binding molecule is administered before, simultaneously with, and / or after administration of the at least one other anticancer agent. (E-15) Any one of the pharmaceutical compositions (E-1) to (E-14), wherein the at least one other anticancer agent is at least one selected from the group consisting of a chemotherapeutic agent, an immune checkpoint inhibitor, and a PARP inhibitor. (E-16) The pharmaceutical composition of any one of (E-1) to (E-15), wherein the at least one other anticancer agent is an agent that enhances the expression of CLDN6 in cells. (E-17) The pharmaceutical composition of any one of (E-1) to (E-16), wherein the at least one other anticancer agent is an agent that induces the expression of TGFβ in cells. (E-18) The pharmaceutical composition of any one of (E-1) to (E-17), wherein the at least one other anticancer agent is an agent that induces the expression of TGFβ1 in cells. (E-19) The pharmaceutical composition of any one of (E-15) to (E-18), wherein the at least one other anticancer agent is a platinum agent, a plant alkaloid, or an antimetabolite. (E-20) The pharmaceutical composition of any one of (E-15) to (E-19), wherein the at least one other anticancer agent is a platinum preparation. (E-21) The pharmaceutical composition of any one of (E-15) to (E-19), wherein the at least one other anticancer agent is an alkaloid. (E-22) The pharmaceutical composition of any one of (E-15) to (E-19), wherein the at least one other anticancer agent is a topoisomerase inhibitor. (E-23) The pharmaceutical composition of any one of (E-15) to (E-19), wherein the at least one other anticancer agent is an antimetabolite. (E-24) The pharmaceutical composition of any one of (E-15) to (E-19), wherein the at least one other anticancer agent is carboplatin or cisplatin. (E-25) The pharmaceutical composition of any one of (E-15) to (E-19), wherein the at least one other anticancer agent is irinotecan. (E-26) The pharmaceutical composition of any one of (E-15) to (E-19), wherein the at least one other anticancer agent is gemcitabine. (E-27) The pharmaceutical composition of (E-15), wherein the at least one other anticancer agent is an anti-PD-L1 antibody. (E-28) The pharmaceutical composition of (E-15), wherein the at least one other anticancer drug is olaparib. (E-29) A cytotoxicity inducer, a cell proliferation inhibitor, a cell proliferation inhibitor, an immune response activator, a cancer therapeutic agent, or a cancer preventive agent, comprising any one of the pharmaceutical compositions (E-1) to (E-28). (E2-1) A pharmaceutical composition for treating or preventing cancer, comprising the following multispecific antigen-binding molecule in combination with at least one TGFβ inducer: A multispecific antigen-binding molecule comprising: (i) a first antigen-binding portion capable of binding to CD3 and CD137, the first antigen-binding portion binding to either CD3 or CD137; and (ii) a second antigen-binding portion capable of binding to claudin 6 (CLDN6). (E2-2) The pharmaceutical composition of (E2-1), wherein the multispecific antigen-binding molecule is the multispecific antigen-binding molecule of any of (A-2) to (A-39). (E2-3) The pharmaceutical composition of any one of (E2-1) or (E2-2), wherein the cancer is a CLDN6-positive cancer. (E2-4) The pharmaceutical composition of any one of (E2-1) to (E2-3), wherein the cancer is any cancer selected from the group consisting of ovarian cancer, non-small cell lung cancer, gastric cancer, liver cancer, endometrial cancer, germ cell tumor, colorectal cancer, bladder cancer, and atypical teratoid rhabdoid tumor. (E2-5) The pharmaceutical composition according to any one of (E2-1) to (E2-4), wherein the cancer is cancer that has metastasized to the peritoneum. (E2-6) The pharmaceutical composition according to any one of (E2-1) to (E2-5), wherein the cancer is a peritoneal disseminated cancer. (E2-7) Any one of the pharmaceutical compositions (E2-1) to (E2-6) for treating a patient having cancer that is refractory to treatment with the at least one TGFβ inducer or a TGFβ inducer different from the at least one TGFβ inducer. (E2-8) Any one of the pharmaceutical compositions (E2-1) to (E2-7), wherein the cancer is a cancer that has previously been treated with the at least one TGFβ inducer or a TGFβ inducer different from the at least one TGFβ inducer. (E2-9) The pharmaceutical composition of any one of (E2-1) to (E2-8), wherein the cancer is a cancer for which the desired effect has not been achieved by treatment with the administration of the at least one TGFβ inducer alone. (E2-10) The pharmaceutical composition of any one of (E2-1) to (E2-9), wherein the antitumor effect of the multispecific antigen-binding molecule is enhanced by administering the at least one TGFβ inducer. (E2-11) The pharmaceutical composition according to any one of (E2-1) to (E2-10), wherein the pharmaceutical composition is a combination drug. (E2-12) The pharmaceutical composition of any one of (E2-1) to (E2-11), wherein the multispecific antigen-binding molecule and the at least one TGFβ inducer are administered separately or sequentially. (E2-13) The pharmaceutical composition according to (E2-12), wherein the multispecific antigen-binding molecule is administered before, simultaneously with, and / or after administration of the at least one TGFβ inducer. (E2-14) A cytotoxicity inducer, a cell proliferation suppressor, a cell proliferation inhibitor, an immune response activator, a cancer therapeutic agent, or a cancer preventive agent, comprising the pharmaceutical composition of any one of (E2-1) to (E2-13). (E3-1) A pharmaceutical composition for treating or preventing cancer, comprising a combination of the following multispecific antigen-binding molecule and at least one CLDN6 expression inducer: A multispecific antigen-binding molecule comprising: (i) a first antigen-binding portion capable of binding to CD3 and CD137, the first antigen-binding portion binding to either CD3 or CD137; and (ii) a second antigen-binding portion capable of binding to claudin 6 (CLDN6). (E3-2) The pharmaceutical composition of (E3-1), wherein the multispecific antigen-binding molecule is the multispecific antigen-binding molecule of any one of (A-2) to (A-39). (E3-3) The pharmaceutical composition of any one of (E3-1) or (E3-2), wherein the cancer is a CLDN6-positive cancer. (E3-4) Any one of the pharmaceutical compositions (E3-1) to (E3-3), wherein the cancer is any cancer selected from the group consisting of ovarian cancer, non-small cell lung cancer, gastric cancer, liver cancer, endometrial cancer, germ cell tumor, colorectal cancer, bladder cancer, and atypical teratoid rhabdoid tumor. (E3-5) The pharmaceutical composition according to any one of (E3-1) to (E3-4), wherein the cancer is cancer that has metastasized to the peritoneum. (E3-6) The pharmaceutical composition according to any one of (E3-1) to (E3-5), wherein the cancer is a peritoneal disseminated cancer. (E3-7) Any one of the pharmaceutical compositions (E3-1) to (E3-6) for treating a patient having cancer that is refractory to treatment with the at least one CLDN6 expression inducer or a CLDN6 expression inducer different from the at least one CLDN6 expression inducer. (E3-8) Any one of the pharmaceutical compositions (E3-1) to (E3-7), wherein the cancer is a cancer that has previously been treated with at least one CLDN6 expression inducer or a CLDN6 expression inducer different from the at least one CLDN6 expression inducer. (E3-9) The pharmaceutical composition of any one of (E3-1) to (E3-8), wherein the cancer is a cancer for which the desired effect has not been achieved by treatment with the administration of the at least one TGFβ inducer alone. (E3-10) The pharmaceutical composition of any one of (E3-1) to (E3-9), wherein the antitumor effect of the multispecific antigen-binding molecule is enhanced by administering the at least one CLDN6 expression inducer. (E3-11) The pharmaceutical composition according to any one of (E3-1) to (E3-10), wherein the pharmaceutical composition is a combination drug. (E3-12) The pharmaceutical composition of any one of (E3-1) to (E3-11), wherein the multispecific antigen-binding molecule and the at least one CLDN6 expression inducer are administered separately or sequentially. (E3-13) The pharmaceutical composition of (E3-12), wherein the multispecific antigen-binding molecule is administered before, simultaneously with, and / or after administration of the at least one CLDN6 expression inducer. (E3-14) A cytotoxicity inducer, a cell proliferation suppressor, a cell proliferation inhibitor, an immune response activator, a cancer therapeutic agent, or a cancer preventive agent, comprising the pharmaceutical composition of any one of (E3-1) to (E3-13).

[0013] (F-1) A combination of the following multispecific antigen-binding molecule with at least one other anticancer drug: A multispecific antigen-binding molecule comprising: (i) a first antigen-binding portion capable of binding to CD3 and CD137, the first antigen-binding portion binding to either CD3 or CD137; and (ii) a second antigen-binding portion capable of binding to claudin 6 (CLDN6). (F-2) The combination of (F-1), wherein the multispecific antigen-binding molecule is the multispecific antigen-binding molecule according to any one of (A-2) to (A-39). (F-3) A combination of (F-1) or (F-2), in which the target cancer is CLDN6-positive cancer. (F-4) Any one combination of (F-1) to (F-3), wherein the target cancer is any cancer selected from the group consisting of ovarian cancer, non-small cell lung cancer, gastric cancer, liver cancer, endometrial cancer, germ cell tumor, colorectal cancer, bladder cancer, or atypical teratoid rhabdoid tumor. (F-5) A combination of any one of (F-1) to (F-4), in which the target cancer is cancer that has metastasized to the peritoneum. (F-6) Any one of the combinations (F-1) to (F-5) in which the target cancer is peritoneal dissemination cancer. (F-7) Any one of the combinations (F-1) to (F-6) for treating a patient having cancer that is refractory to treatment with the at least one other anticancer agent or an anticancer agent different from the at least one other anticancer agent. (F-8) Any one of the combinations (F-1) to (F-7), wherein the target cancer is a cancer that has previously been treated with at least one other anticancer drug or an anticancer drug different from the at least one other anticancer drug. (F-9) Any one of the combinations (F-1) to (F-8), wherein the target cancer is a cancer for which the desired effect has not been achieved by treatment with the administration of the at least one other anticancer agent alone. (F-10) Any one of the combinations (F-1) to (F-9), wherein the antitumor effect of the multispecific antigen-binding molecule is enhanced by the at least one other anticancer drug. (F-11) Any one of the combinations (F-1) to (F-9), wherein the multispecific antigen-binding molecule enhances the antitumor effect of the at least one other anticancer agent. (F-12) Any one of the combinations (F-1) to (F-11), wherein the multispecific antigen-binding molecule and the at least one other anticancer agent are administered separately or sequentially. (F-13) Any one of the combinations (F-1) to (F-12), wherein the multispecific antigen-binding molecule is administered before, simultaneously with, and / or after the at least one other anticancer drug. (F-14) Any one of the combinations (F-1) to (F-13), wherein the at least one other anticancer agent is at least one selected from the group consisting of a chemotherapeutic agent, an immune checkpoint inhibitor, and a PARP inhibitor. (F-15) Any one of the combinations (F-1) to (F-14), wherein the at least one other anticancer agent is an agent that enhances the expression of CLDN6 in cells. (F-16) Any one of the combinations (F-1) to (F-15), wherein the at least one other anticancer agent is an agent that induces the expression of TGFβ in cells. (F-17) Any one of the combinations (F-1) to (F-16), wherein the at least one other anticancer agent is an agent that induces the expression of TGFβ1 in cells. (F-18) Any one of the combinations (F-14) to (F-17), wherein the at least one other anticancer drug is a platinum agent, an alkaloid, or an antimetabolite. (F-19) Any one of the combinations (F-14) to (F-18), wherein the at least one other anticancer drug is a platinum preparation. (F-20) Any one of the combinations (F-14) to (F-18), wherein the at least one other anticancer drug is an alkaloid. (F-21) The pharmaceutical composition of any one of (F-14) to (F-18), wherein the at least one other anticancer agent is a topoisomerase inhibitor. (F-22) Any one of the combinations (F-14) to (F-18), wherein the at least one other anticancer drug is an antimetabolite. (F-23) Any one of the combinations (F-14) to (F-17), wherein the at least one other anticancer drug is carboplatin or cisplatin. (F-24) Any one of the combinations (F-14) to (F-17), wherein the at least one other anticancer drug is irinotecan. (F-25) Any one of the combinations (F-14) to (F-17), wherein the at least one other anticancer drug is gemcitabine. (F-26) The combination of (F-14), wherein the at least one other anticancer agent is an anti-PD-L1 antibody. (F-27) The combination of (F-14), wherein the at least one other anticancer drug is olaparib. (F-28) A cytotoxicity inducer, a cell proliferation inhibitor, a cell proliferation inhibitor, an immune response activator, a cancer therapeutic agent, or a cancer preventive agent, comprising a combination of any one of (F-1) to (F-27). (F2-1) Combination of the following multispecific antigen-binding molecules with at least one TGFβ inducer: A multispecific antigen-binding molecule comprising: (i) a first antigen-binding portion capable of binding to CD3 and CD137, the first antigen-binding portion binding to either CD3 or CD137; and (ii) a second antigen-binding portion capable of binding to claudin 6 (CLDN6). (F2-2) The combination of (F2-1), wherein the multispecific antigen-binding molecule is the multispecific antigen-binding molecule according to any one of (A-2) to (A-39). (F2-3) A combination of (F2-1) or (F2-2), in which the target cancer is CLDN6-positive cancer. (F2-4) Any one combination of (F2-1) to (F2-3), wherein the target cancer is any cancer selected from the group consisting of ovarian cancer, non-small cell lung cancer, gastric cancer, liver cancer, endometrial cancer, germ cell tumor, colorectal cancer, bladder cancer, or atypical teratoid rhabdoid tumor. (F2-5) A combination of any one of (F2-1) to (F2-4), in which the target cancer is cancer that has metastasized to the peritoneum. (F2-6) Any one of the combinations (F2-1) to (F2-5) in which the target cancer is peritoneal dissemination cancer. (F2-7) A combination of any one of (F2-1) to (F2-6) for treating a patient having cancer that is refractory to treatment with the at least one TGFβ inducer or a TGFβ inducer different from the at least one TGFβ inducer. (F2-8) Any one of the combinations (F2-1) to (F2-7), wherein the target cancer is a cancer that has previously been treated with at least one TGFβ inducer or a TGFβ inducer different from the at least one TGFβ inducer. (F2-9) Any one of the combinations (F2-1) to (F2-8), wherein the target cancer is a cancer for which the desired effect has not been achieved by treatment with the administration of at least one TGFβ inducer alone. (F2-10) Any one of the combinations (F2-1) to (F2-9), wherein the antitumor effect of the multispecific antigen-binding molecule is enhanced by the at least one TGFβ inducer. (F2-11) Any one of the combinations (F2-1) to (F2-10), wherein the multispecific antigen-binding molecule and the at least one TGFβ inducer are administered separately or sequentially. (F2-12) Any one of the combinations (F2-1) to (F2-11), wherein the multispecific antigen-binding molecule is administered before, simultaneously with, and / or after the at least one TGFβ inducer. (F2-13) Any one of the combinations (F2-1) to (F2-12), wherein the at least one TGFβ inducer is an agent that enhances the expression of CLDN6 in cells. (F2-14) Any one of the combinations (F2-1) to (F2-13), wherein the at least one TGFβ inducer is an agent that induces the expression of TGFβ1 in cells. (F2-15) A cytotoxicity inducer, a cell proliferation suppressor, a cell proliferation inhibitor, an immune response activator, a cancer therapeutic agent, or a cancer preventive agent, comprising a combination of any one of (F2-1) to (F2-14). (F3-1) A combination of the following multispecific antigen-binding molecules and at least one CLDN6 expression inducer: A multispecific antigen-binding molecule comprising: (i) a first antigen-binding portion capable of binding to CD3 and CD137, the first antigen-binding portion binding to either CD3 or CD137; and (ii) a second antigen-binding portion capable of binding to claudin 6 (CLDN6). (F3-2) The combination of (F3-1), wherein the multispecific antigen-binding molecule is the multispecific antigen-binding molecule according to any one of (A-2) to (A-39). (F3-3) A combination of (F3-1) or (F3-2), in which the target cancer is CLDN6-positive cancer. (F3-4) Any one combination of (F3-1) to (F3-3), in which the target cancer is any cancer selected from the group consisting of ovarian cancer, non-small cell lung cancer, gastric cancer, liver cancer, endometrial cancer, germ cell tumor, colorectal cancer, bladder cancer, or atypical teratoid rhabdoid tumor. (F3-5) A combination of any one of (F3-1) to (F3-4), in which the target cancer is cancer that has metastasized to the peritoneum. (F3-6) Any one of the combinations (F3-1) to (F3-5) in which the target cancer is peritoneal dissemination cancer. (F3-7) A combination of any one of (F3-1) to (F3-6) for treating a patient having cancer that is refractory to treatment with the at least one CLDN6 expression inducer or a CLDN6 expression inducer different from the at least one CLDN6 expression inducer. (F3-8) Any one of the combinations (F3-1) to (F3-7), wherein the target cancer is a cancer that has previously been treated with at least one CLDN6 expression inducer or a CLDN6 expression inducer different from the at least one CLDN6 expression inducer. (F3-9) Any one of the combinations (F3-1) to (F3-8), wherein the target cancer is a cancer for which the desired effect was not obtained by treatment with the administration of at least one other CLDN6 expression inducer alone. (F3-10) Any one of the combinations (F3-1) to (F3-9), wherein the antitumor effect of the multispecific antigen-binding molecule is enhanced by the at least one other CLDN6 expression inducer. (F3-11) Any one of the combinations (F3-1) to (F3-10), wherein the multispecific antigen-binding molecule and the at least one CLDN6 expression inducer are administered separately or sequentially. (F3-12) Any one of the combinations (F3-1) to (F3-11), characterized in that the multispecific antigen-binding molecule is administered before, simultaneously with, and / or after the at least one CLDN6 expression inducer. (F3-13) Any one of the combinations (F3-1) to (F3-12), wherein the at least one CLDN6 expression inducer is an agent that enhances the expression of TGFβ in cells. (F3-14) Any one of the combinations (F3-1) to (F3-13), wherein the at least one CLDN6 expression inducer is an agent that induces the expression of TGFβ1 in cells. (F3-15) A cytotoxicity inducer, a cell proliferation suppressor, a cell proliferation inhibitor, an immune response activator, a cancer therapeutic agent, or a cancer preventive agent, comprising a combination of any one of (F3-1) to (F3-14).

[0014] (G-1) A method for inducing cytotoxicity, suppressing cell proliferation, inhibiting cell growth, activating an immune response, treating cancer, or preventing cancer in an individual, comprising administering an effective amount of a multispecific antigen-binding molecule and an effective amount of at least one other anticancer agent, The multispecific antigen-binding molecule comprises: (i) a first antigen-binding moiety capable of binding to CD3 and CD137, the first antigen-binding moiety binding to either CD3 or CD137; and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6). method. (G-2) A method for inducing cytotoxicity, suppressing cell proliferation, inhibiting cell proliferation, activating an immune response, treating cancer, or preventing cancer in an individual in combination with a multispecific antigen-binding molecule, comprising administering to the individual an effective amount of at least one other anticancer agent, The multispecific antigen-binding molecule comprises: (i) a first antigen-binding moiety capable of binding to CD3 and CD137, the first antigen-binding moiety binding to either CD3 or CD137; and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6). method. (G-3) A method for enhancing the effect of a multispecific antigen-binding molecule on inducing cytotoxicity, suppressing cell growth, inhibiting cell proliferation, activating immune responses, treating cancer, or preventing cancer in an individual, comprising administering to the individual an effective amount of at least one other anticancer agent, The multispecific antigen-binding molecule comprises: (i) a first antigen-binding moiety capable of binding to CD3 and CD137, the first antigen-binding moiety binding to either CD3 or CD137; and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6). method. (G-4) A method for enhancing the effect of at least one other anticancer agent on inducing cytotoxicity, suppressing cell growth, inhibiting cell proliferation, activating immune responses, treating cancer, or preventing cancer in an individual, comprising administering to the individual an effective amount of a multispecific antigen-binding molecule, The multispecific antigen-binding molecule comprises: (i) a first antigen-binding moiety capable of binding to CD3 and CD137, the first antigen-binding moiety binding to either CD3 or CD137; and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6). method. (G-5) Any one of the methods (G-1) to (G-4), wherein the multispecific antigen-binding molecule is the multispecific antigen-binding molecule of any one of (A-2) to (A-39). (G-6) Any one of the methods (G-1) to (G-5), wherein the cancer is a CLDN6-positive cancer. (G-7) Any one of the methods (G-1) to (G-6), wherein the cancer is any cancer selected from the group consisting of ovarian cancer, non-small cell lung cancer, gastric cancer, liver cancer, endometrial cancer, germ cell tumor, colorectal cancer, bladder cancer, or atypical teratoid rhabdoid tumor. (G-8) Any one of the methods (G-1) to (G-7), wherein the cancer is cancer that has metastasized to the peritoneum. (G-9) Any one of the methods (G-1) to (G-8), wherein the cancer is a peritoneal disseminated cancer. (G-10) Any one of the methods (G-1) to (G-9), wherein the multispecific antigen-binding molecule and the at least one other anticancer agent are administered separately or sequentially. (G-11) Any one of the methods (G-1) to (G-10), wherein the multispecific antigen-binding molecule is administered before, simultaneously with, and / or after administration of the at least one other anticancer agent. (G-12) Any one of methods (G-1) to (G-11), wherein the at least one other anticancer agent is at least one selected from the group consisting of a chemotherapeutic agent, an immune checkpoint inhibitor, and a PARP inhibitor. (G-13) Any one of the methods (G-1) to (G-12), wherein the at least one other anticancer agent is an agent that enhances the expression of CLDN6 in cells. (G-14) The method according to any one of (G-1) to (G-13), wherein the at least one other anticancer agent is an agent that induces the expression of TGFβ in cells. (G-15) The method according to any one of (G-1) to (G-14), wherein the at least one other anticancer agent is an agent that induces the expression of TGFβ1 in cells. (G-16) Any one of methods (G-12) to (G-15), wherein the at least one other anticancer drug is a platinum agent, an alkaloid, or an antimetabolite. (G-17) Any one of the methods (G-12) to (G-16), wherein the at least one other anticancer drug is a platinum agent. (G-18) Any one of the methods (G-12) to (G-16), wherein the at least one other anticancer agent is an alkaloid. (G-19) The pharmaceutical composition of any one of (G-12) to (G-16), wherein the at least one other anticancer agent is a topoisomerase inhibitor. (G-20) Any one of the methods (G-12) to (G-16), wherein the at least one other anticancer agent is an antimetabolite. (G-21) The method according to (G-12) to (G-16), wherein the at least one other anticancer drug is carboplatin or cisplatin. (G-22) The method according to (G-12) to (G-16), wherein the at least one other anticancer drug is irinotecan. (G-23) The method according to (G-12) to (G-16), wherein the at least one other anticancer drug is gemcitabine. (G-24) The method of (G-12), wherein the at least one other anticancer drug is an anti-PD-L1 antibody. (G-25) The method of (G-12), wherein the at least one other anticancer drug is olaparib. (G2-1) A method for inducing cytotoxicity, suppressing cell proliferation, inhibiting cell growth, activating an immune response, treating cancer, or preventing cancer in an individual, comprising administering an effective amount of a multispecific antigen-binding molecule and an effective amount of at least one TGFβ inducer, The multispecific antigen-binding molecule comprises: (i) a first antigen-binding moiety capable of binding to CD3 and CD137, the first antigen-binding moiety binding to either CD3 or CD137; and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6). method. (G2-2) A method for inducing cytotoxicity, suppressing cell proliferation, inhibiting cell proliferation, activating an immune response, treating cancer, or preventing cancer in an individual, in combination with a multispecific antigen-binding molecule, comprising administering to the individual an effective amount of at least one TGFβ inducer, The multispecific antigen-binding molecule comprises: (i) a first antigen-binding moiety capable of binding to CD3 and CD137, the first antigen-binding moiety binding to either CD3 or CD137; and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6). method. (G2-3) A method for enhancing the effect of a multispecific antigen-binding molecule on inducing cytotoxicity, suppressing cell proliferation, inhibiting cell growth, activating immune responses, treating cancer, or preventing cancer in an individual, comprising administering to the individual an effective amount of at least one TGFβ inducer, The multispecific antigen-binding molecule comprises: (i) a first antigen-binding moiety capable of binding to CD3 and CD137, the first antigen-binding moiety binding to either CD3 or CD137; and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6). method. (G2-4) Any one of the methods (G2-1) to (G2-3), wherein the multispecific antigen-binding molecule is the multispecific antigen-binding molecule of any one of (A-2) to (A-39). (G2-5) Any one of the methods (G2-1) to (G2-4), wherein the cancer is a CLDN6-positive cancer. (G2-6) Any one of the methods (G2-1) to (G2-5), wherein the cancer is any cancer selected from the group consisting of ovarian cancer, non-small cell lung cancer, gastric cancer, liver cancer, endometrial cancer, germ cell tumor, colorectal cancer, bladder cancer, or atypical teratoid rhabdoid tumor. (G2-7) Any one of the methods (G2-1) to (G2-6), wherein the cancer is cancer that has metastasized to the peritoneum. (G2-8) Any one of the methods (G2-1) to (G2-7), wherein the cancer is a peritoneal disseminated cancer. (G2-9) Any one of the methods (G2-1) to (G2-8), wherein the multispecific antigen-binding molecule and the at least one TGFβ inducer are administered separately or sequentially. (G2-10) Any one of the methods (G2-1) to (G2-9), wherein the multispecific antigen-binding molecule is administered before, simultaneously with, and / or after administration of the at least one TGFβ inducer. (G2-11) Any one of the methods (G2-1) to (G2-10), wherein the at least one TGFβ inducer is an agent that enhances the expression of CLDN6 in cells. (G2-12) Any one of the methods (G2-1) to (G2-11), wherein the at least one TGFβ inducer is an agent that induces the expression of TGFβ1 in cancer cells. (G3-1) A method for inducing cytotoxicity, suppressing cell proliferation, inhibiting cell growth, activating an immune response, treating cancer, or preventing cancer in an individual, comprising administering an effective amount of a multispecific antigen-binding molecule and an effective amount of at least one CLDN6 expression inducer, The multispecific antigen-binding molecule comprises: (i) a first antigen-binding moiety capable of binding to CD3 and CD137, the first antigen-binding moiety binding to either CD3 or CD137; and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6). method. (G3-2) A method for inducing cytotoxicity, suppressing cell proliferation, inhibiting cell proliferation, activating an immune response, treating cancer, or preventing cancer in an individual, comprising administering to the individual an effective amount of at least one CLDN6 expression inducer in combination with a multispecific antigen-binding molecule, The multispecific antigen-binding molecule comprises: (i) a first antigen-binding moiety capable of binding to CD3 and CD137, the first antigen-binding moiety binding to either CD3 or CD137; and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6). method. (G3-3) A method for enhancing the effects of a multispecific antigen-binding molecule on induction of cytotoxicity, suppression of cell proliferation, inhibition of cell growth, activation of immune response, treatment of cancer, or prevention of cancer in an individual, comprising administering to the individual an effective amount of at least one CLDN6 expression inducer, The multispecific antigen-binding molecule comprises: (i) a first antigen-binding moiety capable of binding to CD3 and CD137, the first antigen-binding moiety binding to either CD3 or CD137; and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6). method. (G3-4) Any one of the methods (G3-1) to (G3-3), wherein the multispecific antigen-binding molecule is the multispecific antigen-binding molecule of any one of (A-2) to (A-39). (G3-5) Any one of the methods (G3-1) to (G3-4), wherein the cancer is a CLDN6-positive cancer. (G3-6) Any one of the methods (G3-1) to (G3-5), wherein the cancer is any cancer selected from the group consisting of ovarian cancer, non-small cell lung cancer, gastric cancer, liver cancer, endometrial cancer, germ cell tumor, colorectal cancer, bladder cancer, or atypical teratoid rhabdoid tumor. (G3-7) Any one of the methods (G3-1) to (G3-6), wherein the cancer is cancer that has metastasized to the peritoneum. (G3-8) Any one of the methods (G3-1) to (G3-7), wherein the cancer is a peritoneal disseminated cancer. (G3-9) Any one of the methods (G3-1) to (G3-8), wherein the multispecific antigen-binding molecule and the at least one CLDN6 expression inducer are administered separately or sequentially. (G3-10) Any one of the methods (G3-1) to (G3-9), wherein the multispecific antigen-binding molecule is administered before, simultaneously with, and / or after administration of the at least one CLDN6 expression inducer. (G3-11) Any one of the methods (G2-1) to (G2-10), wherein the at least one CLDN6 expression inducer is an agent that enhances the expression of TGFβ in cancer cells. (G3-12) Any one of the methods (G3-1) to (G3-11), wherein the at least one CLDN6 expression inducer is an agent that induces the expression of TGFβ1 in cancer cells.

[0015] (H-1) A method for injuring cancer cells or tumor tissue containing cancer cells, or a method for suppressing the growth of cancer cells or tumor tissue containing cancer cells, by contacting cancer cells with a multispecific antigen-binding molecule and at least one other anticancer agent, comprising: The multispecific antigen-binding molecule comprises: (i) a first antigen-binding moiety capable of binding to CD3 and CD137, the first antigen-binding moiety binding to either CD3 or CD137; and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6). method. (H-2) A method for confirming whether a multispecific antigen-binding molecule and at least one other anticancer drug cause damage to cancer cells or tumor tissue containing cancer cells, or suppress the growth of cancer cells or tumor tissue containing cancer cells, by contacting cancer cells with the multispecific antigen-binding molecule and at least one other anticancer drug, wherein the multispecific antigen-binding molecule is (i) a first antigen-binding moiety capable of binding to CD3 and CD137, wherein the first antigen-binding moiety binds to either CD3 or CD137, and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6). (H-3) The method of (H-1) or (H-2), wherein the multispecific antigen-binding molecule is the multispecific antigen-binding molecule of any of (A-2) to (A-39). (H-4) Any one of the methods (H-1) to (H-3), wherein the cancer cells are CLDN6-positive cancer cells. (H-5) Any one of the methods (H-1) to (H-4), wherein the cancer cells are cancer cells of at least one cancer selected from the group consisting of ovarian cancer, non-small cell lung cancer, gastric cancer, liver cancer, endometrial cancer, germ cell tumor, colorectal cancer, bladder cancer, and atypical teratoid rhabdoid tumor. (H-6) Any one of the methods (H-1) to (H-5), wherein the cancer cells are cancer cells of a peritoneal disseminated cancer. (H-7) Any one of methods (H-1) to (H-6), wherein the at least one other anticancer agent is at least one selected from the group consisting of a chemotherapeutic agent, an immune checkpoint inhibitor, and a PARP inhibitor. (H-8) Any one of the methods (H-1) to (H-7), wherein the at least one other anticancer agent is an agent that enhances the expression of CLDN6 in cells. (H-9) The method according to any one of (H-1) to (H-8), wherein the at least one other anticancer agent is an agent that induces the expression of TGFβ in cancer cells. (H-10) The method according to any one of (H-1) to (H-9), wherein the at least one other anticancer agent is an agent that induces the expression of TGFβ1 in cancer cells. (H-11) Any one of methods (H-7) to (H-10), wherein the at least one other anticancer agent is a platinum agent, an alkaloid, or an antimetabolite. (H-12) Any one of the methods (H-7) to (H-11), wherein the at least one other anticancer agent is a platinum agent. (H-13) Any one of the methods (H-7) to (H-11), wherein the at least one other anticancer agent is an alkaloid. (H-14) The pharmaceutical composition according to any one of (H-7) to (H-11), wherein the at least one other anticancer agent is a topoisomerase inhibitor. (H-15) The method according to any one of (H-7) to (H-11), wherein the at least one other anticancer agent is an antimetabolite. (H-16) Any one of the methods (H-7) to (H-11), wherein the at least one other anticancer drug is carboplatin or cisplatin. (H-17) The pharmaceutical composition according to any one of (H-7) to (H-11), wherein the at least one other anticancer agent is irinotecan. (H-18) The pharmaceutical composition according to any one of (H-7) to (H-11), wherein the at least one other anticancer drug is gemcitabine. (H-19) The method of (H-7), wherein the at least one other anticancer agent is an anti-PD-L1 antibody. (H-20) The method of (H-7), wherein the at least one other anticancer drug is olaparib.

[0016] (I-1) (A) A pharmaceutical composition comprising a multispecific antigen-binding molecule capable of binding to CD3 and CD137, the multispecific antigen-binding molecule comprising a first antigen-binding moiety that binds to either CD3 or CD137, and a second antigen-binding moiety that has binding activity to claudin 6 (CLDN6); (B) a container; and (C) Instructions or a label indicating that the multispecific antigen-binding molecule is administered to an individual in combination with at least one other anti-cancer agent to treat or prevent cancer in the individual. Kit including: (I-2) (A) at least one other anticancer drug; (B) a container; and (C) Instructions or a label indicating that the at least one other anticancer agent is administered to an individual in combination with a pharmaceutical composition comprising a multispecific antigen-binding molecule capable of binding to CD3 and CD137, the multispecific antigen-binding molecule comprising a first antigen-binding moiety that binds to either CD3 or CD137, and a second antigen-binding moiety that has binding activity to claudin 6 (CLDN6), for treating or preventing cancer in the individual. Kit including: (I-3) The kit according to (I-1) or (I-2), wherein the multispecific antigen-binding molecule or at least one other anticancer drug is packed in the container. (I-4) (A) A pharmaceutical composition comprising a multispecific antigen-binding molecule capable of binding to CD3 and CD137, the multispecific antigen-binding molecule comprising a first antigen-binding moiety that binds to either CD3 or CD137, and a second antigen-binding moiety that has binding activity to claudin 6 (CLDN6); (B) a container; and (C) at least one other anticancer drug Kit including: (I-5) The kit according to (I-4), wherein the multispecific antigen-binding molecule and at least one other anticancer drug are filled in a single container or each is filled in a separate container. (I-6) The kit of any one of (I-1) to (I-5), wherein the multispecific antigen-binding molecule is the multispecific antigen-binding molecule of any one of (A-2) to (A-39). (I-7) Any one of the kits (I-1) to (I-6), wherein the target cancer is a CLDN6-positive cancer. (I-8) Any one of the kits (I-1) to (I-7), wherein the target cancer is any cancer selected from the group consisting of ovarian cancer, non-small cell lung cancer, gastric cancer, liver cancer, endometrial cancer, germ cell tumor, colorectal cancer, bladder cancer, or atypical teratoid rhabdoid tumor. (I-9) The kit according to any one of (I-1) to (I-8), wherein the cancer is cancer that has metastasized to the peritoneum. (I-10) Any one of the kits (I-1) to (I-9) wherein the target cancer is peritoneal disseminated cancer. (I-11) The kit according to any one of (I-1) to (I-10), wherein the multispecific antigen-binding molecule and the at least one other anticancer agent are administered separately or sequentially. (I-12) The kit of any one of (I-1) to (I-11), wherein the multispecific antigen-binding molecule is administered before, simultaneously with, and / or after the at least one other anticancer drug. (I-13) Any one of the kits (I-1) to (I-12), wherein the at least one other anticancer agent is at least one selected from the group consisting of a chemotherapeutic agent, an immune checkpoint inhibitor, and a PARP inhibitor. (I-14) The kit according to any one of (I-1) to (I-13), wherein the at least one other anticancer agent is an agent that enhances the expression of CLDN6 in cells. (I-15) The kit according to any one of (I-1) to (I-14), wherein the at least one other anticancer agent is an agent that induces the expression of TGFβ in cancer cells. (I-16) The kit according to any one of (I-1) to (I-15), wherein the at least one other anticancer agent is an agent that induces the expression of TGFβ1 in cancer cells. (I-17) The kit according to any one of (I-1) to (I-16), wherein the at least one other anticancer agent is a platinum agent, an alkaloid, or an antimetabolite. (I-18) The kit according to any one of (I-1) to (I-17), wherein the at least one other anticancer agent is a platinum preparation. (I-19) The kit according to any one of (I-1) to (I-17), wherein the at least one other anticancer agent is an alkaloid. (I-20) The kit according to any one of (I-1) to (I-17), wherein the at least one other anticancer agent is a topoisomerase inhibitor. (I-21) The kit according to any one of (I-1) to (I-17), wherein the at least one other anticancer agent is an antimetabolite. (I-22) The kit according to any one of (I-1) to (I-17), wherein the at least one other anticancer drug is carboplatin or cisplatin. (I-23) The kit according to (I-1) to (I-17), wherein the at least one other anticancer drug is irinotecan. (I-24) The kit according to (I-1) to (I-17), wherein the at least one other anticancer drug is gemcitabine. (I-25) The kit according to any one of (I-1) to (I-17), wherein the at least one other anticancer agent is an anti-PD-L1 antibody. (I-26) The kit according to (I-1) to (I-17), wherein the at least one other anticancer drug is olaparib. (I2-1) (A) at least one TGFβ inducer; (B) a container; and (C) Instructions or a label indicating that the at least one other anticancer agent is administered to an individual in combination with a pharmaceutical composition comprising a multispecific antigen-binding molecule capable of binding to CD3 and CD137, the multispecific antigen-binding molecule comprising a first antigen-binding moiety that binds to either CD3 or CD137, and a second antigen-binding moiety that has binding activity to claudin 6 (CLDN6), for treating or preventing cancer in the individual. Kit including: (I2-2) The kit according to (I2-1), wherein the multispecific antigen-binding molecule or at least one TGFβ inducer is packed in the container. (I2-3) (A) A pharmaceutical composition comprising a multispecific antigen-binding molecule capable of binding to CD3 and CD137, the multispecific antigen-binding molecule comprising a first antigen-binding moiety that binds to either CD3 or CD137, and a second antigen-binding moiety that has binding activity to claudin 6 (CLDN6); (B) a container; and (C) at least one TGFβ inducer; Kit including: (I2-4) The kit according to (I2-3), wherein the multispecific antigen-binding molecule and the at least one TGFβ inducer are contained in a single container or in separate containers. (I2-5) The kit of any one of (I2-1) to (I2-4), wherein the multispecific antigen-binding molecule is the multispecific antigen-binding molecule of any one of (A-2) to (A-39). (I2-6) Any one of the kits (I2-1) to (I2-5), wherein the target cancer is a CLDN6-positive cancer. (I2-7) Any one of the kits (I2-1) to (I2-6), wherein the target cancer is any cancer selected from the group consisting of ovarian cancer, non-small cell lung cancer, gastric cancer, liver cancer, endometrial cancer, germ cell tumor, colorectal cancer, bladder cancer, or atypical teratoid rhabdoid tumor. (I2-8) The kit according to any one of (I2-1) to (I2-7), wherein the cancer is cancer that has metastasized to the peritoneum. (I2-9) Any one of the kits (I2-1) to (I2-8) wherein the target cancer is peritoneal disseminated cancer. (I2-10) The kit according to any one of (I2-1) to (I2-9), wherein the multispecific antigen-binding molecule is administered separately or consecutively with the at least one TGFβ inducer. (I2-11) The kit according to any one of (I2-1) to (I2-10), wherein the multispecific antigen-binding molecule is administered before, simultaneously with, and / or after the at least one TGFβ inducer. (I2-12) The kit according to any one of (I2-1) to (I2-11), wherein the at least one TGFβ inducer is an agent that enhances the expression of CLDN6 in cells. (I2-13) The kit according to any one of (I2-1) to (I2-12), wherein the at least one TGFβ inducer is an agent that induces the expression of TGFβ in cells. (I2-14) The kit according to any one of (I2-1) to (I2-13), wherein the at least one TGFβ inducer is an agent that induces the expression of TGFβ1 in cells. (I3-1) (A) at least one CLDN6 expression inducer; (B) a container; and (C) Instructions or a label indicating that the at least one other anticancer agent is administered to an individual in combination with a pharmaceutical composition comprising a multispecific antigen-binding molecule capable of binding to CD3 and CD137, the multispecific antigen-binding molecule comprising a first antigen-binding moiety that binds to either CD3 or CD137, and a second antigen-binding moiety that has binding activity to claudin 6 (CLDN6), for treating or preventing cancer in the individual. Kit including: (I3-2) The kit according to (I3-1), wherein the multispecific antigen-binding molecule or the at least one CLDN6 expression inducer is filled in the container. (I3-3) (A) A pharmaceutical composition comprising a multispecific antigen-binding molecule capable of binding to CD3 and CD137, the multispecific antigen-binding molecule comprising a first antigen-binding moiety that binds to either CD3 or CD137, and a second antigen-binding moiety that has binding activity to claudin 6 (CLDN6); (B) a container; and (C) at least one CLDN6 expression inducer; Kit including: (I3-4) The kit according to (I3-3), wherein the multispecific antigen-binding molecule and the at least one CLDN6 expression inducer are packed in a single container or in separate containers. (I3-5) The kit of any one of (I3-1) to (I3-4), wherein the multispecific antigen-binding molecule is the multispecific antigen-binding molecule of any one of (A-2) to (A-39). (I3-6) Any one of the kits (I3-1) to (I3-5), wherein the target cancer is a CLDN6-positive cancer. (I3-7) Any one of the kits (I3-1) to (I3-6), wherein the target cancer is any cancer selected from the group consisting of ovarian cancer, non-small cell lung cancer, gastric cancer, liver cancer, endometrial cancer, germ cell tumor, colorectal cancer, bladder cancer, or atypical teratoid rhabdoid tumor. (I3-8) The kit according to any one of (I3-1) to (I3-7), wherein the cancer is cancer that has metastasized to the peritoneum. (I3-9) Any one of the kits (I3-1) to (I3-8), wherein the target cancer is peritoneal disseminated cancer. (I3-10) Any one of the kits (I3-1) to (I3-9), wherein the multispecific antigen-binding molecule is administered separately or consecutively with the at least one CLDN6 expression inducer. (I3-11) Any one of the kits (I3-1) to (I3-10), wherein the multispecific antigen-binding molecule is administered before, simultaneously with, and / or after the at least one CLDN6 expression inducer. (I3-12) The kit according to any one of (I3-1) to (I3-11), wherein the at least one CLDN6 expression inducer is an agent that enhances the expression of CLDN6 in cells. (I3-13) The kit according to any one of (I3-1) to (I3-12), wherein the at least one CLDN6 expression inducer is an agent that induces the expression of TGFβ in cells. (I3-14) The kit according to any one of (I3-1) to (I3-13), wherein the at least one CLDN6 expression inducer is an agent that induces the expression of TGFβ1 in cells.

[0017] (J-1) A multispecific antigen-binding molecule for use in cancer treatment, comprising: A multispecific antigen-binding molecule comprising: (i) a first antigen-binding portion capable of binding to CD3 and CD137, and binding to either CD3 or CD137; and (ii) a second antigen-binding portion having binding activity for claudin 6 (CLDN6). (J-2) The multispecific antigen-binding molecule of (J-1), which is the multispecific antigen-binding molecule of any of (A-2) to (A-39). (J-3) The multispecific antigen-binding molecule of any one of (J-1) and (J-2), which is used to treat cancer in combination with at least one agent selected from the group consisting of other anticancer agents, TGFβ inducers, and CLDN6 expression inducers. (J-4) A combination of a multispecific antigen-binding molecule and at least one agent selected from the group consisting of other anticancer agents, TGFβ inducers, and CLDN6 expression inducers for use in cancer treatment, wherein the multispecific antigen-binding molecule: A combination comprising: (i) a first antigen-binding portion capable of binding to CD3 and CD137, and which binds to either CD3 or CD137; and (ii) a second antigen-binding portion having binding activity for claudin 6 (CLDN6). (J-5) The combination of (J-4), wherein the multispecific antigen-binding molecule is the multispecific antigen-binding molecule according to any one of (A-2) to (A-39). (J-6) The multispecific antigen-binding molecule or combination of any one of (J-3) to (J-5), wherein the multispecific antigen-binding molecule and the at least one agent are administered separately or sequentially. (J-7) The multispecific antigen-binding molecule or combination of (J-6), wherein the multispecific antigen-binding molecule is administered before, simultaneously with, and / or after the at least one agent. (J-8) The multispecific antigen-binding molecule or combination of any one of (J-3) to (J-7), wherein the at least one agent is at least one selected from the group consisting of a chemotherapeutic agent, an immune checkpoint inhibitor, and a PARP inhibitor. (J-9) The multispecific antigen-binding molecule or combination of any one of (J-3) to (J-8), wherein the at least one agent is an agent that enhances cellular CLDN6 expression. (J-10) The multispecific antigen-binding molecule or combination of any one of (J-3) to (J-9), wherein the at least one agent is an agent that induces the expression of TGFβ in cancer cells. (J-11) The multispecific antigen-binding molecule or combination of any one of (J-3) to (J-10), wherein the at least one agent is an agent that induces the expression of TGFβ1 in cancer cells. (J-12) The multispecific antigen-binding molecule or combination of any one of (J-8) to (J-11), wherein the at least one agent is a platinum agent, an alkaloid, or an antimetabolite. (J-13) The multispecific antigen-binding molecule or combination of any one of (J-8) to (J-12), wherein the at least one agent is a platinum agent. (J-14) The multispecific antigen-binding molecule or combination of any one of (J-8) to (J-12), wherein the at least one agent is an alkaloid. (J-15) The multispecific antigen-binding molecule or combination of any one of (J-8) to (J-12), wherein the at least one agent is a topoisomerase inhibitor. (J-16) The multispecific antigen-binding molecule or combination of any one of (J-8) to (J-12), wherein the at least one agent is an antimetabolite. (J-17) The multispecific antigen-binding molecule or combination of any one of (J-8) to (J-12), wherein the at least one agent is carboplatin or cisplatin. (J-18) The multispecific antigen-binding molecule or combination of any one of (J-8) to (J-12), wherein the at least one agent is irinotecan. (J-19) The multispecific antigen-binding molecule or combination of (J-8) to (J-12), wherein the at least one agent is gemcitabine. (J-20) The multispecific antigen-binding molecule or combination of (J-8), wherein the at least one agent is an anti-PD-L1 antibody. (J-21) The multispecific antigen-binding molecule or combination of (J-8), wherein said at least one agent is olaparib. (J-22) The multispecific antigen-binding molecule or combination of any one of (J-1) to (J-21), wherein the cancer is a CLDN6-positive cancer. (J-23) The multispecific antigen-binding molecule or combination of any one of (J-1) to (J-22), wherein the cancer is any cancer selected from the group consisting of ovarian cancer, non-small cell lung cancer, gastric cancer, liver cancer, endometrial cancer, germ cell tumor, colorectal cancer, bladder cancer, and atypical teratoid rhabdoid tumor. (J-24) The multispecific antigen-binding molecule or combination of any one of (J-1) to (J-23), wherein the cancer is cancer that has metastasized to the peritoneum. (J-25) The multispecific antigen-binding molecule or combination of any one of (J-1) to (J-24), wherein the cancer is peritoneal disseminated cancer. (J-26) A multispecific antigen-binding molecule or combination of any one of (J-1) to (J-25) for treating a patient having cancer that is refractory to treatment with an immune checkpoint inhibitor. (J-27) The multispecific antigen-binding molecule or combination of any one of (J-1) to (J-26), wherein the cancer has previously been treated with the other anticancer agent or an anticancer agent different from the other anticancer agent. (J-28) The multispecific antigen-binding molecule or combination of any one of (J-1) to (J-27), wherein the cancer is a cancer for which the desired effect was not obtained in treatment with the other anticancer agent alone.

[0018] (K-1) Use of the following multispecific antigen-binding molecule in the manufacture of a medicament for treating cancer: (i) a multispecific antigen-binding molecule comprising a first antigen-binding portion capable of binding to CD3 and CD137 and binding to either CD3 or CD137, and (ii) a second antigen-binding portion having binding activity for claudin 6 (CLDN6). (K-2) Use of (K-1), wherein the multispecific antigen-binding molecule is the multispecific antigen-binding molecule according to any one of (A-2) to (A-39). (K-3) Use of any one of (K-1) and (K-2), wherein the multispecific antigen-binding molecule is used in combination with at least one agent selected from the group consisting of other anticancer agents, TGFβ inducers, and CLDN6 expression inducers. (K-4) Use of a combination of the following multispecific antigen-binding molecule and at least one agent selected from the group consisting of other anticancer agents, TGFβ inducers, and CLDN6 expression inducers in the manufacture of a medicament for treating cancer: A multispecific antigen-binding molecule comprising: (i) a first antigen-binding portion capable of binding to CD3 and CD137, and binding to either CD3 or CD137; and (ii) a second antigen-binding portion having binding activity for claudin 6 (CLDN6). (K-5) Use of (K-4), wherein the multispecific antigen-binding molecule is the multispecific antigen-binding molecule according to any one of (A-2) to (A-39). (K-6) Use of (K-4) or (K-5), characterized in that the pharmaceutical is a combination of a multispecific antigen-binding molecule and the at least one agent. (K-7) Use of any one of (K-3) to (K-5), wherein the multispecific antigen-binding molecule and the at least one agent are administered separately or sequentially. (K-8) The use of (K-7), wherein the multispecific antigen-binding molecule is administered before, simultaneously with, and / or after the at least one agent. (K-9) Use of any one of (K-3) to (K-8), wherein the at least one agent is at least one selected from the group consisting of a chemotherapeutic agent, an immune checkpoint inhibitor, and a PARP inhibitor. (K-10) Use of any one of (K-3) to (K-9), wherein the at least one agent is an agent that enhances the expression of CLDN6 in cells. (K-11) Use of any one of (K-3) to (K-10), wherein the at least one agent is an agent that induces the expression of TGFβ in cancer cells. (K-12) Use of any one of (K-3) to (K-11), wherein the at least one agent is an agent that induces the expression of TGFβ1 in cancer cells. (K-13) Use of any one of (K-9) to (K-12), wherein the at least one agent is a platinum agent, an alkaloid, or an antimetabolite. (K-14) Use of any one of (K-9) to (K-13), wherein the at least one agent is a platinum agent. (K-15) Use of any one of (K-9) to (K-13), wherein the at least one agent is an alkaloid. (K-16) Use of any one of (K-9) to (K-13), wherein the at least one agent is a topoisomerase inhibitor. (K-17) Use of any one of (K-9) to (K-13), wherein the at least one agent is an antimetabolite. (K-18) Use of any one of (K-9) to (K-13), wherein the at least one other agent is carboplatin or cisplatin. (K-19) Use of any one of (K-9) to (K-13), wherein the at least one other agent is irinotecan. (K-20) Use of any one of (K-9) to (K-13), wherein the at least one other agent is gemcitabine. (K-21) Use of (K-9), wherein the at least one other agent is an anti-PD-L1 antibody. (K-22) The use of (K-9), wherein the at least one other agent is olaparib. (K-23) Use of any one of (K-1) to (K-22), wherein the cancer is a CLDN6-positive cancer. (K-24) Use of any one of (K-1) to (K-23), wherein the cancer is any cancer selected from the group consisting of ovarian cancer, non-small cell lung cancer, gastric cancer, liver cancer, endometrial cancer, germ cell tumor, colorectal cancer, bladder cancer, or atypical teratoid rhabdoid tumor. (K-25) Use of any one of (K-1) to (K-24), wherein the cancer is cancer that has metastasized to the peritoneum. (K-26) Use of any one of (K-1) to (K-25), wherein the cancer is peritoneal disseminated cancer. (K-27) Use of any one of (K-1) to (K-26) for treating a patient having cancer that is refractory to treatment with an immune checkpoint inhibitor. (K-28) Use of any one of (K-1) to (K-27), wherein the cancer is a cancer that has previously been treated with at least one other anticancer drug or an anticancer drug different from the at least one other anticancer drug. (K-29) Use of any one of (K-1) to (K-28), wherein the cancer is a cancer for which the desired effect has not been achieved by treatment with the administration of the at least one other anticancer agent alone. [Brief explanation of the drawings]

[0019] [Figure 1] This is a graph comparing CLDN6 expression in various cancer tissues based on TCGA (The Cancer Genome Atlas) data. [Figure 2]The epitope of the H0868L0581 Fab contact region on CD137 is shown. Epitope mapping in the CD137 amino acid sequence (black: closer than 3.0 Å to H0868L0581; striped: closer than 4.5 Å). [Figure 3] The epitope of the H0868L0581 Fab contact region on CD137 is shown, with epitope mapping in the crystal structure (dark grey spheres: closer than 3.0 Å to H0868L0581, light grey bars: closer than 4.5 Å). [Figure 4] Illustrates various antibody formats. Annotation of each Fv region in Table 4, and naming conventions in Tables 4, 5, and 6. (a) Diagram illustrating 1+1 bispecific antibody by utilizing FAST-Ig; (b) Diagram illustrating 1+1 bispecific antibody by utilizing CrossMab technology. [Figure 5] The binding activity of anti-CLDN6 / CD3 bispecific antibodies (CS2961 and 6PHU3 / TR01) to human CLDN family proteins (CLDN3, CLDN4, CLDN6, and CLDN9) is shown. The binding activity of anti-CLDN6 / CD3 bispecific antibodies to BaF3 transfectants (hCLDN6 / BaF, hCLDN3 / BaF, hCLDN4 / BaF, and hCLDN9 / BaF) was examined at a concentration of 15 μg / ml by flow cytometry and plotted as a histogram. KLH / TR01 was used as a negative control. [Figure 6] 1 shows the evaluation of T cell-dependent cytotoxicity by LDH assay. [Figure 7] Figure 1 shows an amino acid sequence alignment of human CLDN9 and human CLDN6. Human CLDN9 and human CLDN6 contain nearly identical sequences in extracellular domain 1, except for the N-terminal residue (Met / Leu at position 29). In extracellular domain 2, two amino acids differ between human CLDN9 and human CLDN6 (Arg / Leu at position 145 and Glu / Leu at position 156). [Figure 8]1 shows the results of evaluating T cell-dependent cytotoxicity of an antibody (PPU4135) against various cancer cell lines (NUGC-3, PA-1, SNG-M, NEC8, NEC14, CHLA-02-ATRT, HT-1197, and OUMS-23) by LDH assay. [Figure 9] 1 shows the results of a real-time cell growth inhibition assay of antibodies (CS3348, PPU4135, and PPU4136) against various cancer cell lines using the xCELLigence assay. [Figure 10] Figure 1 shows T cell activation through CD3 binding by antibodies (CS3348, PPU4135, PPU4136, PPU4137, and PPU4138) in coculture with CLDN6-expressing human cell lines (OVCAR3 and NCI-H1435) and a CLDN6-negative cell line (5637). KLH / TR01 was used as a negative control. [Figure 11] This shows the activation of NFκB through CD137 binding by antibodies (CS3348, PPU4134, PPU4135, PPU4136, PPU4137, and PPU4138) in coculture with CLDN6-expressing human cell lines (OVCAR3 and NCI-H1435) and a CLDN6-negative cell line (5637). KLH / TR01 was used as a negative control. [Figure 12] 1 shows the in vivo antitumor effects of antibodies (CS3348, PPU4134, PPU4135, PPU4136, PPU4137, and PPU4138) at a dose of 1 mg / kg using the NCI-H1435 / HuNOG mouse model. [Figure 13] 1 shows the in vivo antitumor effects of antibodies (CS3348 and PPU4135) at doses of 0.05 mg / kg and 0.2 mg / kg using an OV-90 / HuNOG mouse model. [Figure 14] 1 shows changes in AST, ALT, GLDH (liver enzymes), ALP, TBIL, GGT, TBA (hepatobiliary injury parameters), and CRP (inflammatory marker) levels mediated by administration of CS3348 or PPU4135. [Figure 15]FIG. 1 shows the survival rate after administration of anti-CLDN6 / Dual-Fab antibody in a mouse peritoneal dissemination model. [Figure 16] FIG. 1 shows changes in CLDN6 expression levels after treatment of various cancer cells (human ovarian cancer cell line (NIH-OVCAR3), human lung cancer cell line (NCI-H1435), and human endometrial cancer cell line (SNG-M)) with chemotherapeutic agents (cisplatin (CDDP) or carboplatin (CBDCA)). [Figure 17] This figure shows changes in the ability of the CS4135 antibody to activate T cells through CD3 binding after treatment with chemotherapeutic agents (cisplatin (CDDP) or carboplatin (CBDCA)), evaluated using a luciferase assay system with GloResponse NFAT-luc2 Jurkat cells. [Figure 18] This is a graph showing the relative expression level of CLDN6 after carboplatin administration in SNG-M tumor-implanted mice, as determined by real-time PCR using GAPDH as an internal control. [Figure 19] FIG. 10 shows changes in tumor volume following administration of the CS4135 antibody alone, carboplatin alone, and the combination of the CS4135 antibody and carboplatin in a xenograft transplantation model using NOG (huNOG) mice transplanted with the human endometrial cancer cell line SNG-M and human CD34-positive cells. [Figure 20] This figure shows the change in tumor volume following administration of the CS4135 antibody alone, carboplatin alone, and the combined administration of the CS4135 antibody and carboplatin (simultaneous administration) in a xenograft transplantation model using NOG (huNOG) mice transplanted with the ovarian cancer cell line OVCAR3 and human CD34-positive cells. [Figure 21] FIG. 10 shows changes in tumor volume following single-agent administration of the CS4135 antibody, single-agent administration of carboplatin, and sequential administration of the CS4135 antibody after carboplatin administration in a xenograft transplantation model using NOG (huNOG) mice transplanted with the ovarian cancer cell line OVCAR3 and human CD34-positive cells. [Figure 22]FIG. 10 is a graph showing changes in tumor volume following administration of the CS4135 antibody alone, irinotecan hydrochloride alone, and combined administration of the CS4135 antibody and irinotecan hydrochloride in a xenograft transplantation model using NOG (huNOG) mice transplanted with the ovarian cancer cell line OVCAR3 and human CD34-positive cells. [Figure 23] FIG. 10 is a graph showing changes in tumor volume following single-agent administration of the CS4135 antibody in hCD137 KI / hCD3Tg mice transplanted with the lung cancer cell line LLC1, in which claudin 6 (CLDN6) was forcibly expressed. [Figure 24] This figure shows the results of flow cytometry (FCM) analysis of the number of CD8-positive T cells in tumor tissues after administration of the CS4135 antibody to hCD137 KI / hCD3Tg mice transplanted with the lung cancer cell line LLC1, which overexpresses claudin 6 (CLDN6). [Figure 25] This figure shows the change in tumor volume following administration of the CS4135 antibody alone, the anti-mouse PD-L1 antibody alone, and the combination of the CS4135 antibody and the anti-mouse PD-L1 antibody in hCD137 KI / hCD3Tg mice implanted with the lung cancer cell line LLC1, which overexpresses claudin 6 (CLDN6). [Figure 26] FIG. 1 shows the change in CLDN6 expression level after treatment with a PARP inhibitor (olaparib) in the BRCA1-deficient ovarian cancer cell line UWB1.289 expressing human CLDN6 or the BRCA1 wild-type ovarian cancer cell line OV-90 (ATCC), as determined by FACS analysis. [Figure 27] FIG. 1 shows the cytotoxic activity of the CS4135 antibody in the BRCA1-deficient ovarian cancer cell line UWB1.289 or the BRAC1 wild-type ovarian cancer cell line OV-90 expressing human CLDN6, which was treated with a PARP inhibitor (olaparib), assessed by a lactate dehydrogenase (LDH) release assay using human PBMCs as effector cells. [Figure 28]This figure shows the cytotoxic activity of the CS4135 antibody in the presence or absence of the KLH / CD137 bispecific antibody, analyzed using a real-time cell growth inhibition assay (xCELLigence assay). The target cells used were the mouse colon cancer cell line MC38 / CLDN6, which expresses human CLDN6, and the effector cells were T cells derived from hCD3 transgenic mice or hCD3 / hCD137 knock-in mice. [Figure 29] This figure shows the results of treating a human ovarian cancer cell line (NIH:OVCAR-3) with carboplatin, cisplatin, irinotecan, or gemcitabine, measuring the CLDN6 expression level by quantitative PCR, and comparing it with that of untreated cells. [Figure 30] This figure shows the results of treating a human ovarian cancer cell line (NIH:OVCAR-3) with carboplatin, cisplatin, irinotecan, or gemcitabine, measuring the TGFβ1 expression level by quantitative PCR, and comparing it with that of untreated cells. [Figure 31] This figure shows the results of stimulating a human ovarian cancer cell line (NIH:OVCAR-3) with TGFβ1, measuring the CLDN6 expression level by quantitative PCR, and comparing it with that of unstimulated cells. [Figure 32] This figure shows the results of stimulating various ovarian cancer cell lines (NIH: OVCAR-3, COV362, COV413A, COV413B) with TGFβ1, analyzing the CLDN6 expression levels by FACS, and comparing them with unstimulated cells. DETAILED DESCRIPTION OF THE INVENTION

[0020] The techniques and procedures described or referenced herein are generally well understood and can be found, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press;Animal Cell Culture (RI Freshney), ed., 1987);Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press;Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, eds., 1993-8) J. Wiley and Sons;Handbook of Experimental Immunology (DM Weir and CCBlackwell, eds.);Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987);PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994);Current Protocols in Immunology (JE Coligan et al., eds., 1991);Short Protocols in Molecular Biology (Wiley and Sons, 1999);Immunobiology (CA Janeway and P. Travers, 1997);Antibodies (P. Finch, 1997);Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989);Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995); and those commonly employed by those skilled in the art using conventional methodologies such as those widely used in Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993).

[0021] The following definitions and detailed description are provided to facilitate understanding of the disclosure described herein.

[0022] definition amino acid As used herein, amino acids are described by one-letter or three-letter codes or both, for example, Ala / A, Leu / L, Arg / R, Lys / K, Asn / N, Met / M, Asp / D, Phe / F, Cys / C, Pro / P, Gln / Q, Ser / S, Glu / E, Thr / T, Gly / G, Trp / W, His / H, Tyr / Y, Ile / I, or Val / V.

[0023] Amino acid modification

[0024] For amino acid modification (herein also referred to as "amino acid substitution" or "amino acid mutation") in the amino acid sequence of an antigen-binding molecule, known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR can be appropriately employed. Furthermore, several known methods for amino acid modification to substitute with unnatural amino acids can also be employed (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249; and Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, it is suitable to use a cell-free translation system (Clover Direct (Protein Express)) containing a tRNA in which an unnatural amino acid is bound to a complementary amber suppressor tRNA for the UAG codon (amber codon), which is a type of stop codon.

[0025] As used herein, the term "and / or" when describing the site of an amino acid modification includes any combination of "and" and "or." Specifically, for example, "amino acids at positions 33, 55, and / or 96 are substituted" includes the following variations of amino acid modification: (a) 33, (b) 55, (c) 96, (d) 33 and 55, (e) 33 and 96, (f) 55 and 96, and (g) 33, 55, and 96 amino acids.

[0026] Furthermore, as used herein, expressions indicating amino acid modifications may be appropriately expressed by indicating the one-letter or three-letter code of the amino acid before and after the modification, respectively, before and after the number indicating a specific position. For example, the modification N100bL or Asn100bLeu used to substitute an amino acid contained in an antibody variable region represents a substitution of Asn at position 100b (according to Kabat numbering) with Leu. That is, the number indicates the amino acid position according to Kabat numbering, the one-letter or three-letter amino acid code written before the number represents the amino acid before substitution, and the one-letter or three-letter amino acid code written after the number represents the amino acid after substitution. Similarly, the modification P238D or Pro238Asp used to substitute an amino acid in the Fc region contained in an antibody constant region represents a substitution of Pro at position 238 (according to EU numbering) with Asp. That is, the numbers indicate the amino acid positions according to EU numbering, the one-letter or three-letter amino acid code written before the number indicates the amino acid before substitution, and the one-letter or three-letter amino acid code written after the number indicates the amino acid after substitution.

[0027] Polypeptides As used herein, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linked in a linear chain by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain of two or more amino acids and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to a chain of two or more amino acids is included within the definition of "polypeptide," and the term "polypeptide" may be used in place of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to products of post-expression modifications of the polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but need not necessarily be translated from a designated nucleic acid. It may be generated by any method, including chemical synthesis. The polypeptides described herein may be about 3 or more amino acids, 5 or more amino acids, 10 or more amino acids, 20 or more amino acids, 25 or more amino acids, 50 or more amino acids, 75 or more amino acids, 100 or more amino acids, 200 or more amino acids, 500 or more amino acids, 1,000 or more amino acids, or 2,000 or more amino acids in size. Polypeptides may have a defined three-dimensional structure, but they do not necessarily have such a structure. Polypeptides that have a defined three-dimensional structure are said to be folded, while polypeptides that do not have a defined three-dimensional structure but can adopt multiple different conformations are said to be unfolded.

[0028] Percent (%) amino acid sequence identity "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences to achieve the maximum percent sequence identity and introducing gaps, if necessary, and excluding any conservative substitutions from the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved by a variety of methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is the copyright of Genentech, Inc., and its source code, together with user documentation, has been filed with the U.S. Copyright Office, Washington, DC 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code. The ALIGN-2 program is compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (alternatively, one can say that a given amino acid sequence A has or contains a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in its alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0029] Recombination methods and constructs Antibodies and antigen-binding molecules can be produced using recombinant methods and constructs, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an antibody described herein is provided. Such a nucleic acid may encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of the antibody (e.g., the light chain and / or heavy chain of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In a further embodiment, a host cell comprising such nucleic acids is provided. In one such embodiment, the host cell comprises (e.g., is transformed with) (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic (e.g., a Chinese hamster ovary (CHO) cell) or a lymphoid cell (e.g., a Y0, NS0, or Sp2 / 0 cell)). In one embodiment, there is provided a method of making a multispecific antigen-binding molecule of the present disclosure, comprising culturing a host cell comprising nucleic acid encoding the antibody, as described above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0030] For recombinant production of the antibodies described herein, nucleic acid encoding the antibody (e.g., such as those described above) is isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes capable of binding specifically to genes encoding the antibody heavy and light chains).

[0031] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibody may be isolated in a soluble fraction from the bacterial cell paste or further purified.

[0032] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of antibodies with partial or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0033] Host cells derived from multicellular organisms (invertebrates and vertebrates) are also suitable for expressing glycosylated antibodies. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified for use in conjugation with insect cells, particularly for transformation of Spodoptera frugiperda cells.

[0034] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0035] Vertebrate cells can also be used as hosts. For example, mammalian cell lines that have been adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 (COS-7); human embryonic kidney (293 or 293 cells, e.g., as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney (BHK) cells; mouse Sertoli cells (TM4 cells, e.g., as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney (CV1); African green monkey kidney (VERO-76); human cervical carcinoma (HELA); canine kidney (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary carcinoma (MMT 060562); TRI cells (e.g., as described in Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)). Other useful mammalian host cell lines include DHFR cells; MRC5 cells; and FS4 cells. - Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0036] Recombinant production of the antigen-binding molecules described herein can be carried out in a similar manner to that described above, by using host cells containing (e.g., transformed with) one or more vectors containing nucleic acids encoding an amino acid sequence comprising the entire antigen-binding molecule or a portion of the antigen-binding molecule.

[0037] Antigen-binding molecules and multispecific antigen-binding molecules As used herein, the term "antigen-binding molecule" refers to any molecule containing an antigen-binding site or any molecule that has binding activity to an antigen, and may further refer to molecules such as peptides or proteins having a length of about 5 amino acids or more. Peptides and proteins are not limited to those derived from living organisms; for example, they may be polypeptides produced from artificially designed sequences. They may be natural polypeptides, synthetic polypeptides, recombinant polypeptides, etc. Scaffold molecules that contain a known stable three-dimensional structure such as an α / β barrel as a scaffold, and a portion of the molecule serves as the antigen-binding site, are also an embodiment of the antigen-binding molecules described herein.

[0038] A "multispecific antigen-binding molecule" refers to an antigen-binding molecule that specifically binds to two or more antigens. "Multispecific antigen-binding molecules" include antibodies and antibody fragments that specifically bind to two or more antigens. The term "bispecific" means that an antigen-binding molecule can specifically bind to at least two different antigenic determinants. The term "trispecific" means that an antigen-binding molecule can specifically bind to at least three different antigenic determinants.

[0039] In certain embodiments, the multispecific antigen-binding molecules of the present disclosure are trispecific antigen-binding molecules, i.e., trispecific antigen-binding molecules that can specifically bind to three different antigens, i.e., that can bind to CD3 and CD137 but not both antigens simultaneously, and that can specifically bind to CLDN6.

[0040] In one aspect, the present disclosure provides a method for producing a cellular membrane comprising: (i) a first antigen-binding moiety capable of binding to CD3 and CD137, which binds to either CD3 or CD137; and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6), preferably human CLDN6. The present invention provides an anti-cancer agent comprising, as an active ingredient, a multispecific antigen-binding molecule comprising:

[0041] In one aspect, the present disclosure provides a method for producing a cellular membrane comprising: (i) a first antigen-binding moiety capable of binding to CD3 and CD137, which binds to either CD3 or CD137; and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6), preferably human CLDN6. The present invention provides a pharmaceutical composition for use in combination with at least one other anticancer agent, which comprises a multispecific antigen-binding molecule as an active ingredient, comprising: In one aspect, the present disclosure also provides a method for manufacturing a semiconductor device comprising: (i) a first antigen-binding moiety capable of binding to CD3 and CD137, which binds to either CD3 or CD137; and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6), preferably human CLDN6. The present invention provides a pharmaceutical composition for use in combination with at least one TGFβ inducer, which comprises a multispecific antigen-binding molecule as an active ingredient, comprising: In one aspect, the present disclosure also provides a method for manufacturing a semiconductor device comprising: (i) a first antigen-binding moiety capable of binding to CD3 and CD137, which binds to either CD3 or CD137; and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6), preferably human CLDN6. The present invention provides a pharmaceutical composition for use in combination with at least one CLDN6 expression inducer, which comprises a multispecific antigen-binding molecule as an active ingredient, comprising:

[0042] In one aspect, the present disclosure provides a multispecific antigen-binding molecule comprising (i) a first antigen-binding moiety capable of binding to both CD3 and CD137, and binding to either CD3 or CD137, and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6), wherein the first antigen-binding moiety has a higher cytotoxic activity than an antigen-binding moiety capable of binding only to CD3. The present invention provides an anti-cancer agent comprising the above as an active ingredient. Also, in one aspect, the present disclosure provides a multispecific antigen-binding molecule comprising (i) a first antigen-binding moiety capable of binding to both CD3 and CD137, and binding to either CD3 or CD137, and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6), wherein the first antigen-binding moiety has reduced toxicity compared to an antigen-binding moiety capable of binding only to CD3. The present invention provides an anti-cancer agent comprising the above as an active ingredient. In one aspect, the present disclosure also provides a method for manufacturing a semiconductor device comprising: A multispecific antigen-binding molecule comprising (1) a first antigen-binding portion capable of binding to a T cell receptor complex, and (2) a second antigen-binding portion capable of binding to CLDN6, wherein the multispecific antigen-binding molecule has a T cell cytotoxic activity that is equivalent to or greater than that of a multispecific antibody (CS3348) comprising an antigen-binding portion capable of binding to a T cell receptor complex, the antigen-binding portion comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 194 and a light chain comprising the amino acid sequence of SEQ ID NO: 192, and an antigen-binding portion capable of binding to CLDN6, the antigen-binding portion comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 193 and a light chain comprising the amino acid sequence of SEQ ID NO: 195. The present invention provides an anti-cancer agent comprising the above as an active ingredient.

[0043] In one aspect, the present disclosure provides a pharmaceutical composition comprising at least one other anticancer agent as an active ingredient, for use in combination with a multispecific antigen-binding molecule comprising (i) a first antigen-binding moiety capable of binding to CD3 and CD137, the first antigen-binding moiety binding to either CD3 or CD137, and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6).

[0044] In one aspect, the present disclosure provides a method for producing a cellular membrane comprising: A pharmaceutical composition for treating or preventing cancer, comprising the following multispecific antigen-binding molecule in combination with at least one other anticancer agent: A multispecific antigen-binding molecule comprising: (i) a first antigen-binding moiety capable of binding to CD3 and CD137, the first antigen-binding moiety binding to either CD3 or CD137; and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6). to provide. In one aspect, the present disclosure also provides a method for manufacturing a semiconductor device comprising: A pharmaceutical composition for treating or preventing cancer, comprising in combination the following multispecific antigen-binding molecule and at least one TGFβ-inducing therapy: A multispecific antigen-binding molecule comprising: (i) a first antigen-binding moiety capable of binding to CD3 and CD137, the first antigen-binding moiety binding to either CD3 or CD137; and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6). to provide. In one aspect, the present disclosure also provides a method for manufacturing a semiconductor device comprising: A pharmaceutical composition for treating or preventing cancer, comprising a combination of the following multispecific antigen-binding molecule and at least one CLDN6 expression inducer: A multispecific antigen-binding molecule comprising: (i) a first antigen-binding moiety capable of binding to CD3 and CD137, the first antigen-binding moiety binding to either CD3 or CD137; and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6). to provide.

[0045] In one aspect, the present disclosure provides a combination of a multispecific antigen-binding molecule comprising (i) a first antigen-binding moiety capable of binding to CD3 and CD137, the first antigen-binding moiety binding to either CD3 or CD137, and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6), and at least one other anti-cancer agent. Also, in one aspect, the present disclosure provides a combination of a multispecific antigen-binding molecule comprising (i) a first antigen-binding moiety capable of binding to CD3 and CD137, and which binds to either CD3 or CD137, and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6), and at least one TGFβ inducer. Also, in one aspect, the present disclosure provides a combination of a multispecific antigen-binding molecule comprising (i) a first antigen-binding portion capable of binding to CD3 and CD137, and binding to either CD3 or CD137, and (ii) a second antigen-binding portion capable of binding to claudin 6 (CLDN6), and at least one CLDN6 expression inducer.

[0046] In one aspect, the present disclosure provides a method for inducing cytotoxicity, suppressing cell proliferation, inhibiting cell proliferation, activating an immune response, treating cancer, or preventing cancer in an individual, comprising administering an effective amount of a multispecific antigen-binding molecule comprising (i) a first antigen-binding moiety capable of binding to CD3 and CD137, the first antigen-binding moiety binding to either CD3 or CD137, and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6), and an effective amount of at least one other anti-cancer agent. Also, in one aspect, the present disclosure provides a method for inducing cytotoxicity, suppressing cell proliferation, inhibiting cell proliferation, activating an immune response, treating cancer, or preventing cancer in an individual, comprising administering to the individual an effective amount of at least one other anti-cancer agent in combination with a multispecific antigen-binding molecule comprising (i) a first antigen-binding moiety capable of binding to CD3 and CD137 and that binds to either CD3 or CD137, and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6). Furthermore, in one aspect, the present disclosure provides a method for enhancing the effect of inducing cytotoxicity, suppressing cell growth, inhibiting cell proliferation, activating an immune response, treating cancer, or preventing cancer in an individual by a multispecific antigen-binding molecule comprising (i) a first antigen-binding moiety capable of binding to CD3 and CD137 and binding to either CD3 or CD137, and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6), comprising administering to the individual an effective amount of at least one other anti-cancer agent. In one aspect, the present disclosure provides a method for enhancing the effect of at least one other anticancer agent in inducing cytotoxicity, suppressing cell growth, inhibiting cell proliferation, activating an immune response, treating cancer, or preventing cancer in an individual, comprising administering to the individual an effective amount of a multispecific antigen-binding molecule comprising: (i) a first antigen-binding moiety capable of binding to CD3 and CD137, the first antigen-binding moiety binding to either CD3 or CD137, and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6).

[0047] Also, in one aspect, the present disclosure provides a method for inducing cytotoxicity, suppressing cell proliferation, inhibiting cell proliferation, activating an immune response, treating cancer, or preventing cancer in an individual, comprising administering an effective amount of a multispecific antigen-binding molecule comprising (i) a first antigen-binding portion capable of binding to CD3 and CD137 and binding to either CD3 or CD137, and (ii) a second antigen-binding portion capable of binding to claudin 6 (CLDN6), and an effective amount of at least one TGFβ inducer. Also, in one aspect, the present disclosure provides a method for inducing cytotoxicity, suppressing cell proliferation, inhibiting cell proliferation, activating an immune response, treating or preventing cancer in an individual, comprising administering to the individual an effective amount of at least one TGFβ inducer in combination with a multispecific antigen-binding molecule comprising (i) a first antigen-binding moiety capable of binding to CD3 and CD137, the first antigen-binding moiety binding to either CD3 or CD137, and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6). Furthermore, in one aspect, the present disclosure provides a method for enhancing the effect of inducing cytotoxicity, suppressing cell growth, inhibiting cell proliferation, activating an immune response, treating cancer, or preventing cancer in an individual by a multispecific antigen-binding molecule comprising (i) a first antigen-binding moiety capable of binding to CD3 and CD137 and binding to either CD3 or CD137, and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6), comprising administering to the individual an effective amount of at least one TGFβ inducer.

[0048] Also, in one aspect, the present disclosure provides a method for inducing cytotoxicity, suppressing cell proliferation, inhibiting cell proliferation, activating an immune response, treating cancer, or preventing cancer in an individual, comprising administering an effective amount of a multispecific antigen-binding molecule comprising (i) a first antigen-binding portion capable of binding to CD3 and CD137 and binding to either CD3 or CD137, and (ii) a second antigen-binding portion capable of binding to claudin 6 (CLDN6), and an effective amount of at least one CLDN6 expression inducer. Also, in one aspect, the present disclosure provides a method for inducing cytotoxicity, suppressing cell proliferation, inhibiting cell proliferation, activating an immune response, treating or preventing cancer in an individual, comprising administering to the individual an effective amount of at least one CLDN6 expression inducer in combination with a multispecific antigen-binding molecule comprising (i) a first antigen-binding moiety capable of binding to CD3 and CD137 and binding to either CD3 or CD137, and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6). Furthermore, in one aspect, the present disclosure provides a method for enhancing the effect of inducing cytotoxicity, suppressing cell growth, inhibiting cell proliferation, activating immune responses, treating cancer, or preventing cancer in an individual by a multispecific antigen-binding molecule comprising (i) a first antigen-binding moiety capable of binding to CD3 and CD137 and binding to either CD3 or CD137, and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6), comprising administering to the individual an effective amount of at least one CLDN6 expression inducer.

[0049] In one aspect, the present disclosure provides a method for causing damage to cancer cells or tumor tissue containing cancer cells, or a method for suppressing the growth of cancer cells or tumor tissue containing cancer cells, by contacting the cancer cells with a multispecific antigen-binding molecule comprising (i) a first antigen-binding moiety capable of binding to CD3 and CD137 and binding to either CD3 or CD137, and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6), and at least one other anti-cancer agent. In one aspect, the present disclosure provides a method for determining whether the multispecific antigen-binding molecule and at least one other anticancer agent cause damage to or suppress the growth of cancer cells or tumor tissue containing cancer cells, by contacting cancer cells with a multispecific antigen-binding molecule comprising (i) a first antigen-binding moiety capable of binding to CD3 and CD137 and binding to either CD3 or CD137, and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6), and at least one other anticancer agent.

[0050] In one aspect, the present disclosure provides a method for producing a cellular membrane comprising: (A) A pharmaceutical composition comprising a multispecific antigen-binding molecule capable of binding to CD3 and CD137, the multispecific antigen-binding molecule comprising a first antigen-binding portion that binds to either CD3 or CD137, and a second antigen-binding portion that has binding activity to claudin 6 (CLDN6); (B) a container; and (C) Instructions or a label indicating that the multispecific antigen-binding molecule is administered to an individual in combination with at least one other anti-cancer agent to treat or prevent cancer in the individual. A kit comprising: In one aspect, the present disclosure provides a method for producing a cellular membrane comprising: (A) at least one other anticancer agent; (B) a container; and (C) Instructions or a label indicating that the at least one other anticancer agent is administered to an individual in combination with a pharmaceutical composition comprising a multispecific antigen-binding molecule capable of binding to CD3 and CD137, the multispecific antigen-binding molecule comprising a first antigen-binding moiety that binds to either CD3 or CD137, and a second antigen-binding moiety that has binding activity to claudin 6 (CLDN6), for treating or preventing cancer in the individual. A kit comprising: In one aspect, the present disclosure provides a method for producing a cellular membrane comprising: (A) A pharmaceutical composition comprising a multispecific antigen-binding molecule capable of binding to CD3 and CD137, the multispecific antigen-binding molecule comprising a first antigen-binding portion that binds to either CD3 or CD137, and a second antigen-binding portion that has binding activity to claudin 6 (CLDN6); (B) a container; and (C) at least one other anticancer drug A kit comprising:

[0051] In another aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (A) at least one TGFβ inducer; (B) a container; and (C) Instructions or a label indicating that the at least one other anticancer agent is administered to an individual in combination with a pharmaceutical composition comprising a multispecific antigen-binding molecule capable of binding to CD3 and CD137, the multispecific antigen-binding molecule comprising a first antigen-binding moiety that binds to either CD3 or CD137, and a second antigen-binding moiety that has binding activity to claudin 6 (CLDN6), for treating or preventing cancer in the individual. A kit comprising: In one aspect, the present disclosure provides a method for producing a cellular membrane comprising: (A) A pharmaceutical composition comprising a multispecific antigen-binding molecule capable of binding to CD3 and CD137, the multispecific antigen-binding molecule comprising a first antigen-binding portion that binds to either CD3 or CD137, and a second antigen-binding portion that has binding activity to claudin 6 (CLDN6); (B) a container; and (C) at least one TGFβ inducer A kit comprising:

[0052] In another aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (A) at least one CLDN6 expression inducer; (B) a container; and (C) Instructions or a label indicating that the at least one other anticancer agent is administered to an individual in combination with a pharmaceutical composition comprising a multispecific antigen-binding molecule capable of binding to CD3 and CD137, the multispecific antigen-binding molecule comprising a first antigen-binding moiety that binds to either CD3 or CD137, and a second antigen-binding moiety that has binding activity to claudin 6 (CLDN6), for treating or preventing cancer in the individual. A kit comprising: In one aspect, the present disclosure provides a method for producing a cellular membrane comprising: (A) A pharmaceutical composition comprising a multispecific antigen-binding molecule capable of binding to CD3 and CD137, the multispecific antigen-binding molecule comprising a first antigen-binding portion that binds to either CD3 or CD137, and a second antigen-binding portion that has binding activity to claudin 6 (CLDN6); (B) a container; and (C) at least one CLDN6 expression inducer A kit comprising:

[0053] In one aspect, the present disclosure provides a method for treating cancer comprising: Provided is a multispecific antigen-binding molecule comprising: (i) a first antigen-binding moiety capable of binding to CD3 and CD137, and which binds to either CD3 or CD137; and (ii) a second antigen-binding moiety having binding activity for claudin 6 (CLDN6). Also, in one aspect, the present disclosure provides a combination of a multispecific antigen-binding molecule, comprising (i) a first antigen-binding portion capable of binding to CD3 and CD137 and binding to either CD3 or CD137, and (ii) a second antigen-binding portion having binding activity for claudin 6 (CLDN6), and at least one agent selected from the group consisting of other anti-cancer agents, TGFβ inducers, and CLDN6 expression inducers, for use in treating cancer.

[0054] In one aspect, the present disclosure provides use of a multispecific antigen-binding molecule comprising (i) a first antigen-binding moiety capable of binding to CD3 and CD137, and which binds to either CD3 or CD137, and (ii) a second antigen-binding moiety having binding activity for claudin 6 (CLDN6), in the manufacture of a medicament for treating cancer. Also, in one aspect, the present disclosure provides use of a combination of a multispecific antigen-binding molecule comprising (i) a first antigen-binding moiety capable of binding to CD3 and CD137 and binding to either CD3 or CD137, and (ii) a second antigen-binding moiety having binding activity to claudin 6 (CLDN6), and at least one agent selected from the group consisting of other anticancer agents, TGFβ inducers, and CLDN6 expression inducers, in the manufacture of a medicament for treating cancer.

[0055] In one aspect, the multispecific antigen-binding molecule contained in the anticancer agent, pharmaceutical composition, combination, kit, or used in the method or use of the present disclosure is (iii) an Fc domain that exhibits reduced binding affinity to human Fcγ receptors compared to a native human IgG1 Fc domain; It may further include:

[0056] The components of the multispecific antigen-binding molecules contained in the anti-cancer agents, pharmaceutical compositions, combinations, kits, or used in the methods or uses of the present disclosure can be fused to each other in various configurations. Exemplary configurations are illustrated in Figure 4. In certain embodiments, the multispecific antigen-binding molecule comprises an Fc domain composed of a first Fc region subunit and a second Fc region subunit that can stably associate.

[0057] According to any of the above aspects, the components of the multispecific antigen-binding molecule (e.g., antigen-binding portion, Fc domain) may be fused directly or through various linkers, particularly peptide linkers comprising one or more amino acids, typically about 2 to 20 amino acids, described herein or known in the art. Suitable non-immunogenic peptide linkers include, for example, (G4S)n, (SG4)n, (G4S)n, or G4(SG4)n peptide linkers, where n is generally a number between 1 and 10, typically between 2 and 4.

[0058] In one aspect, the multispecific antigen-binding molecule contained in the anticancer agent, pharmaceutical composition, combination, kit, or used in the method or use of the present disclosure is (i) a first antigen-binding moiety capable of binding to CD3 and CD137, which binds to either CD3 or CD137; and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6); A multispecific antigen-binding molecule comprising: The antigen-binding molecule may be a multispecific antigen-binding molecule in which a first antibody variable region of the first antigen-binding moiety is fused to a first heavy chain constant region, a second antibody variable region of the first antigen-binding moiety is fused to a first light chain constant region, a third antibody variable region of the second antigen-binding moiety is fused to a second heavy chain constant region, and a fourth antibody variable region of the second antigen-binding moiety is fused to a second light chain constant region.

[0059] In one aspect, the multispecific antigen-binding molecule contained in the anticancer agent, pharmaceutical composition, combination, kit, or used in the method or use of the present disclosure is (i) a first antigen-binding moiety capable of binding to CD3 and CD137, which binds to either CD3 or CD137; and (ii) a second antigen-binding moiety capable of binding to claudin 6 (CLDN6); a first antibody variable region of the first antigen-binding portion is fused to a first heavy chain constant region, a second antibody variable region of the first antigen-binding portion is fused to a first light chain constant region, a third antibody variable region of the second antigen-binding portion is fused to a second heavy chain constant region, and a fourth antibody variable region of the second antigen-binding portion is fused to a second light chain constant region, wherein the constant regions are selected from the following (g1) to (g7): (g1) a first heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 74, a first light chain constant region comprising the amino acid sequence of SEQ ID NO: 87, a second heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 73, and a second light chain constant region comprising the amino acid sequence of SEQ ID NO: 88; (g2) a first heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 74, a first light chain constant region comprising the amino acid sequence of SEQ ID NO: 85, a second heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 81, and a second light chain constant region comprising the amino acid sequence of SEQ ID NO: 86; (g3) a first heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 79, a first light chain constant region comprising the amino acid sequence of SEQ ID NO: 72, a second heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 80, and a second light chain constant region comprising the amino acid sequence of SEQ ID NO: 89; (g4) a first heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 83, a first light chain constant region comprising the amino acid sequence of SEQ ID NO: 87, a second heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 82, and a second light chain constant region comprising the amino acid sequence of SEQ ID NO: 88; (g5) a first heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 83, a first light chain constant region comprising the amino acid sequence of SEQ ID NO: 85, a second heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 84, and a second light chain constant region comprising the amino acid sequence of SEQ ID NO: 86; (g6) a first heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 77, a first light chain constant region comprising the amino acid sequence of SEQ ID NO: 72, a second heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 78, and a second light chain constant region comprising the amino acid sequence of SEQ ID NO: 89; (g7) A first heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 75, a first light chain constant region comprising the amino acid sequence of SEQ ID NO: 72, a second heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 76, and a second light chain constant region comprising the amino acid sequence of SEQ ID NO: 89. The multispecific antigen-binding molecule may be any one of:

[0060] In one aspect, a multispecific antigen-binding molecule comprised in an anticancer agent, pharmaceutical composition, combination, or kit of the present disclosure, or used in a method or use of the present disclosure, is a multispecific antigen-binding molecule comprising four polypeptide chains, wherein the four polypeptide chains are selected from the group consisting of the following (h01) to (h18): (h01) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 42 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 51, and a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 56 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 69; (h02) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 41 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 50, and a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 54 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 68; (h03) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 41 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 50, and a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 55 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 68; (h04) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 42 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 51, and a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 57 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 69; (h05) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 44 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 52, and a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 60 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 70; (h06) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 44 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 52, and a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 61 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 70; (h07) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 45 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 50, and a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 62 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 68; (h08) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 45 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 50, and a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 63 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 68; (h09) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 46 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 51, and a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 64 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 69; (h10) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 46 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 51, and a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 65 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 69; (h11) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 47 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 52, and a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 66 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 70; (h12) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 47 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 52, and a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 67 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 70; (h13) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 48 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 53, and a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 56 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 71; (h14) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 48 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 53, and a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 57 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 71; (h15) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 49 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 53, and a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 64 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 71; (h16) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 49 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 53, and a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 65 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 71; (h17) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 43 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 52, and a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 58 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 70; (h18) A heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 43 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 52, and a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 59 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 70. The multispecific antigen-binding molecule may be any one of:

[0061] Pyroglutamylation It is known that when antibody is expressed in cells, antibody is modified after translation.Examples of post-translational modifications include cleavage of lysine at the C-terminus of heavy chain by carboxypeptidase; modification of glutamine or glutamic acid at the N-terminus of heavy chain and light chain to pyroglutamic acid by pyroglutamylation; glycosylation; oxidation; deamidation; and glycation, and such post-translational modifications are known to occur in various antibodies (Journal of Pharmaceutical Sciences, 2008, Vol. 97, p. 2426-2447).

[0062] The multispecific antigen-binding molecules contained in the anticancer agents, pharmaceutical compositions, combinations, and kits of the present disclosure, or used in the methods or uses of the present disclosure, also include multispecific antibodies that have undergone post-translational modifications. Examples of the multispecific antigen-binding molecules of the present disclosure that have undergone post-translational modifications include multispecific antibodies that have undergone 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 such post-translational modifications by pyroglutamylation at the N-terminus and deletion of lysine at the C-terminus have no effect on the activity of antibodies (Analytical Biochemistry, 2006, Vol. 348, pp. 24-39).

[0063] antigen binding part As used herein, the term "antigen-binding moiety" refers to a polypeptide molecule that specifically binds to an antigen. In one embodiment, an antigen-binding moiety can direct the entity to which it is bound (e.g., a second antigen-binding moiety) to a target site, such as a specific type of tumor cell expressing a cancer antigen (CLDN6). In another embodiment, an antigen-binding moiety can activate signaling through its target antigen, such as a T-cell receptor complex antigen (CD3) or the costimulatory molecule CD137. Antigen-binding moieties include antibodies and fragments thereof as further defined herein. Specific antigen-binding moieties include antibody antigen-binding domains or antibody variable regions, including antibody heavy chain variable regions and antibody light chain variable regions. In certain embodiments, an antigen-binding moiety may comprise an antibody constant region as further defined herein and known in the art. Useful heavy chain constant regions include any of the five isotypes: α, δ, ε, γ, or μ. Useful light chain constant regions include any of the two isotypes: κ and λ.

[0064] As used herein, the terms "first," "second," "third," and "fourth" with respect to antigen-binding moieties, etc., are used for the convenience of distinguishing between two or more different types of moieties, etc. The use of these terms is not intended to confer a particular order or orientation of the multispecific antigen-binding molecules, unless otherwise specified.

[0065] an antigen-binding moiety capable of binding to CD3 and CD137 The multispecific antigen-binding molecules described herein comprise at least one antigen-binding portion capable of binding to CD3 and CD137 (also referred to herein as a "dual antigen-binding portion" or "first antigen-binding portion" or "Dual-Ig" or "Dual-Fab"). The first antigen-binding portion described herein is capable of binding to CD3 and CD137 and binds to either CD3 or CD137. That is, the first antigen-binding portion described herein binds to CD3. Alternatively, the first antigen-binding portion described herein binds to CD137. In certain embodiments, the multispecific antigen-binding molecule is capable of specifically binding to CD3 and CD137, but comprises no more than two antigen-binding portions that bind to either CD3 or CD137. In one embodiment, the multispecific antigen-binding molecule is capable of binding to CD3 and CD137 and provides monovalent binding to either CD3 or CD137. In one embodiment, the first antigen-binding moiety has binding activity for CD3 and binding activity for CD137, but when binding to an antigen, it binds to either CD3 or CD137. In one embodiment, the first antigen-binding moiety is an antigen-binding moiety that can bind to both CD3 and CD137, but does not bind to CD3 and CD137 simultaneously.

[0066] an antigen-binding moiety capable of binding to the T-cell receptor complex The multispecific antigen-binding molecules described herein comprise at least one antigen-binding portion (also referred to herein as a "first antigen-binding portion") capable of binding to a T cell receptor complex. The first antigen-binding portion described herein is capable of binding to a T cell receptor complex. An antigen-binding portion capable of binding to a T cell receptor complex refers to a portion of an anti-T cell receptor complex antibody comprising a region that specifically binds to and is complementary to a part or all of the T cell receptor complex. The T cell receptor complex may be the T cell receptor itself, or an adapter molecule that forms the T cell receptor complex together with the T cell receptor. A preferred adapter is CD3.

[0067] In certain embodiments, the dual antigen-binding moiety ("first antigen-binding moiety") is generally a Fab molecule, particularly a conventional Fab molecule. In certain embodiments, the dual antigen-binding moiety ("first antigen-binding moiety") is a domain comprising antibody light and heavy chain variable regions (VL and VH). Suitable examples of such domains comprising antibody light and heavy chain variable regions include "single-chain Fv (scFv)", "single-chain antibody", "Fv", "single-chain Fv2 (scFv2)", "Fab", "F(ab')2", etc.

[0068] In certain embodiments, the dual antigen-binding moiety ("first antigen-binding moiety") specifically binds to all or part of a partial peptide of CD3. In certain embodiments, the CD3 is human CD3 or cynomolgus CD3, particularly human CD3. In certain embodiments, the first antigen-binding moiety is cross-reactive with (i.e., specifically binds to) human CD3 and cynomolgus CD3. In some embodiments, the first antigen-binding moiety can specifically bind to the epsilon subunit of CD3, particularly the human CD3ε subunit of CD3 set forth in SEQ ID NO: 170 (NP_000724.1) (RefSeq accession number shown in parentheses). In some embodiments, the first antigen-binding moiety can specifically bind to the CD3ε chain expressed on the surface of eukaryotic cells. In some embodiments, the first antigen-binding moiety binds to the CD3ε chain expressed on the surface of T cells.

[0069] In certain embodiments, the CD137 is human CD137. In some embodiments, suitable examples of antigen-binding molecules of the present disclosure include antigen-binding molecules that bind to the same epitope as the human CD137 epitope bound by an antibody selected from the group consisting of: an antibody recognizing a region including the sequence SPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGC (SEQ ID NO: 182); An antibody recognizing a region containing the sequence DCTPGFHCLGAGCSMCEQDCKQGQELTKKGC (SEQ ID NO: 181); An antibody that recognizes a region containing the sequence LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAEC (SEQ ID NO: 183), and An antibody that recognizes a region containing the sequence LQDPCSNCPAGTFCDNNRNQIC (sequence number: 180) in the human CD137 protein.

[0070] In certain embodiments, the dual antigen-binding moiety ("first antigen-binding moiety") comprises the following antibody variable regions (a1)-(a4): (a1) a heavy chain variable region (first antibody variable region) comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 9, CDR 2 of SEQ ID NO: 15, and CDR 3 of SEQ ID NO: 21, and a light chain variable region (second antibody variable region) comprising CDR 1 of SEQ ID NO: 31, CDR 2 of SEQ ID NO: 35, and CDR 3 of SEQ ID NO: 39; (a2) a heavy chain variable region (first antibody variable region) comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 10, CDR 2 of SEQ ID NO: 16, and CDR 3 of SEQ ID NO: 22, and a light chain variable region (second antibody variable region) comprising CDR 1 of SEQ ID NO: 31, CDR 2 of SEQ ID NO: 35, and CDR 3 of SEQ ID NO: 39; (a3) a heavy chain variable region (first antibody variable region) comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 11, CDR 2 of SEQ ID NO: 17, and CDR 3 of SEQ ID NO: 23, and a light chain variable region (second antibody variable region) comprising CDR 1 of SEQ ID NO: 32, CDR 2 of SEQ ID NO: 36, and CDR 3 of SEQ ID NO: 40; (a4) A heavy chain variable region (first antibody variable region) comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 12, CDR 2 of SEQ ID NO: 18, and CDR 3 of SEQ ID NO: 24, and a light chain variable region (second antibody variable region) comprising CDR 1 of SEQ ID NO: 32, CDR 2 of SEQ ID NO: 36, and CDR 3 of SEQ ID NO: 40. Contains one of the following:

[0071] In certain embodiments, the dual antigen-binding portion ("first antigen-binding portion") comprises an antibody variable region comprising a human antibody framework or a humanized antibody framework.

[0072] In certain embodiments, the dual antigen-binding moiety ("first antigen-binding moiety") comprises one of the following (c1) to (c4): (c1) a heavy chain variable region (first antibody variable region) comprising the amino acid sequence of SEQ ID NO: 3, and a light chain variable region (second antibody variable region) comprising the amino acid sequence of SEQ ID NO: 27; (c2) a heavy chain variable region (first antibody variable region) comprising the amino acid sequence of SEQ ID NO: 4, and a light chain variable region (second antibody variable region) comprising the amino acid sequence of SEQ ID NO: 27; (c3) a heavy chain variable region (first antibody variable region) comprising the amino acid sequence of SEQ ID NO: 5, and a light chain variable region (second antibody variable region) comprising the amino acid sequence of SEQ ID NO: 28; (c4) A heavy chain variable region (first antibody variable region) comprising the amino acid sequence of SEQ ID NO: 6, and a light chain variable region (second antibody variable region) comprising the amino acid sequence of SEQ ID NO: 28. Contains one of the following:

[0073] In one embodiment, the dual antigen-binding portion ("first antigen-binding portion") comprises a heavy chain variable region (first antibody variable region) sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:3, and a light chain variable region (second antibody variable region) sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:27.

[0074] In one embodiment, the dual antigen-binding portion ("first antigen-binding portion") comprises a heavy chain variable region (first antibody variable region) sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:4, and a light chain variable region (second antibody variable region) sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:27.

[0075] In one embodiment, the dual antigen-binding portion ("first antigen-binding portion") comprises a heavy chain variable region (first antibody variable region) sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:5, and a light chain variable region (second antibody variable region) sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:28.

[0076] In one embodiment, the dual antigen-binding portion ("first antigen-binding portion") comprises a heavy chain variable region (first antibody variable region) sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:6, and a light chain variable region (second antibody variable region) sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:28.

[0077] In certain embodiments, the dual antigen-binding moiety ("first antigen-binding moiety") is selected from the group consisting of (j01) to (j18) below: (j01) a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 54 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 68; (j02) a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 55 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 68; (j03) a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 56 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 69; (j04) a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 57 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 69; (j05) a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 60 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 70; (j06) a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 61 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 70; (j07) a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 62 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 68; (j08) a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 63 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 68; (j09) a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 64 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 69; (j10) a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 65 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 69; (j11) a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 66 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 70; (j12) a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 67 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 70; (j13) a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 56 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 71; (j14) a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 57 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 71; (j15) a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 64 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 71; (j16) a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 65 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 71; (j17) a heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 58 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 70; (j18) A heavy chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 59 and a light chain (chain 4) comprising the amino acid sequence of SEQ ID NO: 70 Contains one of the following:

[0078] In certain embodiments, the dual antigen-binding moiety ("first antigen-binding moiety") comprises an antibody variable region. In certain embodiments, the dual antigen-binding moiety ("first antigen-binding moiety") comprises a first antibody variable region and a second antibody variable region as described above.

[0079] The multispecific antigen-binding molecules of the present disclosure also include multispecific antibodies that have undergone post-translational modifications. Examples of the multispecific antigen-binding molecules of the present disclosure that have undergone post-translational modifications include multispecific antigen-binding molecules that have undergone 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 such post-translational modifications, such as pyroglutamylation at the N-terminus and deletion of lysine at the C-terminus, have no effect on the activity of antibodies (Analytical Biochemistry, 2006, Vol. 348, pp. 24-39).

[0080] Antigen-binding moieties capable of binding to CLDN6 The multispecific antigen-binding molecules described herein comprise at least one antigen-binding moiety capable of binding to CLDN6 (also referred to herein as a "CLDN6 antigen-binding moiety" or a "second antigen-binding moiety"). In certain embodiments, the multispecific antigen-binding molecules comprise one antigen-binding moiety capable of binding to CLDN6.

[0081] In certain embodiments, the CLDN6 antigen-binding portion ("second antigen-binding portion") is generally a Fab molecule, particularly a conventional Fab molecule. In certain embodiments, the CLDN6 antigen-binding portion ("second antigen-binding portion") is a domain comprising antibody light and heavy chain variable regions (VL and VH). Suitable examples of such domains comprising antibody light and heavy chain variable regions include "single-chain Fv (scFv)", "single-chain antibody", "Fv", "single-chain Fv2 (scFv2)", "Fab", "F(ab')2", etc.

[0082] In certain embodiments, the CLDN6 antigen-binding portion ("second antigen-binding portion") specifically binds to all or part of a partial peptide of CLDN6. In certain embodiments, the CLDN6 is human CLDN6, cynomolgus monkey CLDN6, or mouse CLDN6, particularly human CLDN6. In certain embodiments, the CLDN6 antigen-binding portion ("second antigen-binding portion") is cross-reactive with (i.e., specifically binds to) human CLDN6 and cynomolgus monkey CLDN6.

[0083] In certain embodiments, the CLDN6 antigen-binding portion ("second antigen-binding portion") specifically binds to the first extracellular domain of CLDN6 (amino acids 29-81 of SEQ ID NO: 196 or 197) or the second extracellular domain of CLDN6 (amino acids 138-159 of SEQ ID NO: 196 or 197). In certain embodiments, the CLDN6 antigen-binding portion ("second antigen-binding portion") specifically binds to human CLDN6 expressed on the surface of eukaryotic cells. In certain embodiments, the binding activity to CLDN6 is binding activity to CLDN6 protein expressed on the surface of cancer cells.

[0084] In certain embodiments, the CLDN6 antigen binding portion (the "second antigen binding portion") does not substantially bind to human CLDN9.

[0085] In certain embodiments, the CLDN6 antigen binding portion (the "second antigen binding portion") does not substantially bind to human CLDN4.

[0086] In certain embodiments, the CLDN6 antigen binding portion (the "second antigen binding portion") does not substantially bind to human CLDN3.

[0087] In certain embodiments, the CLDN6 antigen binding portion (the "second antigen binding portion") does not substantially bind to the CLDN6 variant set forth in SEQ ID NO:205.

[0088] In certain embodiments, the CLDN6 antigen-binding portion ("second antigen-binding portion") is a crossover Fab molecule, i.e., a Fab molecule in which either the variable or constant regions of the Fab heavy and Fab light chains have been exchanged.

[0089] In certain embodiments, the CLDN6 antigen-binding portion ("second antigen-binding portion") comprises the following antibody variable region (b1) or (b2): (b1) a heavy chain variable region (third antibody variable region) comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 8, CDR 2 of SEQ ID NO: 14, and CDR 3 of SEQ ID NO: 20, and a light chain variable region (fourth antibody variable region) comprising CDR 1 of SEQ ID NO: 30, CDR 2 of SEQ ID NO: 34, and CDR 3 of SEQ ID NO: 38; (b2) a heavy chain variable region (third antibody variable region) comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 7, CDR 2 of SEQ ID NO: 13, and CDR 3 of SEQ ID NO: 19, and a light chain variable region (fourth antibody variable region) comprising CDR 1 of SEQ ID NO: 29, CDR 2 of SEQ ID NO: 33, and CDR 3 of SEQ ID NO: 37. (b3) a heavy chain variable region (third antibody variable region) comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 29, CDR 2 of SEQ ID NO: 33, and CDR 3 of SEQ ID NO: 37, and a light chain variable region (fourth antibody variable region) comprising CDR 1 of SEQ ID NO: 7, CDR 2 of SEQ ID NO: 13, and CDR 3 of SEQ ID NO: 19. Includes.

[0090] In certain embodiments, the CLDN6 antigen-binding portion (the "second antigen-binding portion") comprises an antibody variable region comprising a human antibody framework or a humanized antibody framework.

[0091] In certain embodiments, the CLDN6 antigen-binding portion ("second antigen-binding portion") is (d1) or (d2) of the following: (d1) a heavy chain variable region (third antibody variable region) comprising the amino acid sequence of SEQ ID NO: 2, and a light chain variable region (fourth antibody variable region) comprising the amino acid sequence of SEQ ID NO: 26; (d2) a heavy chain variable region (third antibody variable region) comprising the amino acid sequence of SEQ ID NO: 1, and a light chain variable region (fourth antibody variable region) comprising the amino acid sequence of SEQ ID NO: 25 (d3) A heavy chain variable region (third antibody variable region) comprising the amino acid sequence of SEQ ID NO: 25, and a light chain variable region (fourth antibody variable region) comprising the amino acid sequence of SEQ ID NO: 1. Includes.

[0092] In one embodiment, the CLDN6 antigen-binding portion ("second antigen-binding portion") comprises a heavy chain variable region (third antibody variable region) sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:2, and a light chain variable region (fourth antibody variable region) sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:26.

[0093] In one embodiment, the CLDN6 antigen-binding portion ("second antigen-binding portion") comprises a heavy chain variable region (third antibody variable region) sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:1, and a light chain variable region (fourth antibody variable region) sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:25. In one embodiment, the CLDN6 antigen-binding portion ("second antigen-binding portion") comprises a heavy chain variable region (third antibody variable region) sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:25, and a light chain variable region (fourth antibody variable region) sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:1.

[0094] In certain embodiments, the CLDN6 antigen-binding portion ("second antigen-binding portion") is selected from the group consisting of (k01) to (k09) below: (k01) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 41 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 50; (k02) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 42 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 51; (k03) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 44 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 52; (k04) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 45 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 50; (k05) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 46 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 51; (k06) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 47 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 52; (k07) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 48 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 53; (k08) a heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 49 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 53; (k09) A heavy chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 43 and a light chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 52 Contains one of the following:

[0095] In certain embodiments, the CLDN6 antigen-binding portion ("second antigen-binding portion") comprises an antibody variable region. In certain embodiments, the CLDN6 antigen-binding portion ("second antigen-binding portion") comprises the third and fourth antibody variable regions described above.

[0096] In the above-mentioned multispecific antigen-binding molecules, the amino acid sequences of the heavy and light chain CDR1, CDR2, CDR3, heavy chain variable region, light chain variable region, full-length heavy chain, and full-length light chain may have one or more amino acid substitutions, deletions, additions, and / or insertions, as long as they have binding activity to CD3, CD137, the T cell receptor complex, or CLDN6. Methods for introducing mutations into proteins are well known to those skilled in the art for preparing amino acid sequences in which one or more amino acids have been substituted, deleted, added, and / or inserted.For example, those skilled in the art will be familiar with site-directed mutagenesis (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, and Nakagawa, M. (1995) An oligodeoxyribonucleotide-directed dual amber method for site-directed mutagenesis. Gene 152, 271-275; Zoller, MJ, and Smith, M. (1983) Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 100, 468-500; Kramer, W, Drutsa, V, Jansen, HW, Kramer, B, Pflugfelder, M, and Fritz, HJ (1984) The gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 12, 9441-9456; Kramer, W, and Fritz, HJ (1987) By appropriately introducing mutations into the amino acid sequence of an antibody that has binding activity to CD3, CD137, the T cell receptor complex, or CLDN6 using techniques such as oligonucleotide-directed construction of mutations via gapped duplex DNA methods. Enzymol. 154, 350-367; Kunkel, TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 82, 488-492), mutant multispecific antigen-binding molecules can be prepared that contain combinations of antibody variable regions that are functionally equivalent to the combination of antibody variable regions in the original multispecific antigen-binding molecule.Here, in the present invention, "functionally equivalent" means that the binding affinity to the antigen is equivalent, or that, when used as a multispecific antigen-binding molecule, the cytotoxic activity against cells expressing claudin 6 or tissues containing such cells is equivalent. The binding affinity and cytotoxic activity can be measured based on the descriptions herein, as described in detail below.

[0097] There is no limit to the number of amino acids to be modified, but it may be, for example, 40 or less, 30 or less, 20 or less, preferably 18 or less, 16 or less, 15 or less, 12 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.

[0098] When modifying an amino acid residue, it is desirable to mutate it to another amino acid that preserves the properties of the amino acid side chain. For example, the properties of the amino acid side chain include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), amino acids with aliphatic side chains (G, A, V, L, I, P), amino acids with hydroxyl-containing side chains (S, T, Y), amino acids with sulfur-containing side chains (C, M), amino acids with carboxylic acid- and amide-containing side chains (D, N, E, Q), amino acids with base-containing side chains (R, K, H), and amino acids with aromatic-containing side chains (H, F, Y, W) (the characters in parentheses represent the single-letter symbols of the amino acids). Substitution of amino acids within each of these groups is referred to as a conservative substitution. It is already known that polypeptides having an amino acid sequence modified by deletion, addition, and / or substitution of one or more amino acid residues with other amino acids can retain their biological activity (Mark, D.F. et al., Proc. Natl. Acad. Sci. USA (1984) 81:5662-6; Zoller, M.J. and Smith, M., Nucleic Acids Res. (1982) 10:6487-500; Wang, A. et al., Science (1984) 224:1431-3; Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. USA (1982) 79:6409-13).

[0099] antigen As used herein, the term "antigen" refers to a site on a polypeptide macromolecule to which an antigen-binding moiety binds (e.g., a three-dimensional structure composed of a continuous stretch of amino acids or a discrete region of non-contiguous amino acids), forming an antigen-binding moiety-antigen complex. Useful antigenic determinants can be found, for example, on the surface of tumor cells, virus-infected cells, other diseased cells, immune cells, free in serum, and / or in the extracellular matrix (ECM). Unless otherwise indicated, proteins (e.g., CD3, CD137, CLDN6) referred to herein as antigens can be any naturally occurring form of the protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). In certain embodiments, the antigen is human CD3, human CD137, or human CLDN6. When a specific protein is referred to herein, the term encompasses the "full-length," unprocessed protein as well as any form of the protein produced by processing in the cell. The term also encompasses naturally occurring variants of the protein, such as splice variants or allelic variants.

[0100] In certain embodiments, the multispecific antigen-binding molecules described herein bind to epitopes of CD3, CD137, or CLDN6 that are conserved among CD3, CD137, or CLDN6 of different species. In certain embodiments, the multispecific antigen-binding molecules of the present application are trispecific antigen-binding molecules, i.e., trispecific antigen-binding molecules that can specifically bind to three different antigens, i.e., that can bind to CD3 and CD137 but not both antigens simultaneously, and that can specifically bind to CLDN6.

[0101] Claudin 6 (CLDN6) and other claudin family proteins As used herein, the term "CLDN6," unless otherwise specified, refers to any native claudin 6 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The amino acid sequence of human CLDN6 (hCLDN6) is set forth in SEQ ID NO: 196 or 197, and the amino acid sequence of mouse CLDN6 (mCLDN6) is set forth in SEQ ID NO: 201.

[0102] In addition to CLDN6, there are many other proteins in the claudin family, such as CLDN3, CLDN4, and CLDN9. The amino acid sequences of human CLDN3 (hCLDN3), human CLDN4 (hCLDN4), and human CLDN9 (hCLDN9) are shown in SEQ ID NOs: 199, 200, and 198, respectively. The amino acid sequences of mouse CLDN3 (mCLDN3), mouse CLDN4 (mCLDN4), and mouse CLDN9 (mCLDN9) are shown in SEQ ID NOs: 203, 204, and 202, respectively.

[0103] CD3 In certain embodiments, the multispecific antigen-binding molecule specifically binds to all or part of a partial peptide of CD3. In certain embodiments, the CD3 is human CD3 or cynomolgus monkey CD3, particularly human CD3. In certain embodiments, the multispecific antigen-binding molecule is cross-reactive with (i.e., specifically binds to) human CD3 and cynomolgus monkey CD3. In some embodiments, the multispecific antigen-binding molecule can specifically bind to the ε subunit of CD3, particularly the human CD3ε subunit of CD3 set forth in SEQ ID NO: 170 (NP_000724.1) (RefSeq accession number shown in parentheses). In some embodiments, the multispecific antigen-binding molecule can specifically bind to the CD3ε chain expressed on the surface of eukaryotic cells. In some embodiments, the multispecific antigen-binding molecule binds to the CD3ε chain expressed on the surface of T cells.

[0104] CD137 In certain embodiments, the CD137 is human CD137. In some embodiments, suitable examples of antigen-binding molecules of the present disclosure include antigen-binding molecules that bind to the same epitope as the human CD137 epitope bound by an antibody selected from the group consisting of: an antibody recognizing a region including the sequence SPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGC (SEQ ID NO: 182); An antibody recognizing a region containing the sequence DCTPGFHCLGAGCSMCEQDCKQGQELTKKGC (SEQ ID NO: 181); An antibody that recognizes a region containing the sequence LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAEC (SEQ ID NO: 183), and An antibody that recognizes a region containing the sequence LQDPCSNCPAGTFCDNNRNQIC (sequence number: 180) in the human CD137 protein.

[0105] antigen-binding domain The term "antigen-binding domain" refers to a portion of an antibody comprising the area that specifically binds to and is complementary to part or all of an antigen. An antigen-binding domain may be provided, for example, by one or more antibody variable domains (also called antibody variable regions). Preferably, the antigen-binding domain comprises both an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). Such preferred antigen-binding domains include, for example, "single-chain Fv (scFv)," "single-chain antibody," "Fv," "single-chain Fv2 (scFv2)," "Fab," and "F(ab')2." An antigen-binding domain may also be provided by a single-domain antibody.

[0106] Single Domain Antibodies As used herein, the term "single-domain antibody" is not limited by its structure, as long as the domain can exhibit antigen-binding activity by itself. While typical antibodies, such as IgG antibodies, exhibit antigen-binding activity when their variable regions are formed by pairing VH and VL, it is known that the domain structure of a single-domain antibody can exhibit antigen-binding activity by itself, without pairing with another domain. Single-domain antibodies typically have a relatively low molecular weight and exist in the form of a monomer.

[0107] Examples of single domain antibodies include, but are not limited to, antigen-binding molecules such as camelid VHH and shark VNAR, which naturally lack light chains, and antibody fragments containing the entire or a portion of an antibody VH domain or the entire or a portion of an antibody VL domain. Examples of single domain antibodies, which are antibody fragments containing the entire or a portion of an antibody VH domain or an antibody VL domain, include, but are not limited to, artificially prepared single domain antibodies derived from human antibody VH or human antibody VL, such as those described in U.S. Patent No. 6,248,516 B1. In some embodiments of the present invention, a single domain antibody has three CDRs (CDR1, CDR2, and CDR3).

[0108] Single-domain antibodies can be obtained from animals capable of producing single-domain antibodies or by immunization of animals capable of producing single-domain antibodies. Examples of animals capable of producing single-domain antibodies include, but are not limited to, camelids and transgenic animals carrying genes capable of producing single-domain antibodies. Camelids include camels, llamas, alpacas, dromedaries, and guanacos. Examples of transgenic animals carrying genes capable of producing single-domain antibodies include, but are not limited to, the transgenic animals described in International Publication No. WO2015 / 143414 and U.S. Patent Publication No. US2011 / 0123527 A1. The framework sequences of single-domain antibodies obtained from such animals may be converted to human germline sequences or sequences similar thereto to obtain humanized single-domain antibodies. Humanized single-domain antibodies (e.g., humanized VHHs) are also an embodiment of the single-domain antibodies of the present invention.

[0109] Alternatively, single domain antibodies can be obtained by ELISA, panning, or the like from a polypeptide library containing single domain antibodies. Examples of polypeptide libraries containing single domain antibodies include, but are not limited to, naive antibody libraries obtained from various animals or humans (e.g., Methods in Molecular Biology 2012 911 (65-78); and Biochimica et Biophysica Acta - Proteins and Proteomics 2006 1764: 8 (1307-1319)), antibody libraries obtained by immunization of various animals (e.g., Journal of Applied Microbiology 2014 117: 2 (528-536)), and synthetic antibody libraries prepared from antibody genes of various animals or humans (e.g., Journal of Biomolecular Screening 2016 21: 1 (35-43); Journal of Biological Chemistry 2016 291:24 (12641-12657); and AIDS 2016 30: 11 (1691-1701)).

[0110] Variable region The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies typically have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen may be isolated by screening a complementary library of VL or VH domains, respectively, using a VH or VL domain from an antibody that binds to that antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0111] HVR or CDR As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence ("complementarity determining region" or "CDR") and / or forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contacts"). Hypervariable regions (HVRs) are also referred to as "complementarity determining regions" (CDRs), and these terms are used interchangeably herein with respect to the portions of the variable domain that form the antigen binding region. Typically, antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs herein include the following: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigenic contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and (d) A combination of (a), (b), and / or (c), comprising HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

[0112] Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0113] HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 are also referred to as "H-CDR1," "H-CDR2," "H-CDR3," "L-CDR1," "L-CDR2," and "L-CDR3," respectively.

[0114] Able to bind to CD3 and CD137 Whether an antibody variable region of the present disclosure is "capable of binding to CD3 and CD137" can be determined by methods known in the art.

[0115] This can be determined, for example, by electrochemiluminescence (ECL) (BMC Research Notes 2011, 4:281).

[0116] Specifically, for example, a region of a biotin-labeled test antigen-binding molecule capable of binding to CD3 and CD137, such as a small antibody composed of the Fab region, or a monovalent antibody thereof (an antibody lacking one of the two Fab regions of a normal antibody), is mixed with CD3 or CD137 labeled with a sulfo-tag (Ru complex), and the mixture is added to a streptavidin-immobilized plate. During this procedure, the biotin-labeled test antigen-binding molecule binds to the streptavidin on the plate. Light is generated from the sulfo-tag, and the luminescence signal is detected using a Sector Imager 600 or 2400 (MSD KK), etc., thereby confirming the binding of the above-mentioned region of the test antigen-binding molecule to CD3 or CD137.

[0117] Alternatively, the assay may be performed by ELISA, FACS (fluorescence activated cell sorting), ALPHAScreen (amplified luminescence proximity homogeneous assay screen), BIACORE method based on the surface plasmon resonance (SPR) phenomenon, etc. (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

[0118] Specifically, the assay can be performed using, for example, Biacore (GE Healthcare Japan Corp.), an interaction analysis instrument based on the surface plasmon resonance (SPR) phenomenon. Biacore analysis instruments include any model, such as the Biacore T100, T200, X100, A100, 4000, 3000, 2000, 1000, or C. Any Biacore sensor chip, such as a CM7, CM5, CM4, CM3, C1, SA, NTA, L1, HPA, or Au chip, can be used as the sensor chip. Proteins for capturing the antigen-binding molecules of the present disclosure, such as protein A, protein G, protein L, anti-human IgG antibody, anti-human IgG-Fab, anti-human L chain antibody, anti-human Fc antibody, antigen protein, or antigen peptide, are immobilized on the sensor chip by a coupling method such as amine coupling, disulfide coupling, or aldehyde coupling. CD3 or CD137 is injected onto the sensor chip as an analyte, and the interaction is measured to obtain a sensorgram. In this procedure, the concentration of CD3 or CD137 can be selected within the range of several μM to several pM according to the strength of the interaction (eg, KD) of the assay sample.

[0119] Alternatively, CD3 or CD137 may be immobilized on a sensor chip instead of an antigen-binding molecule, and then the antibody sample to be evaluated may be allowed to interact. Whether the antibody variable region of the antigen-binding molecule of the present disclosure has binding activity to CD3 or CD137 can be confirmed based on the dissociation constant (KD) value calculated from the sensorgram of the interaction, or based on the degree of increase in the sensorgram after the action of the antigen-binding molecule sample above the level before the action. In some embodiments, the binding activity or affinity of the antibody variable region of the present disclosure to the target antigen (i.e., CD3 or CD137) is evaluated, for example, using a Biacore T200 instrument (GE Healthcare) or a Biacore 8K instrument (GE Healthcare) at 37 ° C (for CD137) or 25 ° C (for CD3). Anti-human Fc (e.g., GE Healthcare) is immobilized on all flow cells of a CM4 sensor chip using an amine coupling kit (e.g., GE Healthcare). Antigen-binding molecules or antibody variable regions are captured on an anti-Fc sensor surface, and then antigen (CD3 or CD137) is injected onto the flow cell. The capture level of the antigen-binding molecules or antibody variable regions may be aimed for at 200 resonance units (RU). Recombinant human CD3 or CD137 may be injected at 400 to 25 nM, prepared by two-fold serial dilution, followed by dissociation. All antigen-binding molecules or antibody variable regions and analytes are prepared in ACES pH 7.4 containing 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, and 0.005% NaN3. The sensor surface is regenerated with 3 M MgCl2 after each cycle. Binding affinity is determined by processing the data and fitting to a 1:1 binding model using, for example, Biacore T200 Evaluation software, version 2.0 (GE Healthcare) or Biacore 8K Evaluation software (GE Healthcare). To assess the specific binding activity or affinity of an antigen-binding domain of the present disclosure, a KD value is calculated.

[0120] ALPHAScreen is implemented using ALPHA technology, which uses two types of beads (donor and acceptor), based on the following principle: luminescence signals are detected only when a biological interaction between a molecule bound to a donor bead and a molecule bound to an acceptor bead brings these two beads into close proximity. A photosensitizer in the donor bead, excited by a laser, converts ambient oxygen into excited singlet oxygen. The singlet oxygen diffuses around the donor bead and reaches the nearby acceptor bead, thereby triggering a chemiluminescent reaction in the bead, ultimately resulting in the emission of light. If there is no interaction between the molecules bound to the donor bead and the molecules bound to the acceptor bead, the singlet oxygen produced by the donor bead will not reach the acceptor bead. Therefore, the chemiluminescent reaction will not occur.

[0121] One of the substances (ligands) whose interaction is to be observed is immobilized on a thin gold film on a sensor chip. Light is shone on the back of the sensor chip to induce total internal reflection at the interface between the gold film and the glass. As a result, a region of reduced reflection intensity (SPR signal) is formed in a portion of the reflected light. The other substance (analyte) whose interaction is to be observed is injected onto the surface of the sensor chip. When the analyte binds to the ligand, the mass of the immobilized ligand molecule increases, causing a change in the refractive index of the solvent on the sensor chip surface. This change in refractive index causes a shift in the position of the SPR signal (conversely, when the bound molecule dissociates, the signal returns to its original position). The Biacore system plots the amount of shift, i.e., the change in mass on the sensor chip surface, on the ordinate, and displays the time-dependent change in mass as assay data (sensorgram). The amount of analyte bound to the ligand captured on the sensor chip surface (the amount of change in response on the sensorgram before and after analyte interaction) can be determined from the sensorgram. However, because the amount of binding also depends on the amount of ligand, comparisons must be performed under conditions using substantially the same amount of ligand. Kinetics, i.e., association rate constant (ka) and dissociation rate constant (kd) can be determined from the curve of the sensorgram, while affinity (KD) can be determined from the ratio of these constants. Inhibition assays are also suitable for use in the BIACORE method. An example of inhibition assay is described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010.

[0122] Does not bind to CD3 and CD137 (4-1BB) simultaneously As described above, binding to either CD3 or CD137 includes not simultaneously binding to CD3 and CD137(4-1BB). The terms "not simultaneously binding to CD3 and CD137(4-1BB)" or "not simultaneously binding to CD3 and CD137(4-1BB)" mean that an antigen-binding portion or antibody variable region of the present disclosure cannot bind to CD137 when bound to CD3, and conversely, an antigen-binding portion or antibody variable region cannot bind to CD3 when bound to CD137. Here, the phrase "not simultaneously binding to CD3 and CD137" also includes not cross-linking cells expressing CD3 and cells expressing CD137, or not simultaneously binding to CD3 and CD137, each expressed on different cells. Such antibody variable regions are not particularly limited, as long as they have these functions. Examples of such antibodies include a variable region derived from an IgG antibody variable region, in which some of the amino acids have been modified to bind to a desired antigen. The modified amino acids are selected from, for example, amino acids in the variable region of an antibody that binds to CD3 or CD137, such that the modification does not abolish antigen binding.

[0123] Here, the phrase "expressed on different cells" simply means that the antigens are expressed on separate cells, and such cell pairs may be of the same type, such as a T cell and another T cell, or may be of different types, such as a T cell and an NK cell.

[0124] Whether a multispecific antigen-binding molecule contained in an anticancer agent, pharmaceutical composition, combination, or kit of the present disclosure, or used in a method or use of the present disclosure, "binds to either CD3 or CD137" upon antigen binding can be confirmed by confirming that the antigen-binding molecule has binding activity to both CD3 and CD137; then, pre-binding either CD3 or CD137 to an antigen-binding molecule containing a variable region having this binding activity; and then determining the presence or absence of its binding activity to the other antigen by the above-mentioned method. Alternatively, this can also be confirmed by determining whether the binding of the antigen-binding molecule to either CD3 or CD137 immobilized on an ELISA plate or sensor chip is inhibited by the addition of the other antigen to the solution. In some embodiments, the binding of the antigen-binding molecule of the present disclosure to either CD3 or CD137 is inhibited by the binding of the antigen-binding molecule to the other antigen by at least 50%, preferably 60% or more, more preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, or even more preferably 95% or more.

[0125] In one aspect, while one antigen (e.g., CD3) is immobilized, inhibition of binding of the antigen-binding molecule to CD3 can be determined in the presence of another antigen (e.g., CD137) by a method known in the prior art (i.e., ELISA, BIACORE, etc.). In another aspect, while CD137 is immobilized, inhibition of binding of the antigen-binding molecule to CD137 can also be determined in the presence of CD3. When either one of the above two aspects is performed, if binding is inhibited by at least 50%, preferably 60% or more, preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, or even more preferably 95% or more, it is determined that the antigen-binding molecule of the present disclosure does not simultaneously bind to CD3 and CD137.

[0126] In some embodiments, the concentration of the antigen injected as the analyte is at least 1-fold, 2-fold, 5-fold, 10-fold, 30-fold, 50-fold, or 100-fold higher than the concentration of the other antigen that is immobilized.

[0127] In a preferred mode, the concentration of the antigen injected as analyte is 100 times higher than the concentration of the other antigen to be immobilized, and binding is inhibited by at least 80%.

[0128] In one embodiment, the ratio of the KD value for the CD3 (analyte)-binding activity of the antigen-binding molecule to the KD value for the CD137 (immobilized)-binding activity of the antigen-binding molecule (KD(CD3) / KD(CD137)) is calculated, and a CD3 (analyte) concentration that is 10-fold, 50-fold, 100-fold, or 200-fold higher than the CD137 (immobilized) concentration by the KD value ratio (KD(CD3) / KD(CD137)) can be used for the above-mentioned competitive measurement. (For example, if the KD value ratio is 0.1, a concentration 1-fold, 5-fold, 10-fold, or 20-fold higher can be selected. Furthermore, if the KD value ratio is 10, a concentration 100-fold, 500-fold, 1000-fold, or 2000-fold higher can be selected.)

[0129] In one aspect, while one antigen (e.g., CD3) is immobilized, the attenuation of the binding signal of the antigen-binding molecule to CD3 can be determined in the presence of another antigen (e.g., CD137) by a method known in the prior art (i.e., ELISA, ECL, etc.). In another aspect, while CD137 is immobilized, the attenuation of the binding signal of the antigen-binding molecule to CD137 can also be determined in the presence of CD3. When either one of the above two aspects is performed, if the binding signal is attenuated by at least 50%, preferably 60% or more, preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, or even more preferably 95% or more, it is determined that the antigen-binding molecule of the present disclosure does not simultaneously bind to CD3 and CD137.

[0130] In some embodiments, the concentration of the antigen injected as the analyte is at least 1-fold, 2-fold, 5-fold, 10-fold, 30-fold, 50-fold, or 100-fold higher than the concentration of the other antigen that is immobilized.

[0131] In a preferred mode, the concentration of the antigen injected as analyte is 100 times higher than the concentration of the other antigen to be immobilized, and binding is inhibited by at least 80%.

[0132] In one embodiment, the ratio of the KD value for the CD3 (analyte)-binding activity of the antigen-binding molecule to the KD value for the CD137 (immobilized)-binding activity of the antigen-binding molecule (KD(CD3) / KD(CD137)) is calculated, and a CD3 (analyte) concentration that is 10-fold, 50-fold, 100-fold, or 200-fold higher than the CD137 (immobilized) concentration by this KD value ratio (KD(CD3) / KD(CD137)) can be used for the above measurement. (For example, if the KD value ratio is 0.1, a concentration 1-fold, 5-fold, 10-fold, or 20-fold higher can be selected. Furthermore, if the KD value ratio is 10, a concentration 100-fold, 500-fold, 1000-fold, or 2000-fold higher can be selected.)

[0133] Specifically, for example, when using the ECL method, a biotin-labeled test antigen-binding molecule, CD3 labeled with a sulfo-tag (Ru complex), and unlabeled CD137 are prepared. If the test antigen-binding molecule can bind to CD3 and CD137, and binds to either CD3 or CD137, the mixture of the test antigen-binding molecule and labeled CD3 is added to a streptavidin-immobilized plate, and then the luminescence signal of the sulfo-tag is detected in the absence of unlabeled CD137 by light emission. In contrast, the luminescence signal decreases in the presence of unlabeled CD137. The decrease in the luminescence signal can be quantified to determine the relative binding activity. This analysis can be performed similarly using labeled CD137 and unlabeled CD3.

[0134] In ALPHAScreen, a test antigen-binding molecule interacts with CD3 in the absence of competing CD137, generating a signal at 520-620 nm. Untagged CD137 competes with CD3 for interaction with the test antigen-binding molecule. The resulting decrease in fluorescence is quantified, thereby determining relative binding activity. Biotinylation of polypeptides using sulfo-NHS-biotin or similar techniques is known in the art. For example, CD3 can be tagged with GST by any suitable method, including fusing a polynucleotide encoding CD3 in frame with a polynucleotide encoding GST; expressing the resulting fusion gene in cells carrying a vector capable of expressing it; and then purifying it using a glutathione column. The resulting signal is preferably analyzed using, for example, the software GRAPHPAD PRISM (GraphPad Software, Inc., San Diego), which is fitted with a one-site competition model based on nonlinear regression analysis. This analysis can be performed similarly using tagged CD137 and untagged CD3.

[0135] Alternatively, a method using fluorescence resonance energy transfer (FRET) may be used. FRET is a phenomenon in which excitation energy is directly transferred between two closely spaced fluorescent molecules due to electronic resonance. When FRET occurs, the excitation energy of the donor (a fluorescent molecule in an excited state) is transferred to the acceptor (another fluorescent molecule located near the donor), resulting in quenching of the fluorescence emitted from the donor (more precisely, a shortening of the fluorescence lifetime), and instead, fluorescence emission from the acceptor. This phenomenon can be used to analyze whether an antibody simultaneously binds to CD3 and CD137. For example, when CD3 containing a fluorescent donor and CD137 containing a fluorescent acceptor simultaneously bind to a test antigen-binding molecule, the fluorescence of the donor is quenched, while fluorescence is emitted from the acceptor. Therefore, a change in fluorescence wavelength is observed. Such an antibody is confirmed to simultaneously bind to CD3 and CD137. On the other hand, if mixing CD3, CD137, and the test antigen-binding molecule does not change the fluorescence wavelength of the fluorescent donor bound to CD3, the test antigen-binding molecule can bind to both CD3 and CD137 and can be considered to be an antigen-binding domain that binds to either CD3 or CD137.

[0136] For example, a biotin-labeled test antigen-binding molecule is bound to streptavidin on donor beads, while glutathione S-transferase (GST)-tagged CD3 is bound to acceptor beads. The test antigen-binding molecule interacts with CD3 in the absence of a competing second antigen, generating a signal at 520-620 nm. The untagged second antigen competes with CD3 for interaction with the test antigen-binding molecule. The resulting decrease in fluorescence is quantified, thereby determining relative binding activity. Biotinylation of polypeptides using sulfo-NHS-biotin or similar is known in the art. For example, CD3 can be tagged with GST by any suitable method, including fusing a polynucleotide encoding CD3 in frame with a polynucleotide encoding GST; expressing the resulting fusion gene in cells carrying a vector capable of expressing it; and then purifying it using a glutathione column. The resulting signals are preferably analyzed using, for example, the software GRAPHPAD PRISM (GraphPad Software, Inc., San Diego) fitted to a one-site competition model based on nonlinear regression analysis.

[0137] Tagging is not limited to GST tagging, and may be performed with any tag, including, but not limited to, histidine tag, MBP, CBP, Flag tag, HA tag, V5 tag, c-myc tag, etc. Binding of the test antigen-binding molecule to the donor beads is not limited to binding using biotin-streptavidin reaction. In particular, when the test antigen-binding molecule contains Fc, possible methods include binding the test antigen-binding molecule via an Fc-recognizing protein such as protein A or protein G on the donor beads.

[0138] The inability to simultaneously bind to CD3 and CD137 expressed on different cells can also be assayed by methods known in the art.

[0139] Specifically, a test antigen-binding molecule that has been confirmed to be positive in ECL-ELISA for detecting simultaneous binding to CD3 and CD137 is also mixed with cells expressing CD3 and cells expressing CD137. Unless the antigen-binding molecule and these cells simultaneously bind to each other, it can be shown that the test antigen-binding molecule cannot simultaneously bind to CD3 and CD137 expressed on different cells. This assay can be performed, for example, by cell-based ECL-ELISA. CD3-expressing cells are immobilized on a plate in advance. After the test antigen-binding molecule binds to it, CD137-expressing cells are added to the plate. A different antigen expressed only on CD137-expressing cells is detected using an antibody labeled with a sulfo-tag against this antigen. If the antigen-binding molecule simultaneously binds to two antigens expressed on two cells, respectively, a signal is observed. If the antigen-binding molecule does not simultaneously bind to these antigens, no signal is observed.

[0140] Alternatively, this assay can be carried out by the ALPHAScreen method. Test antigen-binding molecule is mixed with the cells expressing CD3 bound to donor beads and the cells expressing CD137 bound to acceptor beads. When the antigen-binding molecule simultaneously binds to the two antigens expressed on the two cells, respectively, a signal is observed. When the antigen-binding molecule does not simultaneously bind to these antigens, no signal is observed.

[0141] Alternatively, this assay can be performed using Octet interaction analysis. First, cells expressing peptide-tagged CD3 are bound to a biosensor that recognizes the peptide tag. CD137-expressing cells and a test antigen-binding molecule are placed in a well and analyzed for interaction. If the antigen-binding molecule simultaneously binds to two antigens expressed on two cells, respectively, a large wavelength shift is observed due to the binding of the test antigen-binding molecule and the CD137-expressing cells to the biosensor. If the antigen-binding molecule does not simultaneously bind to these antigens, a small wavelength shift is observed due to the binding of only the test antigen-binding molecule to the biosensor.

[0142] Instead of these binding activity-based methods, biological activity-based assays can be performed. For example, CD3-expressing cells and CD137-expressing cells are mixed and cultured with a test antigen-binding molecule. When the antigen-binding molecule simultaneously binds to the two antigens, the two antigens expressed on the two cells are mutually activated via the test antigen-binding molecule. Therefore, changes in activation signals, such as increases in the phosphorylation levels downstream of each antigen, can be detected. Alternatively, cytokine production is induced as a result of activation. Therefore, the amount of cytokine produced can be measured, thereby confirming simultaneous binding to the two cells. Alternatively, cytotoxic activity against CD137-expressing cells can be induced as a result of activation. Alternatively, reporter gene expression can be induced by a promoter activated downstream of the CD137 or CD3 signaling pathway as a result of activation. Therefore, the cytotoxic activity or the amount of reporter protein produced can be measured, thereby confirming simultaneous binding to the two cells.

[0143] The multispecific antigen-binding molecules contained in the anticancer agents, pharmaceutical compositions, combinations, and kits, or used in the methods or uses of the present disclosure, do not simultaneously bind to CD3 and CD137 (i.e., do not simultaneously bind to CD3 and CD137), thereby preventing simultaneous binding of the same antigen-binding molecule to CD3 and / or CD137 expressed on different immune cells (e.g., T cells). This avoids toxicity caused by undesired cross-linking between different immune cells, which is thought to be responsible for adverse reactions when conventional multispecific antigen-binding molecules that can simultaneously bind to CD3 expressed on T cells and a second molecule (e.g., CD137) are administered in vivo. The toxicity of multispecific antigen-binding molecules administered in vivo can be measured by cytokine production, etc. Low toxicity refers to the absence of immune activation induction, such as CLDN6-independent cytokine production, compared to a control multispecific antibody.

[0144] Fab molecules A "Fab molecule" refers to a protein consisting of the VH and CH1 domains of an immunoglobulin heavy chain (a "Fab heavy chain") and the VL and CL domains of a light chain (a "Fab light chain").

[0145] to be fused "Fused" means that the components (eg, a Fab molecule and an Fc domain subunit) are linked by peptide bonds, either directly or via one or more peptide linkers.

[0146] "Crossover" Fab A "crossover" Fab molecule (also referred to as "Crossfab") refers to a Fab molecule in which either the variable or constant regions of the Fab heavy and Fab light chains have been exchanged; i.e., the crossover Fab molecule comprises a peptide chain composed of a light chain variable region and a heavy chain constant region, and a peptide chain composed of a heavy chain variable region and a light chain constant region. For clarity, in a crossover Fab molecule in which the variable regions of the Fab light and Fab heavy chains have been exchanged, the peptide chain comprising the heavy chain constant region is referred to herein as the "heavy chain" of the crossover Fab molecule. Conversely, in a crossover Fab molecule in which the constant regions of the Fab light and Fab heavy chains have been exchanged, the peptide chain comprising the heavy chain variable region is referred to herein as the "heavy chain" of the crossover Fab molecule.

[0147] "Traditional" Fab In contrast, a "conventional" Fab molecule refers to a Fab molecule in its native form, i.e., a Fab molecule comprising a heavy chain (VH-CH1) composed of the variable and constant regions of the heavy chain, and a light chain (VL-CL) composed of the variable and constant regions of the light chain. The term "immunoglobulin molecule" refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of approximately 150,000 daltons composed of two light chains and two heavy chains linked by disulfide bonds. Each heavy chain has, from N- to C-terminus, a variable region (VH), also known as the variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also known as the heavy chain constant region. Similarly, each light chain has, from N- to C-terminus, a variable region (VL), also known as the variable light domain or light chain variable domain, followed by a constant light (CL) domain, also known as the light chain constant region. Immunoglobulin heavy chains may be assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which may be further classified into subtypes, e.g., γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). Immunoglobulin light chains may be assigned to one of two types, called κ and λ, based on the amino acid sequence of their constant domains. Immunoglobulins essentially consist of two Fab molecules and an Fc domain linked via an immunoglobulin hinge region.

[0148] affinity "Affinity" refers to the strength of the total non-covalent interactions between one binding site of a molecule (e.g., an antigen-binding molecule or antibody) and the molecule's binding partner (e.g., an antigen). Unless otherwise specified, "binding affinity" as used herein refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antigen-binding molecule and an antigen, or an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD), which is the ratio of the dissociation rate constant to the association rate constant (koff and kon, respectively). Thus, equivalent affinities may involve different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by established methods known in the art, including those described herein. A specific method for measuring affinity is surface plasmon resonance (SPR).

[0149] Methods for determining affinity In certain embodiments, the antigen-binding molecules or antibodies provided herein have a binding affinity to their antigen of ≦1 μM, ≦120 nM, ≦100 nM, ≦80 nM, ≦70 nM, ≦50 nM, ≦40 nM, ≦30 nM, ≦20 nM, ≦10 nM, ≦2 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, 10 -8 M~10 -13 M, 10 -9 M~10 -13 In certain embodiments, the KD value of the antibody / antigen-binding molecule for CD3, CD137, or CLDN6 falls within the range of 1 to 40, 1 to 50, 1 to 70, 1 to 80, 30 to 50, 30 to 70, 30 to 80, 40 to 70, 40 to 80, or 60 to 80 nM.

[0150] In one embodiment, KD is measured by radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed using a Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of the Fab for the antigen is measured at the lowest concentration ( 125 I) Measurement is performed by equilibrating Fab with labeled antigen and then capturing the bound antigen using a plate coated with anti-Fab antibody. (See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish measurement conditions, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125 [I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., as in the evaluation of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight, although this incubation can be continued for longer periods (e.g., approximately 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed, and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate has dried, 150 μl / well of scintillant (MICROSCINT-20™, Packard) is added, and the plate is counted for 10 minutes in a TOPCOUNT™ gamma counter (Packard). The concentration of each Fab that gives 20% or less of maximum binding is selected for use in the competitive binding assay.

[0151] In another embodiment, Kd is measured using a BIACORE® surface plasmon resonance assay. For example, measurements using a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) are performed at 25°C using a CM5 chip with approximately 10 response units (RU) of antigen immobilized. In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate, pH 4.8, before injection at a flow rate of 5 μl / min to achieve approximately 10 response units (RU) of protein binding. After injection of the antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS containing 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25°C and a flow rate of approximately 25 μl / min. The association rate (k on ) and dissociation rate (k off ) is calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software Version 3.2). The equilibrium dissociation constant (Kd) is given by k off / k on See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate is 10 by the surface plasmon resonance assay described above, 6 M -1 s -1If the on-rate exceeds , the on-rate can be determined by using a fluorescence quenching technique to measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, bandpass 16 nm) at 25°C of 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2 in the presence of increasing concentrations of antigen, as measured in a spectrometer (e.g., a stopped-flow spectrophotometer (Aviv Instruments) or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) using a stirred cuvette).

[0152] Following the above-described methods for measuring the affinity of an antigen-binding molecule or antibody, those skilled in the art can measure the affinity of other antigen-binding molecules or antibodies for various antigens.

[0153] antibody The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0154] In certain embodiments, the multispecific antibodies described herein bind to epitopes of CD3, CD137, or CLDN6 that are conserved among CD3, CD137, or CLDN6 of different species. In certain embodiments, the multispecific antibodies of the present disclosure are trispecific antibodies, which are antibodies that can specifically bind to three different antigens. That is, in certain embodiments, the multispecific antibodies of the present disclosure are trispecific antibodies that can bind to CD3 and CD137, and bind to either CD3 or CD137, i.e., do not simultaneously bind to both CD3 and CD137 antigens, and can specifically bind to CLDN6.

[0155] Antibody classes The "class" of an antibody refers to the type of constant domain or constant region present in the antibody's heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0156] Unless otherwise indicated, amino acid residues in the light chain constant region are numbered herein according to Kabat et al., and numbering of amino acid residues in the heavy chain constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0157] Framework "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences typically appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0158] Human Consensus Framework A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Typically, the subgroup of sequences is a subgroup in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda, MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup κI according to Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III according to Kabat et al., supra.

[0159] chimeric antibodies The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species. Similarly, the term "chimeric antibody variable domain" refers to an antibody variable region in which a portion of the heavy and / or light chain variable region is derived from a particular source or species, while the remainder of the heavy and / or light chain variable region is derived from a different source or species.

[0160] humanized antibodies A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization. A "humanized antibody variable region" refers to the variable region of a humanized antibody.

[0161] Human antibodies A "human antibody" is an antibody with an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or an antibody derived from a human antibody repertoire or other non-human source that uses human antibody coding sequences. This definition of human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues. A "human antibody variable region" refers to the variable region of a human antibody.

[0162] Polynucleotides (nucleic acids) "Polynucleotide" or "nucleic acid," used interchangeably herein, refers to a polymer of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substance that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides can include modified nucleotides, such as methylated nucleotides and their analogs. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can include modifications made after synthesis, such as conjugation to a label. Other types of modifications include, for example, "caps," substitutions of one or more naturally occurring nucleotides with analogs, internucleotide modifications, such as those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) and charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalating agents (e.g., acridine, psoralens, etc.), those containing chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.), those containing alkylating agents, modified linkages (e.g., alpha-anomeric nucleic acids, etc.), and unmodified forms of polynucleotides. Additionally, any hydroxyl groups normally present on the sugar can be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to generate additional linkages to additional nucleotides, or conjugated to solid or semi-solid supports. The 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of 1 to 20 carbon atoms. Other hydroxyls can also be derivatized to standard protecting groups.Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl-, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose or xylose or lyxose, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs, and basic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages can be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which phosphate is replaced by: P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where each R or R' is independently H or substituted or unsubstituted alkyl (1-20C), optionally including an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all linkages in a polynucleotide need be identical. The above description applies to all polynucleotides referred to herein, including RNA and DNA.

[0163] Isolated (nucleic acid) An "isolated" nucleic acid molecule is one that is separated from a component of its original environment. Isolated nucleic acid molecules further include nucleic acid molecules contained in cells that normally contain the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.

[0164] vector As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. This term includes vectors as self-replicating nucleic acid structures and vectors that are integrated into the genome of a host cell into which it is introduced. Some vectors are capable of conferring expression of a nucleic acid to which they are operatively linked. Such vectors are also referred to herein as "expression vectors." Vectors can be introduced into host cells using viruses or electroporation. However, vector introduction is not limited to in vitro methods. For example, vectors can also be introduced directly into a subject using in vivo methods.

[0165] host cell The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the originally transformed cell and progeny derived from that cell regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as that for which the original transformed cell was screened or selected are also included herein.

[0166] specificity "Specific" means that a molecule that specifically binds to one or more binding partners does not exhibit any significant binding to molecules other than the partners. Furthermore, "specific" is also used when an antigen-binding site is specific to a particular epitope among multiple epitopes contained in an antigen. When an antigen-binding molecule specifically binds to an antigen, it is also described as "the antigen-binding molecule has / exhibits specificity for / for the antigen." When the epitope to which the antigen-binding site binds is contained in multiple different antigens, the antigen-binding molecule containing the antigen-binding site can bind to various antigens that have the epitope.

[0167] antibody fragment An "antibody fragment" refers to a molecule other than a complete antibody that contains a portion of the complete antibody that binds to the antigen to which the complete antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and single-domain antibodies. For a review of specific antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); in addition, WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. See U.S. Patent No. 5,869,046 for a discussion of Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues and exhibiting increased in vivo half-lives. Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat Med 9, 129-134 (2003); Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). A single-domain antibody is an antibody fragment that contains all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of whole antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0168] Variable fragment (Fv) As used herein, the term "variable fragment (Fv)" refers to the smallest unit of an antibody-derived antigen-binding site consisting of a pair of an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). In 1988, Skerra and Pluckthun discovered that homogeneous and active antibodies could be prepared from the periplasmic fraction of E. coli by inserting an antibody gene downstream of a bacterial signal sequence and inducing expression of the gene in E. coli (Science (1988) 240 (4855), 1038-1041). In the Fv prepared from the periplasmic fraction, the VH and VL are associated in such a manner that they bind to antigens.

[0169] scFv, single chain antibodies, and sc(Fv) 2 As used herein, the terms "scFv," "single-chain antibody," and "sc(Fv)2" all refer to a single polypeptide chain antibody fragment that contains variable regions derived from heavy and light chains but no constant region. Generally, single-chain antibodies further contain a polypeptide linker between the VH and VL domains that allows the formation of the desired structure that will enable antigen binding. Single-chain antibodies are discussed in detail by Pluckthun in "The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)." See also International Publication WO 1988 / 001649, U.S. Patent Nos. 4,946,778 and 5,260,203. In certain embodiments, single-chain antibodies can be bispecific and / or humanized.

[0170] scFv is a single-chain low molecular weight antibody in which the VH and VL that form the Fv are linked together by a peptide linker (Proc. Natl. Acad. Sci. USA (1988) 85 (16), 5879-5883). The peptide linker can hold the VH and VL in close proximity. sc(Fv)2 is a single-chain antibody in which four variable regions, two VLs and two VHs, are linked by a linker such as a peptide linker to form a single chain (J Immunol. Methods (1999) 231 (1-2), 177-189). The two VHs and two VLs may be derived from different monoclonal antibodies. Suitable examples of such sc(Fv)2 include bispecific sc(Fv)2s that recognize two epitopes present in a single antigen, as disclosed in Journal of Immunology (1994) 152 (11), 5368-5374. sc(Fv)2s can be produced by methods known to those skilled in the art. For example, sc(Fv)2s can be produced by linking scFvs with a linker such as a peptide linker.

[0171] As used herein, sc(Fv)2 comprises two VH units and two VL units arranged in the following order, starting from the N-terminus of the single-chain polypeptide: VH, VL, VH, VL ([VH]-linker-[VL]-linker-[VH]-linker-[VL]). The order of the two VH units and two VL units is not limited to the above configuration and may be arranged in any order. Examples of configurations are listed below. [VL]-linker-[VH]-linker-[VH]-linker-[VL] [VH]-linker-[VL]-linker-[VL]-linker-[VH] [VH]-linker-[VH]-linker-[VL]-linker-[VL] [VL]-linker-[VL]-linker-[VH]-linker-[VH] [VL]-linker-[VH]-linker-[VL]-linker-[VH]

[0172] The molecular form of sc(Fv)2 is also described in detail in WO2006 / 132352. Those skilled in the art can follow these descriptions to appropriately prepare the desired sc(Fv)2 for producing the polypeptide complexes disclosed herein. Furthermore, the antigen-binding molecules or antibodies of the present disclosure may be conjugated with carrier polymers such as PEG or organic compounds such as anticancer drugs. Alternatively, a glycosylation sequence is suitably inserted into the antigen-binding molecules or antibodies so that the sugar chains exert the desired effect.

[0173] Linkers used to link antibody variable regions include any peptide linker that can be introduced by genetic engineering, synthetic linkers, and linkers such as those disclosed in Protein Engineering, 9 (3), 299-305, 1996. However, in the present disclosure, peptide linkers are preferred. The length of the peptide linker is not particularly limited and can be appropriately selected by those skilled in the art depending on the purpose. The length is preferably 5 amino acids or more (although not particularly limited, the upper limit is usually 30 amino acids or less, preferably 20 amino acids or less), and particularly preferably 15 amino acids. When sc(Fv)2 contains three peptide linkers, the lengths of these linkers may all be the same or different.

[0174] For example, such peptide linkers include: Ser, Gly-Ser, Gly-Gly-Ser, Ser-Gly-Gly, Gly-Gly-Gly-Ser (SEQ ID NO: 171), Ser-Gly-Gly-Gly (SEQ ID NO: 172), Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 173), Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 174), Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 175), Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 176), Gly-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 177), Ser-Gly-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 178), (Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 173)), and (Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 174))n. Here, n is an integer equal to or greater than 1. The length and sequence of the peptide linker can be appropriately selected by those skilled in the art depending on the purpose.

[0175] Synthetic linkers (chemical cross-linkers) are commonly used to cross-link peptides, including N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS3), dithiobis(succinimidyl propionate) (DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES). These cross-linkers are commercially available.

[0176] Three linkers are usually required to link four antibody variable regions, and the linkers used may be of the same type or different types.

[0177] Fab, F(ab') 2 , and Fab' "Fab" consists of one light chain and the CH1 domain and variable region of one heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0178] "F(ab')2" or "Fab" refers to an antibody fragment produced by treating an immunoglobulin (monoclonal antibody) with a protease such as pepsin or papain, digesting the immunoglobulin (monoclonal antibody) near the disulfide bond between the hinge regions of the two heavy chains. For example, papain cleaves IgG upstream of the disulfide bond between the hinge regions of the two heavy chains, producing two homologous antibody fragments in which an L chain containing a VL (light chain variable region) and a CL (light chain constant region) is linked by a disulfide bond at their C-terminal regions to an H chain fragment containing a VH (heavy chain variable region) and a CHγ1 (γ1 region of the heavy chain constant region). Each of these two homologous antibody fragments is called Fab'.

[0179] "F(ab')2" consists of two light chains and two heavy chains containing constant regions, such as the CH1 domain and a portion of the CH2 domain, such that disulfide bonds are formed between the two heavy chains. The F(ab')2 disclosed herein can be conveniently produced as follows: a monoclonal whole antibody or the like containing the desired antigen-binding site is partially digested with a protease such as pepsin, and the Fc fragment is removed by adsorption onto a protein A column. The protease used is not particularly limited, as long as it can selectively cleave the whole antibody to yield F(ab')2 under appropriately set enzymatic reaction conditions, such as pH. Examples of such proteases include pepsin and ficin.

[0180] Fc area As used herein, the term "Fc region" or "Fc domain" refers to a region of an antibody molecule comprising a hinge or a portion thereof, and a fragment consisting of the CH2 and CH3 domains. The Fc region of an IgG class refers, for example, but is not limited to, the region from cysteine ​​226 (EU numbering, also referred to herein as the EU index) to the C-terminus, or from proline 230 (EU numbering) to the C-terminus. The Fc region can be obtained, for example, by partially digesting an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody with a protease such as pepsin, followed by re-elution of the fraction adsorbed to a protein A or protein G column. The protease is not particularly limited, as long as it can digest a full-length antibody to form Fab or F(ab')2 under appropriately selected enzyme reaction conditions (e.g., pH). Examples include pepsin and papain.

[0181] In the present invention, for example, an Fc region derived from a native IgG can be used as the "Fc region" of the present disclosure. Here, native IgG refers to a polypeptide that contains the same amino acid sequence as an IgG found in nature and belongs to the class of antibodies substantially encoded by the immunoglobulin γ gene. Native human IgG refers to, for example, native human IgG1, native human IgG2, native human IgG3, or native human IgG4. Native IgG also includes naturally occurring variants thereof. Multiple allotype sequences based on genetic polymorphisms are described in "Sequences of proteins of immunological interest," NIH Publication No. 91-3242, for the constant regions of human IgG1, human IgG2, human IgG3, and human IgG4 antibodies, and any of these sequences can be used in the present disclosure. In particular, the sequence of human IgG1 may have DEL or EEM as the amino acid sequence at positions 356 to 358 (EU numbering).

[0182] In some embodiments, the Fc domain of a multispecific antigen-binding molecule is composed of a pair of polypeptide chains comprising the heavy chain domain of an immunoglobulin molecule.For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which comprises CH2 and CH3 IgG heavy chain constant domains.The two subunits of an Fc domain can stably associate with each other.In one embodiment, the multispecific antigen-binding molecule described herein comprises no more than one Fc domain.

[0183] In one embodiment described herein, the Fc domain of the multispecific antigen-binding molecule is an IgG Fc domain. In a specific embodiment, the Fc domain is an IgG1 Fc domain. In another embodiment, the Fc domain is an IgG1 Fc domain. In a further specific embodiment, the Fc domain is a human IgG1 Fc region.

[0184] In one aspect, the multispecific antigen-binding molecule contained in the anticancer agent, pharmaceutical composition, combination, kit, or used in the method or use of the present disclosure is (iii) an Fc domain that exhibits reduced binding affinity to human Fcγ receptors compared to a native human IgG1 Fc domain; wherein the Fc domain is composed of a first Fc region subunit and a second Fc region subunit that are capable of stably associating.

[0185] In one aspect, the multispecific antigen-binding molecule contained in the anticancer agent, pharmaceutical composition, combination, kit, or used in the method or use of the present disclosure is (iii) an Fc domain that exhibits reduced binding affinity to human Fcγ receptors compared to a native human IgG1 Fc domain; wherein the Fc domain is one of the following (e1) or (e2): (e1) a first Fc region subunit comprising a Cys at position 349, a Ser at position 366, an Ala at position 368, and a Val at position 407, and a second Fc region comprising a Cys at position 354 and a Trp at position 366; (e2) a first Fc region subunit containing Glu at position 439 and a second Fc region containing Lys at position 356 wherein the amino acid positions are numbered according to the EU index.

[0186] In one aspect, the multispecific antigen-binding molecule contained in the anticancer agent, pharmaceutical composition, combination, kit, or used in the method or use of the present disclosure is (iii) an Fc domain that exhibits reduced binding affinity to human Fcγ receptors compared to a native human IgG1 Fc domain; wherein the first and / or second Fc region subunit comprised in the Fc domain is (f1) or (f2) below: (f1) Ala at position 234 and Ala at position 235; (f2) Ala at 234th place, Ala at 235th place, and Ala at 297th place wherein the amino acid positions are numbered according to the EU index.

[0187] In one aspect, the multispecific antigen-binding molecule contained in the anticancer agent, pharmaceutical composition, combination, kit, or used in the method or use of the present disclosure is (iii) an Fc domain that exhibits reduced binding affinity to human Fcγ receptors compared to a native human IgG1 Fc domain; wherein the Fc domain further exhibits stronger FcRn-binding affinity for human FcRn compared to a native human IgG1 Fc domain.

[0188] In one aspect, the multispecific antigen-binding molecule contained in the anticancer agent, pharmaceutical composition, combination, kit, or used in the method or use of the present disclosure is (iii) an Fc domain that exhibits reduced binding affinity to human Fcγ receptors compared to a native human IgG1 Fc domain; wherein the first and / or second Fc region subunit comprise Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440, and the amino acid positions are numbered according to the EU index.

[0189] In one aspect, in the anticancer agent, pharmaceutical composition, combination, kit, method, or use of the present disclosure, a chemotherapeutic agent and the multispecific antigen-binding molecule are used in combination. In some embodiments, a platinum agent and the multispecific antigen-binding molecule are used in combination. In certain embodiments, carboplatin or cisplatin and the multispecific antigen-binding molecule are used in combination. In some embodiments, an alkaloid and the multispecific antigen-binding molecule are used in combination. In some embodiments, a plant alkaloid and the multispecific antigen-binding molecule are used in combination. In some embodiments, a topoisomerase inhibitor and the multispecific antigen-binding molecule are used in combination. In certain embodiments, irinotecan and the multispecific antigen-binding molecule are used in combination. In some embodiments, an antimetabolite and the multispecific antigen-binding molecule are used in combination. In certain embodiments, gemcitabine and the multispecific antigen-binding molecule are used in combination.

[0190] In one aspect, the anti-cancer agents, pharmaceutical compositions, combinations, kits, methods, or uses of the present disclosure use an immune checkpoint inhibitor in combination with the multispecific antigen-binding molecule described above. In one embodiment, an anti-PD-L1 antibody is used in combination with the multispecific antigen-binding molecule described above.

[0191] In one aspect, in the anticancer agent, pharmaceutical composition, combination, kit, method, or use of the present disclosure, a PARP inhibitor and the multispecific antigen-binding molecule described above are used in combination. In one embodiment, olaparip and the multispecific antigen-binding molecule described above are used in combination.

[0192] Fc region with reduced Fc receptor (Fcγ receptor) binding activity In certain embodiments, the Fc domain of the multispecific antigen-binding molecule described herein exhibits reduced binding affinity to Fc receptors compared to a native IgG1 Fc domain. In one such embodiment, the Fc domain (or a multispecific antigen-binding molecule comprising the Fc domain) exhibits less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% of the binding affinity to Fc receptors compared to a native IgG1 Fc domain (or a multispecific antigen-binding molecule comprising a native IgG1 Fc domain). In one embodiment, the Fc domain (or a multispecific antigen-binding molecule comprising the Fc domain) does not substantially bind to Fc receptors. In a certain embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In a particular embodiment, the Fc receptor is an activating human Fcγ receptor, more particularly human Fcγ RIIIa, Fcγ RI, or Fcγ RIIa, most particularly human Fcγ RIIIa.

[0193] In certain embodiments, the Fc domain of the multispecific antigen-binding molecule contains one or more amino acid mutations that reduce the binding affinity of the Fc domain to an Fc receptor. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In one embodiment, the amino acid mutations reduce the binding affinity of the Fc domain to an Fc receptor. In one embodiment, the amino acid mutations reduce the binding affinity of the Fc domain to an Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In embodiments where two or more amino acid mutations that reduce the binding affinity of the Fc domain to an Fc receptor are present, the combination of these amino acid mutations can reduce the binding affinity of the Fc domain to an Fc receptor by at least 10-fold, at least 20-fold, or even at least 50-fold. In one embodiment, a multispecific antigen-binding molecule comprising an engineered Fc domain exhibits less than 20%, particularly less than 10%, and more particularly less than 5% of the binding affinity of a multispecific antigen-binding molecule comprising a non-engineered Fc domain. In certain embodiments, the Fc receptor is an Fcγ receptor. In some embodiments, the Fc receptor is a human Fc receptor. In some embodiments, the Fc receptor is an activating Fc receptor. In certain embodiments, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, most specifically human FcγRIIIa. Preferably, binding to each of these receptors is reduced.

[0194] In one embodiment, the amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor is an amino acid substitution. In one embodiment, the Fc domain comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331, and P329. In a more specific embodiment, the Fc domain comprises an amino acid substitution at a position selected from the group of L234, L235, and P329. In some embodiments, the Fc domain comprises amino acid substitutions L234A and L235A. In one such embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. In one embodiment, the Fc domain comprises an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G. In one embodiment, the Fc domain comprises an amino acid substitution at position P329 and an additional amino acid substitution at a position selected from E233, L234, L235, N297, and P331. In a more particular embodiment, the additional amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a particular embodiment, the Fc domain comprises amino acid substitutions at positions P329, L234, and L235. In a more particular embodiment, the Fc domain comprises the amino acid mutations L234A, L235A, and P329G ("P329G LALA"). In one such embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. The "P329G LALA" combination of amino acid substitutions almost completely abolishes Fcγ receptor (and complement) binding of human IgG1 Fc domains, as described in PCT Publication No. WO2012 / 130831. WO2012 / 130831 also describes methods for preparing such mutant Fc domains and determining their properties, such as Fc receptor binding or effector function.

[0195] IgG4 antibodies exhibit reduced binding affinity to Fc receptors and reduced effector function compared to IgG1 antibodies. Thus, in some embodiments, the Fc domain of the bispecific antigen-binding molecule that activates T cells described herein is an IgG4 Fc domain, particularly a human IgG4 Fc domain. In one embodiment, the IgG4 Fc domain comprises an amino acid substitution at position S228, particularly the amino acid substitution S228P. To further reduce its binding affinity to Fc receptors and / or its effector function, in one embodiment, the IgG4 Fc domain comprises an amino acid substitution at position L235, particularly the amino acid substitution L235E. In another embodiment, the IgG4 Fc domain comprises an amino acid substitution at position P329, particularly the amino acid substitution P329G. In a specific embodiment, the IgG4 Fc domain comprises amino acid substitutions at positions S228, L235, and P329, particularly the amino acid substitutions S228P, L235E, and P329G. Such IgG4 Fc domain variants and their Fcγ receptor binding properties are described in PCT Publication No. WO2012 / 130831.

[0196] In certain embodiments, the N-glycosylation of the Fc domain is ablated. In one such embodiment, the Fc domain comprises an amino acid mutation at position N297, in particular an amino acid substitution substituting asparagine with alanine (N297A) or aspartic acid (N297D).

[0197] In a particularly preferred embodiment, the Fc domain that exhibits reduced binding affinity to Fc receptors compared to a native IgG1 Fc domain is a human IgG1 Fc domain that comprises the amino acid substitutions L234A, L235A, and N297A.

[0198] Mutant Fc domains can be prepared by amino acid deletion, substitution, insertion, or modification using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis of the coding DNA sequence, PCR, gene synthesis, etc. The exact nucleotide changes can be verified, for example, by sequencing.

[0199] Binding to Fc receptors can be easily determined, for example, by ELISA or surface plasmon resonance (SPR) using standard instrumentation such as a BIAcore instrument (GE Healthcare), and Fc receptors may also be obtained by recombinant expression. Suitable such binding assays are described herein. Alternatively, the binding affinity of an Fc domain or a cell-activating bispecific antigen-binding molecule containing an Fc domain to an Fc receptor can be assessed using a cell line known to express a particular Fc receptor, such as human NK cells expressing the FcγIIIa receptor.

[0200] Fc receptors The term "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, th...

Claims

1. A method for damaging cancer cells or tumor tissues containing cancer cells, or for suppressing the growth of cancer cells or tumor tissues containing cancer cells, comprising contacting CLDN6-positive cancer cells with at least one anticancer agent to increase CLDN6 expression, and further contacting the cancer cells with increased CLDN6 expression with a multispecific antigen-binding molecule. including multispecific antigen-binding molecules, containing at least one other anticancer drug, or A combination of a multispecific antigen-binding molecule and at least one other anticancer drug. A pharmaceutical composition comprising: The multispecific antigen-binding molecule comprises: (i) a first antigen-binding portion capable of binding to CD3 and CD137, the first antigen-binding portion binding to either CD3 or CD137; and (ii) a second antigen-binding portion capable of binding to claudin 6 (CLDN6); The at least one other anticancer agent is at least one selected from the group consisting of a chemotherapeutic agent, an immune checkpoint inhibitor, and a PARP inhibitor. Medicine.

2. The pharmaceutical composition of claim 1, wherein the at least one other anticancer agent is a platinum agent, an alkaloid, or an antimetabolite.

3. The pharmaceutical composition of claim 1, wherein the at least one other anticancer drug is any one of carboplatin, cisplatin, irinotecan, gemcitabine, and olaparib.

4. 2. The pharmaceutical composition according to claim 1, wherein the cancer cells are cancer cells of at least one cancer selected from the group consisting of ovarian cancer, non-small cell lung cancer, gastric cancer, liver cancer, endometrial cancer, germ cell tumor, colorectal cancer, bladder cancer, and atypical teratoid rhabdoid tumor.

5. The pharmaceutical composition according to claim 1 , wherein the cancer cells are cancer cells of peritoneal disseminated cancer.

6. The pharmaceutical composition of claim 1, wherein the at least one other anticancer agent is an agent that induces expression of TGFβ in cancer cells.

7. The pharmaceutical composition of claim 1, wherein the at least one other anticancer agent is an agent that induces expression of TGFβ1 in cancer cells.

8. The pharmaceutical composition of claim 1, wherein the multispecific antigen-binding molecule is a multispecific antigen-binding molecule according to any one of the following (1) to (6): (1) A multispecific antigen-binding molecule comprising: a first antibody variable region comprising a complementarity-determining region (CDR) 1 of SEQ ID NO: 11, a CDR 2 of SEQ ID NO: 17, and a CDR 3 of SEQ ID NO: 23; a second antibody variable region comprising a CDR 1 of SEQ ID NO: 32, a CDR 2 of SEQ ID NO: 36, and a CDR 3 of SEQ ID NO: 40; a third antibody variable region comprising a complementarity-determining region (CDR) 1 of SEQ ID NO: 7, a CDR 2 of SEQ ID NO: 13, and a CDR 3 of SEQ ID NO: 19; and a fourth antibody variable region comprising a CDR 1 of SEQ ID NO: 29, a CDR 2 of SEQ ID NO: 33, and a CDR 3 of SEQ ID NO: 37; (2) A multispecific antigen-binding molecule comprising: a first antibody variable region comprising a complementarity-determining region (CDR) 1 of SEQ ID NO: 9, a CDR 2 of SEQ ID NO: 15, and a CDR 3 of SEQ ID NO: 21; a second antibody variable region comprising a CDR 1 of SEQ ID NO: 31, a CDR 2 of SEQ ID NO: 35, and a CDR 3 of SEQ ID NO: 39; a third antibody variable region comprising a complementarity-determining region (CDR) 1 of SEQ ID NO: 8, a CDR 2 of SEQ ID NO: 14, and a CDR 3 of SEQ ID NO: 20; and a fourth antibody variable region comprising a CDR 1 of SEQ ID NO: 30, a CDR 2 of SEQ ID NO: 34, and a CDR 3 of SEQ ID NO: 38; (3) A multispecific antigen-binding molecule comprising: a first antibody variable region comprising a complementarity-determining region (CDR) 1 of SEQ ID NO: 10, a CDR 2 of SEQ ID NO: 16, and a CDR 3 of SEQ ID NO: 22; a second antibody variable region comprising a CDR 1 of SEQ ID NO: 31, a CDR 2 of SEQ ID NO: 35, and a CDR 3 of SEQ ID NO: 39; a third antibody variable region comprising a complementarity-determining region (CDR) 1 of SEQ ID NO: 8, a CDR 2 of SEQ ID NO: 14, and a CDR 3 of SEQ ID NO: 20; and a fourth antibody variable region comprising a CDR 1 of SEQ ID NO: 30, a CDR 2 of SEQ ID NO: 34, and a CDR 3 of SEQ ID NO: 38; (4) A multispecific antigen-binding molecule comprising: a first antibody variable region comprising a complementarity-determining region (CDR) 1 of SEQ ID NO: 12, a CDR 2 of SEQ ID NO: 18, and a CDR 3 of SEQ ID NO: 24; a second antibody variable region comprising a CDR 1 of SEQ ID NO: 32, a CDR 2 of SEQ ID NO: 36, and a CDR 3 of SEQ ID NO: 40; a third antibody variable region comprising a complementarity-determining region (CDR) 1 of SEQ ID NO: 7, a CDR 2 of SEQ ID NO: 13, and a CDR 3 of SEQ ID NO: 19; and a fourth antibody variable region comprising a CDR 1 of SEQ ID NO: 29, a CDR 2 of SEQ ID NO: 33, and a CDR 3 of SEQ ID NO: 37; (5) A multispecific antigen-binding molecule comprising: a first antibody variable region comprising a complementarity-determining region (CDR) 1 of SEQ ID NO: 11, a CDR 2 of SEQ ID NO: 17, and a CDR 3 of SEQ ID NO: 23; a second antibody variable region comprising a CDR 1 of SEQ ID NO: 32, a CDR 2 of SEQ ID NO: 36, and a CDR 3 of SEQ ID NO: 40; a third antibody variable region comprising a complementarity-determining region (CDR) 1 of SEQ ID NO: 29, a CDR 2 of SEQ ID NO: 33, and a CDR 3 of SEQ ID NO: 37; and a fourth antibody variable region comprising a CDR 1 of SEQ ID NO: 7, a CDR 2 of SEQ ID NO: 13, and a CDR 3 of SEQ ID NO: 19; (6) A multispecific antigen-binding molecule comprising: a first antibody variable region comprising a complementarity-determining region (CDR) 1 of SEQ ID NO: 12, a CDR 2 of SEQ ID NO: 18, and a CDR 3 of SEQ ID NO: 24; a second antibody variable region comprising a CDR 1 of SEQ ID NO: 32, a CDR 2 of SEQ ID NO: 36, and a CDR 3 of SEQ ID NO: 40; a third antibody variable region comprising a complementarity-determining region (CDR) 1 of SEQ ID NO: 29, a CDR 2 of SEQ ID NO: 33, and a CDR 3 of SEQ ID NO: 37; and a fourth antibody variable region comprising a CDR 1 of SEQ ID NO: 7, a CDR 2 of SEQ ID NO: 13, and a CDR 3 of SEQ ID NO:

19.

9. The pharmaceutical of claim 8, wherein the multispecific antigen-binding molecule comprises at least one selected from the group consisting of the first antibody variable region, the second antibody variable region, the third antibody variable region, and the fourth antibody variable region comprising a human antibody framework or a humanized antibody framework.

10. The pharmaceutical composition of claim 1, wherein the multispecific antigen-binding molecule is a multispecific antigen-binding molecule of any one of the following (I) to (VI): (I) A multispecific antigen-binding molecule comprising: a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 5; a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 28; a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 1; and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 25; (II) a multispecific antigen-binding molecule comprising: a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 3; a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 27; a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 2; and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 26; (III) a multispecific antigen-binding molecule comprising: a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 4; a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 27; a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 2; and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 26; (IV) a multispecific antigen-binding molecule comprising: a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 6; a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 28; a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 1; and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 25; (V) a multispecific antigen-binding molecule comprising: a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 5; a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 28; a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 25; and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 1; (VI) A multispecific antigen-binding molecule comprising: a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 6; a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 28; a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 25; and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO:

1.

11. The pharmaceutical according to claim 10, wherein the first antibody variable region and the second antibody variable region are capable of binding to CD3 and CD137 and constitute a first antigen-binding portion that binds to either CD3 or CD137, and the third antibody variable region and the fourth antibody variable region constitute a second antigen-binding portion that can bind to CLDN6.

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