Combination of multi-specific molecule and immune checkpoint inhibitor

Abstract

Provided is a new combination of medicines for treating cancer. A pharmaceutical composition for treating and / or preventing cancer comprises a multi-specific antibody described in [i] below and is used in combination with a compound described in [ii] below: [i] a multi-specific antibody or an antigen-binding fragment thereof; and [ii] an immune checkpoint inhibitor or a chemotherapeutic agent.

Description

Combining multispecific molecules with immune checkpoint inhibitors

[0001] The present invention relates to pharmaceutical compositions, therapeutic or prophylactic methods, etc., comprising multispecific molecules used in combination with immune checkpoint inhibitors, etc.

[0002] Cancer treatment involves surgical removal of the cancerous tissue and the killing of cancer cells with anticancer drugs or radiation, with the hope of curing the disease or prolonging life. However, for many cancers, single anticancer drug treatments are insufficient, so treatments using a variety of drugs in combination are being attempted. Anticancer drugs include chemotherapeutic agents such as doxorubicin, carboplatin, taxol, and camptothecin; molecularly targeted drugs such as imatinib, crizotinib, dasatinib, and lapatinib; and cancer treatment antibodies such as trastuzumab, bevacizumab, cetuximab, and ramucirumab. The type of cancer, stage, and treatment status determine the anticancer drug and combination therapy used.

[0003] Immune checkpoint inhibitors, which have recently become standard therapeutic agents, are drugs that inhibit the immunosuppressive system and activate anti-tumor immunity (Non-Patent Documents 1 to 3). Examples of immune checkpoint inhibitors include anti-PD-1 antibodies such as nivolumab (Patent Document 1) and pembrolizumab (Patent Document 2), anti-PD-L1 antibodies such as atezolizumab (Patent Document 3), durvalumab (Patent Document 4), and avelumab (Patent Document 5), and anti-CTLA-4 antibodies such as ipilimumab (Patent Document 6), tremelimumab (Patent Document 7), and spartalizumab. Known therapeutic agents include Spartalizumab (Patent Document 8), Cemiplimab (Patent Document 9), and anti-TIGIT antibodies, Tiragolumab (Patent Document 10) and Vibostolimab (Patent Document 11). There are also cases where a life-prolonging effect has been observed when combined with a chemotherapeutic agent (Non-Patent Document 4). Furthermore, cases have been reported in which the combined effect of a bispecific antibody targeting a cancer antigen and CD3 was investigated (Non-Patent Documents 5 and 6).

[0004] However, little is known about drugs whose anti-cancer effects are enhanced when used in combination with a bispecific antibody composed of an anti-HLA-A2 / NY-ESO antibody and an anti-CD3 antibody (Patent Document 12).

[0005] Patent Document 1: WO 2006 / 121168 Patent Document 2: WO 2008 / 156712 Patent Document 3: WO 2010 / 077634 Patent Document 4: WO 2011 / 066389 Patent Document 5: WO 2013 / 079174 Patent Document 6: WO 2001 / 014424 Patent Document 7: WO 2000 / 037504 Patent Document 8: WO 2015 / 112900 Patent Document 9: WO 2015 / 196051 Patent Document 10: WO 2017 / 053748 Patent Document 11: WO 2016 / 028656 Patent Document 12: WO 2021 / 200857

[0006] Non-patent document 1: Menon S. , et al. , Cancers (2016) 8, 106. Non-Patent Document 2: Pardoll DM. , Nat Rev Cancer (2012) 12, 252-264. Non-Patent Document 3: Wolchok JD. , Cell (2015) 162, 937. Non-patent document 4: Motzer, R. , et al. , New England Journal of Medicine. (2021) 384:1289-300. Non-patent document 5: Deegen P. , et al. , Clinical Cancer Research. (2021) 27:2928-37. Non-patent document 6: Tevernero J. , et al. , Journal of Clinical Oncology. (2017) 35:15_suppl, 3002-3002.

[0007] One object of the present invention is to provide a combination of a multispecific antibody consisting of an anti-HLA-A2 / NY-ESO antibody and an anti-CD3 antibody and another drug, as well as the antibody or a pharmaceutical composition containing the antibody, which is used in combination with another drug.

[0008] Another object of the present invention is to provide a multispecific antibody composed of an anti-HLA-A2 / NY-ESO antibody and an anti-CD3 antibody, which is used in the treatment or prevention of cancer, or a pharmaceutical composition containing the antibody, which is used in the treatment or prevention of cancer in combination with another agent.

[0009] Furthermore, another object of the present invention is to provide a method for treating or preventing cancer by administering a multispecific antibody composed of an anti-HLA-A2 / NY-ESO antibody and an anti-CD3 antibody in combination with another agent, or by administering the composition.

[0010] The inventors conducted studies to solve the above problems and found that an excellent antitumor effect can be obtained by combining a bispecific antibody composed of an anti-HLA-A2 / NY-ESO antibody and an anti-CD3 antibody, which have anticancer activity, with various anticancer agents (e.g., chemotherapeutic agents such as carboplatin, paclitaxel, and Nab-paclitaxel, and immune checkpoint inhibitors such as pembrolizumab), and thus completed the present invention.

[0011] The present invention relates to: (1) a pharmaceutical composition for the treatment and / or prevention of cancer, comprising the multispecific antibody described in [i] below, used in combination with the compound described in [ii] below: [i] a heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 1; a heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 2; a heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 3, or a light chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 3 in which the sixth amino acid is N; a light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 4, or a light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 4 in which the seventh amino acid is W and / or the eighth amino acid is K; a light chain CDRL2 consisting of the amino acid sequence represented by Asp Asn Asn (the fifth sequence from the top in Figure 11 : SEQ ID NO: 5 in Figure 82 ); and a light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 6, or a light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 6 in which the second amino acid is A or S. [ii] a multispecific antibody comprising: an antibody or antigen-binding fragment thereof that binds to HLA / NY-ESO, comprising: a heavy chain CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 7; a heavy chain CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 8 or a heavy chain CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 8 in which the third amino acid is N or S; a heavy chain CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 9; a light chain CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 10; a light chain CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 11 of Arg Asp Asp (the fifth sequence from the top in Figure 12; SEQ ID NO: 11 of Figure 82) or a light chain CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 11 of Arg Asp Asp (the fifth sequence from the top in Figure 12; SEQ ID NO: 11 of Figure 82) in which the second amino acid is G, Q, N, S, or A; and a light chain CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 12; (2) A compound that inhibits an immune checkpoint molecule or a chemotherapeutic agent. (3) The pharmaceutical composition according to (1), wherein the multispecific antibody is a bispecific antibody.

[0012] (3) The pharmaceutical composition according to (1) or (2), wherein the antibody or antigen-binding fragment thereof that binds to HLA / NY-ESO comprises one or more sets of CDRH1 to CDRH3 and CDRL1 to CDRL3 selected from the group consisting of the following sets (i) to (v): (i) a heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 2, a heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 3, a light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 4, a light chain CDRL2 consisting of the amino acid sequence represented by Asp Asn Asn (the fifth sequence from the top in Figure 11: SEQ ID NO: 5 in Figure 82), and a light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 6; (ii) a heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 2, a heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 3, a light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO:4, in which the 7th amino acid is W; a light chain CDRL2 consisting of the amino acid sequence represented by Asp Asn Asn (the fifth sequence from the top in Figure 11 : SEQ ID NO:5 in Figure 82 ); and a light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO:6; (iii) a heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO:1; a heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO:2; a heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO:3, in which the 6th amino acid is N; a light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO:4; a light chain CDRL2 consisting of the amino acid sequence represented by Asp Asn Asn (the fifth sequence from the top in Figure 11 : SEQ ID NO:5 in Figure 82 ); and a light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO:6, in which the 2nd amino acid is A; (iv) a heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO:1; a heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 2; a heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 3; a light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 4;(v) a light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 1; a heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 2; a heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 3; a light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 4, in which the seventh amino acid is W and the eighth amino acid is K; a light chain CDRL2 consisting of the amino acid sequence represented by Asp Asn Asn (the fifth sequence from the top in FIG. 11 ; SEQ ID NO: 5 in FIG. 82 ); and a light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 6, in which the second amino acid is A; (4) The pharmaceutical composition according to any one of (1) to (3), wherein the antibody or antigen-binding fragment thereof that binds to HLA / NY-ESO comprises a heavy chain variable region consisting of an amino acid sequence of amino acid numbers 21 to 140 in the amino acid sequence represented by SEQ ID NO: 18, or an amino acid sequence having 95% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 69 or SEQ ID NO: 70, and a light chain variable region consisting of an amino acid sequence of amino acid numbers 156 to 266 in the amino acid sequence represented by SEQ ID NO: 18 or SEQ ID NO: 28, or an amino acid sequence having 95% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 23;

[0013] (5) An antibody or antigen-binding fragment thereof that binds to HLA / NY-ESO comprises: a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 13; a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 15; a heavy chain variable region consisting of the amino acid sequence from the 21st to the 140th positions of the amino acid sequence represented by SEQ ID NO: 20; a heavy chain variable region consisting of the amino acid sequence from the 21st to the 140th positions of the amino acid sequence represented by SEQ ID NO: 17; a heavy chain variable region consisting of the amino acid sequence from the 21st to the 140th positions of the amino acid sequence represented by SEQ ID NO: 18; a heavy chain variable region consisting of the amino acid sequence from the 21st to the 140th positions of the amino acid sequence represented by SEQ ID NO: 19; a heavy chain variable region consisting of the amino acid sequence from the 21st to the 140th positions of the amino acid sequence represented by SEQ ID NO: 22; a heavy chain variable region consisting of the amino acid sequence from the 21st to the 140th positions of the amino acid sequence represented by SEQ ID NO: 24; a heavy chain variable region consisting of the amino acid sequence from the 21st to the 140th positions of the amino acid sequence represented by SEQ ID NO: 25; a heavy chain variable region consisting of the amino acid sequence from the 21st to the 140th positions of the amino acid sequence represented by SEQ ID NO: 26; a heavy chain variable region consisting of the amino acid sequence of positions 21 to 140 in the amino acid sequence represented by SEQ ID NO:27, a heavy chain variable region consisting of the amino acid sequence of positions 21 to 140 in the amino acid sequence represented by SEQ ID NO:28, a heavy chain variable region consisting of the amino acid sequence of positions 21 to 140 in the amino acid sequence represented by SEQ ID NO:29, a heavy chain variable region consisting of the amino acid sequence of positions 21 to 140 in the amino acid sequence represented by SEQ ID NO:21, a heavy chain variable region consisting of the amino acid sequence of positions 21 to 140 in the amino acid sequence represented by SEQ ID NO:55, or a heavy chain variable region consisting of the amino acid sequence of positions 21 to 140 in the amino acid sequence represented by SEQ ID NO:71, and a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO:14, a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO:16, a light chain variable region consisting of the amino acid sequence of positions 156 to 266 in the amino acid sequence represented by SEQ ID NO:20, a light chain variable region consisting of the amino acid sequence of positions 156 to 266 in the amino acid sequence represented by SEQ ID NO:17, a light chain variable region consisting of the amino acid sequence of positions 156 to 266 of the amino acid sequence represented by SEQ ID NO: 18;a light chain variable region consisting of the amino acid sequence of positions 156 to 266 in the amino acid sequence represented by SEQ ID NO: 19; a light chain variable region consisting of the amino acid sequence of positions 156 to 266 in the amino acid sequence represented by SEQ ID NO: 22; a light chain variable region consisting of the amino acid sequence of positions 156 to 266 in the amino acid sequence represented by SEQ ID NO: 24; a light chain variable region consisting of the amino acid sequence of positions 156 to 266 in the amino acid sequence represented by SEQ ID NO: 25; a light chain variable region consisting of the amino acid sequence of positions 156 to 266 in the amino acid sequence represented by SEQ ID NO: 26; a light chain variable region consisting of the amino acid sequence of positions 156 to 266 in the amino acid sequence represented by SEQ ID NO: 27; a light chain variable region consisting of the amino acid sequence of positions 156 to 266 in the amino acid sequence represented by SEQ ID NO: 28; a light chain variable region consisting of the amino acid sequence of positions 156 to 266 in the amino acid sequence represented by SEQ ID NO: 29; a light chain variable region consisting of the amino acid sequence of positions 156 to 266 in the amino acid sequence represented by SEQ ID NO: 21; (6) The pharmaceutical composition according to any one of (1) to (4), comprising a light chain variable region consisting of the amino acid sequence of positions 161 to 271 of the amino acid sequence represented by SEQ ID NO: 55, or a light chain variable region consisting of the amino acid sequence of positions 156 to 266 of the amino acid sequence represented by SEQ ID NO: 71; (7) A heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 13 and a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 14; A heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 15 and a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 16; A heavy chain variable region consisting of the amino acid sequence of positions 21 to 140 of the amino acid sequence represented by SEQ ID NO: 20 and a light chain variable region consisting of the amino acid sequence of positions 156 to 266 of the amino acid sequence represented by SEQ ID NO: 20; A heavy chain variable region consisting of the amino acid sequence of positions 21 to 140 of the amino acid sequence represented by SEQ ID NO: 17 and a light chain variable region consisting of the amino acid sequence of positions 156 to 266 of the amino acid sequence represented by SEQ ID NO: 17; a heavy chain variable region consisting of the amino acid sequence of positions 21 to 140 in the amino acid sequence represented by SEQ ID NO: 18, and a light chain variable region consisting of the amino acid sequence of positions 156 to 266 in the amino acid sequence represented by SEQ ID NO: 18;a heavy chain variable region consisting of the amino acid sequence at positions 21 to 140 of the amino acid sequence set forth in SEQ ID NO: 19, and a light chain variable region consisting of the amino acid sequence at positions 156 to 266 of the amino acid sequence set forth in SEQ ID NO: 19; a heavy chain variable region consisting of the amino acid sequence at positions 21 to 140 of the amino acid sequence set forth in SEQ ID NO: 22, and a light chain variable region consisting of the amino acid sequence at positions 156 to 266 of the amino acid sequence set forth in SEQ ID NO: 22; a heavy chain variable region consisting of the amino acid sequence at positions 21 to 140 of the amino acid sequence set forth in SEQ ID NO: 24, and a light chain variable region consisting of the amino acid sequence at positions 156 to 266 of the amino acid sequence set forth in SEQ ID NO: 24; a heavy chain variable region consisting of the amino acid sequence at positions 21 to 140 of the amino acid sequence set forth in SEQ ID NO: 25, and a light chain variable region consisting of the amino acid sequence at positions 156 to 266 of the amino acid sequence set forth in SEQ ID NO: 25; a heavy chain variable region consisting of the amino acid sequence at positions 21 to 140 of the amino acid sequence set forth in SEQ ID NO: 26, and a light chain variable region consisting of the amino acid sequence at positions 156 to 266 of the amino acid sequence set forth in SEQ ID NO: 26; a heavy chain variable region consisting of the amino acid sequence of positions 21 to 140 of the amino acid sequence set forth in SEQ ID NO:27, and a light chain variable region consisting of the amino acid sequence of positions 156 to 266 of the amino acid sequence set forth in SEQ ID NO:27; a heavy chain variable region consisting of the amino acid sequence of positions 21 to 140 of the amino acid sequence set forth in SEQ ID NO:28, and a light chain variable region consisting of the amino acid sequence of positions 156 to 266 of the amino acid sequence set forth in SEQ ID NO:28; a heavy chain variable region consisting of the amino acid sequence of positions 21 to 140 of the amino acid sequence set forth in SEQ ID NO:29, and a light chain variable region consisting of the amino acid sequence of positions 156 to 266 of the amino acid sequence set forth in SEQ ID NO:29; a heavy chain variable region consisting of the amino acid sequence of positions 21 to 140 of the amino acid sequence set forth in SEQ ID NO:21, and a light chain variable region consisting of the amino acid sequence of positions 156 to 266 of the amino acid sequence set forth in SEQ ID NO:21; a heavy chain variable region consisting of the amino acid sequence of positions 21 to 140 of the amino acid sequence set forth in SEQ ID NO:55, and a light chain variable region consisting of the amino acid sequence of positions 161 to 271 of the amino acid sequence set forth in SEQ ID NO:55;The pharmaceutical composition according to any one of (1) to (5), comprising a heavy chain variable region consisting of the amino acid sequence of positions 21 to 140 in the amino acid sequence represented by SEQ ID NO: 71 and a light chain variable region consisting of the amino acid sequence of positions 156 to 266 in the amino acid sequence represented by SEQ ID NO: 71;

[0014] (7) The pharmaceutical composition according to any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof that binds to HLA / NY-ESO is an scFv. (8) An antibody or antigen-binding fragment thereof that binds to HLA / NY-ESO is selected from the group consisting of: an scFv consisting of the amino acid sequence of positions 21 to 266 of the amino acid sequence represented by SEQ ID NO: 30; an scFv comprising a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 15 and a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 16; an scFv consisting of the amino acid sequence of positions 21 to 266 of the amino acid sequence represented by SEQ ID NO: 20; an scFv consisting of the amino acid sequence of positions 21 to 266 of the amino acid sequence represented by SEQ ID NO: 17; an scFv consisting of the amino acid sequence of positions 21 to 266 of the amino acid sequence represented by SEQ ID NO: 18; an scFv consisting of the amino acid sequence of positions 21 to 266 of the amino acid sequence represented by SEQ ID NO: 19; an scFv consisting of the amino acid sequence of positions 21 to 266 of the amino acid sequence represented by SEQ ID NO: 22; an scFv consisting of the amino acid sequence of positions 21 to 266 of the amino acid sequence represented by SEQ ID NO: 24; an scFv consisting of the amino acid sequence of positions 21 to 266 of the amino acid sequence represented by SEQ ID NO: 25; The pharmaceutical composition according to any one of (1) to (7), which is an scFv consisting of the amino acid sequence of the 21st to 266th positions in the amino acid sequence represented by SEQ ID NO: 26, an scFv consisting of the amino acid sequence of the 21st to 266th positions in the amino acid sequence represented by SEQ ID NO: 27, an scFv consisting of the amino acid sequence of the 21st to 266th positions in the amino acid sequence represented by SEQ ID NO: 28, an scFv consisting of the amino acid sequence of the 21st to 266th positions in the amino acid sequence represented by SEQ ID NO: 29, an scFv consisting of the amino acid sequence of the 21st to 266th positions in the amino acid sequence represented by SEQ ID NO: 21, an scFv consisting of the amino acid sequence of the 21st to 271st positions in the amino acid sequence represented by SEQ ID NO: 55, or an scFv consisting of the amino acid sequence of the 21st to 266th positions in the amino acid sequence represented by SEQ ID NO: 71,

[0015] (9) The pharmaceutical composition according to any one of (1) to (8), wherein the antibody or antigen-binding fragment thereof that binds to CD3 comprises one or more sets of CDRH1 to CDRH3 and CDRL1 to CDRL3 selected from the group consisting of the following sets (i) to (X): (i) a heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7, a heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 8, a heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9, a light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10, a light chain CDRL2 consisting of the amino acid sequence represented by Arg Asp Asp (the fifth sequence from the top in Figure 12: SEQ ID NO: 11 in Figure 82), and a light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 12. (ii) a heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7, a heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 8 in which the third amino acid is N, a heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9, a light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10, a light chain CDRL2 consisting of the amino acid sequence represented by Arg Asp Asp (the fifth sequence from the top in Figure 12 : SEQ ID NO: 11 in Figure 82 ), and a light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 12; (iii) a heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7, a heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 8 in which the third amino acid is S, a heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9, a light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10, a light chain CDRL2 consisting of the amino acid sequence represented by Arg Asp Asp (the fifth sequence from the top in Figure 12 : SEQ ID NO: 11 in Figure 82 ), and a light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 12; (iv) a heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7, a heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 8, a heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9; a light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10;a light chain CDRL2 consisting of the amino acid sequence represented by SEQ ID NO: 12, in which the second amino acid is G in the amino acid sequence represented by SEQ ID NO: 12; and a light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 12; (v) a heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7; a heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 8; a heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9; a light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10; a light chain CDRL2 consisting of the amino acid sequence represented by SEQ ID NO: 11, in which the second amino acid is Q in the amino acid sequence represented by SEQ ID NO: 12; and a light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 12; (vi) a heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7; a heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 8; a heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9; a light chain CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 10; a light chain CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 11 of Figure 82, in which the second amino acid is N in the amino acid sequence set forth in SEQ ID NO: 12; and a light chain CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 12; (vii) a heavy chain CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 7; a heavy chain CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 8; a heavy chain CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 9; a light chain CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 10; a light chain CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 11 of Figure 82, in which the second amino acid is S in the amino acid sequence set forth in SEQ ID NO: 12; and a light chain CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 12; (viii) a heavy chain CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 7; a heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 8; a heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9; a light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10;(i) a light chain CDRL2 consisting of the amino acid sequence represented by SEQ ID NO: 12 in which the second amino acid is A in the amino acid sequence represented by Arg Asp Asp (the fifth sequence from the top in Figure 12 ; SEQ ID NO: 11 in Figure 82 ), and a light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 12; (iX) a heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7; a heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 8 in which the third amino acid is S; a heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9; a light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10; a light chain CDRL2 consisting of the amino acid sequence represented by SEQ ID NO: 12 in which the second amino acid is N in the amino acid sequence represented by Arg Asp Asp (the fifth sequence from the top in Figure 12 ; SEQ ID NO: 11 in Figure 82 ), and a light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 12; and (X) a heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7; a heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 8 in which the third amino acid is S; a heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9; a light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 10; a light chain CDRL2 consisting of the amino acid sequence represented by Arg Asp Asp (the fifth sequence from the top in Figure 12; SEQ ID NO: 11 in Figure 82) in which the second amino acid is S; and a light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 12.

[0016] (10) An antibody or antigen-binding fragment thereof that binds to CD3 comprises: a heavy chain variable region consisting of the amino acid sequence from the 2nd to the 119th positions in the amino acid sequence represented by SEQ ID NO: 46; a heavy chain variable region consisting of the amino acid sequence from the 2nd to the 119th positions in the amino acid sequence represented by SEQ ID NO: 47; a heavy chain variable region consisting of the amino acid sequence from the 2nd to the 119th positions in the amino acid sequence represented by SEQ ID NO: 51; a heavy chain variable region consisting of the amino acid sequence from the 2nd to the 119th positions in the amino acid sequence represented by SEQ ID NO: 48; a heavy chain variable region consisting of the amino acid sequence from the 2nd to the 119th positions in the amino acid sequence represented by SEQ ID NO: 49; a heavy chain variable region consisting of the amino acid sequence from the 2nd to the 119th positions in the amino acid sequence represented by SEQ ID NO: 50; a heavy chain variable region consisting of the amino acid sequence from the 272nd to the 389th positions in the amino acid sequence represented by SEQ ID NO: 54; a heavy chain variable region consisting of the amino acid sequence from the 277th to the 394th positions in the amino acid sequence represented by SEQ ID NO: 55; or a heavy chain variable region consisting of the amino acid sequence from the 277th to the 394th positions in the amino acid sequence represented by SEQ ID NO: 56; (11) The pharmaceutical composition according to (9), comprising: a light chain variable region consisting of the amino acid sequence of positions 135 to 243 in the amino acid sequence represented by SEQ ID NO: 46; a light chain variable region consisting of the amino acid sequence of positions 135 to 241 in the amino acid sequence represented by SEQ ID NO: 47; a light chain variable region consisting of the amino acid sequence of positions 135 to 243 in the amino acid sequence represented by SEQ ID NO: 51; a light chain variable region consisting of the amino acid sequence of positions 135 to 241 in the amino acid sequence represented by SEQ ID NO: 48; a light chain variable region consisting of the amino acid sequence of positions 135 to 243 in the amino acid sequence represented by SEQ ID NO: 49; a light chain variable region consisting of the amino acid sequence of positions 135 to 243 in the amino acid sequence represented by SEQ ID NO: 50; a light chain variable region consisting of the amino acid sequence of positions 405 to 511 in the amino acid sequence represented by SEQ ID NO: 54; a light chain variable region consisting of the amino acid sequence of positions 410 to 516 in the amino acid sequence represented by SEQ ID NO: 55; or a light chain variable region consisting of the amino acid sequence of positions 410 to 516 in the amino acid sequence represented by SEQ ID NO: 56; The antibody or antigen-binding fragment thereof that binds to CD3 isa heavy chain variable region consisting of the amino acid sequence at positions 2 to 119 of the amino acid sequence set forth in SEQ ID NO: 46, and a light chain variable region consisting of the amino acid sequence at positions 135 to 243 of the amino acid sequence set forth in SEQ ID NO: 46; a heavy chain variable region consisting of the amino acid sequence at positions 2 to 119 of the amino acid sequence set forth in SEQ ID NO: 47, and a light chain variable region consisting of the amino acid sequence at positions 135 to 241 of the amino acid sequence set forth in SEQ ID NO: 47; a heavy chain variable region consisting of the amino acid sequence at positions 2 to 119 of the amino acid sequence set forth in SEQ ID NO: 51, and a light chain variable region consisting of the amino acid sequence at positions 135 to 243 of the amino acid sequence set forth in SEQ ID NO: 51; a heavy chain variable region consisting of the amino acid sequence at positions 2 to 119 of the amino acid sequence set forth in SEQ ID NO: 48, and a light chain variable region consisting of the amino acid sequence at positions 135 to 241 of the amino acid sequence set forth in SEQ ID NO: 48; a heavy chain variable region consisting of the amino acid sequence at positions 2 to 119 of the amino acid sequence set forth in SEQ ID NO: 49, and a light chain variable region consisting of the amino acid sequence at positions 135 to 243 of the amino acid sequence set forth in SEQ ID NO: 49; the pharmaceutical composition according to (8) or (9), comprising: a heavy chain variable region consisting of the amino acid sequence of positions 2 to 119 in the amino acid sequence represented by SEQ ID NO: 50 and a light chain variable region consisting of the amino acid sequence of positions 135 to 243 in the amino acid sequence represented by SEQ ID NO: 50; a heavy chain variable region consisting of the amino acid sequence of positions 272 to 389 in the amino acid sequence represented by SEQ ID NO: 54 and a light chain variable region consisting of the amino acid sequence of positions 405 to 511 in the amino acid sequence represented by SEQ ID NO: 54; a heavy chain variable region consisting of the amino acid sequence of positions 277 to 394 in the amino acid sequence represented by SEQ ID NO: 55 and a light chain variable region consisting of the amino acid sequence of positions 410 to 516 in the amino acid sequence represented by SEQ ID NO: 55; or a heavy chain variable region consisting of the amino acid sequence of positions 277 to 394 in the amino acid sequence represented by SEQ ID NO: 56 and a light chain variable region consisting of the amino acid sequence of positions 410 to 516 in the amino acid sequence represented by SEQ ID NO: 56;

[0017] (12) The pharmaceutical composition according to any one of (1) to (11), wherein the antibody or antigen-binding fragment thereof that binds to CD3 is an scFv; (13) The antibody or antigen-binding fragment thereof that binds to CD3 is an scFv consisting of the amino acid sequence from the 2nd to the 243rd positions of the amino acid sequence represented by SEQ ID NO: 46; an scFv consisting of the amino acid sequence from the 2nd to the 241st positions of the amino acid sequence represented by SEQ ID NO: 47; an scFv consisting of the amino acid sequence from the 2nd to the 243rd positions of the amino acid sequence represented by SEQ ID NO: 51; an scFv consisting of the amino acid sequence from the 2nd to the 241st positions of the amino acid sequence represented by SEQ ID NO: 48; an scFv consisting of the amino acid sequence from the 2nd to the 243rd positions of the amino acid sequence represented by SEQ ID NO: 49; an scFv consisting of the amino acid sequence from the 2nd to the 243rd positions of the amino acid sequence represented by SEQ ID NO: 50; an scFv consisting of the amino acid sequence from the 272nd to the 511th positions of the amino acid sequence represented by SEQ ID NO: 54; an scFv consisting of the amino acid sequence from the 277th to the 516th positions of the amino acid sequence represented by SEQ ID NO: 55; or (14) A pharmaceutical composition according to any one of (1) to (12), which is an scFv consisting of the amino acid sequence of positions 277 to 516 of the amino acid sequence represented by SEQ ID NO: 56; (14) A first polypeptide comprising, from the N-terminus to the C-terminus, an HLA / NY-ESO-binding antibody or antigen-binding fragment thereof which is an scFv, an CD3-binding antibody or antigen-binding fragment thereof which is an scFv, and an Fc region (i), in that order;and a second polypeptide comprising an Fc region (ii), preferably comprising an Fc region (ii) associated with the Fc region (i); (15) the pharmaceutical composition according to any one of (1) to (13), wherein the first polypeptide is selected from the group consisting of the amino acid sequence of positions 21 to 511 of the amino acid sequence represented by SEQ ID NO: 32, the amino acid sequence of positions 21 to 511 of the amino acid sequence represented by SEQ ID NO: 34, the amino acid sequence of positions 21 to 511 of the amino acid sequence represented by SEQ ID NO: 35, the amino acid sequence of positions 21 to 511 of the amino acid sequence represented by SEQ ID NO: 36, the amino acid sequence of positions 21 to 511 of the amino acid sequence represented by SEQ ID NO: 37, the amino acid sequence of positions 21 to 511 of the amino acid sequence represented by SEQ ID NO: 38, the amino acid sequence of positions 21 to 511 of the amino acid sequence represented by SEQ ID NO: 39, the amino acid sequence of positions 21 to 511 of the amino acid sequence represented by SEQ ID NO: 40, and the amino acid sequence of positions 21 to 511 of the amino acid sequence represented by SEQ ID NO: 41. the pharmaceutical composition according to (14), comprising the amino acid sequence of positions 21 to 511 of the amino acid sequence represented by SEQ ID NO: 42, the amino acid sequence of positions 21 to 511 of the amino acid sequence represented by SEQ ID NO: 43, the amino acid sequence of positions 21 to 511 of the amino acid sequence represented by SEQ ID NO: 33, the amino acid sequence of positions 21 to 511 of the amino acid sequence represented by SEQ ID NO: 52, the amino acid sequence of positions 21 to 511 of the amino acid sequence represented by SEQ ID NO: 53, the amino acid sequence of positions 21 to 511 of the amino acid sequence represented by SEQ ID NO: 54, the amino acid sequence of positions 21 to 516 of the amino acid sequence represented by SEQ ID NO: 55, or the amino acid sequence of positions 21 to 516 of the amino acid sequence represented by SEQ ID NO: 56; (16) The pharmaceutical composition according to (14) or (15), wherein the first polypeptide comprises an amino acid sequence from the 529th to the 745th positions of the amino acid sequence represented by SEQ ID NO: 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 33, 52, 53, or 54, or an amino acid sequence from the 534th to the 750th positions of the amino acid sequence represented by SEQ ID NO: 55 or 56;

[0018] (17) The first polypeptide is an amino acid sequence of the 20th to 745th amino acids of the amino acid sequence represented by SEQ ID NO: 32, an amino acid sequence of the 20th to 745th amino acids of the amino acid sequence represented by SEQ ID NO: 34, an amino acid sequence of the 20th to 745th amino acids of the amino acid sequence represented by SEQ ID NO: 35, an amino acid sequence of the 20th to 745th amino acids of the amino acid sequence represented by SEQ ID NO: 36, an amino acid sequence of the 20th to 745th amino acids of the amino acid sequence represented by SEQ ID NO: 37, an amino acid sequence of the 20th to 745th amino acids of the amino acid sequence represented by SEQ ID NO: 38, an amino acid sequence of the 20th to 745th amino acids of the amino acid sequence represented by SEQ ID NO: 39, an amino acid sequence of the 20th to 745th amino acids of the amino acid sequence represented by SEQ ID NO: 40, an amino acid sequence of the 20th to 745th amino acids of the amino acid sequence represented by SEQ ID NO: 41. The pharmaceutical composition according to any one of (14) to (16), which consists of an amino acid sequence, an amino acid sequence of the 20th to 745th amino acids in the amino acid sequence represented by SEQ ID NO: 42, an amino acid sequence of the 20th to 745th amino acids in the amino acid sequence represented by SEQ ID NO: 43, an amino acid sequence of the 20th to 745th amino acids in the amino acid sequence represented by SEQ ID NO: 33, an amino acid sequence of the 20th to 745th amino acids in the amino acid sequence represented by SEQ ID NO: 52, an amino acid sequence of the 20th to 745th amino acids in the amino acid sequence represented by SEQ ID NO: 53, an amino acid sequence of the 20th to 745th amino acids in the amino acid sequence represented by SEQ ID NO: 54, an amino acid sequence of the 20th to 750th amino acids in the amino acid sequence represented by SEQ ID NO: 55, or an amino acid sequence of the 20th to 750th amino acid in the amino acid sequence represented by SEQ ID NO: 56; (18) The pharmaceutical composition according to any one of (14) to (17), wherein the second polypeptide comprises the amino acid sequence from the 20th to the 246th amino acids of the amino acid sequence shown in SEQ ID NO: 31; (19) A pharmaceutical composition comprising a first polypeptide, a second polypeptide, and a third polypeptide, wherein the first polypeptide comprises, from the N-terminus to the C-terminus, an antibody or an antigen-binding fragment thereof that binds to HLA / NY-ESO, which is an scFv; an antibody or an antigen-binding fragment thereof that specifically binds to CD3, which is an scFv; and an Fc region (i), in that order; the second polypeptide comprises an immunoglobulin heavy chain comprising an Fc region (ii); and the third polypeptide comprises an immunoglobulin light chain; and preferably, the second polypeptide and the third polypeptide are associated with each other;(20) The pharmaceutical composition according to any one of (1) to (13), wherein the first polypeptide and the second peptide are associated with each other at their Fc regions; (21) The pharmaceutical composition according to (19) above, wherein the second polypeptide comprises the amino acid sequence of amino acid numbers 20 to 242 of the amino acid sequence represented by SEQ ID NO: 44; (22) The pharmaceutical composition according to (19) or (20), wherein the third polypeptide comprises the amino acid sequence represented by SEQ ID NO: 45; The first polypeptide is selected from the group consisting of the amino acid sequence of positions 21 to 511 in the amino acid sequence represented by SEQ ID NO: 32, the amino acid sequence of positions 21 to 511 in the amino acid sequence represented by SEQ ID NO: 34, the amino acid sequence of positions 21 to 511 in the amino acid sequence represented by SEQ ID NO: 35, the amino acid sequence of positions 21 to 511 in the amino acid sequence represented by SEQ ID NO: 36, the amino acid sequence of positions 21 to 511 in the amino acid sequence represented by SEQ ID NO: 37, the amino acid sequence of positions 21 to 511 in the amino acid sequence represented by SEQ ID NO: 38, the amino acid sequence of positions 21 to 511 in the amino acid sequence represented by SEQ ID NO: 39, the amino acid sequence of positions 21 to 511 in the amino acid sequence represented by SEQ ID NO: 40, and the amino acid sequence of positions 21 to 511 in the amino acid sequence represented by SEQ ID NO: 41. (23) The pharmaceutical composition according to any one of (19) to (21), comprising an amino acid sequence, the amino acid sequence of the 21st to 511th positions of the amino acid sequence represented by SEQ ID NO: 42, the amino acid sequence of the 21st to 511th positions of the amino acid sequence represented by SEQ ID NO: 43, the amino acid sequence of the 21st to 511th positions of the amino acid sequence represented by SEQ ID NO: 33, the amino acid sequence of the 21st to 511th positions of the amino acid sequence represented by SEQ ID NO: 52, the amino acid sequence of the 21st to 511th positions of the amino acid sequence represented by SEQ ID NO: 53, the amino acid sequence of the 21st to 511th positions of the amino acid sequence represented by SEQ ID NO: 54, the amino acid sequence of the 21st to 516th positions of the amino acid sequence represented by SEQ ID NO: 55, or the amino acid sequence of the 21st to 516th positions of the amino acid sequence represented by SEQ ID NO: 56;The first polypeptide is selected from the group consisting of the amino acid sequence of positions 20 to 745 in the amino acid sequence represented by SEQ ID NO: 32, the amino acid sequence of positions 20 to 745 in the amino acid sequence represented by SEQ ID NO: 34, the amino acid sequence of positions 20 to 745 in the amino acid sequence represented by SEQ ID NO: 35, the amino acid sequence of positions 20 to 745 in the amino acid sequence represented by SEQ ID NO: 36, the amino acid sequence of positions 20 to 745 in the amino acid sequence represented by SEQ ID NO: 37, the amino acid sequence of positions 20 to 745 in the amino acid sequence represented by SEQ ID NO: 38, the amino acid sequence of positions 20 to 745 in the amino acid sequence represented by SEQ ID NO: 39, the amino acid sequence of positions 20 to 745 in the amino acid sequence represented by SEQ ID NO: 40, and the amino acid sequence of positions 20 to 745 in the amino acid sequence represented by SEQ ID NO: 41. the pharmaceutical composition according to any one of (19) to (22), consisting of an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 42, the amino acid sequence of positions 20 to 745 of the amino acid sequence represented by SEQ ID NO: 43, and the amino acid sequence of positions 20 to 745 of the amino acid sequence represented by SEQ ID NO: 33, the amino acid sequence of positions 20 to 745 of the amino acid sequence represented by SEQ ID NO: 52, the amino acid sequence of positions 20 to 745 of the amino acid sequence represented by SEQ ID NO: 53, the amino acid sequence of positions 20 to 745 of the amino acid sequence represented by SEQ ID NO: 54, the amino acid sequence of positions 20 to 750 of the amino acid sequence represented by SEQ ID NO: 55, or the amino acid sequence of positions 20 to 750 of the amino acid sequence represented by SEQ ID NO: 56;

[0019] (24) The pharmaceutical composition according to any one of (9) to (11), comprising, from the N-terminus to the C-terminus, a first polypeptide comprising, in that order, an antibody or antigen-binding fragment thereof that specifically binds to HLA / NY-ESO, which is an svFv; a heavy chain variable region and constant region CH1 of an antibody that binds to CD3, and an immunoglobulin Fc region (i); a second polypeptide comprising an immunoglobulin hinge region and Fc region (ii); and a third polypeptide comprising an antibody light chain that binds to CD3, which comprises a variable region and a constant region, wherein preferably the first polypeptide and the second polypeptide are associated with each other at the Fc region (i) and the Fc region (ii), and the first polypeptide is associated with the third polypeptide at the variable region and constant region CH1 of the antibody heavy chain. (25) (26) The pharmaceutical composition according to any one of (9) to (11) and (25), wherein the first polypeptide comprises the amino acid sequence of positions 20 to 724 of the amino acid sequence represented by SEQ ID NO: 57, the amino acid sequence of positions 20 to 719 of the amino acid sequence represented by SEQ ID NO: 60, or the amino acid sequence of positions 20 to 719 of the amino acid sequence represented by SEQ ID NO: 61; (27) The pharmaceutical composition according to any one of (9) to (11), (24) and (26), wherein the second polypeptide comprises the amino acid sequence of positions 20 to 246 of the amino acid sequence represented by SEQ ID NO: 31; (28) The pharmaceutical composition according to any one of (9) to (11) and (24) to (27), wherein the third polypeptide comprises the amino acid sequence of positions 21 to 127 of the amino acid sequence represented by SEQ ID NO: 58; (29) The pharmaceutical composition according to any one of (9) to (11) and (24) to (27), wherein the third polypeptide comprises the amino acid sequence of positions 21 to 233 of the amino acid sequence represented by SEQ ID NO: 58; (29) The pharmaceutical composition according to any one of (1) to (28), wherein one or two amino acids are deleted from the carboxyl terminus of the amino acid sequence of one or more polypeptides contained in the pharmaceutical composition. (30)Cancers include kidney cancer, melanoma, squamous cell carcinoma, basal cell carcinoma, conjunctival cancer, oral cancer, laryngeal cancer, pharyngeal cancer, thyroid cancer, lung cancer (non-small cell lung cancer (adenocarcinoma, squamous cell carcinoma, large cell carcinoma), small cell lung cancer), breast cancer, esophageal cancer, stomach cancer, duodenal cancer, small intestine cancer, colon cancer, rectal cancer, appendix cancer, anal cancer, liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, adrenal cancer, bladder cancer, prostate cancer, uterine cancer, vaginal cancer, liposarcoma, angiosarcoma, chondrosarcoma, and rhabdomyosarcoma. the pharmaceutical composition according to any one of (1) to (29), which is one or more cancers selected from the group consisting of Ewing's sarcoma, osteosarcoma, undifferentiated pleomorphic sarcoma, myxoid fibrosarcoma, malignant peripheral neurilemmoma, retroperitoneal sarcoma, synovial sarcoma, uterine sarcoma, gastrointestinal stromal tumor, leiomyosarcoma, epithelioid sarcoma, B-cell lymphoma, T / NK cell lymphoma, Hodgkin's lymphoma, myeloid leukemia, lymphocytic leukemia, myeloproliferative disorders, myelodysplastic syndrome, multiple myeloma, testicular cancer, and ovarian cancer;

[0020] (31) The pharmaceutical composition according to any one of (1) to (30), which is administered after a compound that inhibits an immune checkpoint molecule or a chemotherapeutic agent. (32) The pharmaceutical composition according to any one of (1) to (30), which is administered prior to a compound or chemotherapeutic agent that inhibits an immune checkpoint molecule; (33) The pharmaceutical composition according to any one of (1) to (30), which is administered simultaneously with a compound or chemotherapeutic agent that inhibits an immune checkpoint molecule; (34) The pharmaceutical composition according to any one of (1) to (30) and (33), which comprises a compound or chemotherapeutic agent that inhibits an immune checkpoint molecule; (35) The pharmaceutical composition according to any one of (1) to (34), which is used in combination with one or more additional medicines and / or treatments; (36) The pharmaceutical composition according to any one of (1) to (35), wherein the compound according to [ii] is a compound that inhibits an immune checkpoint molecule; (37) The pharmaceutical composition according to any one of (1) to (36), wherein the immune checkpoint molecule is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4 and TIGIT; (38) (39) The pharmaceutical composition according to any one of (1) to (37), wherein the immune checkpoint molecule is PD-1; (38) The pharmaceutical composition according to (38), wherein the compound that inhibits an immune checkpoint molecule is nivolumab or pembrolizumab, an antigen-binding fragment thereof, or a compound containing the antigen-binding fragment thereof.(40) The pharmaceutical composition according to any one of (1) to (37), wherein the immune checkpoint molecule is PD-L1; (41) The pharmaceutical composition according to (40), wherein the compound inhibiting the immune checkpoint molecule is atezolizumab, durvalumab, or avelumab, an antigen-binding fragment thereof, or a compound comprising the antigen-binding fragment thereof; (42) The pharmaceutical composition according to any one of (1) to (37), wherein the immune checkpoint molecule is CTLA-4; (43) The pharmaceutical composition according to (42), wherein the compound inhibiting the immune checkpoint molecule is ipilimumab, tremelimumab, spartalizumab, or cemiplimab, an antigen-binding fragment thereof, or a compound comprising the antigen-binding fragment thereof; (44) The pharmaceutical composition according to any one of (1) to (37), wherein the immune checkpoint molecule is TIGIT; (45) (44) The pharmaceutical composition according to (44), wherein the compound inhibiting an immune checkpoint molecule is tiragolumab or vibostolimab, an antigen-binding fragment thereof, or a compound comprising the antigen-binding fragment thereof; (46) The pharmaceutical composition according to any one of (1) to (36), wherein the compound inhibiting an immune checkpoint molecule is pembrolizumab, nivolumab, spartalizumab, cemiplimab, avelumab, atezolizumab, durvalumab, ipilimumab, or tremelimumab; (47) The pharmaceutical composition according to any one of (1) to (39), wherein the compound inhibiting an immune checkpoint molecule is pembrolizumab or an antigen-binding fragment of pembrolizumab, or a compound comprising the antigen-binding fragment thereof; (48) The pharmaceutical composition according to any one of (1) to (35), wherein the compound according to [ii] is a chemotherapeutic agent; (49) The pharmaceutical composition according to any one of (1) to (35) and (48), wherein the chemotherapeutic agent is a microtubule formation inhibitor.

[0021] (50) The pharmaceutical composition according to any one of (1) to (35), (48) and (49), wherein the chemotherapeutic agent is a taxane-based microtubule formation inhibitor; (51) The pharmaceutical composition according to any one of (1) to (35) and (48) to (50), wherein the chemotherapeutic agent is selected from the group consisting of paclitaxel, docetaxel, vincristine, vinblastine, vindesine, eribulin, vinorelbine, albumin-suspended paclitaxel, oxaliplatin, carboplatin, cisplatin, nedaplatin, azacitidine, decitabine, doxorubicin, bleomycin, liposomal doxorubicin, ifosfamide, cyclophosphamide and dacarbazine; (52) The pharmaceutical composition according to any one of (1) to (35) and (48) to (51), wherein the chemotherapeutic agent is paclitaxel or albumin-suspended paclitaxel; (53) The pharmaceutical composition according to any one of (1) to (52), which is further used in combination with a multikinase inhibitor; (54) The pharmaceutical composition according to (53), wherein the multikinase inhibitor is one or more selected from the group consisting of sorafenib, sunitinib, pazopanib, regorafenib, and lenvatinib; (55) The pharmaceutical composition according to (53) or (54), wherein the multikinase inhibitor is lenvatinib; (56) The pharmaceutical composition according to any one of (53) to (55), wherein the compound according to [ii] is a compound that inhibits an immune checkpoint molecule, and is preferably pembrolizumab or an antigen-binding fragment of pembrolizumab, or a compound comprising the antigen-binding fragment thereof; (57) (58) The pharmaceutical composition according to any one of (1) to (35) and (48) to (52), wherein the chemotherapeutic agent is a taxane microtubule formation inhibitor selected from the group consisting of paclitaxel, docetaxel, vincristine, vinblastine, vindesine, eribulin, vinorelbine, and albumin-suspended paclitaxel; (59) The pharmaceutical composition according to any one of (1) to (35) and (48) to (52), wherein the compound inhibiting an immune checkpoint molecule is pembrolizumab or an antigen-binding fragment of pembrolizumab, or a compound containing the antigen-binding fragment thereof, and the multikinase inhibitor is lenvatinib; (59) The pharmaceutical composition according to any one of (1) to (35) and (48) to (52), wherein the chemotherapeutic agent is a taxane microtubule formation inhibitor selected from the group consisting of paclitaxel, docetaxel, vincristine, vinblastine, vindesine, eribulin, vinorelbine, and albumin-suspended paclitaxel;(60) A compound or chemotherapeutic agent that inhibits an immune checkpoint molecule or a multikinase inhibitor, comprising a multispecific antibody [i] according to any one of (1) to (47) and a compound or chemotherapeutic agent that inhibits an immune checkpoint molecule or a multikinase inhibitor, comprising a multispecific antibody [i] according to any one of (53) to (57) and a compound or chemotherapeutic agent that inhibits an immune checkpoint molecule or a multikinase inhibitor, comprising a multispecific antibody [i] according to any one of (1) to (4 ...1) to (47) and a compound or chemotherapeutic agent that inhibits an immune checkpoint molecule or a multikinase inhibitor, comprising a multispecific antibody [i] according to any one of (1) to (47) and a compound or chemotherapeutic agent that inhibits an immune checkpoint molecule or a multikinase inhibitor, comprising a multispecific antibody [i] according to any one of (1) to (47) and a compound or chemotherapeutic agent that inhibits an immune checkpoint molecule or a multikinase inhibitor, comprising a multispecific antibody [i] according to (63) A method for treating and / or preventing cancer, comprising administering a compound or a chemotherapeutic agent that inhibits an immune checkpoint molecule; (64) A method for treating and / or preventing cancer, comprising administering [i] the multispecific antibody according to any one of (53) to (57) and [ii] a compound or a chemotherapeutic agent that inhibits an immune checkpoint molecule, and a multikinase inhibitor; and

[0022] By using a combination of the antibody provided by the present invention and other drugs, it becomes possible to treat or prevent various cancers.

[0023] Figure 1(1) shows the antitumor activity of Fc-tagged anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules, NYZ-1010 monotherapy, Paclitaxel monotherapy, and the combination of NYZ-1010 and Paclitaxel in a human T cell transfer model. Error bars in the figure indicate standard error (n=5). Figure 1(2) shows the percent change in tumor volume from Day 12 to Day 31 in each individual mouse treated with Fc-tagged anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules, NYZ-1010 monotherapy, Paclitaxel monotherapy, and the combination of NYZ-1010 and Paclitaxel in a human T cell transfer model. Figure 2(1) shows the antitumor activity of the Fc-tagged anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, Nab Paclitaxel alone, and the combination of NYZ-1010 and Nab Paclitaxel in a human T cell transfer model. Error bars in the figure represent standard error (n=5). Figure 2(2) shows the percent change in tumor volume from Day 13 to Day 38 for each individual in the human T cell transfer model, using the tumor volume on Day 13 as the baseline. Figure 3 shows the antitumor activity of an Fc-tagged anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 monotherapy, pembrolizumab monotherapy, and a combination of NYZ-1010 and pembrolizumab in a human T cell transfer model. Error bars in the figure indicate standard error (n=5). Figure 4 shows the antitumor activity of an Fc-tagged anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, NYF-0016 monotherapy, anti-mouse PD-1 antibody monotherapy, and a combination of NYF-0016 and anti-mouse PD-1 antibody in a human CD3ε knock-in mouse model. Error bars in the figure indicate standard error (n=5). Figure 5 shows the format of the antibody described in the present invention.(5a) scFv: A format in which an antibody heavy chain variable region (VH, described below) and an antibody light chain variable region (VL, described below) (both white) are linked by a linker. In the examples of the present invention, anti-HLA-A2 / NY-ESO and CD3 scFvs were used for evaluation. (5b) Fab: A format consisting of VH (white) and an antibody heavy chain constant region (CH1: checkered pattern) and VL (white) and an antibody light chain constant region (horizontal line). In the examples of the present invention, anti-HLA-A2 / NY-ESO Fab, etc. were used for evaluation. (5c) taFv: A format in which two types of scFv (white and upper right diagonal line) are linked by a linker. In the examples of the present invention, taFvs comprising anti-HLA-A2 / NY-ESO scFv and anti-CD3 scFv were used for evaluation. (5d) taFv-heterodimer Fc type: This format comprises an Fc (upper left diagonal line) containing a mutation that allows heterodimer formation at the C-terminus of taFv (also referred to as the first polypeptide), which is heterodimerized with another Fc (filled in black, also referred to as the second polypeptide). In the examples of the present invention, a taFv-heterodimer Fc comprising an anti-HLA-A2 / NY-ESO scFv and an anti-CD3 scFv was used for evaluation. (5e) taFv-Fab-heterodimer Fc type: This format comprises the above-mentioned taFv-heterodimer Fc type with a Fab added. In the examples of the present invention, a taFv comprising an anti-HLA-A2 / NY-ESO scFv and an anti-CD3 scFv, and a taFv-Fab-heterodimer Fc using HLA-A2 / NY-ESO Fab were used for evaluation. Figure 1 shows the formats of the antibodies described in the present invention. (6a) Shows a first polypeptide common to the taFv-heterodimer Fc type and the taFv-Fab-heterodimer Fc type. The first polypeptide comprises, from the N-terminus to the C-terminus, an scFv that specifically binds to human HLA / NY-ESO, an scFv that specifically binds to CD3, and an Fc region (i), in that order. (6b) Shows a second polypeptide of the taFv-heterodimer Fc type. The second polypeptide comprises a hinge region and an Fc region (ii). (6c) Shows a second polypeptide of the taFv-Fab-heterodimer Fc type. The second polypeptide comprises an immunoglobulin heavy chain comprising the hinge region and the Fc region (ii).(6d) The third polypeptide of a taFv-Fab-heterodimer Fc type is shown. The third polypeptide comprises an immunoglobulin light chain. A diagram showing the format of an antibody included in the present invention. (7a) Hybrid type: A format in which an Fc (hatched and filled in black) with a heterodimer-forming mutation is added to the C-terminus of each of the Fab and scFv, resulting in the association of two Fc molecules. In the examples of the present invention, a hybrid type comprising an anti-HLA-A2 / NY-ESO Fab and an anti-CD3 scFv was used for evaluation. (7b) Dual type: A format in which an Fc (hatched and filled in black) with a heterodimer-forming mutation is added to the C-terminus of each of two different scFvs, resulting in the association of two Fc molecules. In the examples of the present invention, a dual type comprising an anti-HLA-A2 / NY-ESO scFv and an anti-CD3 scFv was used for evaluation. (7c) scFv-Fab-heterodimer Fc type: This format comprises an Fc (upper left diagonal line) with a heterodimer-forming mutation attached to the C-terminus of a scFv and Fab linked by a linker, and then associated with another Fc (filled in black). In the examples of the present invention, an scFv-Fab-heterodimer Fc type comprising an anti-CD3 scFv (upper right diagonal line) and an anti-HLA-A2 / NY-ESO Fab was used for evaluation. An scFv-Fab-heterodimer Fc type comprising an anti-HLA-A2 / NY-ESO scFv (upper right diagonal line) and an anti-CD3 Fab was also used for evaluation. A diagram showing the antibody formats described in the present invention. (8a) taFv-heterodimer Fc type: Same as Figure 5 (5d). This format comprises an Fc (upper left diagonal line) with a mutation that allows heterodimer formation at the C-terminus of taFv, heterodimerized with another Fc (filled in black). In the examples of the present invention, a taFv-heterodimer Fc type consisting of an anti-HLA-A2 / NY-ESO scFv and an anti-CD3 scFv was used for evaluation. (8b) taFv(inversed)-heterodimer Fc type: The order of the two scFvs in the above taFv-heterodimer Fc type has been swapped. A diagram showing the antibody format of the present invention. (9a) The first polypeptide of the taFv(inversed)-heterodimer Fc type is shown.The first polypeptide comprises, from the N-terminus to the C-terminus, an scFv that specifically binds to CD3, an scFv that specifically binds to human HLA / NY-ESO, and an Fc region (i), in that order. (9b) The second polypeptide is a taFv (inversed)-heterodimer Fc type. The second polypeptide comprises a hinge region and an Fc region (ii). The amino acid sequence of human CD3ε is registered at NCBI / GenPept under accession number NP_000724 (NM_000724.1). The amino acid sequence of CDRH1-3 and CDRL1-3 contained in NYA-0001 (SEQ ID NOS: 1-4 and 6; the fifth position is a DNN, SEQ ID NOS: 5 in Figure 82). Amino acid sequences of heavy chain CDR H1 to CDR H3 and light chain CDR L1 to CDR L3 of C3E-7085 (SEQ ID NOs:7 to 10 and 12; the 11th position is RDD, SEQ ID NO:11 in Figure 82). Amino acid sequence of the heavy chain variable region of NYA-0001 (SEQ ID NO:13). Amino acid sequence of the light chain variable region of NYA-0001 (SEQ ID NO:14). Amino acid sequence of the heavy chain variable region of NYA-0082 (SEQ ID NO:15). Amino acid sequence of the light chain variable region of NYA-0082 (SEQ ID NO:16). Amino acid sequence of full-length NYA-1163 (SEQ ID NO:17). Signal sequence (1-19), NYA-1163 (21-266), FLAG-His tag (267-292). Amino acid sequence of full-length NYA-2023 (SEQ ID NO:18). Signal sequence (1-19), NYA-2023 (21-266), FLAG-His tag (267-292). Full-length amino acid sequence of NYA-2027 (SEQ ID NO: 19). Signal sequence (1-19), NYA-2027 (21-266), FLAG-His tag (267-292). Full-length amino acid sequence of NYA-1143 (SEQ ID NO: 20). Signal sequence (1-19), NYA-1143 (21-266), FLAG-His tag (267-292). Full-length amino acid sequence of NYA-2143 (SEQ ID NO: 21). Signal sequence (1-19), NYA-2143 (21-266), FLAG-His tag (267-292). Full-length amino acid sequence of NYA-2035 (SEQ ID NO: 22). Signal sequence (1-19), NYA-2035 (21-266), FLAG-His tag (267-292). Amino acid sequence of NYA-1143-VL01 (SEQ ID NO: 23).Full-length amino acid sequence of NYA-2044 (SEQ ID NO: 24). Signal sequence (1-19), NYA-2044 (21-266), FLAG-His tag (267-292). Full-length amino acid sequence of NYA-2045 (SEQ ID NO: 25). Signal sequence (1-19), NYA-2045 (21-266), FLAG-His tag (267-292). Full-length amino acid sequence of NYA-2047 (SEQ ID NO: 26). Signal sequence (1-19), NYA-2047 (21-266), FLAG-His tag (267-292). Full-length amino acid sequence of NYA-2048 (SEQ ID NO: 27). The amino acid sequence of NYA-2060 (full length) (SEQ ID NO: 28), signal sequence (1-19), NYA-2048 (21-266), FLAG-His tag (267-292), signal sequence (1-19), NYA-2060 (21-266), FLAG-His tag (267-292), the amino acid sequence of NYA-2061 (full length) (SEQ ID NO: 29), signal sequence (1-19), NYA-2061 (21-266), FLAG-His tag (267-292), the amino acid sequence of NYA-0001 (full length) (SEQ ID NO: 30), the amino acid sequence of HC1 (full length) (SEQ ID NO: 31), signal sequence (1-19), NYA-0001 (21-266), FLAG-His tag (267-292), signal sequence (1-19), NYA-0001 (21-266), FLAG-His tag (267-292), the amino acid sequence of HC1 (full length) (SEQ ID NO: 31). Signal sequence (1-19), Hinge (20-29), CH2 (30-139), CH3 (140-246). NYF-0016-HC2 full-length amino acid sequence (SEQ ID NO: 32). Signal sequence (1-19), NYA-1143 (21-266), linker (267-271), C3E-7085 (272-511), linker (512-513), Hinge (514-528), CH2 (529-638), CH3 (639-745). NYF-0019-HC2 full-length amino acid sequence (SEQ ID NO: 33). Signal sequence (1-19), NYA-2143 (21-266), linker (267-271), C3E-7085 (272-511), linker (512-513), Hinge (514-528), CH2 (529-638), CH3 (639-745). NYF-0022-HC2 full-length amino acid sequence (SEQ ID NO: 34).Signal sequence (1-19), NYA-1163 (21-266), linker (267-271), C3E-7085 (272-511), linker (512-513), Hinge (514-528), CH2 (529-638), CH3 (639-745). NYF-0023-HC2 full-length amino acid sequence (SEQ ID NO: 35). Signal sequence (1-19), NYA-2023 (21-266), linker (267-271), C3E-7085 (272-511), linker (512-513), Hinge (514-528), CH2 (529-638), CH3 (639-745). The amino acid sequence of the full-length NYF-0027-HC2 (SEQ ID NO: 36). Signal sequence (1-19), NYA-2027 (21-266), linker (267-271), C3E-7085 (272-511), linker (512-513), Hinge (514-528), CH2 (529-638), CH3 (639-745). The amino acid sequence of the full-length NYF-0035-HC2 (SEQ ID NO: 37). Signal sequence (1-19), NYA-2035 (21-266), linker (267-271), C3E-7085 (272-511), linker (512-513), Hinge (514-528), CH2 (529-638), CH3 (639-745). NYF-0044-HC2 full-length amino acid sequence (SEQ ID NO: 38). Signal sequence (1-19), NYA-2044 (21-266), linker (267-271), C3E-7085 (272-511), linker (512-513), Hinge (514-528), CH2 (529-638), CH3 (639-745). The amino acid sequence of the full-length NYF-0045-HC2 (SEQ ID NO: 39). Signal sequence (1-19), NYA-2045 (21-266), linker (267-271), C3E-7085 (272-511), linker (512-513), Hinge (514-528), CH2 (529-638), CH3 (639-745). The amino acid sequence of the full-length NYF-0047-HC2 (SEQ ID NO: 40).Signal sequence (1-19), NYA-2047 (21-266), linker (267-271), C3E-7085 (272-511), linker (512-513), Hinge (514-528), CH2 (529-638), CH3 (639-745). NYF-0048-HC2 full-length amino acid sequence (SEQ ID NO: 41). Signal sequence (1-19), NYA-2048 (21-266), linker (267-271), C3E-7085 (272-511), linker (512-513), Hinge (514-528), CH2 (529-638), CH3 (639-745). The amino acid sequence of the full-length NYF-0060-HC2 (SEQ ID NO: 42): Signal sequence (1-19), NYA-2060 (21-266), linker (267-271), C3E-7085 (272-511), linker (512-513), Hinge (514-528), CH2 (529-638), CH3 (639-745). The amino acid sequence of the full-length NYF-0061-HC2 (SEQ ID NO: 43). Signal sequence (1-19), NYA-2061 (21-266), linker (267-271), C3E-7085 (272-511), linker (512-513), hinge (514-528), CH2 (529-638), CH3 (639-745). Amino acid sequence of full-length NYA-0001-Fab-HC1-κ delete (SEQ ID NO: 44). Signal sequence (1-19), NYA-0001_VH (20-139), CH1 (140-237), hinge (238-252), CH2 (253-362), CH3 (363-468). Amino acid sequence of full-length NYA-0001-LC (SEQ ID NO: 45). Signal sequence (1-20), NYA-0001_VL (21-131), CL (132-237). Amino acid sequence of C3E-7034 (SEQ ID NO: 46). C3E-7034 (1-267), scFv (2-241), FLAG-His tag (242-267). Amino acid sequence of C3E-7036 (SEQ ID NO: 47). C3E-7036 (1-269), FLAG-His tag (244-269). Amino acid sequence of C3E-7085 (SEQ ID NO: 48). C3E-7085 (1-267), scFv (2-241), FLAG-His tag (242-267).Amino acid sequence of C3E-7088 (SEQ ID NO: 49). C3E-7088 (1-269), FLAG-His tag (244-269). Amino acid sequence of C3E-7093 (SEQ ID NO: 50). C3E-7093 (1-269), FLAG-His tag (244-269). Amino acid sequence of C3E-7078 (SEQ ID NO: 51). C3E-7078 (1-269), FLAG-His tag (244-269). Amino acid sequence of full-length NYF-0014-HC2 (SEQ ID NO: 52). Signal sequence (1-19), NYA-0001 (21-266), linker (267-271), C3E-7085 (272-511), linker (512-513), Hinge (514-528), CH2 (529-638), CH3 (639-745). NYF-0082-HC2 full-length amino acid sequence (SEQ ID NO: 53). Signal sequence (1-19), NYA-0082 (21-266), linker (267-271), C3E-7085 (272-511), linker (512-513), Hinge (514-528), CH2 (529-638), CH3 (639-745). The amino acid sequence of the full-length NYF-0083-HC2 (SEQ ID NO: 54). Signal sequence (1-19), NYA-2061 (21-266), linker (267-271), C3E-7095 (272-511), linker (512-513), Hinge (514-528), CH2 (529-638), CH3 (639-745). The amino acid sequence of the full-length NYZ-0082-HC2 (SEQ ID NO: 55). Signal sequence (1-19), NYA-3061 (21-271), linker (272-276), C3E-7096 (277-516), linker (517-518), Hinge (519-533), CH2 (534-643), CH3 (644-750). NYZ-0083-HC2 full-length amino acid sequence (SEQ ID NO: 56). Signal sequence (1-19), NYA-3061 (21-271), linker (272-276), C3E-7097 (277-516), linker (517-518), Hinge (519-533), CH2 (534-643), CH3 (644-750). Full-length amino acid sequence of NYZ-1010-HC2 (SEQ ID NO: 57).Signal sequence (1-19), NYA-3061 (21-271), linker (272-276), C3E-7085-VH (277-394), CH1 (395-492), Hinge (493-507), CH2 (508-617), CH3 (618-724). Amino acid sequence of full-length C3E-7085-LC (SEQ ID NO: 58). Signal sequence (1-20), C3E-7085_VL (21-127), CL (128-233). Amino acid sequence of full-length NYA-3061 (SEQ ID NO: 59). Signal sequence (1-19), NYA-3061 (21-271), FLAG-His tag (272-297). The amino acid sequence of the full-length NYZ-1007-HC2 (SEQ ID NO: 60). Signal sequence (1-19), NYA-2061 (21-266), linker (267-271), C3E-7085-VH (272-389), CH1 (390-487), Hinge (488-502), CH2 (503-612), CH3 (613-719). The amino acid sequence of the full-length NYZ-1017-HC2 (SEQ ID NO: 61). Signal sequence (1-19), NYA-2047 (21-266), linker (267-271), C3E-7085-VH (272-389), CH1 (390-487), Hinge (488-502), CH2 (503-612), CH3 (613-719). Full-length amino acid sequence of C3E-7096 (SEQ ID NO: 62). Full-length amino acid sequence of C3E-7097 (SEQ ID NO: 63). Full-length amino acid sequence of C3E-7098 (SEQ ID NO: 64). Full-length amino acid sequence of C3E-7099 (SEQ ID NO: 65). Amino acid sequence of NY-ESO (SEQ ID NO: 66). Amino acid sequence of a truncated form of HLA-A*0201 (GenBank: ASA47534.1) (SEQ ID NO: 67). Amino acid sequence of β2-microglobulin (SEQ ID NO: 68). Amino acid sequence of NYA-1143-VH02 (SEQ ID NO: 69), amino acid sequence of NYA-1143-VH03 (SEQ ID NO: 70), amino acid sequence of full-length NYA-1154 (SEQ ID NO: 71), signal sequence (1-19), NYA-1154 (21-266), FLAG-His tag (267-292).Figure 72(1) is a diagram showing the antitumor activity of the Fc-tagged anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 administered alone, in combination with lenvatinib and pembrolizumab, and in combination with NYZ-1010, lenvatinib, and pembrolizumab in a human T cell transfer model. Error bars in the figure indicate standard error (n=5). Figure 72(2) is a diagram showing the rate of change in tumor volume on Day 52 for each individual, with the tumor volume on Day 28 as the baseline, in a human T cell transfer model using Fc-tagged anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, co-administration of lenvatinib and pembrolizumab, and co-administration of NYZ-1010, levanib, and pembrolizumab. Figure 73(1) is a diagram showing the anti-tumor activity of Fc-tagged anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, anti-mouse PD-1 antibody alone, and co-administration of NYZ-1010 and anti-mouse PD-1 antibody in a BALB / C human CD3ε knock-in mouse model. Error bars in the figure indicate standard error (n = 5 to 10). Figure 73(2) shows the estimated tumor volume for each individual in a BALB / C strain human CD3ε knock-in mouse model treated with an Fc-tagged anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, an anti-mouse PD-1 antibody alone, and a combination of NYZ-1010 and an anti-mouse PD-1 antibody, as well as the estimated tumor volume after re-implantation of CT26 NY-SCT in mice with complete regression and naive mice. Figure 74(1) shows the anti-tumor activity of an Fc-tagged anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, durvalumab alone, and a combination of NYZ-1010 and durvalumab in a human T cell transfer model. The error bars in the figure indicate the standard error (n=5).Figure 74(2) shows the percent change in tumor volume on Day 49 for each individual, with the baseline tumor volume on Day 24, in a human T cell transfer model treated with an Fc-tagged anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, Durvalumab alone, and a combination of NYZ-1010 and Durvalumab. Figure 75(1) shows the anti-tumor activity of an Fc-tagged anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, an anti-mouse PD-L1 antibody alone, and a combination of NYZ-1010 and anti-mouse PD-L1 antibody in a BALB / C human CD3ε knock-in mouse model. Error bars in the figure indicate standard error (n = 5 to 10). Figure 75(2) shows the estimated tumor volume for each individual in a BALB / C human CD3ε knock-in mouse model treated with an Fc-tagged anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, an anti-mouse CTLA-4 antibody alone, and a combination of NYZ-1010 and an anti-mouse CTLA-4 antibody, as well as the estimated tumor volume after re-implantation of CT26 Mock tumors in mice that showed complete regression and naive mice. Figure 76(1) shows the anti-tumor activity of a BALB / C human CD3ε knock-in mouse model treated with an Fc-tagged anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, an anti-mouse CTLA-4 antibody alone, and a combination of NYZ-1010 and an anti-mouse CTLA-4 antibody. Error bars in the figure indicate standard error (n = 5 to 10). Figure 76(2) shows the estimated tumor volume for each individual in a BALB / C human CD3ε knock-in mouse model treated with an Fc-tagged anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, an anti-mouse CTLA-4 antibody alone, and a combination of NYZ-1010 and an anti-mouse CTLA-4 antibody, as well as the estimated tumor volume after re-implantation of CT26 NY-SCT in mice that showed complete regression and naive mice.Figure 77(1) shows the antitumor activity of Fc-tagged anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, anti-mouse TIGIT antibody alone, and combined administration of NYZ-1010 and anti-mouse TIGIT antibody in a BALB / C human CD3ε knock-in mouse model. Error bars in the figure indicate standard error (n = 5 to 10). Figure 77(2) shows the estimated tumor volume for each individual in a BALB / C human CD3ε knock-in mouse model treated with an Fc-tagged anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, anti-mouse TIGIT antibody alone, and a combination of NYZ-1010 and anti-mouse TIGIT antibody, as well as the estimated tumor volume after re-implantation of CT26 NY-SCT in mice with complete regression and naive mice. Figure 78 shows the antitumor activity of an Fc-tagged anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, carboplatin alone, and a combination of NYZ-1010 and carboplatin in a human T cell transfer model. Error bars in the figure indicate standard error (n=5). Figure 79(1) shows the antitumor activity of Fc-tagged anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, sacituzumab govitecan-hziy alone, and the combination of NYZ-1010 and sacituzumab govitecan-hziy in a human T cell transfer model. Error bars in the figure indicate standard error (n=5). Figure 79(2) shows the percent change in tumor volume on Day 21 for each individual, using the baseline tumor volume on Day 6, in a human T cell transfer model administered with an Fc-tagged anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, sacituzumab govitecan-hziy alone, and a combination of NYZ-1010 and sacituzumab govitecan-hziy. Figure 80(1) shows the antitumor activity of an Fc-tagged anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, doxorubicin alone, and a combination of NYZ-1010 and doxorubicin in a human T cell transfer model.Error bars in the figure indicate standard error (n=5). Figure 80(2) is a diagram showing the rate of change in tumor volume on Day 55 for each individual, with the tumor volume on Day 40 as the baseline, in a human T cell transfer model administered with an Fc-tagged anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, doxorubicin alone, and a combination of NYZ-1010 and doxorubicin. Figure 81(1) is a diagram showing the antitumor activity of an Fc-tagged anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, decitabine alone, and a combination of NYZ-1010 and decitabine in a human T cell transfer model. Error bars in the figure indicate standard error (n=5). Figure 81 (2) is a diagram showing the rate of change in tumor volume on Day 32 for each individual in a human T cell transfer model using the tumor volume on Day 7 as the baseline in the administration of an Fc-tagged anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, NYZ-1010 alone, Decitabine alone, and NYZ-1010 and Decitabine in combination. Sequence listing of Japanese Patent Application No. 2022-041253 filed on March 16, 2022.

[0024] The present invention is described in detail below. 1. Definitions In the present invention, the term "gene" refers to a nucleotide chain containing a base sequence encoding the amino acids of a protein, or its complementary chain. For example, the term "gene" includes polynucleotides, oligonucleotides, DNA, mRNA, cDNA, cRNA, etc., which are nucleotide chains containing a base sequence encoding the amino acids of a protein, or their complementary chains. Such genes are single-stranded, double-stranded, triple- or more-stranded nucleotides, and the term "gene" also includes an assembly of a DNA chain and an RNA chain, a nucleotide chain in which ribonucleotides (RNA) and deoxyribonucleotides (DNA) are mixed, and double-stranded, triple- or more-stranded nucleotides containing such nucleotide chains. In the present invention, the terms "base sequence" and "nucleotide sequence" have the same meaning.

[0025] In the present invention, "polynucleotide," "nucleotide chain," "nucleic acid," and "nucleic acid molecule" are synonymous, and for example, DNA, RNA, probes, oligonucleotides, primers, etc. are also included in the meaning of "polynucleotide." Such polynucleotides are single-stranded, double-stranded, or composed of three or more strands, and the meaning of "polynucleotide" also includes an aggregate of a DNA strand and an RNA strand, a polynucleotide in which ribonucleotides (RNA) and deoxyribonucleotides (DNA) are mixed on a single polynucleotide strand, and an aggregate of two or more strands containing such a polynucleotide chain.

[0026] In the present invention, the terms "polypeptide," "peptide," and "protein" have the same meaning.

[0027] In the present invention, the term "antigen" may be used to mean "immunogen."

[0028] In the present invention, the term "cells" also includes various cells derived from individual animals, subcultured cells, primary cultured cells, cell lines, recombinant cells, microorganisms, and the like.

[0029] In the present invention, the term "antibody" is synonymous with immunoglobulin. However, when referring to the anti-HLA / NY-ESO antibody of the present invention, the term "antibody" is used to mean an immunoglobulin having a constant region and a variable region. There are no particular limitations on whether the antibody is a natural antibody or a partially or completely synthetically produced immunoglobulin. The anti-HLA / NY-ESO antibody of the present invention is included in the "molecule" described below.

[0030] In the present invention, the term "NY-ESO peptide" refers to a peptide consisting of 9 amino acids from the 157th to the 165th positions of NY-ESO-1 and LAGE-1 (SLLMWITQC: SEQ ID NO: 1).

[0031] In the present invention, "HLA-A2 / NY-ESO" means a complex of NY-ESO peptide and Histocompatibility Leukocyte Antigen-A2 (HLA-A2), and is also referred to as "HLA / NY-ESO."

[0032] In the present invention, "anti-HLA-A2 / NY-ESO antibody" refers to an antibody that binds to HLA-A2 / NY-ESO, in other words, an antibody that recognizes HLA-A2 / NY-ESO. Similarly, "anti-HLA-A2 / NY-ESO scFv" refers to an scFv that binds to HLA / NY-ESO, in other words, that recognizes HLA-A2 / NY-ESO. "Anti-HLA-A2 / NY-ESO antibody" and "anti-HLA-A2 / NY-ESO scFv" are also referred to as "anti-HLA / NY-ESO antibody" and "anti-HLA / NY-ESO scFv," respectively.

[0033] The basic four-chain antibody structure consists of two identical light chains (L chains) and two identical heavy chains (H chains). The light chains are linked to the heavy chains by a single covalent disulfide bond. The two heavy chains are linked to each other by one or more disulfide bonds, depending on the heavy chain isotype. Each light chain and heavy chain has regularly spaced intrachain disulfide bonds. Heavy and light chains have constant regions with very similar amino acid sequences and variable regions with less similar amino acid sequences. Light chains have a variable region (VL) at the amino terminus followed by a constant region (CL). Heavy chains have a variable region (VH) at the amino terminus followed by three constant regions (CH1 / CH2 / CH3). The VL and VH are paired, and the CL is aligned with the first constant region (CH1) of the heavy chain. The paired VL and VH form a single antigen-binding site.

[0034] The constant region of the antibody of the present invention is not particularly limited, but is preferably a human antibody when used to treat or prevent a human disease. Examples of heavy chain constant regions of human antibodies include Cγ1, Cγ2, Cγ3, Cγ4, Cμ, Cδ, Cα1, Cα2, and Cε. Examples of light chain constant regions of human antibodies include Cκ and Cλ.

[0035] Fab consists of a heavy chain VH followed by CH1 and a light chain VL followed by CL, which contain the complementarity-determining regions (CDRs).

[0036] The Fc (also referred to as the Fc region) is the carboxyl-terminal region of the heavy chain constant region, comprising CH2 and CH3, and is a dimer. The Fc of the present invention may be a native sequence Fc or a mutant Fc in which the native sequence has been mutated (referred to as a "mutant Fc"). In the multispecific and bispecific molecules of the present invention, a preferred Fc region is a mutant Fc, more preferably a pair of Fc that can form a heterodimer. Examples of the pair of Fc include a combination of Fc(i) contained in the first polypeptide and Fc(ii) contained in the second polypeptide, as described below, but are not limited thereto, as long as the pair of Fc can associate (form a heterodimer).

[0037] Examples of variant Fc include modified Fc regions (including heterodimeric Fc regions) contained in heteromultimers with improved stability, as disclosed in WO 2013 / 063702, Fc containing a CH3 region of an immunoglobulin derived from an IgG antibody having "protrusions" and "cavities" contained in heteromultimers, as disclosed in WO 1996 / 27011, Fc containing a CH3 domain contained in a heterodimer that is electrostatically favored by substituting one or more amino acid residues with charged amino acids, as disclosed in WO 2009 / 089004, heterodimeric Fc regions contained in heterodimers using conformational mutations and / or pI (isoelectric point) mutations, as disclosed in WO 2014 / 110601, and heterodimeric Fc containing a CH3 domain containing modifications that eliminate or reduce binding to Protein A, as disclosed in WO 2010 / 151792, and the like, but are not limited thereto.

[0038] The variable region consists of highly variable regions called hypervariable regions (HVRs) and relatively invariant regions called framework regions (FRs) separated by the hypervariable regions. Natural heavy and light chain variable regions contain four FRs connected by three hypervariable regions, and the hypervariable regions of each chain are held in close proximity with the hypervariable regions of the other chains by the FRs, contributing to the formation of the antigen-binding site of antibodies.

[0039] It is known that the heavy and light chains of an antibody molecule each contain three complementarity determining regions (CDRs). The complementarity determining regions, also known as hypervariable regions, are located within the variable regions of the heavy and light chains of an antibody and are regions with particularly high variability in their primary structure. They are usually separated into three regions in the primary structure of the heavy and light polypeptide chains. In the present invention, the complementarity determining regions of an antibody are referred to as CDRH1, CDRH2, and CDRH3 from the amino-terminal end of the heavy chain amino acid sequence, and CDRL1, CDRL2, and CDRL3 from the amino-terminal end of the light chain amino acid sequence. These regions are adjacent to each other in the three-dimensional structure and determine the specificity for the antigen to which they bind.

[0040] In the present invention, the positions and lengths of CDRs were determined according to the IMGT definition (Developmental and Comparative Immunology 27 (2003) 55-77).

[0041] FR refers to the variable region other than the CDR residues. A variable region generally has four FRs: FR1, FR2, FR3, and FR4.

[0042] The CDRs and FRs in the heavy and light chains are arranged from the amino terminus to the carboxyl terminus in the following order: FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4 and FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4, respectively.

[0043] The positions of CDR and FR can be determined by various definitions known in the art, such as the definitions of Kabat, Chothia, AbM, contact, etc., in addition to the IMGT.

[0044] In the present invention, the term "antigen-binding fragment of an antibody" refers to a partial fragment of an antibody that is composed of a heavy chain variable region and a light chain variable region and has antigen-binding activity. Examples of "antibody antigen-binding fragments" include Fab, F(ab') and the like. ２Examples of antigen-binding fragments include, but are not limited to, scFv, Fab', Fv, and single-domain antibodies (sdAb). Such antibody antigen-binding fragments may be obtained by treating the full-length antibody protein molecule with an enzyme such as papain or pepsin, as well as recombinant proteins produced in suitable host cells using recombinant genes. In the present invention, the term "antibody-binding fragment" is synonymous with the term "antigen-binding fragment of an antibody."

[0045] In the present invention, the "site" to which an antibody binds, i.e., the "site" recognized by an antibody, refers to a partial peptide or partial higher-order structure on an antigen to which the antibody binds or recognizes. In the present invention, such a site is also referred to as an epitope or antibody-binding site. In the present invention, a "mutated antibody" refers to a polypeptide that has an amino acid sequence obtained by substitution, deletion, or addition (addition includes insertion) (hereinafter collectively referred to as "mutation") of amino acids in the amino acid sequence of the original antibody, and that binds to the HLA / NY-ESO of the present invention. The number of mutated amino acids in such a mutant antibody is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, or 50. Such mutant antibodies are also encompassed by the "antibody" of the present invention.

[0046] In the present invention, the term "several" in "one or several" refers to 2 to 10.

[0047] In the present invention, the term "multispecific molecule" refers to any molecule containing two or more portions capable of binding to multiple, mutually distinct epitopes and / or different epitopes on two or more molecules. Preferably, two or more of these portions are "multispecific antibodies," each of which is an antibody or antigen-binding fragment thereof, or a molecule containing a "multispecific antibody." Multispecific antibodies may contain one or more heavy chain variable regions (VH) and / or light chain variable regions (VL). They may be full-length antibodies having two or more different heavy and light chains, i.e., IgG-type multispecific antibodies, or antibody-related molecules consisting of antigen-binding fragments having two or more VLs and VHs, such as combinations of Fab, Fab', Fv, scFv, sdAb, etc., or molecules derived therefrom (e.g., tandem scFv, diabodies, single-chain diabodies, triabodies, etc.). Multispecific antibodies may also contain portions that do not have an immunoglobulin scaffold.

[0048] Examples of the activities and properties exhibited by the anti-HLA / NY-ESO antibody of the present invention or an antigen-binding fragment of the antibody, or the molecule of the present invention include biological activities and physicochemical properties (also referred to as physical properties). Specific examples include various biological activities such as cytotoxic activity, ADCC activity, and antitumor activity (all of which will be described later), binding activity to antigens or epitopes, stability during production and storage, and thermal stability.

[0049] In the present invention, "hybridizing under stringent conditions" means hybridization at 65°C in a solution containing 5xSSC, followed by washing at 65°C for 20 minutes in an aqueous solution containing 2xSSC-0.1% SDS, at 65°C for 20 minutes in an aqueous solution containing 0.5xSSC-0.1% SDS, and at 65°C for 20 minutes in an aqueous solution containing 0.2xSSC-0.1% SDS, or hybridization under equivalent conditions. SSC is an aqueous solution of 150 mM NaCl and 15 mM sodium citrate, and nxSSC means n-fold concentrated SSC.

[0050] In the present invention, "cytotoxicity" refers to bringing about some form of pathological change in cells, and refers not only to direct trauma but also to any damage to the structure or function of cells, such as DNA breakage, formation of base dimers, chromosome breakage, damage to the cell division apparatus, and reduced activity of various enzymes. In the present invention, "cytotoxic activity" refers to causing the above-mentioned cell damage.

[0051] In the present invention, the term "antibody-dependent cellular cytotoxicity" refers to "antibody-dependent cellular cytotoxicity (ADCC) activity" and means the activity of NK cells to damage target cells such as tumor cells via antibodies.

[0052] In the present invention, "cytotoxic activity through T cell redirection" means that the above-mentioned cytotoxicity is caused via a multispecific molecule comprising an anti-tumor antigen antibody and an anti-HLA / NY-ESO antibody. That is, the anti-tumor antigen antibody binds to the target tumor cell, and the anti-HLA / NY-ESO antibody binds to the T cell, thereby shortening the distance between the target tumor cell and the T cell, thereby inducing cytotoxicity via T cell activation. The molecule can be contained in a pharmaceutical composition.

[0053] 2. Antigens 2-1. HLA / NY-ESO Antigen In the present invention, "HLA / NY-ESO" is used synonymously with HLA / NY-ESO protein.

[0054] HLA / NY-ESO is a ternary complex of HLA-A2, β2-microglobulin, and NY-ESO peptide. HLA-A2 is a type of HLA allele and is known to be the most frequent allele in Caucasians. HLA forms a ternary complex of β2-microglobulin and a peptide fragment of a self-protein in the endoplasmic reticulum of a cell, presents it extracellularly, and is recognized by the TCR (T cell receptor) of a T cell. NY-ESO peptide (SLLMWITQC: SEQ ID NO: 66) is a peptide consisting of nine amino acids from positions 157 to 165 of NY-ESO-1 and LAGE-1, and has been reported to be presented by HLA-A2.

[0055] 2-2. CD3 Antigen In the present invention, "CD3" is used synonymously with CD3 protein. CD3 is expressed on T cells as part of the multimolecular T cell receptor complex and is a complex of five polypeptides: γ chain, δ chain, ε chain, ζ chain, and η chain (molecular weights of 25,000-28,000, 21,000, 20,000, 16,000, and 22,000, respectively). The CD3 complex includes γ, δ, ε, ζ, and η chains. These are also referred to as subunits. Binding of anti-CD3 antibodies to T cells induces cytotoxicity via T cell activation. Many anti-CD3 antibodies bind to CD3ε. The nucleotide sequence of cDNA encoding human CD3ε has been registered in NCBI / GenBank under accession number NM_000733 (NM_000733.3), and the amino acid sequence of human CD3ε has been registered in NCBI / GenPept under accession number NP_000724 (NM_000724.1). The nucleotide sequence of cDNA encoding cynomolgus monkey CD3 has been registered in GenBank under accession number NM_001283615.1. The amino acid sequence of human CD3ε is shown in Figure 10.

[0056] 2-3. Preparation of Antigens The antigen proteins used in the present invention, namely HLA / NY-ESO and CD3 (hereinafter, HLA / NY-ESO and CD3 are also collectively referred to as the antigen proteins), can be prepared by purification and isolation from animal tissues (including body fluids), cells derived from the tissues, or cell cultures thereof, genetic recombination, in vitro translation, chemical synthesis, etc. The cDNA of the antigen protein can be obtained, for example, by the so-called PCR method, in which a cDNA library of an organ expressing the mRNA of the antigen protein is used as a template and primers that specifically amplify the cDNA of the antigen protein are used to perform a polymerase chain reaction (hereinafter referred to as "PCR") (Saiki, R. K., et al., Science (1988) 239, 487-49). The cDNA of the antigen protein also includes a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence encoding the antigen protein expressed in humans or rats and encodes a protein having biological activity equivalent to that of the antigen protein. Furthermore, the cDNA of the antigen protein also includes a splicing variant transcribed from the antigen protein gene locus expressed in humans or rats, or a polynucleotide that hybridizes thereto under stringent conditions and encodes a protein having biological activity equivalent to that of the antigen protein. The nucleotide sequence of the antigen protein gene also includes a nucleotide sequence that encodes a protein having biological activity equivalent to that of the antigen protein, consisting of an amino acid sequence in which one to several amino acids have been substituted, deleted, or added in the amino acid sequence of the human or rat antigen protein, or an amino acid sequence from which the signal sequence has been removed. The antigen protein also includes an amino acid sequence encoded by a splicing variant transcribed from the gene locus of the antigen protein in humans or rats, or an amino acid sequence in which one or several amino acids have been substituted, deleted, or added in the amino acid sequence, and which has biological activity equivalent to that of the antigen protein.

[0057] 2-4 Binding Specificity to Antigen Protein The anti-HLA / NY-ESO antibodies and antigen-binding fragments thereof, etc. of the present invention recognize HLA / NY-ESO. That is, they bind to the HLA / NY-ESO antigen. HLA / NY-ESO is not known to exist in non-human animals such as mice, rats, and cynomolgus monkeys. The anti-CD3 antibodies and binding fragments thereof, etc. included in the multispecific antibodies of the present invention recognize, i.e., bind to the CD3 antigen. Such anti-CD3 antibodies, etc. preferably bind to human CD3, monkey CD3, etc., and more preferably bind to human CD3 and cynomolgus monkey CD3. On the other hand, such preferred anti-CD3 antibodies do not bind to rat and / or mouse CD3.

[0058] In the present invention, "recognition", i.e., "binding", means binding that is not nonspecific adsorption. The criterion for determining whether or not recognition has occurred, i.e., whether or not binding has occurred, can be, for example, the dissociation constant (hereinafter referred to as "KD"). The KD value of the preferred antibodies of the present invention for HLA / NY-ESO or CD3 is 1 x 10 －５ M or less, 5×10 －６ M or less, 2×10 －６ M or less, 1×10 －６ M or less, and the preferred KD value for HLA / NY-ESO is 5×10 －７ M or less, 2×10 －７ M or less, 1×10 －７ M or less, 5×10 －８ M or less, 2×10 －８ M or less, 1×10 －８ M or less, 5×10 －９ M or less, or 2 x 10 －９ M or less, and more preferably 1×10 －９ Examples of anti-HLA / NY-ESO scFv of the present invention having excellent antigen-binding activity include NYA-1143, NYA-2023, NYA-2143, NYA-2044, NYA-2045, NYA-2060, NYA-2061, and NYA-3061. The KD values ​​for HLA / NY-ESO of NYA-1143, NYA-2044, NYA-2045, NYA-2143, and NYA-1154 are 1 x 10 －９M or less. The binding between the antigen and the antibody in the present invention can be measured or determined by a biomolecular interaction analysis system such as SPR or BLI, or by ELISA, RIA, or the like (see WO2021 / 200857). Among the anti-HLA / NY-ESO antibodies of the present invention, examples of those having excellent antigen binding specificity include the anti-HLA / NY-ESO scFvs NYA-0001, NYA-1143, NYA-1163, NYA-2023, NYA-2027, NYA-2035, NYA-2044, NYA-2045, NYA-2047, NYA-2048, NYA-2060, NYA-2061, NYA-2143, and NYA-3061.

[0059] 3. Antibodies or binding fragments thereof that specifically bind to HLA / NY-ESO 3-1 Anti-HLA / NY-ESO or binding fragments thereof The present invention provides antibodies or binding fragments thereof that recognize and bind to HLA / NY-ESO. As described above, HLA / NY-ESO is a complex containing HLA-A2 and the 9-mer NY-ESO peptide (SLLMWITQC: SEQ ID NO: 66). The NY-ESO peptide is a peptide derived from NY-ESO-1 or LAGE-1, which are intracellular proteins and cancer-testis antigens. HLA / NY-ESO is presented on the surface of cancer cells. The anti-HLA / NY-ESO antibodies and antigen-binding fragments thereof of the present invention (hereinafter also referred to as "antibodies of the present invention") may be either monoclonal or polyclonal antibodies. The isotype of the monoclonal antibody of the present invention is not particularly limited, and examples thereof include IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgM, IgA1, IgA2), IgD, and Ig. The isotype and subclass of a monoclonal antibody can be determined, for example, by the Ouchterlony method, ELISA, or RIA. Examples of the monoclonal antibody of the present invention include antibodies derived from non-human animals (non-human animal antibodies), human antibodies, chimeric antibodies (also referred to as "chimeric antibodies"), and humanized antibodies, with human antibodies being preferred. The scope of the antibodies of the present invention also includes antibody mutants ("mutant antibodies" described below). For example, the scope of human antibodies also includes human mutant antibodies. Examples of non-human animal antibodies include antibodies derived from vertebrates such as mammals and birds. Examples of mammal-derived antibodies include antibodies derived from rodents such as mouse antibodies and rat antibodies. Examples of avian-derived antibodies include chicken antibodies. Examples of chimeric antibodies include, but are not limited to, antibodies formed by combining a variable region derived from a non-human animal antibody with a constant region of a human antibody (human immunoglobulin).Examples of humanized antibodies include, but are not limited to, antibodies in which the CDRs in the variable regions of a non-human animal antibody have been transplanted onto a human antibody (the variable regions of human immunoglobulins), antibodies in which not only the CDRs but also part of the framework region sequence of a non-human animal antibody has been transplanted onto a human antibody, and antibodies in which one or more amino acids derived from any of these non-human animal antibodies have been replaced with human amino acids.

[0060] Antibodies can be produced by various known methods. Known methods include methods using hybridomas, cell-based immunization, and gene recombination techniques. Methods for obtaining human antibodies derived from phage display selected from a human antibody library are also known. For example, a phage display method can be used in which the variable regions of human antibodies are expressed on the surface of phages as scFvs and phages that bind to the antigen are selected. The DNA sequence encoding the variable regions of human antibodies that bind to the antigen can be determined by analyzing the genes of phages selected by binding to the antigen. Once the DNA sequence of an scFv that binds to an antigen has been determined, an expression vector containing that sequence can be constructed and introduced into an appropriate host for expression to obtain a human antibody (WO92 / 01047, WO92 / 20791, WO93 / 06213, WO93 / 11236, WO93 / 19172, WO95 / 01438, WO95 / 15388, Annu. Rev. Immunol (1994) 12, 433-455). Highly active antibodies thus obtained can also be used as lead antibodies, and the gene encoding the lead antibody can be mutated to produce mutants with even higher activity (the "mutated antibodies" described below).

[0061] Preferred examples of CDRH1 to CDRH3 contained in the heavy chain of the anti-HLA / NY-ESO antibody or antigen-binding fragment thereof of the present invention include a combination of CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 1, CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 2, and heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 3 or the amino acid sequence represented by SEQ ID NO: 3 in which the sixth amino acid is N (Asn).More preferably, the heavy chain variable region is a NYA-0001 heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 13, a NYA-0082 heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 15, a NYA-2023 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence represented by SEQ ID NO: 18, a NYA-2027 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence represented by SEQ ID NO: 19, a NYA-1143 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence represented by SEQ ID NO: 20, a NYA-1163 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence represented by SEQ ID NO: 17 ... a NYA-2035 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence represented by SEQ ID NO: 24; a NYA-2044 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence represented by SEQ ID NO: 24; a NYA-2045 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence represented by SEQ ID NO: 25; a NYA-2047 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence represented by SEQ ID NO: 26; a NYA-2048 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence represented by SEQ ID NO: 27; a NYA-2060 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence represented by SEQ ID NO: 28; a NYA-2061 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence represented by SEQ ID NO: 53; or a combination of CDRH1 to CDRH3 contained in a NYA-2143 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence represented by SEQ ID NO: 21. Further examples include a combination of CDRH1 to CDRH3 contained in the NYA-3061 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence represented by SEQ ID NO: 55, and a combination of CDRH1 to CDRH3 contained in the NYA-1154 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence represented by SEQ ID NO: 71.

[0062] Preferred examples of CDRL1 to CDRL3 contained in the light chain of the anti-HLA / NY-ESO antibody or antigen-binding fragment thereof of the present invention include a combination of CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 4 or the amino acid sequence set forth in SEQ ID NO: 4 in which the seventh amino acid is W (Trp) and / or the eighth amino acid is K (Lys); CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5; and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6 or the amino acid sequence set forth in SEQ ID NO: 6 in which the second amino acid is A (Ala) or S (Ser).

[0063] More preferably, the NYA-0001 light chain variable region consists of the amino acid sequence represented by SEQ ID NO: 14, the NYA-0082 light chain variable region consists of the amino acid sequence represented by SEQ ID NO: 16, the NYA-1143 light chain variable region consists of amino acid numbers 156 to 266 of the amino acid sequence represented by SEQ ID NO: 20, the NYA-1163 light chain variable region consists of amino acid numbers 156 to 266 of the amino acid sequence represented by SEQ ID NO: 26, the NYA-2023 light chain variable region consists of amino acid numbers 156 to 266 of the amino acid sequence represented by SEQ ID NO: 18, the NYA-2027 light chain variable region consists of amino acid numbers 156 to 266 of the amino acid sequence represented by SEQ ID NO: 19, the NYA-2035 light chain variable region consists of amino acid numbers 156 to 266 of the amino acid sequence represented by SEQ ID NO: 2 ...4, a NYA-2044 light chain variable region consisting of amino acid numbers 156 to 266 in the amino acid sequence represented by SEQ ID NO:25; a NYA-2045 light chain variable region consisting of amino acid numbers 156 to 266 in the amino acid sequence represented by SEQ ID NO:25; a NYA-2047 light chain variable region consisting of amino acid numbers 156 to 266 in the amino acid sequence represented by SEQ ID NO:26; a NYA-2048 light chain variable region consisting of amino acid numbers 156 to 266 in the amino acid sequence represented by SEQ ID NO:27; a NYA-2060 light chain variable region consisting of amino acid numbers 156 to 266 in the amino acid sequence represented by SEQ ID NO:28; a NYA-2061 light chain variable region consisting of amino acid numbers 156 to 266 in the amino acid sequence represented by SEQ ID NO:29; or a combination of CDRL1 to CDRL3 contained in a NYA-2143 light chain variable region consisting of amino acid numbers 156 to 266 in the amino acid sequence represented by SEQ ID NO:21. Further examples include a combination of CDRL1 to CDRL3 contained in the NYA-3061 light chain variable region consisting of amino acid numbers 161 to 271 of the amino acid sequence represented by SEQ ID NO:55, and a combination of CDRL1 to CDRL3 contained in the NYA-1154 light chain variable region consisting of amino acid numbers 156 to 266 of the amino acid sequence represented by SEQ ID NO:71.

[0064] Preferred examples of CDRH1 to CDRH3 contained in the heavy chain and CDRL1 to CDRL3 contained in the light chain of the anti-HLA / NY-ESO antibody or antigen-binding fragment thereof of the present invention include a combination of: CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 1; CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 2; heavy chain CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 3 or the amino acid sequence set forth in SEQ ID NO: 3 in which the sixth amino acid is N (Asn); CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 4 or the amino acid sequence set forth in SEQ ID NO: 4 in which the seventh amino acid is W (Trp) and / or the eighth amino acid is K (Lys); CDRL2 consisting of the amino acid sequence set forth in DNN (the fifth sequence from the top in Figure 11; SEQ ID NO: 5 in Figure 82); and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6 or the amino acid sequence set forth in SEQ ID NO: 6 in which the second amino acid is A (Ala) or S (Ser).More preferably, the heavy and light chain variable regions of NYA-0001 are composed of the amino acid sequences represented by SEQ ID NOs: 13 and 14, respectively; the heavy and light chain variable regions of NYA-1143 are composed of the amino acid sequences represented by SEQ ID NO: 20, respectively, and the heavy and light chain variable regions of NYA-1163 are composed of the amino acid sequences represented by SEQ ID NO: 17, respectively, and the heavy and light chain variable regions of NYA-1163 are composed of the amino acid sequences represented by SEQ ID NO: 18, respectively, and the heavy and light chain variable regions of NYA-0001 are composed of the amino acid sequences represented by SEQ ID NO: 13 and 14, respectively, and the light chain variable regions of NYA-1143 are composed of the amino acid sequences represented by SEQ ID NO: 20, respectively, and the light chain variable regions of NYA-1163 are composed of the amino acid sequences represented by SEQ ID NO: 17, respectively, and the light chain variable regions of NYA-1163 are composed of the amino acid sequences represented by SEQ ID NO: 18 ... NYA-2023 heavy and light chain variable regions consisting of amino acid numbers 21 to 140 and 156 to 266 of the amino acid sequence represented by SEQ ID NO: 19; NYA-2027 heavy and light chain variable regions consisting of amino acid numbers 21 to 140 and 156 to 266 of the amino acid sequence represented by SEQ ID NO: 19; NYA-2035 heavy and light chain variable regions consisting of amino acid numbers 21 to 140 and 156 to 266 of the amino acid sequence represented by SEQ ID NO: 22; and NYA-2044 heavy and light chain variable regions consisting of amino acid numbers 21 to 140 and 156 to 266 of the amino acid sequence represented by SEQ ID NO: 24. NYA-2045 heavy and light chain variable regions consisting of amino acid numbers 21 to 140 and 156 to 266 of the amino acid sequence represented by SEQ ID NO: 25; NYA-2047 heavy and light chain variable regions consisting of amino acid numbers 21 to 140 and 156 to 266 of the amino acid sequence represented by SEQ ID NO: 26; NYA-2048 heavy and light chain variable regions consisting of amino acid numbers 21 to 140 and 156 to 266 of the amino acid sequence represented by SEQ ID NO: 27; Examples of such a combination include the combinations of CDRH1 to CDRH3 and CDRL1 to CDRL3 contained in the NYA-2060 heavy and light chain variable regions consisting of amino acid numbers 21 to 140 and 156 to 266 of the amino acid sequence represented by SEQ ID NO:29, the NYA-2061 heavy and light chain variable regions consisting of amino acid numbers 21 to 140 and 156 to 266 of the amino acid sequence represented by SEQ ID NO:29, and the NYA-2143 heavy and light chain variable regions consisting of amino acid numbers 21 to 140 and 156 to 266 of the amino acid sequence represented by SEQ ID NO:21.Further examples include the combination of CDRH1 to CDRH3 and CDRL1 to CDRL3 contained in the NYA-3061 heavy chain variable region and light chain variable region, respectively, consisting of amino acid numbers 21 to 140 and 161 to 271 of the amino acid sequence represented by SEQ ID NO: 55, and the combination of CDRH1 to CDRH3 and CDRL1 to CDRL3 contained in the NYA-1154 heavy chain variable region and light chain variable region, respectively, consisting of amino acid numbers 21 to 140 and 156 to 266 of the amino acid sequence represented by SEQ ID NO: 71.

[0065] Preferred examples of the heavy chain variable region of the anti-HLA / NY-ESO antibody or antigen-binding fragment thereof of the present invention include those containing the above-mentioned heavy chain CDRs or a combination thereof. More preferred examples include the NYA-0001 heavy chain variable region, NYA-0082 heavy chain variable region, NYA-1143 heavy chain variable region, NYA-1163 heavy chain variable region, NYA-2023 heavy chain variable region, and NYA-2027 heavy chain variable region. Examples of heavy chain variable regions include NYA-2035 heavy chain variable region, NYA-2044 heavy chain variable region, NYA-2045 heavy chain variable region, NYA-2047 heavy chain variable region, NYA-2048 heavy chain variable region, NYA-2060 heavy chain variable region, NYA-2061 heavy chain variable region, NYA-2143 heavy chain variable region, NYA-3061 heavy chain variable region, and NYA-1154 heavy chain variable region. The amino acid sequences of the respective heavy chain variable regions are as described above.

[0066] Preferred examples of the light chain variable region of the anti-HLA / NY-ESO antibody or antigen-binding fragment thereof of the present invention include those containing the above-mentioned light chain CDRs or a combination thereof. More preferred examples include the NYA-0001 light chain variable region, NYA-0082 light chain variable region, NYA-1143 light chain variable region, NYA-1163 light chain variable region, NYA-2023 light chain variable region, and NYA-2027 light chain variable region. Examples of light chain variable regions include the NYA-2035 light chain variable region, the NYA-2044 light chain variable region, the NYA-2045 light chain variable region, the NYA-2047 light chain variable region, the NYA-2048 light chain variable region, the NYA-2060 light chain variable region, the NYA-2061 light chain variable region, the NYA-2143 light chain variable region, the NYA-3061 light chain variable region, and the NYA-1154 light chain variable region. The amino acid sequences of the respective light chain variable regions are as described above.

[0067] Preferred examples of the heavy chain variable region and light chain variable region of the anti-HLA / NY-ESO antibody or antigen-binding fragment thereof of the present invention include those comprising the above-mentioned heavy chain and light chain CDRs or a combination thereof. More preferred examples include the NYA-0001 heavy chain variable region and light chain variable region, NYA-0082 heavy chain variable region and light chain variable region, NYA-1143 heavy chain variable region and light chain variable region, NYA-1163 heavy chain variable region and light chain variable region, NYA-2023 heavy chain variable region and light chain variable region, NYA-2027 heavy chain variable region and light chain variable region, and NYA-2035 heavy chain variable region. Examples of such a combination include a heavy chain variable region and a light chain variable region, a NYA-2044 heavy chain variable region and a light chain variable region, a NYA-2045 heavy chain variable region and a light chain variable region, a NYA-2047 heavy chain variable region and a light chain variable region, a NYA-2048 heavy chain variable region and a light chain variable region, a NYA-2060 heavy chain variable region and a light chain variable region, a NYA-2061 heavy chain variable region and a light chain variable region, a NYA-2143 heavy chain variable region and a light chain variable region, a NYA-3061 heavy chain variable region and a light chain variable region, and a NYA-1154 heavy chain variable region and a light chain variable region.

[0068] Preferred examples of the heavy chain of the anti-HLA / NY-ESO antibody or antigen-binding fragment thereof of the present invention include those comprising the above-mentioned preferred or more preferred heavy chain variable regions, and more preferred examples include NYA-0001 heavy chain, NYA-0082 heavy chain, NYA-1143 heavy chain, NYA-1163 heavy chain, NYA-2023 heavy chain, NYA-2027 heavy chain, NYA-2035 heavy chain, NYA-2044 heavy chain, NYA-2045 heavy chain, NYA-2047 heavy chain, NYA-2048 heavy chain, NYA-2060 heavy chain, NYA-2061 heavy chain, NYA-2143 heavy chain, NYA-3061 heavy chain, and NYA-1154 heavy chain.

[0069] Preferred examples of the light chain of the anti-HLA / NY-ESO antibody or antigen-binding fragment thereof of the present invention include those comprising the above-mentioned preferred or more preferred light chain variable region, and more preferred examples include NYA-0001 light chain, NYA-0082 light chain, NYA-1143 light chain, NYA-1163 light chain, NYA-2023 light chain, NYA-2027 light chain, NYA-2035 light chain, NYA-2044 light chain, NYA-2045 light chain, NYA-2047 light chain, NYA-2048 light chain, NYA-2060 light chain, NYA-2061 light chain, NYA-2143 light chain, NYA-3061 light chain, and NYA-1154 light chain.

[0070] Preferred examples of the heavy and light chains of the anti-HLA / NY-ESO antibody or antigen-binding fragment thereof of the present invention include those containing the above-mentioned preferred or more preferred heavy chain variable regions and light chain variable regions, respectively. More preferred examples include the heavy chain and light chain combinations of NYA-0001, NYA-1143, NYA-1163, NYA-2023, NYA-2027, NYA-2035, NYA-2044, NYA-2045, NYA-2047, NYA-2048, NYA-2060, NYA-2061, NYA-2143, NYA-3061, and NYA-1154.

[0071] An antigen-binding fragment of an antibody refers to a fragment or modified product thereof that retains at least the antigen-binding ability of the antibody. Such antibody functions generally include antigen-binding activity, activity to regulate antigen activity, antibody-dependent cellular cytotoxicity, and complement-dependent cytotoxicity. Examples of functions of the antibodies of the present invention and multispecific molecules comprising the antibodies of the present invention include T cell redirection, T cell activation, and cytotoxicity against cancer cells by activating T cells.

[0072] The antigen-binding fragment of an antibody is not particularly limited as long as it is a fragment of the antibody that retains at least the antigen-binding activity of the antibody, and examples thereof include Fab, Fab', F(ab') ２ Examples of such antibodies include, but are not limited to, Fvs, single-chain Fvs (scFvs) in which heavy and light chain Fvs are linked via a suitable linker, and single-domain antibodies (sdAbs). Molecules containing portions other than the antigen-binding fragment of the antibody of the present invention, such as scFvs having a linker moiety, are also encompassed in the meaning of the antigen-binding fragment of the antibody of the present invention.

[0073] Molecules in which one or several or more amino acids have been deleted from the amino and / or carboxyl terminals of an antibody protein and which retain at least some of the functions of the antibody are also encompassed within the meaning of an antigen-binding fragment of an antibody. Modified forms of such antibody antigen-binding fragments are also encompassed within the scope of the antibodies or antigen-binding fragments thereof, or modified forms thereof (described below) of the present invention.

[0074] One embodiment of the antibody or antigen-binding fragment thereof of the present invention is an scFv. An scFv can be obtained by linking the heavy chain variable region and light chain variable region of an antibody with a polypeptide linker (Pluckthun A., The Pharmacology of Monoclonal Antibodies 113, edited by Rosenburg and Moore, Springer Verlag, New York, pp. 269-315 (1994); Nature Biotechnology (2005), 23, 1126-1136). Tandem scFvs, which are prepared by linking two scFvs with a polypeptide linker, can also be used as bispecific molecules. Furthermore, triabodies composed of three or more scFvs can also be used as multispecific molecules.

[0075] The scFv specific to HLA / NY-ESO (also referred to as "anti-HLA / NY-ESO scFv") preferably comprises the above-mentioned CDRH1 to CDRH3 and CDRL1 to CDRL3, more preferably comprises the above-mentioned heavy chain variable region and light chain variable region, and even more preferably comprises NYA-0001 (amino acid numbers 21 to 266 of the amino acid sequence represented by SEQ ID NO: 30), NYA-0082 (comprising the amino acid sequence represented by SEQ ID NO: 15 and the amino acid sequence represented by SEQ ID NO: 16), NYA-1143 (amino acid numbers 21 to 266 of the amino acid sequence represented by SEQ ID NO: 20), NYA-1163 (amino acid numbers 21 to 266 of the amino acid sequence represented by SEQ ID NO: 17), NYA-2023 (amino acid numbers 21 to 266 of the amino acid sequence represented by SEQ ID NO: 18), NYA-2027 (amino acid numbers 21 to 266 of the amino acid sequence represented by SEQ ID NO: 19), Examples of such amino acids include NYA-2035 (amino acid numbers 21 to 266 in the amino acid sequence represented by SEQ ID NO:22), NYA-2044 (amino acid numbers 21 to 266 in the amino acid sequence represented by SEQ ID NO:24), NYA-2045 (amino acid numbers 21 to 266 in the amino acid sequence represented by SEQ ID NO:25), NYA-2047 (amino acid numbers 21 to 266 in the amino acid sequence represented by SEQ ID NO:26), NYA-2048 (amino acid numbers 21 to 266 in the amino acid sequence represented by SEQ ID NO:27), NYA-2060 (amino acid numbers 21 to 266 in the amino acid sequence represented by SEQ ID NO:28), NYA-2061 (amino acid numbers 21 to 266 in the amino acid sequence represented by SEQ ID NO:29), and NYA-2143 (amino acid numbers 21 to 266 in the amino acid sequence represented by SEQ ID NO:21). Further examples include NYA-3061 (amino acid numbers 21 to 271 in the amino acid sequence represented by SEQ ID NO: 55) and NYA-1154 (amino acid numbers 21 to 266 in the amino acid sequence represented by SEQ ID NO: 71).

[0076] A preferred embodiment of the anti-HLA / NY-ESO scFv includes one to which a FLAG-His tag is fused at the carboxyl terminal side (also simply referred to as a "tagged form"). Suitable tagged products include NYA-0001 tagged product (amino acids 20-292 of SEQ ID NO:30), NYA-1143 tagged product (amino acids 20-292 of SEQ ID NO:20), NYA-1163 tagged product (amino acids 20-292 of SEQ ID NO:17), NYA-2023 tagged product (amino acids 20-292 of SEQ ID NO:18), NYA-2027 tagged product (amino acids 20-292 of SEQ ID NO:19), NYA-2035 tagged product (amino acids 20-292 of SEQ ID NO:22), NYA-2044 tagged product (amino acids 20-292 of SEQ ID NO:24), NYA-2045 tagged product (amino acids 20-292 of SEQ ID NO:25), NYA-2047 tagged product (amino acids 20-292 of SEQ ID NO:26), NYA-2048 tagged product (amino acids 20-292 of SEQ ID NO:27), NYA-2049 tagged product (amino acids 20-292 of SEQ ID NO:28), NYA-2050 tagged product (amino acids 20-292 of SEQ ID NO:29), NYA-2051 tagged product (amino acids 20-292 of SEQ ID NO:29), NYA-2052 tagged product (amino acids 20-292 of SEQ ID NO:29), NYA-2053 tagged product (amino acids 20-292 of SEQ ID NO:29), NYA-2054 tagged product (amino acids 20-292 of SEQ ID NO:29), NYA-2055 tagged product (amino acids 20-292 of SEQ ID NO:29), NYA-2056 tagged product (amino acids Examples of such tagged products include a tagged product (amino acid numbers 20 to 292 of SEQ ID NO:26), a NYA-2048 tagged product (amino acid numbers 20 to 292 of SEQ ID NO:27), a NYA-2060 tagged product (amino acid numbers 20 to 292 of SEQ ID NO:28), a NYA-2061 tagged product (amino acid numbers 20 to 292 of SEQ ID NO:29), a NYA-2143 tagged product (amino acid numbers 20 to 292 of SEQ ID NO:21), a NYA-3061 FLAG-His tagged product (an amino acid sequence comprising amino acid numbers 267 to 292 of SEQ ID NO:22 added to the carboxyl terminus of amino acid numbers 21 to 271 of SEQ ID NO:56), and a NYA-1154 tagged product (amino acid numbers 21 to 292 of SEQ ID NO:71).

[0077] Of these, NYA-2023 and tagged derivatives thereof, NYA-2047 and tagged derivatives thereof, NYA-2048 and tagged derivatives thereof, NYA-2060 and tagged derivatives thereof, and NYA-2061 and tagged derivatives thereof have excellent biological activity, physical properties, etc., and are more preferred as Fc-tagged bispecific molecules.

[0078] Furthermore, when anti-HLA / NY-ESO scFv and its tagged products are expressed in host cells, a signal peptide can be added to the amino terminus. Examples of the amino acid sequences of anti-HLA / NY-ESO scFv tagged products to which a signal peptide has been added include amino acid sequences of SEQ ID NOS: 30, 20, 17 to 19, 22, 24, 25, 26 to 29, and 21, and amino acid sequences in which a FLAG-His tag (amino acid numbers 267 to 292 of SEQ ID NO: 22) has been added to the carboxyl terminus of amino acid numbers 21 to 271 of SEQ ID NO: 56.

[0079] scFv can be obtained by the phage display method (Nature Biotechnology (2005), 23, (9), pp. 1105-1116), in which the variable region of an antibody is expressed on the surface of a phage as a single-chain antibody (scFv), and phages that bind to the antigen are selected. By analyzing the genes of phages selected by binding to the antigen, the DNA sequence encoding the variable region of a human antibody that binds to the antigen can be determined. Once the DNA sequence of an scFv that binds to an antigen is determined, an expression vector containing that sequence can be constructed and introduced into an appropriate host for expression to obtain a human antibody (WO92 / 01047, WO92 / 20791, WO93 / 06213, WO93 / 11236, WO93 / 19172, WO95 / 01438, WO95 / 15388, Annu. Rev. Immunol (1994) 12, pp. 433-455, Nature Biotechnology (2005) 23(9), pp. 1105-1116).

[0080] The antibody of the present invention may be an antibody having a single heavy chain variable region and no light chain sequence. Such antibodies are called single domain antibodies (sdAb) or nanobodies, and have been reported to retain antigen-binding ability (Muyldemans S. et al., Protein Eng., (1994) 7(9), 1129-35, Hamers-Casterman C. et al., Nature (1993) 363(6428), 446-448). These antibodies are also encompassed within the meaning of the antigen-binding fragment of an antibody of the present invention.

[0081] The present invention also includes single-chain immunoglobulins in which the full-length sequences of the heavy and light chains of an antibody are linked using an appropriate linker (Lee, H-S, et. al., Molecular Immunology (1999) 36, 61-71; Shirrmann, T. et. al., mAbs (2010), 2(1), 1-4). By dimerizing, such single-chain immunoglobulins can retain a structure and activity similar to that of antibodies that are originally tetrameric. The anti-HLA / NY-ESO antibody of the present invention may be a single-chain immunoglobulin.

[0082] In the scFv of the present invention, the heavy chain variable region and the light chain variable region may be disulfide-bonded.

[0083] The anti-HLA / NY-ESO antibodies of the present invention may be antibodies composed of portions derived from multiple different antibodies, as long as they bind to HLA / NY-ESO. Examples include antibodies in which the heavy and / or light chains are exchanged between multiple different antibodies, antibodies in which the entire heavy and / or light chains are exchanged, antibodies in which only the variable regions or only the constant regions are exchanged, and antibodies in which only all or part of the CDRs are exchanged. The heavy chain variable region and light chain variable region of a chimerized antibody may be derived from different anti-HLA / NY-ESO antibodies of the present invention. The heavy chain CDR1 to CDR3 and light chain CDR1 to CDR3 in the heavy and light chain variable regions of a humanized antibody may be derived from two or more anti-HLA / NY-ESO antibodies of the present invention. The heavy chain CDR1 to CDR3 and light chain CDR1 to CDR3 in the heavy and light chain variable regions of a human antibody may be a combination of CDRs possessed by two or more anti-HLA / NY-ESO antibodies of the present invention.

[0084] The anti-HLA / NY-ESO antibodies of the present invention also include antibodies that contain an amino acid sequence encoded by a nucleotide sequence contained in a polynucleotide that hybridizes under stringent conditions with a complementary strand of a polynucleotide that contains a nucleotide sequence encoding an amino acid sequence contained in the anti-HLA / NY-ESO antibody of the present invention, and that bind to HLA / NY-ESO.

[0085] An amino acid sequence contained in the heavy chain variable region of the anti-HLA / NY-ESO antibody or antigen-binding fragment thereof of the present invention (preferably the amino acid sequence of amino acid numbers 21 to 140 in the amino acid sequence represented by SEQ ID NO: 18, the amino acid sequence represented by SEQ ID NO: 69 or SEQ ID NO: 70, the amino acid sequence represented by SEQ ID NO: 39, or the amino acid sequence of amino acid numbers 21 to 140 in the amino acid sequence represented by SEQ ID NO: 57, SEQ ID NO: 60, or SEQ ID NO: 61), and / or an amino acid sequence contained in the light chain variable region (preferably the amino acid sequence of amino acid numbers 156 to 26 in the amino acid sequence represented by SEQ ID NO: 18). the amino acid sequence of SEQ ID NO:6, the amino acid sequence of amino acid numbers 156 to 266 in the amino acid sequence represented by SEQ ID NO:28, the amino acid sequence represented by SEQ ID NO:23, the amino acid sequence of amino acid numbers 161 to 271 in the amino acid sequence represented by SEQ ID NO:57, or the amino acid sequence of amino acid numbers 156 to 266 in the amino acid sequence represented by SEQ ID NO:60 or SEQ ID NO:61), and which binds to HLA / NY-ESO, or an antigen-binding fragment thereof.

[0086] When the position and length of the light chain variable region are determined using a definition different from that of IMGT (e.g., Kabat, Chothia, AbM, contact, etc.), the carboxyl terminus of the light chain variable region amino acid sequence determined according to the IMGT definition may further contain one or more amino acids, such as arginine or glycine. Antibodies or binding fragments thereof having such light chain variable regions are also encompassed by the antibodies or binding fragments thereof of the present invention.

[0087] The antibodies of the present invention may be those in which the binding ability to HLA / NY-ESO, particularly human and / or cynomolgus monkey HLA / NY-ESO, has been optimized by introducing mutations into the binding fragment of the anti-HLA / NY-ESO antibody of the present invention. Specific methods for introducing mutations include random mutagenesis using error-prone PCR, site-specific amino acid mutagenesis using an NNK library, site-specific mutagenesis using structural information, and combinations thereof.

[0088] 3-2. Anti-HLA / NY-ESO Antibody Mutants (Mutant Antibodies) Mutant anti-HLA / NY-ESO antibodies of the present invention may preferably be modified to reduce susceptibility to proteolysis or oxidation, maintain, improve, or inhibit decline or change in biological activity or function, improve or regulate antigen-binding ability, or be endowed with physicochemical or functional properties. It is known that changes in specific amino acid side chains on the surface of proteins can alter the function or activity of the protein; examples of such changes include deamidation of asparagine side chains and isomerization of aspartic acid side chains. Mutant antibodies of the present invention also include those in which a different amino acid is substituted to prevent such changes in amino acid side chains.

[0089] An example of a mutant antibody of the present invention is an antibody having an amino acid sequence in which conservative amino acid substitutions have been made within a group of amino acids that are related in their side chains.

[0090] Preferred amino acid groups are as follows: acidic group = aspartic acid, glutamic acid; basic group = lysine, arginine, histidine; nonpolar group = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and uncharged polar group = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Other preferred amino acid groups are as follows: aliphatic hydroxy group = serine and threonine; amide-containing group = asparagine and glutamine; aliphatic group = alanine, valine, leucine, and isoleucine; and aromatic group = phenylalanine, tryptophan, and tyrosine. The amino acid substitutions in such mutant antibodies are preferably made within a range that does not reduce the antigen-binding activity of the original antibody.

[0091] The anti-HLA / NY-ESO antibodies, antigen-binding fragments thereof, variants thereof (mutated antibodies or antigen-binding fragments thereof), or molecules of the present invention also include mutant antibodies, antigen-binding fragments thereof, and molecules comprising them, which have amino acid sequences in which conservative amino acid substitutions and / or other mutations have been made in the amino acid sequence of the antibodies of the present invention, such as NYA-2023, and which bind to HLA / NY-ESO; and chimerized antibodies, humanized antibodies, human antibodies, antigen-binding fragments thereof, and molecules comprising them, which have amino acid sequences in which conservative amino acid substitutions and / or other mutations have been made in the amino acid sequence of any of CDRH1 to CDRH3 and CDRL1 to CDRL3 derived from the antibodies of the present invention, including NYA-2023, and which contain the CDRs that bind to HLA / NY-ESO.

[0092] 3-3. Anti-HLA / NY-ESO Antibody Binding Fragment One aspect of the present invention provides an antigen-binding fragment of the anti-HLA / NY-ESO antibody of the present invention (hereinafter simply referred to as "binding fragment"). The anti-HLA / NY-ESO antibody binding fragment of the present invention includes binding fragments of chimeric antibodies, humanized antibodies, or human antibodies. An antibody binding fragment refers to a fragment or modified product thereof that retains at least the antigen-binding ability of the antibody. Such antibody functions generally include antigen-binding activity, activity that regulates antigen activity (e.g., agonist activity), activity that internalizes antigens into cells, and activity that inhibits or promotes interaction between antigens and substances that interact with them.

[0093] The antibody binding fragment is not particularly limited as long as it is a fragment of the antibody that retains at least the antigen-binding activity of the antibody. Examples of such antibody binding fragments include Fab, Fab', F(ab') and the like. ２Examples of such antibodies include, but are not limited to, single-chain Fab (scFab), in which the carboxyl terminus of a Fab light chain and the amino terminus of a Fab heavy chain are linked via a suitable linker; single-chain Fv (scFv), in which the heavy and light chain Fvs are linked via a suitable linker; single-domain antibodies (sdAb), which have a single heavy-chain variable region and no light-chain sequence; or nanobodies (Muyldemans S. et al., Protein Eng., (1994) 7(9), 1129-35; Hamers-Casterman C. et al., Nature (1993) 363(6428), 446-448). Molecules containing moieties other than the binding fragment of the antibody of the present invention, such as scFab and scFv having a linker moiety, are also encompassed within the meaning of the binding fragment of the antibody of the present invention.

[0094] 3-4. Modified Anti-HLA / NY-ESO Antibody or Binding Fragment Thereof, and Conjugates The present invention provides modified antibodies or binding fragments thereof. Modified antibodies or binding fragments thereof of the present invention refer to antibodies or binding fragments thereof that have been chemically or biologically modified. Chemical modifications include attachment of chemical moieties to the amino acid backbone, chemical modifications of N- or O-linked carbohydrate chains, etc. Biological modifications include post-translational modifications (e.g., N- or O-linked glycosylation, processing of the amino- or carboxyl-terminal region, deamidation, aspartic acid isomerization, and methionine oxidation), and modifications in which a methionine residue is added to the amino terminus by expression in a prokaryotic host cell. Also included within the meaning of such modifications are those labeled to enable detection or isolation of the antibodies or antigens of the present invention, such as enzyme-labeled, fluorescent-labeled, and affinity-labeled antibodies. Such modified antibodies or binding fragments thereof of the present invention are useful for improving the stability and blood retention of the original antibodies or binding fragments thereof of the present invention, reducing antigenicity, detecting or isolating such antibodies or antigens, etc.

[0095] Examples of chemical moieties contained in the chemically modified substance include water-soluble polymers such as polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymer, carboxymethyl cellulose, dextran, and polyvinyl alcohol.

[0096] Examples of biologically modified products include those modified by enzymatic treatment or cell treatment, fusion products to which other peptides such as tags have been added by genetic recombination, and products prepared using cells that express endogenous or exogenous glycosylation enzymes as hosts.

[0097] Such modifications may be made at any or desired positions in the antibody or binding fragment thereof, and the same or two or more different modifications may be made at one or more positions.

[0098] However, deletion of these heavy chain sequences or modification of the heavy or light chain sequences does not significantly affect the antigen-binding ability and effector functions (such as complement activation and antibody-dependent cellular cytotoxicity) of the antibody.

[0099] Thus, the present invention also encompasses antibodies that have undergone such deletion or modification. Examples include deletion variants in which one or two amino acids are deleted from the carboxyl terminus of the heavy chain (Journal of Chromatography A; 705; 129-134 (1995)), deletion variants in which two amino acid residues, glycine and lysine, are deleted from the carboxyl terminus of the heavy chain and amidation of a new proline residue at the carboxyl terminus (Analytical Biochemistry, 360: 75-83 (2007)), and antibodies in which glutamine or glutamic acid residues at the amino termini of the heavy or light chain of the antibody are modified by pyroglutamylation (International Patent Publication WO 2013 / 147153) (collectively referred to as "deletion variants"). However, as long as the antigen-binding ability and effector function are maintained, the deletion variants of the carboxyl termini of the heavy and light chains of the antibody of the present invention are not limited to the above types. When the antibody of the present invention comprises two or more chains (e.g., heavy chains), the two or more chains (e.g., heavy chains) may be any one type of heavy chain selected from the group consisting of full-length and the above-mentioned deletion forms, or a combination of any two types. Although the quantitative or molecular ratio of each deletion form may be affected by the type of cultured mammalian cells producing the antibody of the present invention and the culture conditions, an example of a major component of the antibody of the present invention is one in which one amino acid residue is deleted at the carboxyl terminus in both of the two heavy chains.

[0100] Furthermore, even if one to several amino acids derived from an expression vector and / or a signal sequence, etc., are added to the amino and / or carboxyl terminus of an antibody or antigen-binding fragment thereof (such as those included in the multispecific antibody of the present invention) (and some or all of these are modified as described above), so long as the desired antigen-binding activity is maintained, the modified antibody or antigen-binding fragment thereof is included in the scope of the present invention, and multispecific antibodies comprising such modified antibodies or antigen-binding fragments are also included in the scope of the present invention.

[0101] In the present invention, the term "antibody or antigen-binding fragment thereof" also includes within its meaning "modified antibody or antigen-binding fragment thereof." Furthermore, the term "antibody or antigen-binding fragment thereof" included in the multispecific antibody of the present invention also includes within its meaning such "modified antibody or antigen-binding fragment thereof."

[0102] Furthermore, antibody-dependent cellular cytotoxicity can be enhanced by modulating the sugar chain modification (glycosylation, defucosylation, etc.) attached to the antibody of the present invention. Techniques for modulating the sugar chain modification of antibodies are known, including, but not limited to, those disclosed in International Patent Publications WO99 / 54342, WO00 / 61739, and WO02 / 31140.

[0103] The present invention also includes conjugates (immunoconjugates) in which the above-mentioned antibodies are linked to other molecules via a linker. Examples of antibody-drug conjugates in which the antibodies are linked to radioactive substances or pharmacological compounds (drugs) include ADCs (Antibody-Drug Conjugates) (Methods Mol Biol. (2013) 1045:1-27; Nature Biotechnology (2005) 23, pp. 1137-1146).

[0104] Furthermore, the present invention also includes conjugates in which these antibodies are linked to other functional polypeptides. Examples of such antibody-peptide conjugates include conjugates in which the antibodies are linked to albumin-binding polypeptides (Protein Eng Des Sel. (2012) (2): 81-8).

[0105] The antibodies of the present invention include modified antibodies, antibodies with regulated glycosylation, and conjugates, and binding fragments of the modified antibodies, antibodies with regulated glycosylation, and conjugates are included in binding fragments of the antibodies of the present invention. 4. Method for Producing Antibodies The antibodies of the present invention can be produced in cells as recombinant antibodies, for example, by inserting DNA encoding the heavy chain variable region or DNA encoding the light chain variable region into an expression vector, transforming host cells for expression with the vector, and culturing the host cells.

[0106] The DNA encoding the antibody can be obtained by linking DNA encoding a heavy chain variable region with DNA encoding a heavy chain constant region, and can be obtained by linking DNA encoding a light chain variable region with DNA encoding a light chain constant region.

[0107] The anti-HLA / NY-ESO antibody of the present invention can be produced by inserting the DNA encoding the heavy chain and the DNA encoding the light chain into an expression vector, transforming a host cell with the vector, and culturing the host cell. In this case, the DNA encoding the heavy chain and the DNA encoding the light chain may be introduced into the same expression vector and used to transform the host cell, or the DNA encoding the heavy chain and the DNA encoding the light chain may be inserted into separate vectors and used to transform the host cell. In this case, the DNA encoding the heavy chain variable region and the DNA encoding the light chain variable region may be introduced into a vector previously containing DNA encoding the heavy chain constant region and DNA encoding the light chain constant region. The vector may also contain DNA encoding a signal peptide that promotes antibody secretion from the host cell. In this case, the DNA encoding the signal peptide and the DNA encoding the antibody are linked in-frame. After antibody production, the signal peptide is removed to obtain the antibody as a mature protein.

[0108] In this case, the DNA encoding the heavy chain variable region, the DNA encoding the light chain variable region, the DNA in which the DNA encoding the heavy chain variable region and the DNA encoding the heavy chain constant region are linked, and the DNA in which the DNA encoding the light chain variable region and the DNA encoding the light chain constant region are linked may be functionally linked to elements such as a promoter, an enhancer, a polyadenylation signal, etc. Here, "functionally linked" means that the elements are linked so as to perform their functions.

[0109] The expression vector is not particularly limited as long as it can be replicated in a host such as an animal cell, bacteria, or yeast, and examples thereof include known plasmids and phages. Examples of vectors used to construct an expression vector include pcDNA™ (ThermoFisher SCIENTIFIC), Flexi™ Vector (Promega), pUC19, pUEX2 (Amersham), pGEX-4T, pKK233-2 (Pharmacia), and pMAM-neo (Clontech). Prokaryotic cells such as Escherichia coli and Bacillus subtilis, as well as eukaryotic cells such as yeast and animal cells, can be used as host cells, but eukaryotic cells are preferred. For example, HEK293 cells, a human embryonic kidney cell line, or Chinese hamster ovary (CHO) cells may be used as animal cells. The expression vector can be introduced into host cells by known methods, resulting in transformation of the host cells. Examples of such methods include electroporation, calcium phosphate precipitation, and DEAE-dextran transfection. The produced antibodies can be purified using separation and purification methods commonly used for proteins. For example, affinity chromatography, other chromatographies, filters, ultrafiltration, salting out, dialysis, and the like may be appropriately selected and combined.

[0110] 5. Multispecific Antibodies that Bind to HLA / NY-ESO The multispecific antibodies of the present invention include the anti-HLA / NY-ESO antibodies of the present invention or antigen-binding fragments thereof. The multispecific antibodies of the present invention are preferably multispecific antibodies having two or more antigen-binding sites. That is, they are multispecific antibodies capable of binding to two or more different epitopes on a single molecule or to different epitopes on two or more molecules, and encompass multiple different antigen-binding fragments. Such multispecific antibodies include, but are not limited to, IgG-type multispecific antibodies, multispecific antibodies having two or more types of variable regions, antibody fragments such as tandem scFv (taFv), single-chain diabodies, diabodies, and triabodies, and antibody fragments linked by covalent or non-covalent bonds. The multispecific antibodies may also contain an Fc domain.

[0111] The multispecific antibody of the present invention may comprise, in addition to the anti-HLA / NY-ESO antibody of the present invention or an antigen-binding fragment thereof, one or more additional antibodies or antigen-binding fragments thereof. Examples of the antigen-binding fragments of the additional antibodies include Fab, F(ab)', Fv, scFv, and sdAb.

[0112] Preferred multispecific antibodies of the invention further comprise anti-CD3 antibodies or antigen-binding fragments thereof, which also specifically bind to CD3.

[0113] The anti-CD3 antibody or antigen-binding fragment thereof included in the multispecific antibody of the present invention is not particularly limited as long as it binds to human CD3, but preferably also binds to CD3 of non-human primates such as cynomolgus monkeys. More preferred examples of anti-CD3 antibodies or antigen-binding fragments thereof include antibodies or antigen-binding fragments thereof comprising a heavy chain variable region CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO:7, a heavy chain variable region CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO:8, a heavy chain variable region CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO:9, a light chain variable region CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO:10, a light chain variable region CDRL2 consisting of the amino acid sequence set forth in RDD (the fifth sequence from the top in Figure 12 : SEQ ID NO:11 in Figure 82 ), and a light chain variable region CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO:12.

[0114] More preferred antibodies or antigen-binding fragments thereof comprising CDRH1 to CDRH3 and CDRL1 to CDRL3 include a C3E-7034 heavy chain variable region consisting of the amino acid sequence of amino acid numbers 2 to 119 in the amino acid sequence represented by SEQ ID NO:46, a C3E-7036 heavy chain variable region consisting of the amino acid sequence of amino acid numbers 2 to 119 in the amino acid sequence represented by SEQ ID NO:47, a C3E-7085 heavy chain variable region consisting of the amino acid sequence of amino acid numbers 2 to 119 in the amino acid sequence represented by SEQ ID NO:48, and a C3E-7088 heavy chain variable region consisting of the amino acid sequence of amino acid numbers 2 to 119 in the amino acid sequence represented by SEQ ID NO:49. Examples of antibodies or antigen-binding fragments thereof include a heavy chain variable region, a C3E-7093 heavy chain variable region consisting of the amino acid sequence of amino acids 2 to 119 in the amino acid sequence represented by SEQ ID NO: 140, a C3E-7096 heavy chain variable region consisting of the amino acid sequence of amino acids 272 to 389 in the amino acid sequence represented by SEQ ID NO: 54, a C3E-7096 heavy chain variable region consisting of the amino acid sequence of amino acids 277 to 394 in the amino acid sequence represented by SEQ ID NO: 55, and a C3E-7097 heavy chain variable region consisting of the amino acid sequence of amino acids 277 to 394 in the amino acid sequence represented by SEQ ID NO: 56.

[0115] Furthermore, more preferred antibodies or antigen-binding fragments thereof comprising CDRH1 to CDRH3 and CDRL1 to CDRL3 include a C3E-7034 light chain variable region consisting of the amino acid sequence of amino acid numbers 135 to 243 in the amino acid sequence represented by SEQ ID NO: 46, a C3E-7036 light chain variable region consisting of the amino acid sequence of amino acid numbers 135 to 241 in the amino acid sequence represented by SEQ ID NO: 47, a C3E-7085 light chain variable region consisting of the amino acid sequence of amino acid numbers 135 to 241 in the amino acid sequence represented by SEQ ID NO: 48, a C3E-7086 light chain variable region consisting of the amino acid sequence of amino acid numbers 135 to 243 in the amino acid sequence represented by SEQ ID NO: 49, and a C3E-7090 light chain variable region consisting of the amino acid sequence of amino acid numbers 135 to 243 in the amino acid sequence represented by SEQ ID NO: 50. Examples of antibodies or antigen-binding fragments thereof include a C3E-7088 light chain variable region, a C3E-7093 light chain variable region consisting of the amino acid sequence of amino acids 135 to 243 in the amino acid sequence represented by SEQ ID NO: 50, a C3E-7096 light chain variable region consisting of the amino acid sequence of amino acids 405 to 511 in the amino acid sequence represented by SEQ ID NO: 54, a C3E-7096 light chain variable region consisting of the amino acid sequence of amino acids 410 to 516 in the amino acid sequence represented by SEQ ID NO: 55, and a C3E-7097 light chain variable region consisting of the amino acid sequence of amino acids 410 to 516 in the amino acid sequence represented by SEQ ID NO: 56.

[0116] Furthermore, more preferred antibodies or antigen-binding fragments thereof comprising CDRH1 to CDRH3 and CDRL1 to CDRL3 include a combination of C3E-7034 heavy chain variable region and light chain variable region consisting of amino acid numbers 2 to 119 and 135 to 243 represented by SEQ ID NO: 46, a combination of C3E-7036 heavy chain variable region and light chain variable region consisting of amino acid numbers 2 to 119 and 135 to 241 represented by SEQ ID NO: 47, a combination of C3E-7078 heavy chain variable region and light chain variable region consisting of amino acid numbers 2 to 119 and 135 to 243 represented by SEQ ID NO: 51, a combination of C3E-7085 heavy chain variable region and light chain variable region consisting of amino acid numbers 2 to 119 and 135 to 241 represented by SEQ ID NO: 48, a combination of C3E-7085 heavy chain variable region and light chain variable region consisting of amino acid numbers 2 to 119 and 135 to 241 represented by SEQ ID NO: 49, a combination of C3E-7086 heavy chain variable region and light chain variable region consisting of amino acid numbers 2 to 119 and 135 to 241 represented by SEQ ID NO: 51, a combination of C3E-7087 heavy chain variable region and light chain variable region consisting of amino acid numbers 2 to 119 and 135 to 241 represented by SEQ ID NO: 52, a combination of C3E-7088 heavy chain variable region and light chain variable region consisting of amino acid numbers 2 to 119 and 135 to 241 represented by SEQ ID NO: 53, a combination of C3E-7089 heavy chain variable region and light chain variable region consisting of amino acid numbers 2 to 119 and 135 to 241 represented by SEQ ID NO: 54, a combination of C3 and 135 to 243 of the C3E-7088 heavy chain variable region and light chain variable region represented by SEQ ID NO:50; a combination of a C3E-7093 heavy chain variable region and light chain variable region represented by SEQ ID NO:50; a combination of a C3E-7096 heavy chain variable region and light chain variable region represented by SEQ ID NO:54; a combination of a C3E-7096 heavy chain variable region and light chain variable region represented by SEQ ID NO:55; and a combination of a C3E-7097 heavy chain variable region and light chain variable region represented by SEQ ID NO:56.

[0117] Further, examples of more suitable antibodies or antigen-binding fragments thereof comprising CDRH1 to CDRH3 and CDRL1 to CDRL3 include C3E-7034scFv consisting of the amino acid sequence of amino acid numbers 2 to 243 in the amino acid sequence represented by SEQ ID NO: 46; C3E-7036scFv consisting of the amino acid sequence of amino acid numbers 2 to 241 in the amino acid sequence represented by SEQ ID NO: 47; C3E-7078scFv consisting of the amino acid sequence of amino acid numbers 2 to 243 in the amino acid sequence represented by SEQ ID NO: 51; C3E-7085scFv consisting of the amino acid sequence of amino acid numbers 2 to 241 in the amino acid sequence represented by SEQ ID NO: 48; C3E-7088scFv consisting of the amino acid sequence of amino acid numbers 2 to 243 in the amino acid sequence represented by SEQ ID NO: 49; C3E-7093scFv consisting of the amino acid sequence of amino acid numbers 2 to 243 in the amino acid sequence represented by SEQ ID NO: 50; and antibodies or antibody-binding fragments comprising any one of these scFvs. A preferred embodiment of the CD3-specific scFv (also referred to as "anti-CD3 scFv") includes one to which a FLAG-His tag has been added at the carboxyl terminus (also simply referred to as "tagged form"). Preferred tagged forms include C3E-7034 (SEQ ID NO: 46), C3E-7036 (SEQ ID NO: 47), C3E-7085 (SEQ ID NO: 48), C3E-7088 (SEQ ID NO: 49), and C3E-7093 (SEQ ID NO: 50), with C3E-7085 being more preferred.

[0118] A suitable example of a multispecific molecule of the present invention is a bispecific molecule. "Bispecific" means capable of binding to two different epitopes on the same molecule or to different epitopes on two molecules, and includes antibodies or antigen-binding fragments having such dual specificities. The bispecific molecule of the present invention binds to HLA / NY-ESO and also binds to CD3.

[0119] Bispecific molecules of the present invention include those having the following structures (formats):

[0120] In a dual scFv-type bispecific molecule, two scFvs that bind to different epitopes are linked to one of the Fc moieties of the dimer via a linker or directly linked without a linker. Alternatively, two scFvs that bind to different epitopes are linked to the CH and CL via a linker, and then to one of the Fc moieties of the dimer via a linker. This bispecific molecule is a heterodimer-associated format in which two different scFvs each have an Fc downstream that has a mutation that allows them to form a heterodimer. Dual scFv-type bispecific molecules are referred to as dual-type bispecific molecules, or simply as dual-type molecules (Figure 7(7b)).

[0121] In the present invention, for example, a dual-type bispecific molecule consisting of an anti-HLA-A2 / NY-ESO scFv and an anti-CD3 scFv may be used.

[0122] Alternatively, the bispecific molecule of the present invention may be a bispecific molecule in which a Fab and an scFv that bind to different epitopes are linked via a linker, with a Fab of a first antibody attached to one side of a dimeric Fc and an scFv of a second antibody attached to the other side. This bispecific molecule is a heterodimer-associated format in which Fc fragments containing mutations that allow heterodimer formation are attached downstream of the Fab and scFv, respectively. Such bispecific molecules are referred to as hybrid bispecific molecules or hybrid types (Figure 7(7a)). In the present invention, for example, a hybrid type consisting of an anti-HLA-A2 / NY-ESO Fab and an anti-CD3 scFv can be used.

[0123] Furthermore, the bispecific molecule may be one in which the Fab of a first antibody and the scFv of a second antibody are linked via a linker to one of the Fc segments of a dimer. In this case, the Fab may be linked to the Fc and the scFv may be linked to the Fab, or the scFv may be linked to the Fc and the Fab may be linked to the scFv. Preferably, the Fab is linked to the Fab and the scFv is linked to the Fab. The Fab and scFv may be linked to the variable region of the Fab via a linker. This bispecific molecule is in a format in which an Fc containing a mutation that forms a heterodimer is associated downstream of the scFv and Fab linked by a linker. Such a bispecific molecule is referred to as an scFv-Fab-heterodimer Fc-type bispecific molecule or an scFv-Fab-heterodimer Fc-type (Figure 7(7c)).

[0124] In the present invention, for example, an scFv-Fab-heterodimer Fc type consisting of an anti-CD3 scFv and an anti-HLA-A2 / NY-ESO Fab may be used.

[0125] Furthermore, a taFv (Figure 5(5c)), which has a format in which two types of scFv, a first antibody and a second antibody, are linked by a linker, may be directly linked to one of the Fc domains of the dimer, with or without a linker. Such a bispecific molecule is called a taFv-heterodimer Fc type bispecific molecule or a taFv-heterodimer Fc type (Figure 5(5d)). This bispecific molecule is in a format in which an Fc having a mutation that allows heterodimer formation downstream of the taFv is heterodimer-associated. The order in which the first antibody and the second antibody are linked in the taFv is not limited. However, if the order in which the first antibody and the second antibody are linked in the taFv is reversed, the initial bispecific molecule will be called a taFv-heterodimer Fc type, while the resulting molecule will be called a taFv(inversed)-heterodimer Fc type (also called a taFv(inversed)-Fc type).

[0126] Figure 7(7a) shows the structure of a hybrid bispecific molecule, Figure 7(7b) shows the structure of a dual bispecific antibody, and Figure 7(7c) shows the structure of an scFv-Fab-heterodimer Fc bispecific antibody. Figure 5(5a) shows the structure of an scFv, Figure 5(5b) shows the structure of an Fab, Figure 5(5c) shows the structure of a taFv, Figure 5(5d) shows the structure of a taFv-heterodimer Fc bispecific antibody, and Figure 5(5e) shows the structure of a taFv-Fab-heterodimer Fc bispecific antibody. Furthermore, Figure 8 (8a) shows the structure of a taFv-heterodimer Fc-type bispecific antibody (same as Figure 5 (5d)), Figure 8 (8b) shows the structure of a taFv(inversed)-heterodimer Fc-type bispecific antibody, Figure 9 (9a) shows the structure of the first polypeptide contained in the taFv(inversed)-heterodimer Fc-type bispecific antibody, and Figure 9 (9b) shows the structure of the second polypeptide contained in the taFv(inversed)-heterodimer Fc-type bispecific antibody.

[0127] The bispecific antibody of the present invention has a structure in which multiple polypeptides are associated.

[0128] In the present invention, for example, taFvs may be composed of an anti-HLA-A2 / NY-ESO scFv and an anti-CD3 scFv. A taFv-heterodimer Fc-type bispecific antibody preferably comprises (a) a first polypeptide comprising, from the N-terminus to the C-terminus, an scFv that specifically binds to HLA / NY-ESO, an scFv that specifically binds to CD3, and an immunoglobulin Fc region (i), in that order, and a second polypeptide comprising an immunoglobulin hinge region and an Fc region (ii). More preferably, (b) the first polypeptide and the second polypeptide are associated with each other at the Fc region (i) and the Fc region (ii). The Fc regions of the first polypeptide and the second polypeptide may contain mutations that allow for heterodimer formation. An example of a taFv-heterodimer Fc-type bispecific antibody is shown in Figure 5 (5d). As shown in Figure 5(5d), the Fc region (i) of the first polypeptide binds to the Fc region (ii) of the second polypeptide, which is shown in black, thereby associating the first and second polypeptides. Figure 6(6a) shows the first polypeptide, and Figure 6(6b) shows the second polypeptide. For example, in Figure 5(5d), the scFv shown in white is an anti-HLA-A2 / NY-ESO scFv, and the scFv shown with a diagonal line in the upper right corner is an anti-CD3 scFv.

[0129] More preferred first polypeptides included in the taFv-heterodimer Fc-type bispecific antibodies of the present invention include the amino acid sequence of positions 21 to 511 in the amino acid sequence represented by SEQ ID NO: 32, the amino acid sequence of positions 21 to 511 in the amino acid sequence represented by SEQ ID NO: 34, the amino acid sequence of positions 21 to 511 in the amino acid sequence represented by SEQ ID NO: 35, the amino acid sequence of positions 21 to 511 in the amino acid sequence represented by SEQ ID NO: 36, the amino acid sequence of positions 21 to 511 in the amino acid sequence represented by SEQ ID NO: 37, the amino acid sequence of positions 21 to 511 in the amino acid sequence represented by SEQ ID NO: 38, the amino acid sequence of positions 21 to 511 in the amino acid sequence represented by SEQ ID NO: 39, the amino acid sequence of positions 21 to 511 in the amino acid sequence represented by SEQ ID NO: 40, the amino acid sequence of positions 21 to 511 in the amino acid sequence represented by SEQ ID NO: 41, the amino acid sequence of positions 21 to 511 in the amino acid sequence represented by SEQ ID NO: 42, the amino acid sequence of positions 21 to 511 in the amino acid sequence represented by SEQ ID NO: 43, the amino acid sequence of positions 21 to 511 in the amino acid sequence represented by SEQ ID NO: 33, and the amino acid sequence represented by SEQ ID NO: 52. the amino acid sequence of positions 21 to 511 of the amino acid sequence represented by SEQ ID NO: 53, the amino acid sequence of positions 20 to 511 of the amino acid sequence represented by SEQ ID NO: 54, the amino acid sequence of positions 20 to 516 of the amino acid sequence represented by SEQ ID NO: 55, or the amino acid sequence of positions 20 to 516 of the amino acid sequence represented by SEQ ID NO: 56; and more preferably, the first polypeptide contained in the taFv-heterodimer Fc-type bispecific molecule is selected from the group consisting of SEQ ID NOs: 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 32, 52, and 53 comprise the amino acid sequence of positions 529 to 745 of the amino acid sequence represented by SEQ ID NO: 54, or the amino acid sequence of positions 534 to 750 of the amino acid sequence represented by SEQ ID NO: 55 or 56; and even more preferably, the first polypeptide contained in the taFv-heterodimer Fc-type bispecific molecule comprises the amino acid sequence of positions 21 to 745 of the amino acid sequence represented by SEQ ID NO: 32, the amino acid sequence of positions 21 to 745 of the amino acid sequence represented by SEQ ID NO: 34, the amino acid sequence of positions 21 to 745 of the amino acid sequence represented by SEQ ID NO: 35,The amino acid sequence of amino acids 21 to 745 of the amino acid sequence represented by SEQ ID NO: 36, the amino acid sequence of amino acids 21 to 745 of the amino acid sequence represented by SEQ ID NO: 37, the amino acid sequence of amino acids 21 to 745 of the amino acid sequence represented by SEQ ID NO: 38, the amino acid sequence of amino acids 21 to 745 of the amino acid sequence represented by SEQ ID NO: 39, the amino acid sequence of amino acids 21 to 745 of the amino acid sequence represented by SEQ ID NO: 40, the amino acid sequence of amino acids 21 to 745 of the amino acid sequence represented by SEQ ID NO: 41, the amino acid sequence of amino acids 21 to 745 of the amino acid sequence represented by SEQ ID NO: 42, the amino acid sequence of amino acids 21 to 745 of the amino acid sequence represented by SEQ ID NO: 43, the amino acid sequence of amino acids 21 to 745 of the amino acid sequence represented by SEQ ID NO: 33), the amino acid sequence of amino acids 21 to 745 of the amino acid sequence represented by SEQ ID NO: 52, the amino acid sequence of amino acids 21 to 745 of the amino acid sequence represented by SEQ ID NO: 53, or the amino acid sequence of amino acids 20 to 745 of the amino acid sequence represented by SEQ ID NO: 54. Alternatively, it consists of the amino acid sequence from the 20th to the 750th amino acid of the amino acid sequence represented by SEQ ID NO: 55 or the amino acid sequence from the 20th to the 750th amino acid of the amino acid sequence represented by SEQ ID NO: 56.

[0130] A preferred second polypeptide comprised in a taFv-heterodimer Fc-type bispecific molecule of the present invention comprises a hinge region and a variant Fc derived from a human antibody, and an even more preferred second polypeptide comprised in a taFv-heterodimer Fc-type bispecific molecule comprises the amino acid sequence from positions 20 to 246 of the amino acid sequence set forth in SEQ ID NO: 31.

[0131] Among these, NYF-0016 is formed by associating a first polypeptide consisting of the amino acid sequence of positions 21 to 745 in the amino acid sequence shown in SEQ ID NO: 32 with a second polypeptide consisting of positions 21 to 246 in the amino acid sequence shown in SEQ ID NO: 31; NYF-0022 is formed by associating a first polypeptide consisting of the amino acid sequence of positions 21 to 745 in the amino acid sequence shown in SEQ ID NO: 84 with a second polypeptide consisting of positions 21 to 246 in the amino acid sequence shown in SEQ ID NO: 31; NYF-0023 is formed by associating a first polypeptide consisting of the amino acid sequence of positions 21 to 745 in the amino acid sequence shown in SEQ ID NO: 35 with a second polypeptide consisting of positions 21 to 246 in the amino acid sequence shown in SEQ ID NO: 31; NYF-0027 is formed by associating a first polypeptide consisting of the amino acid sequence of positions 21 to 745 in the amino acid sequence shown in SEQ ID NO: 36 with a second polypeptide consisting of positions 21 to 246 in the amino acid sequence shown in SEQ ID NO: 31; NYF-0035 is formed by associating a first polypeptide consisting of the amino acid sequence of positions 21 to 246 of the amino acid sequence shown in SEQ ID NO: 31 with a second polypeptide consisting of positions 21 to 246 of the amino acid sequence shown in SEQ ID NO: 31; NYF-0044 is formed by associating a first polypeptide consisting of the amino acid sequence of positions 21 to 745 of the amino acid sequence shown in SEQ ID NO: 38 with a second polypeptide consisting of positions 21 to 246 of the amino acid sequence shown in SEQ ID NO: 31; NYF-0045, which is formed by associating a first polypeptide consisting of the amino acid sequence of positions 21 to 745 in the amino acid sequence shown in SEQ ID NO:40 with a second polypeptide consisting of positions 21 to 246 in the amino acid sequence shown in SEQ ID NO:31; NYF-0047, which is formed by associating a first polypeptide consisting of the amino acid sequence of positions 21 to 745 in the amino acid sequence shown in SEQ ID NO:41 with a second polypeptide consisting of positions 21 to 246 in the amino acid sequence shown in SEQ ID NO:31;NYF-0060 is an association of a first polypeptide consisting of the amino acid sequence of positions 21 to 745 of the amino acid sequence shown in SEQ ID NO:42 and a second polypeptide consisting of positions 21 to 246 of the amino acid sequence shown in SEQ ID NO:31; NYF-0061 is an association of a first polypeptide consisting of the amino acid sequence of positions 21 to 745 of the amino acid sequence shown in SEQ ID NO:43 and a second polypeptide consisting of positions 21 to 246 of the amino acid sequence shown in SEQ ID NO:31; and NYF-0062 is an association of a first polypeptide consisting of the amino acid sequence of positions 21 to 745 of the amino acid sequence shown in SEQ ID NO:33 and a second polypeptide consisting of positions 21 to 246 of the amino acid sequence shown in SEQ ID NO:31. NYF-0019, which is formed by associating a second polypeptide consisting of the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 52, and a first polypeptide consisting of the 21st to 246th amino acid sequence represented by SEQ ID NO: 31, which is formed by associating a second polypeptide consisting of the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 31. NYF-0014, which is formed by associating a first polypeptide consisting of the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 53, and a second polypeptide consisting of the 21st to 246th amino acid sequence represented by SEQ ID NO: 31, which can be exemplified as suitable taFv-heterodimer Fc-type bispecific antibodies of the present invention. Further examples of suitable taFv-heterodimer Fc-type bispecific antibodies of the present invention include NYZ-0038, which is formed by associating a first polypeptide consisting of the amino acid sequence of positions 20 to 745 in the amino acid sequence shown in SEQ ID NO:54 with a second polypeptide consisting of positions 20 to 246 in the amino acid sequence shown in SEQ ID NO:31; NYZ-0082, which is formed by associating a first polypeptide consisting of the amino acid sequence of positions 20 to 750 in the amino acid sequence shown in SEQ ID NO:55 with a second polypeptide consisting of positions 20 to 246 in the amino acid sequence shown in SEQ ID NO:31; and NYZ-0083, which is formed by associating a first polypeptide consisting of the amino acid sequence of positions 20 to 750 in the amino acid sequence shown in SEQ ID NO:56 with a second polypeptide consisting of positions 20 to 246 in the amino acid sequence shown in SEQ ID NO:31.

[0132] Of these, NYF-0023, NYF-0047, NYF-0048, NYF-0060, NYF-0061, NYZ-0038, NYZ-0082, and NYZ-0083 have particularly excellent biological activity, physical properties, and the like, and are therefore preferred.

[0133] Furthermore, a taFv of a first antibody may be directly linked to one of the Fc domains of the dimer, with or without a linker, and a Fab of the first antibody or the second antibody may be directly linked to the other Fc domain, with or without a linker. This bispecific molecule is a format in which a Fab is added upstream of the Fc region (ii) (blackened) of the second polypeptide of a taFv-heterodimer Fc type. Such a bispecific molecule is referred to as a taFv-Fab-heterodimer Fc type bispecific molecule or a taFv-Fab-heterodimer Fc type (Figure 5(5e)).

[0134] In the present invention, the taFv contained in the taFv-Fab-heterodimer Fc-type bispecific molecule may be, for example, an anti-HLA-A2 / NY-ESO scFv and an anti-CD3 scFv taFv, and the Fab may be, for example, an HLA / NY-ESO Fab.

[0135] The taFv-Fab-heterodimer Fc-type bispecific antibody preferably comprises (a) a first polypeptide comprising, from the N-terminus to the C-terminus, an scFv that specifically binds to human HLA / NY-ESO, an scFv that specifically binds to CD3, and an immunoglobulin Fc region (i), in that order; a second polypeptide consisting of an immunoglobulin heavy chain comprising an Fc region (ii); and a third polypeptide consisting of an immunoglobulin light chain, more preferably (b) the second polypeptide and the third polypeptide are associated with each other; and (c) the first polypeptide and the second polypeptide are associated with each other at the Fc region (i) and the Fc region (ii). An example of a taFv-Fab-heterodimer Fc-type bispecific antibody is shown in Figure 5(5e), ​​the first polypeptide is shown in Figure 6(6a), the second polypeptide is shown in Figure 6(6c), and the third polypeptide is shown in Figure 6(6d). As shown in Figure 5(5e), ​​a second polypeptide consisting of an immunoglobulin heavy chain comprising the Fc region (i) of a first polypeptide and the blackened Fc region (ii) is linked to the Fc region (i) of the first polypeptide and the Fc region (ii) of the second polypeptide, and an immunoglobulin light chain is further linked to the second polypeptide. Such a preferred taFv-Fab-heterodimer Fc-type bispecific molecule can also be said to be composed of a taFv-heterodimer Fc-type bispecific molecule comprising a taFv and an immunoglobulin Fc region, to which a Fab is linked. Preferred amino acid sequences contained in the first polypeptide included in the taFv-Fab heterodimer Fc-type bispecific molecule include the amino acid sequence of positions 21 to 511 of the amino acid sequence represented by SEQ ID NO: 32, the amino acid sequence of positions 21 to 511 of the amino acid sequence represented by SEQ ID NO: 34, the amino acid sequence of positions 21 to 511 of the amino acid sequence represented by SEQ ID NO: 35, the amino acid sequence of positions 21 to 511 of the amino acid sequence represented by SEQ ID NO: 36, the amino acid sequence of positions 21 to 511 of the amino acid sequence represented by SEQ ID NO: 37, the amino acid sequence of positions 21 to 511 of the amino acid sequence represented by SEQ ID NO: 38, the amino acid sequence of positions 21 to 511 of the amino acid sequence represented by SEQ ID NO: 39, the amino acid sequence of positions 21 to 511 of the amino acid sequence represented by SEQ ID NO: 40, and the amino acid sequence of positions 21 to 511 of the amino acid sequence represented by SEQ ID NO: 41.Examples of the first polypeptide comprised in the taFv-Fab heterodimer Fc-type bispecific molecule include the amino acid sequence of positions 21 to 511 of the amino acid sequence represented by SEQ ID NO:42, the amino acid sequence of positions 21 to 511 of the amino acid sequence represented by SEQ ID NO:43, the amino acid sequence of positions 21 to 511 of the amino acid sequence represented by SEQ ID NO:33, the amino acid sequence of positions 21 to 511 of the amino acid sequence represented by SEQ ID NO:52, the amino acid sequence of positions 21 to 511 of the amino acid sequence represented by SEQ ID NO:53, the amino acid sequence of positions 21 to 511 of the amino acid sequence represented by SEQ ID NO:54, the amino acid sequence of positions 21 to 516 of the amino acid sequence represented by SEQ ID NO:55, and the amino acid sequence of positions 21 to 516 of the amino acid sequence represented by SEQ ID NO:56. More preferred first polypeptides comprised in the taFv-Fab heterodimer Fc-type bispecific molecule include the amino acid sequence of positions 21 to 745 of the amino acid sequence represented by SEQ ID NO:32, the amino acid sequence of positions 21 to 745 of the amino acid sequence represented by SEQ ID NO:34, the amino acid sequence of positions 21 to 745 of the amino acid sequence represented by SEQ ID NO:35, and the amino acid sequence of positions 21 to 745 of the amino acid sequence represented by SEQ ID NO:36. the amino acid sequence of positions 21 to 745 of the amino acid sequence represented by SEQ ID NO:37, the amino acid sequence of positions 21 to 745 of the amino acid sequence represented by SEQ ID NO:38, the amino acid sequence of positions 21 to 745 of the amino acid sequence represented by SEQ ID NO:39, the amino acid sequence of positions 21 to 745 of the amino acid sequence represented by SEQ ID NO:40, the amino acid sequence of positions 21 to 745 of the amino acid sequence represented by SEQ ID NO:41, the amino acid sequence of positions 21 to 745 of the amino acid sequence represented by SEQ ID NO:42, the amino acid sequence of positions 21 to 745 of the amino acid sequence represented by SEQ ID NO:43, and the amino acid sequence of positions 21 to 745 of the amino acid sequence represented by SEQ ID NO:33, the amino acid sequence of positions 21 to 745 of the amino acid sequence represented by SEQ ID NO:52, the amino acid sequence of positions 21 to 745 of the amino acid sequence represented by SEQ ID NO:53, the amino acid sequence of positions 21 to 745 of the amino acid sequence represented by SEQ ID NO:54, the amino acid sequence of positions 21 to 750 of the amino acid sequence represented by SEQ ID NO:55, or the amino acid sequence of positions 21 to 750 of the amino acid sequence represented by SEQ ID NO:56.

[0136] The second polypeptide contained in a preferred taFv-Fab-heterodimer Fc-type bispecific antibody of the present invention comprises the variable region, CH1 region, and hinge region of a human antibody or humanized antibody heavy chain, as well as a variant Fc, and the more preferred second polypeptide contained in a taFv-Fab-heterodimer Fc-type bispecific antibody comprises the amino acid sequence from positions 20 to 242 of the amino acid sequence represented by SEQ ID NO:44.

[0137] A preferred third polypeptide contained in a taFv-Fab-heterodimer Fc-type bispecific antibody of the present invention comprises the variable and constant regions of a human antibody or humanized antibody light chain, and a more preferred third polypeptide contained in a taFv-Fab-heterodimer Fc-type bispecific molecule comprises amino acids 21 to 131 of the amino acid sequence represented by SEQ ID NO:45.

[0138] The variable region and CH1 region of the second polypeptide and the third polypeptide contained in such a preferred taFv-Fab-heterodimer Fc-type bispecific molecule constitute a Fab, and a preferred Fab is a Fab of an anti-HLA / NY-ESO antibody, for example, a Fab of NYA-0001.

[0139] In the present invention, the scFv contained in the scFv-Fab-heterodimer Fc-type bispecific antibody may be, for example, an anti-HLA-A2 / NY-ESO scFv or an anti-CD3 scFv, and the Fab may be, for example, an HLA / NY-ESO Fab or an anti-CD3 Fab.

[0140] An scFv-Fab heterodimer Fc-type bispecific molecule preferably comprises (a) a first polypeptide comprising, from N-terminus to C-terminus, an scFv that specifically binds to human HLA / NY-ESO, a variable region and constant region CH1 of an antibody heavy chain that specifically binds to CD3, and an immunoglobulin Fc region (i), in that order, a second polypeptide comprising an immunoglobulin hinge region and Fc region (ii), and a third polypeptide comprising an antibody light chain consisting of a variable region and a constant region; more preferably, (b) the first polypeptide and the second polypeptide are associated with each other at the Fc region (i) and Fc region (ii), and the first polypeptide is associated with the third polypeptide (the antibody light chain) at the variable region and constant region CH1 of the antibody heavy chain. The Fc regions of the first and second polypeptides may be wild-type or may contain mutations that allow for heterodimer formation. An example of an scFv-Fab heterodimer Fc-type bispecific molecule is shown in Figure 7 (7c). The right half of Figure 7 (7c) shows the first polypeptide and the third polypeptide, and the left half shows the second polypeptide. As shown in Figure 7 (7c), the Fc region (i) of the first polypeptide associates with the Fc region (ii) of the second polypeptide, which is shown in black, thereby associating the first polypeptide with the third polypeptide. For example, in Figure 7 (7c), the scFv shown with diagonal lines in the upper right corner is anti-HLA-A2 / NY-ESO scFv, and the Fab shown with white, checkered, and horizontal lines is anti-CD3 Fab.

[0141] An example of an amino acid sequence contained in the first polypeptide contained in a preferred scFv-Fab-heterodimer Fc-type bispecific antibody is the amino acid sequence from positions 21 to 394 of the amino acid sequence represented by SEQ ID NO:57, and more preferably the amino acid sequence from positions 20 to 724.

[0142] Furthermore, examples of other amino acid sequences contained in the first polypeptide included in a preferred scFv-Fab-heterodimer Fc-type bispecific antibody include the amino acid sequence from positions 21 to 389 of the amino acid sequence shown in SEQ ID NO: 60, and more preferably the amino acid sequence from positions 20 to 719.

[0143] Further, examples of other amino acid sequences contained in the first polypeptide contained in a preferred scFv-Fab-heterodimer Fc-type bispecific antibody include the amino acid sequence from positions 21 to 389 of the amino acid sequence represented by SEQ ID NO: 61, and more preferably the amino acid sequence from positions 20 to 719 of the amino acid sequence represented by SEQ ID NO: 61.

[0144] A preferred second polypeptide contained in a scFv-Fab-heterodimer Fc-type bispecific antibody of the present invention comprises a hinge region and a variant Fc derived from a human antibody, and a more preferred second polypeptide contained in a scFv-Fab-heterodimer Fc-type bispecific molecule comprises the amino acid sequence from positions 20 to 246 of the amino acid sequence set forth in SEQ ID NO: 31.

[0145] The third polypeptide contained in a preferred scFv-Fab-heterodimer Fc-type bispecific molecule of the present invention comprises a light chain derived from a human antibody. A more preferred example of the third polypeptide contained in a scFv-Fab-heterodimer Fc-type bispecific molecule is the amino acid sequence from positions 21 to 127 of the amino acid sequence shown in SEQ ID NO: 58, and even more preferably the amino acid sequence from positions 21 to 233 of the amino acid sequence shown in SEQ ID NO: 58.

[0146] Of these, NYZ-1010, which is formed by associating a first polypeptide consisting of amino acids 20 to 724 of the amino acid sequence shown in SEQ ID NO:57, a second polypeptide consisting of amino acids 20 to 246 of the amino acid sequence shown in SEQ ID NO:31, and a third polypeptide consisting of amino acids 21 to 233 of the amino acid sequence shown in SEQ ID NO:58, can be exemplified as a preferred scFv-Fab-heterodimer Fc-type bispecific molecule of the present invention.

[0147] Furthermore, NYZ-1007, which is formed by associating a first polypeptide consisting of the 20th to 719th amino acids of the amino acid sequence shown in SEQ ID NO: 60, a second polypeptide consisting of the 20th to 246th amino acids of the amino acid sequence shown in SEQ ID NO: 31, and a third polypeptide consisting of the 21st to 233rd amino acids of the amino acid sequence shown in SEQ ID NO: 58, can also be exemplified as a suitable scFv-Fab-heterodimer Fc-type bispecific molecule of the present invention.

[0148] Furthermore, NYZ-1017, which is formed by associating a first polypeptide consisting of the 20th to 719th amino acids of the amino acid sequence shown in SEQ ID NO:61, a second polypeptide consisting of the 20th to 246th amino acids of the amino acid sequence shown in SEQ ID NO:31, and a third polypeptide consisting of the 21st to 233rd amino acids of the amino acid sequence shown in SEQ ID NO:58, can also be exemplified as a suitable scFv-Fab-heterodimer Fc-type bispecific molecule of the present invention.

[0149] One, two, or three or more peptides contained in the bispecific molecules of the present invention may be the aforementioned "deletion variant," i.e., one or two (or more) amino acids may be mutated (including deleted) at their carboxyl termini, particularly at the carboxyl termini derived from the antibody heavy chain. For example, the carboxyl terminus of the amino acid sequence of the first polypeptide contained in NYZ-1010, a preferred scFv-Fab-heterodimer Fc-type bispecific molecule of the present invention, may be Lys at position 724 of SEQ ID NO: 57, Gly at position 723 resulting from a single amino acid deletion, or a mixture containing these. Similarly, the carboxyl terminus of the amino acid sequence of the second polypeptide contained in a preferred scFv-Fab-heterodimer Fc-type bispecific molecule of the present invention may be Lys at position 246 of SEQ ID NO: 84, Gly at position 245 resulting from a single amino acid deletion, or a mixture containing these.

[0150] The scFv and Fab comprised in the bispecific molecules of the present invention are preferably scFv and Fab of humanized or human antibodies, and the Fc is preferably the Fc of a human antibody.

[0151] The variable regions contained in the bispecific molecules of the present invention may be linked in the following order from the amino terminus of the antibody: heavy chain variable region followed by light chain variable region, or alternatively, light chain variable region followed by heavy chain variable region. A linker may be present (optionally) between the two variable regions. Furthermore, a glycine residue may be present (optionally) at the amino terminus of the amino-terminal variable region. In tandem scFv-type bispecific molecules, a linker, FLAG tag, and / or His tag may be attached (optionally) to the carboxyl terminus of the carboxyl-terminal variable region. One preferred embodiment is one in which, from the amino terminus, the heavy chain variable region, first linker, light chain variable region, second linker, FLAG tag, and His tag are attached in this order.

[0152] The linker may be a single-chain polypeptide or single-chain oligopeptide, or a synthetic product such as PEG, a nucleotide, a sugar chain, or a compound. There are no particular limitations on the linker as long as it can link two polypeptides, and any known linker can be used.

[0153] The length of the linker is, for example, 5 to 30 amino acids in the case of a peptide linker. When a bispecific molecule contains multiple linkers, the peptide linkers may all be of the same length, or may be of different lengths.

[0154] An example of a peptide linker is a repeat of (Gly.Gly.Gly.Gly.Gly.Ser), to which one to several amino acid residues other than Gly and Ser may be added.

[0155] Of the structures (formats) that the multispecific antibodies, particularly bispecific antibodies, of the present invention can adopt as described above, preferred are the taFv-heterodimer Fc type, the taFv-Fab-heterodimer Fc type, and the scFv-Fab-heterodimer Fc type, with the taFv-heterodimer Fc type being more preferred. The type in which the anti-HLA / NY-ESO scFv and the anti-CD3 scFv are located in this order from the N-terminus to the C-terminus (taFv-heterodimer Fc type) is even more preferred than the type in which they are located in the reverse order (taFv(inversed)-heterodimer Fc type). Another more preferred type is the scFv-Fab-heterodimer Fc type.

[0156] The present invention also includes molecules that contain an amino acid sequence encoded by a nucleotide sequence contained in a polynucleotide that hybridizes under stringent conditions with a complementary strand of a polynucleotide that contains a nucleotide sequence encoding an amino acid sequence contained in a molecule of the present invention, and that bind to HLA / NY-ESO and preferably also bind to CD3.

[0157] The present invention also includes molecules that contain an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to an amino acid sequence contained in a molecule of the present invention, and that bind to HLA / NY-ESO and preferably also bind to CD3.

[0158] The antibodies, binding fragments thereof, and multispecific antibodies comprising them of the present invention possess excellent biological activity, physicochemical properties (hereinafter referred to as "physical properties"), safety, pharmacokinetics, etc. (a) Examples of biological activity or indicators thereof include antigen-binding activity, in vitro cytotoxic activity, in vivo antitumor activity, etc. For example, the dissociation constant (KD value) for HLA / NY-ESO is 100 nM or less or 50 nM or less, preferably 20 nM or less or 10 nM or less, and more preferably 5 nM or less. Furthermore, for example, the EC2 value of the cytotoxic activity exerted against endogenous human NY-ESO-expressing cell lines U266B1 and / or NCI-H1703 using human peripheral blood mononuclear cells as effector cells is 100 nM or less or 50 nM or less, preferably 20 nM or less or 10 nM or less, and more preferably 5 nM or less. ５０The value is 20 nM or less, preferably 10 nM or less, more preferably 5 nM or less (measurement and calculation of in vitro cytotoxic activity can be exemplified by, but not limited to, the method described in Example 8 of WO2021 / 200857). Furthermore, for example, 6 × 10 ７ 0.1 mL of the cell / mL suspension was subcutaneously implanted into NOG mice, and 4 days later, 3.75 × 10 human peripheral blood mononuclear cells were implanted. ７A 0.2 mL cell / mL suspension was implanted into the tail vein, and starting 14 days later, the antibody was administered three times weekly. The tumor volume was then measured. The tumor growth inhibitory activity compared to the vehicle-administered control group was 50% or more, preferably 75% or more, and more preferably 90% or more. (Examples of in vivo antitumor activity measurement and calculation include, but are not limited to, the method described in Example 9.) (b) Impurities contained in biopharmaceuticals contribute to pharmaceutical safety, and therefore, appropriate specifications must be established and controlled to determine whether or not they increase during manufacturing and storage. Among these, HMWS (aggregates) are one of the major impurities and are involved in immunogenicity risk and reduced efficacy, making them particularly subject to strict control. Impurity management must be performed not only during manufacturing, but also during and after manufacturing, including evaluation of stability over time (whether or not they increase). Since the shelf life of pharmaceuticals is determined based on the results of long-term stability tests, antibodies that are stable over time can be set to have a longer shelf life. Thus, physicochemical properties of the present invention that serve as indicators for selecting suitable antibodies aimed at biopharmaceuticals include acid resistance (e.g., inhibition of HMWS production) and solution stability (e.g., inhibition of HMWS production). Other indicators include a high yield or high yield in a culture of recombinant cells obtained by introducing a gene encoding the amino acid sequence contained in an antibody or a binding fragment thereof of the present invention, or a molecule containing the antibody or binding fragment thereof, into host cells suitable for producing the antibody or binding fragment thereof, or a molecule containing the antibody or binding fragment thereof, for example, Expi293F cells. Suitable antibodies, antigen-binding fragments thereof, and multispecific antibodies containing them of the present invention that possess these physicochemical properties: enable their solutions to be exposed to acidic conditions, making their production possible or easy, for example, by carrying out processes such as protein A, ion exchange, and viral inactivation chromatography; suppress the production of HMWS even when they are in solution, making their production, formulation, distribution, and storage of pharmaceuticals containing them possible or easy; and enable their efficient production.Regarding acid resistance, for example, the resulting HMWS content calculated by incubating at pH 3.5, room temperature, for 1 hour, and then measuring HMWS by size exclusion chromatography is 5% or less, preferably 2% or less, more preferably 1% or less (measurement and calculation of HMWS content for evaluating acid resistance can be exemplified by, but not limited to, the method described in Example 19-1 of WO2021 / 200857). Regarding solution stability, for example, the HMWS content calculated by dissolving in 25 mM histidine and 5% sorbitol, pH 6.0, to a concentration of 25 mg / ml, storing at 25 ° C. for 6 days, and then measuring HMWS by size exclusion chromatography is 20% or less, preferably 10% or less (measurement and calculation of HMWS content for evaluating solution stability can be exemplified by, but not limited to, the method described in Example 19-2 of WO2021 / 200857). Examples of methods for measuring and calculating production efficiency or yield include, but are not limited to, the methods described in Examples 20 and 21 of WO2021 / 200857. (c) Examples of safety or indicators thereof include antigen recognition characteristics, findings upon administration, and the like. For example, a peptide recognizes multiple amino acids on the wild-type NY-ESO peptide and does not bind to homologous peptides with amino acid sequences similar to, but not identical to, the wild-type NY-ESO peptide, thereby reducing the risk of side effects due to off-target effects. Furthermore, ISPRI web-based immunogenicity screening (EpiVax, Inc.) predicts low immunogenicity and a low risk of side effects such as cytokine production due to anti-antibodies. Furthermore, when NYF-0023, NYF-0045, NYF-0047, NYF-0048, NYF-0060, NYF-0061, NYZ-0082, or NYZ-1010, which are included in the bispecific antibodies of the present invention, were administered to Balb / c mice, no findings that would be problematic in terms of blood half-life were observed, and no weight loss or other significant toxic findings were observed. Furthermore, when NYZ-0082 or NYZ-1010 was administered in a single dose to cynomolgus monkeys, no findings that would be problematic in terms of blood half-life were observed, and no changes attributable to administration were observed in general condition, body weight, food intake, body temperature, or plasma cytokine levels. (d) Examples of kinetics or indicators thereof include blood half-life, etc.For example, when several bispecific antibodies obtained in the present invention were administered to Balb / c mice or cynomolgus monkeys, no significant findings were observed regarding the blood half-life. The antibodies, binding fragments, and molecules of the present invention, which have such excellent properties as biological activity, physicochemical properties, safety, and pharmacokinetics, can be suitably incorporated into pharmaceutical compositions. Preferred examples of antibodies or antigen-binding fragments thereof of the present invention having the antigen-binding activity described in (a) and the properties described in (b) include, but are not limited to, NYA-1143, NYA-1163, NYA-2023, NYA-2027, NYA-2035, NYA-2044, NYA-2045, NYA-2047, NYA-2048, NYA-2060, NYA-2031, NYA-2047, NYA-2061, NYA-2143, and NYA-3061, more preferably NYA-2047, NYA-2061, NYA-2143, and NYA-3061. Furthermore, preferred multispecific antibodies of the present invention having the properties described in (a) to (d) include NYF-0016, NYF-0019, NYF-0022, NYF-0023, NYF-0027, NYF-0035, NYF-0044, NYF-0045, NYF-0047, NYF-0048, NYF-0058, and NYF-0060. , NYF-0061, NYZ-0038, NYZ-0082, NYZ-0088 and NYZ-1010, and more preferably NYF-0061, NYZ-0038, NYZ-0082, NYZ-0088, NYZ-1007, NYZ-1010 and NYZ-1017, respectively, but are not limited thereto.

[0159] In the present invention, the "site" to which an antibody binds, i.e., the "site" recognized by an antibody, refers to a partial peptide or partial higher-order structure on an antigen to which the antibody binds or recognizes. In the present invention, such a site is also referred to as an epitope or antibody-binding site. Examples of the site on HLA / NY-ESO to which the anti-HLA / NY-ESO antibody of the present invention binds or recognizes include multiple amino acids in an HLA / NY-ESO peptide, partial higher-order structures, and the like.

[0160] The present invention also encompasses "antibodies or binding fragments thereof that bind to the same site as" the antibodies or binding fragments of the present invention. An "antibody that binds to the same site" as a certain antibody means another antibody that binds to the site on the antigen molecule recognized by that antibody. If a second antibody binds to a partial peptide or partial three-dimensional structure on the antigen molecule to which a first antibody binds, it can be determined that the first and second antibodies bind to the same site. If a first antibody of the present invention has the antigen-binding activity described in (a) above, a second antibody that binds to the same site on HLA / NY-ESO is highly likely to have similar activity, and such second antibodies are also encompassed by the present invention. Furthermore, antibodies that compete with the first antibody of the present invention in binding to HLA / NY-ESO are also encompassed by the present invention, provided that they have the antigen-binding activity described in (a). Such antibodies that bind to the site on HLA / NY-ESO recognized by the monoclonal antibodies of the present invention, antibodies that compete with the monoclonal antibodies of the present invention in binding to HLA / NY-ESO, and binding fragments thereof preferably have the in vitro cytotoxic activity and in vivo antitumor activity described in (a) and one or more, more preferably three or more, and most preferably all of the properties described in (b) to (d).

[0161] The antibody binding site can be determined by methods well known to those skilled in the art, such as immunoassays. For example, a series of peptides can be prepared by appropriately truncating the amino acid sequence of an antigen from the C-terminus or N-terminus, and the reactivity of antibodies to these peptides can be examined to determine the approximate recognition site. The binding site can then be determined by synthesizing shorter peptides and examining the reactivity of antibodies to these peptides. Alternatively, the binding site can be determined by deleting or substituting a specific amino acid sequence or region in the amino acid sequence of an antigen or antigen fragment peptide, or by introducing a mutation into the amino acid sequence, and examining the reactivity of antibodies to these peptides. Antigen fragment peptides can be prepared using techniques such as genetic recombination and peptide synthesis.

[0162] When an antibody binds to or recognizes a partial higher-order structure of an antigen, the binding site of such an antibody can be determined by identifying amino acid residues on the antigen adjacent to the antibody using X-ray structural analysis. For example, amino acid residues on the antigen that have an interaction distance with the antibody can be identified by combining an antibody or a fragment thereof with the antigen or a fragment thereof, crystallizing the combined residues, and then performing structural analysis. The interaction distance is 8 Å or less, preferably 6 Å or less, and more preferably 4 Å or less. One or more amino acid residues that have such an interaction distance with the antibody can constitute the antigen-binding site (epitope) of the antibody. When there are two or more such amino acid residues, the amino acids do not have to be adjacent to each other in the primary sequence.

[0163] The anti-HLA / NY-ESO antibody or binding fragment thereof of the present invention specifically recognizes multiple amino acids present in the amino acid sequence of HLA / NY-ESO. Antibodies or binding fragments thereof that recognize these multiple amino acids, compete with the antibody or binding fragment thereof of the present invention in binding to HLA / NY-ESO, or have an interaction distance with these multiple amino acids are also encompassed by the present invention. Furthermore, multispecific antibodies comprising such antibodies or binding fragments thereof are also encompassed by the present invention.

[0164] The multispecific antibody of the present invention can be used in combination with an immune checkpoint inhibitor. [Immune checkpoint inhibitor] In the present invention, the term "immune checkpoint inhibitor" refers to a drug that inhibits the immunosuppressive system and activates tumor immunity, and is synonymous with "a compound that inhibits an immune checkpoint molecule."

[0165] The immune checkpoint inhibitor used in the present invention is not particularly limited, but preferred examples include anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, and anti-TIGIT antibodies, and more preferred examples include anti-PD-1 antibodies and anti-PD-L1 antibodies. In the present invention, "anti-PD-1 antibodies" refer to antibodies that specifically bind to PD-1 (programmed cell death-1; CD279; PDCD1), and preferably refer to antibodies that have the effect of reducing, inhibiting, and / or interfering with signal transduction resulting from the interaction between PD-1 and its binding partners, PD-L1 and PD-L2. The anti-PD-1 antibody used in the present invention is not particularly limited as long as its clinical efficacy and safety have been confirmed, but preferred examples include nivolumab (WO 2006 / 121168, etc.) and pembrolizumab (WO 2008 / 156712, etc.). Furthermore, for the purpose of confirming the combined effect of a multispecific antibody containing the anti-HLA-A2 / NY-ESO antibody used in the present invention in preclinical studies, commercially available anti-PD-1 antibodies for research use (e.g., clone RMP1-14) and the like can also be used. In the present invention, the term "anti-PD-L1 antibody" refers to an antibody that specifically binds to PD-L1 (Programmed cell death ligand 1; CD274; B7-H1), and preferably refers to an antibody that has the effect of reducing, inhibiting, and / or interfering with signal transduction resulting from the interaction between PD-L1 and its binding partners, PD-1 and B7.1 (CD80). The anti-PD-L1 antibody used in the present invention is not particularly limited as long as its clinical efficacy and safety have been confirmed, but preferred examples include atezolizumab (WO 2010 / 077634, etc.), durvalumab (WO 2011 / 066389, etc.), and avelumab (WO 2013 / 079174, etc.).Furthermore, for the purpose of confirming the combined effect of a multispecific antibody containing the anti-HLA-A2 / NY-ESO antibody used in the present invention in preclinical studies, a commercially available anti-PD-L1 antibody for research use (e.g., clone 10F.9G2) or the like can also be used. In the present invention, the term "anti-CTLA-4 antibody" refers to an antibody that specifically binds to CTLA-4 (cytotoxic T-lymphocyte-associated protein 4; CD152), and preferably refers to an antibody that has the effect of reducing, inhibiting, and / or interfering with signal transduction resulting from the interaction between CTLA-4 and its binding partners, B7.1 (CD80) and B7.2 (CD86). The anti-CTLA-4 antibody used in the present invention is not particularly limited as long as its clinical efficacy and safety have been confirmed, but preferred examples include ipilimumab (WO 2001 / 014424, etc.), tremelimumab (WO 2000 / 037504, etc.), spartalizumab (WO 2015 / 112900, etc.), and cemiplimab (WO 2015 / 196051, etc.). Furthermore, for the purpose of confirming the combined effect of a multispecific antibody containing the anti-HLA-A2 / NY-ESO antibody used in the present invention in preclinical studies, commercially available anti-CTLA-4 antibodies for research use (e.g., clone 9H10) and the like can also be used. In the present invention, the term "anti-TIGIT antibody" refers to an antibody that specifically binds to TIGIT (T cell immunoreceptor with Ig and ITIM domains), and preferably refers to an antibody that has the effect of reducing, inhibiting, and / or interfering with signal transduction resulting from the interaction between TIGIT and its binding partner, CD155. The anti-TIGIT antibody used in the present invention is not particularly limited as long as its clinical efficacy and safety have been confirmed, but preferred examples include tiragolumab (WO 2017 / 053748) and vibostolimab (WO 2016 / 028656).Furthermore, for the purpose of confirming the combined effect of a multispecific antibody containing the anti-HLA-A2 / NY-ESO antibody used in the present invention in preclinical studies, a commercially available anti-TIGIT antibody for research use (e.g., clone 1B4) or the like can also be used. When the "immune checkpoint inhibitor" in the present invention is an antibody, an antigen-binding fragment of the antibody is also encompassed within the scope of "antibody." In the present invention, the immune checkpoint inhibitor used in combination with a multispecific antibody may be an antigen-binding fragment of any of the above antibodies, and the antigen-binding fragment may contain other moieties.

[0166] [Chemotherapeutic Agent] In the present invention, the term "chemotherapeutic agent" refers to a chemically synthesized drug among compounds having anti-cancer and anti-tumor activity.

[0167] The chemotherapeutic agents used in the present invention are not particularly limited, but are preferably topoisomerase inhibitors, microtubule inhibitors, platinum preparations, DNA demethylating agents, anticancer antibiotics, and alkylating agents. Suitable examples of topoisomerase inhibitors include irinotecan, topotecan, etoposide, and sacituzumab govitecan conjugated with SN-38, an active metabolite of irinotecan, as a drug conjugate. Suitable examples of microtubule inhibitors include paclitaxel, docetaxel, vincristine, vinblastine, vindesine, eribulin, vinorelbine, albumin-suspended paclitaxel (Nab-paclitaxel), and the like. Suitable examples of platinum preparations include oxaliplatin, carboplatin, cisplatin, nedaplatin, and the like. Suitable examples of DNA demethylating agents include azacitidine, decitabine, and the like. Suitable examples of anticancer antibiotics include doxorubicin, bleomycin, liposomal doxorubicin, etc. Suitable examples of alkylating agents include ifosfamide, cyclophosphamide, dacarbazine, etc. However, chemotherapeutic agents used in combination with the multispecific antibodies of the present invention are not limited to these.

[0168] [Pharmaceuticals] The present invention provides pharmaceutical compositions and methods of treatment comprising a combination of a multispecific molecule and an immune checkpoint inhibitor or a chemotherapeutic agent.

[0169] The pharmaceutical compositions and methods of treatment of the present invention may be characterized in that the multispecific molecule and the immune checkpoint inhibitor or chemotherapeutic agent are contained as active ingredients in separate formulations and administered simultaneously or at different times, or in that the multispecific molecule and the immune checkpoint inhibitor or chemotherapeutic agent are contained as active ingredients in a single formulation and administered.Furthermore, the multispecific molecule of the present invention may be contained as an active ingredient in a single formulation and administered for the treatment of a disease that is ameliorated by the activity of activating anti-tumor immunity.

[0170] The pharmaceutical composition and treatment method of the present invention can be used for the treatment of cancer, and preferably for the treatment of at least one disease selected from the group consisting of lung cancer (including non-small cell lung cancer), urothelial cancer, colon cancer (sometimes referred to as colorectal cancer, including colon cancer and rectal cancer), prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer (sometimes referred to as gastric adenocarcinoma), gastroesophageal junction adenocarcinoma, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, neuroepithelial tissue tumor, nerve sheath tumor, head and neck cancer, skin cancer, pharyngeal cancer, gallbladder cancer, bile duct cancer, mesothelioma, Paget's disease, and sarcoma.

[0171] The pharmaceutical composition and treatment method of the present invention can be selected and used as a drug for chemotherapy, a major cancer treatment, thereby slowing the growth of cancer cells, suppressing their proliferation, and even destroying them. These actions can relieve cancer patients from cancer-related symptoms, improve their quality of life, and achieve therapeutic effects while preserving their lives. Even if cancer cells are not destroyed, the inhibition and control of cancer cell proliferation can enable cancer patients to achieve a higher quality of life and longer survival. In addition to being used alone in such chemotherapy, the pharmaceutical composition and treatment method of the present invention can also be used as a drug to be combined with other therapies in adjuvant therapy, such as surgery, radiation therapy, and hormone therapy. They can also be used as a drug for neoadjuvant chemotherapy. In addition to the above therapeutic uses, the pharmaceutical composition and treatment method of the present invention can also be expected to have a preventive effect, such as suppressing the proliferation and even destroying micrometastatic cancer cells. For example, they can be expected to have effects such as inhibiting and destroying cancer cells present in body fluids during the metastatic process, and inhibiting and destroying microscopic cancer cells immediately after implantation in any tissue. Therefore, the suppression and prevention of cancer metastasis, particularly after surgical removal of cancer, can be expected. The pharmaceutical composition and treatment method of the present invention can be applied to patients as a systemic therapy, or can be applied locally to cancer tissue to achieve therapeutic effects. The pharmaceutical composition and treatment method of the present invention can be used preferably in mammals, but more preferably in humans. The pharmaceutical composition of the present invention can be administered as a pharmaceutical composition containing one or more pharmaceutically compatible ingredients. The substances used in the pharmaceutical composition of the present invention can be appropriately selected from formulation additives and other substances commonly used in this field in terms of dosage amount and administration concentration. For example, the pharmaceutical composition typically contains one or more pharmaceutical carriers (e.g., a sterilized liquid). Examples of the liquid include water and oil (petroleum, animal-derived, plant-derived, or synthetic oil). The oil may be, for example, peanut oil, soybean oil, mineral oil, sesame oil, etc.Water is a more typical carrier when the pharmaceutical composition is administered intravenously. Saline solution and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients can be appropriately selected from those known in the art. The composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. The formulation corresponds to the mode of administration.

[0172] Various delivery systems are known and can be used to administer the pharmaceutical compositions of the present invention. Routes of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes. Administration can be, for example, by infusion or bolus injection. In certain preferred embodiments, the multispecific molecules and immune checkpoint inhibitors or chemotherapeutic agents used in the present invention are administered by infusion. Parenteral administration is a preferred route of administration. In representative embodiments, the pharmaceutical compositions are formulated in accordance with routine procedures as pharmaceutical compositions adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the pharmaceutical compositions may also include a solubilizing agent and a local anesthetic (e.g., lignocaine) to ease pain at the injection site. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container, such as an ampoule or sachet, indicating the quantity of active agent. Where the pharmaceutical composition is in a form to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. When the pharmaceutical composition is administered by injection, an ampoule of sterile water for injection or saline can be provided, for example, so that the ingredients can be mixed prior to administration.

[0173] The pharmaceutical compositions and treatment methods of the present invention may contain a cancer therapeutic agent other than the multispecific molecule of the present invention and an immune checkpoint inhibitor or chemotherapeutic agent. The pharmaceutical compositions and treatment methods of the present invention can also be administered in combination with other cancer therapeutic agents, thereby enhancing the antitumor effect. The other cancer therapeutic agents used for such purposes may be administered to an individual simultaneously with the pharmaceutical composition of the present invention, separately, or sequentially, or at different administration intervals. Examples of such cancer therapeutic agents include pemetrexed, sorafenib, everolimus, tanespimycin, and bevacizumab, but are not limited thereto as long as they have antitumor activity.

[0174] The molecular targeted drug used in the present invention is not particularly limited, but is preferably a VEGF inhibitor, an FGFR inhibitor, or a multikinase inhibitor. Preferred examples of VEGF inhibitors include bevacizumab, ramucirumab, zaltrap, axitinib, etc. Preferred examples of FGFR inhibitors include pemigatinib and futibatinib, etc. Preferred examples of multikinase inhibitors include sorafenib, sunitinib, pazopanib, regorafenib, lenvatinib, etc. However, the chemotherapeutic agent used in combination with the multispecific antibody of the present invention is not limited to these.

[0175] Such pharmaceutical compositions can be formulated as lyophilized or liquid preparations with a selected composition and required purity. When formulated as a lyophilized preparation, it may be a preparation containing appropriate formulation additives used in this field. Similarly, liquid preparations can be formulated as liquid preparations containing various formulation additives used in this field.

[0176] Although the composition and concentration of the pharmaceutical composition vary depending on the administration method, the multispecific molecules and immune checkpoint inhibitors or chemotherapeutic agents contained in the pharmaceutical compositions of the present invention exhibit medicinal effects at lower doses, i.e., in terms of the affinity for the antigen, i.e., the dissociation constant (Kd value) for the antigen, the higher the affinity (lower the Kd value). Therefore, the dose of the multispecific molecules and immune checkpoint inhibitors or chemotherapeutic agents can be determined based on the affinity for the antigen. When the multispecific molecules and immune checkpoint inhibitors or chemotherapeutic agents of the present invention are administered to humans, the dose is, for example, 0.0001 mg to 100 mg. The predetermined dose may be administered once every 1 to 180 days, or may be administered in divided doses two, three, four, or more times per day at appropriate intervals.

[0177] In the case of the multispecific molecules of the present invention, an example of an administration method is to administer 0.001 mg / kg to 8 mg / kg once every three weeks. Administration may be once every three weeks (q3w), but may also be once every week (q1w), once every two weeks (q2w), or once every four weeks (q4w). The present invention will be specifically explained using the following examples, but the present invention is not limited to these. Furthermore, these examples should not be construed as limiting in any way.

[0178] The present invention will be described in more detail in the following examples, but the present invention is not limited to these examples.

[0179] In the following examples, unless otherwise specified, each operation related to genetic manipulation was performed according to the method described in a laboratory manual used by those skilled in the art, or, when using commercially available reagents or kits, according to the instructions provided with the commercially available products.

[0180] Example 1. In vivo effect of combined use of Fc-tagged anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule (NYZ-1010) and paclitaxel on human squamous cell lung cancer cell lines. Human squamous cell lung cancer cell line NCI-H1703 (ATCC) was diluted to 6 x 10 with PBS containing 50% Matrigel (CORNING). ７The medium was adjusted to 0.1 mL / mL and subcutaneously transplanted into NSG mice (female, 4-6 weeks old) at 0.1 mL on Day 0. Starting approximately one week later (Day 6-7), the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the following formula.

[0181] Estimated Tumor Volume (mm ３ ) = average estimated tumor volume of each individual: estimated tumor volume of each individual (mm ３ ) = 1 / 2 x [tumor long diameter] x [tumor short diameter] x [tumor short diameter]

[0182] On Day 12, the mice were divided into groups based on tumor volume (n = 5 per group), and T cells were stimulated and expanded using human PBMCs with CD3 / CD28 Dynabeads (ThermoFisher) at 5 × 10 ７ The cells were prepared at a concentration of 0.2 mL / mL, and 0.2 mL was transplanted into the tail vein. Three to four hours later, an anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule (NYZ-1010, 0.02 mg / kg: prepared by the method described in WO2021 / 200857) was administered into the tail vein. Paclitaxel (20 mg / kg) was also administered intraperitoneally. Administration was performed on Day 13 and Day 20. Tumor growth inhibition (%) was calculated using the following formula.

[0183] Tumor growth inhibition (%) = 100 - (Estimated tumor volume of each group / Estimated tumor volume of vehicle control × 100)

[0184] The results of NYZ-1010 monotherapy, Paclitaxel monotherapy, and NYZ-1010 and Paclitaxel combination therapy are shown in Figure 1 (1). The TGI at 31 days after implantation was 79.8% in the NYZ-1010 monotherapy group and 74.1% in the Paclitaxel monotherapy group, while it was 99.1% in the NYZ-1010 and Paclitaxel combination group, demonstrating that the antitumor effect was enhanced by the combination therapy. Furthermore, the percent change in tumor volume from Day 31 for each individual (change from baseline, % change) was calculated using the formula below and is shown in Figure 1 (2). The mean change from baseline (% change) for each group is shown in Table 1. The combined use of NYZ-1010 and Paclitaxel demonstrated a tumor regression effect.

[0185] Change from baseline (%) = (Estimated tumor volume on Day 31 - Estimated tumor volume on Day 12) / Estimated tumor volume on Day 12 × 100 for each individual

[0186]

[0187] Example 2. In vivo effect of combined use of Fc-tagged anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule (NYZ-1010) and Nab Paclitaxel on human squamous cell lung cancer cell lines. Human squamous cell lung cancer cell line NCI-H1703 (ATCC) was cultured at 6 x 10 ng / mL in PBS containing 50% Matrigel (CORNING). ７ The medium was adjusted to 0.1 mL / mL and subcutaneously transplanted into NSG mice (female, 4-6 weeks old) at 0.1 mL on Day 0. Starting approximately one week later (Day 6-7), the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the following formula.

[0188] Estimated Tumor Volume (mm ３ ) = average estimated tumor volume of each individual: estimated tumor volume of each individual (mm ３) = 1 / 2 x [tumor long diameter] x [tumor short diameter] x [tumor short diameter]

[0189] On Day 13, the mice were divided into groups based on tumor volume (n = 5 per group), and human PBMCs were stimulated and expanded with CD3 / CD28 Dynabeads (ThermoFisher). T cells were then diluted to 5 × 10 in PBS. ７ The cells were adjusted to a concentration of 1000 cells / mL, and 0.2 mL of the solution was transplanted into the tail vein. Three to four hours later, an anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule (NYZ-1010, 0.02 mg / kg) was administered into the tail vein. Nab Paclitaxel (20 mg / kg) was also administered intraperitoneally. Administration was performed on Day 13 and Day 20. Tumor growth inhibition (%) was calculated using the following formula.

[0190] Tumor growth inhibition (%) = 100 - (Estimated tumor volume of each group / Estimated tumor volume of vehicle control × 100)

[0191] The results of NYZ-1010 administration alone, Nab Paclitaxel administration alone, and the combination of NYZ-1010 and Nab Paclitaxel are shown in Figure 2 (1). The TGI at 38 days after implantation was 55.4% in the NYZ-1010 alone group and 86.3% in the Nab Paclitaxel alone group, while it was 100% in the NYZ-1010 and Nab Paclitaxel combination group, demonstrating that the antitumor effect was enhanced by the combination. In particular, complete disappearance of tumors was observed in 5 out of 5 cases by Day 38 in the combination with Nab Paclitaxel. The percentage change in tumor volume from Day 13 to Day 38 for each individual (Change from baseline (%)) was calculated using the formula below and is shown in Figure 1 (2). The mean change from baseline (%) for each group is shown in Table 2. The combined use of NYZ-1010 and Nab Paclitaxel demonstrated a tumor regression effect.

[0192] Change from baseline (%) = (Estimated tumor volume on Day 38 - Estimated tumor volume on Day 13) / Estimated tumor volume on Day 13 × 100 for each individual

[0193]

[0194] Example 3. In vivo combined effect of Fc-tagged anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule (NYZ-1010) and anti-PD-1 antibody pembrolizumab on human lung adenocarcinoma cell lines. Human lung adenocarcinoma cell line NCI-H522 (ATCC) was cultured at 5 x 10 ng / mL in PBS containing 50% Matrigel (CORNING). ７ The solution was adjusted to give a concentration of 1000 cells / mL, and 0.1 mL of the solution was subcutaneously transplanted into NSG mice (female, 4-6 weeks old) on Day 0. Starting about 2 weeks later, the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the following formula.

[0195] Estimated Tumor Volume (mm ３ ) = average estimated tumor volume of each individual: estimated tumor volume of each individual (mm ３ ) = 1 / 2 x [tumor long diameter] x [tumor short diameter] x [tumor short diameter]

[0196] On Day 25, the mice were divided into groups based on tumor volume (n = 5 per group), and human PBMCs were stimulated and expanded with CD3 / CD28 Dynabeads (ThermoFisher). T cells were then diluted to 5 × 10 in PBS. ７The medium was adjusted to 100 cells / mL, and 0.2 mL was transplanted into the tail vein. Three to four hours later, an anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule (NYZ-1010, 0.01 mg / kg) was administered into the tail vein. Pembrolizumab (Merck, 10 mg / kg) was also administered into the tail vein. Administration was performed on Day 25, Day 32, and Day 39 (NYZ-1010) or on Day 28, Day 32, Day 35, Day 39, and Day 42 (Pembrolizumab). Tumor growth inhibition (%) was calculated using the following formula.

[0197] Tumor growth inhibition (%) = 100 - (Estimated tumor volume of each group / Estimated tumor volume of vehicle control × 100)

[0198] The results of administration of NYZ-1010 alone, administration of pembrolizumab alone, and administration of NYZ-1010 and pembrolizumab in combination are shown in Figure 3. The TGI 53 days after transplantation was 50.5% in the NYZ-1010 alone administration group and 3.1% in the pembrolizumab alone administration group, while it was 79.0% in the NYZ-1010 and pembrolizumab combination group, demonstrating that the antitumor effect was enhanced by the combination administration.

[0199] Example 4. Generation of Human CD3ε Knock-in Mice Although the anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule binds to human CD3ε, it does not cross-react with rodent CD3ε. Therefore, to enable in vivo evaluation of the anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, human CD3ε knock-in mice were generated. A vector was constructed in which the human CD3ε sequence to which CRISPR-Cas9 and the anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule binds was knocked into mouse CD3ε, and the vector was then introduced into fertilized eggs of C57BL / 6J mice by microinjection. After injection, the fertilized eggs were implanted into the oviducts or uteruses of female mice, and the tail tissue of the resulting offspring was biopsied and subjected to PCR and sequencing analysis. Individuals carrying the desired mutation were selected and subjected to subsequent experiments.

[0200] Example 5. In vivo combined effect of Fc-tagged anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule (NYF-0016) and anti-mouse PD-1 antibody using human CD3ε knock-in mice. MC-38 NY-SCT cells were used, which were prepared by introducing HLA-A2, NY-ESO, and β2M Single Chain Trimer (abbreviated as NY-SCT) into the mouse colon cancer cell line MC-38 introduced from the National Cancer Institute using a retroviral vector. These cells express HLA-A2 / NY-ESO on the cell membrane. MC-38 NY-SCT was diluted to 8 x 10 with PBS (Wako). ６ The medium was adjusted to give a concentration of 1000 cells / mL, and 0.1 mL of the medium was subcutaneously transplanted (Day 0) into human CD3ε knock-in mice (female, 6-7 weeks old) prepared in Example 4. From Day 7, the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the following formula.

[0201] Estimated Tumor Volume (mm ３ ) = average estimated tumor volume of each individual: estimated tumor volume of each individual (mm ３ ) = 1 / 2 x [tumor long diameter] x [tumor short diameter] x [tumor short diameter]

[0202] On Day 7, the mice were divided into groups based on tumor volume (n = 5 per group), and an anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule (NYF-0016, 5 mg / kg: prepared by the method described in WO2021 / 200857) was administered intravenously to the tail vein. An anti-mouse PD-1 antibody (clone RMP1-14, Bio X Cell, 5 mg / kg) was also administered intravenously to the tail vein. Administration was performed on Day 7 and Day 10. Tumor Growth Inhibition (%) was calculated using the following formula:

[0203] Tumor growth inhibition (%) = 100 - (Estimated tumor volume of each group / Estimated tumor volume of vehicle control × 100)

[0204] The results of NYF-0016 administration alone, anti-mouse PD-1 antibody administration alone, and NYF-0016 and anti-mouse PD-1 antibody combined administration are shown in Figure 4. The TGI 17 days after transplantation was 31.5% in the NYF-0016 alone administration group and 35.3% in the anti-mouse PD-1 antibody alone administration group, while it was 57.5% in the NYF-0016 and anti-mouse PD-1 antibody combined administration group, demonstrating that the antitumor effect was enhanced by the combination.

[0205] Example 6. In vivo combined effect of Fc-tagged anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule (NYZ-1010) with anti-PD-1 antibody pembrolizumab and multikinase inhibitor lenvatinib on human lung adenocarcinoma cell lines. Human lung adenocarcinoma cell line NCI-H522 (ATCC) was cultured at 5x10 with 50% Matrigel (Corning) in PBS. ７ The solution was adjusted to give a concentration of 1000 cells / mL, and 0.1 mL of the solution was subcutaneously transplanted into NSG mice (female, 4-6 weeks old) on Day 0. Starting approximately 3 weeks later, the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the following formula.

[0206] Estimated Tumor Volume (mm ３) = average estimated tumor volume of each individual: estimated tumor volume of each individual (mm ３ ) = 1 / 2 x [tumor long diameter] x [tumor short diameter] x [tumor short diameter]

[0207] On Day 28, the mice were divided into groups based on tumor volume (n = 5 per group), and T cells were stimulated and expanded using human PBMCs with CD3 / CD28 Dynabeads (ThermoFisher) at 5 × 10 ７ The medium was adjusted to 0.2 mL / mL and transplanted into the tail vein. Three to four hours later, anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule (NYZ-1010, 0.01 mg / kg) was administered intravenously. Pembrolizumab (Merck, 10 mg / kg) was administered intraperitoneally, and lenvatinib (30 mpk) was administered orally. Administration was performed on Days 28, 35, and 42 (NYZ-1010), 32, 39, and 46 (pembrolizumab), or once daily from Days 28 to 32, once daily from Days 35 to 39, and once daily from Days 42 to 36 (lenvatinib). Tumor growth inhibition (%) was calculated according to the following formula.

[0208] Tumor growth inhibition (%) = 100 - (Estimated tumor volume of each group / Estimated tumor volume of vehicle control × 100)

[0209] The results of NYZ-1010 monotherapy, lenvatinib and pembrolizumab combination therapy, and NYZ-1010, lenvatinib, and pembrolizumab combination therapy are shown in Figure 72 (1). The TGI 52 days after transplantation was 51.4% in the NYZ-1010 monotherapy group and 50.6% in the lenvatinib and pembrolizumab combination therapy group, while it was 84.1% in the NYZ-1010, lenvatinib, and pembrolizumab combination therapy group, demonstrating that the antitumor effect was enhanced by the triple drug combination. In addition, the percentage change in tumor volume on Day 52 for each individual (Change from baseline (%)), with the tumor volume on Day 28 used as the baseline, was calculated using the formula shown below and shown in Figure 72 (2). The average Change from baseline (%) for each group is shown in Table 3. The tumor regression effect of the combined use of NYZ-1010 with lenvatinib and pembrolizumab was demonstrated.

[0210] Change from baseline (%) = (Estimated tumor volume on Day 52 - Estimated tumor volume on Day 28) / Estimated tumor volume on Day 28 × 100 for each individual

[0211]

[0212] Example 7. Generation of BALB / C strain human CD3ε knock-in mice The C57BL / 6J strain human CD3ε knock-in mice generated in Example 4 were backcrossed with BALB / C strain to generate BALB / C strain human CD3ε knock-in mice. PCR and sequence analysis were performed on the tail tissue of the resulting offspring. Individuals with a high homozygous substitution rate were selected and bred for subsequent experiments.

[0213] Example 8. In vivo combined effect of Fc-tagged anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule (NYZ-1010) and anti-mouse PD-1 antibody using BALB / C strain human CD3ε knock-in mice. CT26 NY-SCT cells were used, which were derived from the mouse colon cancer cell line CT26.WT introduced from ATCC by retroviral vectors and transfected with HLA-A2, NY-ESO, and β2M Single Chain Trimer (abbreviated as NY-SCT). These cells express HLA-A2 / NY-ESO on the cell membrane. CT26 NY-SCT was diluted to 1 x 10 with PBS (Wako). ７ The medium was adjusted to give a concentration of 1000 cells / mL, and 0.1 mL of the medium was subcutaneously implanted (Day 0) into BALB / C human CD3ε knock-in mice (female, 7 to 9 weeks old) prepared in Example 7. From Day 8, the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the following formula.

[0214] Estimated Tumor Volume (mm ３ ) = average estimated tumor volume of each individual: estimated tumor volume of each individual (mm ３ ) = 1 / 2 x [tumor long diameter] x [tumor short diameter] x [tumor short diameter]

[0215] On Day 8, the mice were divided into groups based on tumor volume, with each group consisting of 5-10 mice, and an anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule (NYZ-1010, 5 mg / kg) was administered intravenously to the tail vein. An anti-mouse PD-1 antibody (clone RMP1-14, Bio X Cell, 5 mg / kg) was also administered intraperitoneally. Administration was performed on Day 8. Tumor Growth Inhibition (%) was calculated using the following formula:

[0216] Tumor growth inhibition (%) = 100 - (Estimated tumor volume of each group / Estimated tumor volume of vehicle control × 100)

[0217] The results of NYZ-1010 administration alone, anti-mouse PD-1 antibody administration alone, and NYZ-1010 and anti-mouse PD-1 antibody combined administration are shown in Figure 73 (1). The TGI 21 days after transplantation was 70.8% in the NYZ-1010 alone administration group and 38.0% in the anti-mouse PD-1 antibody alone administration group, while it was 84.8% in the NYZ-1010 and anti-mouse PD-1 antibody combined administration group, demonstrating that the antitumor effect was enhanced by the combination.

[0218] Figure 73 (2). In each single-administration group, tumors remained and the efficacy of the drug was limited (gray solid line and thin dashed line, respectively), whereas in the group administered NYZ-1010 in combination with anti-mouse PD-1 antibody, the estimated tumor volume was 0 mm in 3 out of 10 cases. ３ The tumors showed a complete regression, and the complete regression was maintained thereafter (black circles, solid line). CT26 NY-SCT was re-implanted into the completely regressed mice, or transplanted into BALB / C human CD3ε knock-in mice (hereinafter referred to as "Naive mice", female, 7-9 weeks old) that had no tumor transplantation experience. CT26 NY-SCT was diluted to 1 x 10 ７ The medium was adjusted to a concentration of 1000 cells / mL, and 0.1 mL was implanted subcutaneously (Day 59). In naive mice, tumors took root and grew (rough dashed line), but in mice with complete regression, tumors did not take root, and complete regression by NYZ-1010 and anti-mouse PD-1 antibody was maintained (black circles, solid line), demonstrating the establishment of immunological memory against tumor cells. Example 9. In vivo combined effect of Fc-tagged anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule (NYZ-1010) and anti-PD-L1 antibody durvalumab on human lung adenocarcinoma cell lines. Human lung adenocarcinoma cell line NCI-H522 (ATCC) was diluted to 5x10 with PBS containing 50% Matrigel (CORNING). ７ The medium was adjusted to 0.1 mL / mL and subcutaneously transplanted into NSG mice (female, 4-6 weeks old) at 0.1 mL on Day 0. Starting approximately one week later (Day 6-7), the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the following formula.

[0219] Estimated Tumor Volume (mm3) = Mean value of estimated tumor volume for each individual: Estimated tumor volume (mm３ ) = 1 / 2 x [tumor long diameter] x [tumor short diameter] x [tumor short diameter]

[0220] On Day 24, the mice were divided into groups based on tumor volume, with each group consisting of n = 5. On Day 25, human PBMCs were stimulated with CD3 / CD28 Dynabeads (ThermoFisher) and expanded. T cells were then diluted to 5 × 10 in PBS. ７ The solution was adjusted to 100 cells / mL, and 0.2 mL was transplanted into the tail vein. Three to four hours later, an anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule (NYZ-1010, 0.02 mg / kg) was administered into the tail vein. Durvalumab (AstraZeneca, 10 mg / kg) was administered intraperitoneally. Administration was performed on Day 25 and Day 32 (NYZ-1010) or on Day 28, Day 32, Day 35, and Day 39 (Durvalumab). Tumor growth inhibition (%) and change from baseline (%) were calculated using the following formula.

[0221] Tumor growth inhibition (%) = 100 - (Estimated tumor volume of each group / Estimated tumor volume of vehicle control × 100)

[0222] The results of NYZ-1010 administration alone, Durvalumab administration alone, and NYZ-1010 and Durvalumab combination administration are shown in Figure 74 (1). The TGI at 49 days after implantation was 67.7% in the NYZ-1010 alone administration group and -0.8% in the Durvalumab alone administration group, while it was 92.5% in the NYZ-1010 and Durvalumab combination group, demonstrating that the antitumor effect was enhanced by the combination. Furthermore, the percent change in tumor volume from Day 49 for each individual (Change from baseline (%)) using the tumor volume on Day 24 as the baseline was calculated using the formula shown below, and is shown in Figure 74 (2). The mean Change from baseline (%) for each group is also shown in Table 4. The combination of NYZ-1010 and durvalumab demonstrated a strong tumor regression effect.

[0223] Change from baseline (%) = (Estimated tumor volume on Day 49 - Estimated tumor volume on Day 24) / Estimated tumor volume on Day 24 × 100 for each individual

[0224]

[0225] Example 10. In vivo combined effect of Fc-tagged anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule (NYZ-1010) and anti-mouse PD-L1 antibody using BALB / C strain human CD3ε knock-in mice. CT26 NY-SCT prepared in Example 8 was diluted to 1 x 10 with PBS (Wako). ７ The medium was adjusted to give a concentration of 1000 cells / mL, and 0.1 mL of the medium was subcutaneously transplanted (Day 0) into BALB / C human CD3ε knock-in mice (female, 6 to 12 weeks old) prepared in Example 7. From Day 8, the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the following formula.

[0226] Estimated Tumor Volume (mm ３ ) = average estimated tumor volume of each individual: estimated tumor volume of each individual (mm ３ ) = 1 / 2 x [tumor long diameter] x [tumor short diameter] x [tumor short diameter]

[0227] On Day 8, the mice were divided into groups based on tumor volume, with each group consisting of 5-10 mice, and an anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule (NYZ-1010, 5 mg / kg) was administered intravenously to the tail vein. An anti-mouse PD-L1 antibody (clone 10F.9G2, Bio X Cell, 5 mg / kg) was also administered intraperitoneally. Administration was performed on Day 8. Tumor growth inhibition (%) was calculated using the following formula:

[0228] Tumor growth inhibition (%) = 100 - (Estimated tumor volume of each group / Estimated tumor volume of vehicle control × 100)

[0229] The results of administration of NYZ-1010 alone, anti-mouse PD-L1 antibody alone, and combination administration of NYZ-1010 and anti-mouse PD-L1 antibody are shown in Figure 75 (1). TGI 21 days after transplantation was 68.7% in the NYZ-1010 alone administration group, 36.4% in the anti-mouse PD-L1 antibody alone administration group, and 77.4% in the NYZ-1010 and anti-mouse PD-L1 antibody combined administration group, demonstrating that the antitumor effect was enhanced by the combination.

[0230] Figure 75 (2). In each single-administration group, tumors remained and the efficacy of the drug was limited (gray solid line and thin dashed line, respectively), whereas in the NYZ-1010 and anti-mouse PD-L1 antibody combination group, the estimated tumor volume in 2 out of 10 cases was 0 mm ３ The tumors showed a complete regression and continued to show complete regression thereafter (black circle, solid line). CT26.WT cells transfected with only the retroviral vector (hereafter referred to as CT26 Mock) were re-transplanted into mice with complete regression and into naive mice (female, 15-17 weeks old). CT26 Mock cells were re-transplanted into mice with complete regression and into naive mice (female, 15-17 weeks old). CT26 Mock cells were re-transplanted into mice with 1 x 10 ７ The medium was adjusted to 100 cells / mL and 0.1 mL was implanted subcutaneously (Day 64). Tumors took root and grew in naive mice (rough dashed line), but in completely regressed mice, tumors did not take root and complete regression was maintained with NYZ-1010 and anti-mouse PD-L1 antibody (black circles, solid line), demonstrating the establishment of immune memory against the tumor cells.

[0231] Example 11. In vivo combined effect of Fc-tagged anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule (NYZ-1010) and anti-mouse CTLA-4 antibody using BALB / C strain human CD3ε knock-in mice. CT26 NY-SCT prepared in Example 8 was diluted to 1 x 10 with PBS (Wako). ７ The medium was adjusted to give a concentration of 1000 cells / mL, and 0.1 mL of the medium was subcutaneously implanted (Day 0) into BALB / C human CD3ε knock-in mice (female, 7 to 9 weeks old) prepared in Example 7. From Day 8, the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the following formula.

[0232] Estimated Tumor Volume (mm ３ ) = average estimated tumor volume of each individual: estimated tumor volume of each individual (mm ３ ) = 1 / 2 x [tumor long diameter] x [tumor short diameter] x [tumor short diameter]

[0233] On Day 8, the mice were divided into groups based on tumor volume, with each group consisting of 5-10 mice, and an anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule (NYZ-1010, 5 mg / kg) was administered intravenously through the tail vein. An anti-mouse CTLA-4 antibody (clone 9D9, Bio X Cell, 5 mg / kg) was also administered intraperitoneally. Administration was performed on Day 8. Tumor growth inhibition (%) was calculated using the following formula:

[0234] Tumor Growth Inhibition (%) = 100 - (Estimated Tumor Volume of each group / Estimated Tumor Volume of Vehicle Control × 100) The results of administration of NYZ-1010 alone, administration of anti-mouse CTLA-4 antibody alone, and administration of NYZ-1010 and anti-mouse CTLA-4 antibody in combination are shown in Figure 76 (1). The TGI 21 days after transplantation was 70.8% in the NYZ-1010 alone administration group, 12.1% in the anti-mouse CTLA-4 antibody alone administration group, and 80.4% in the NYZ-1010 and anti-mouse CTLA-4 antibody combined administration group, demonstrating that the anti-tumor effect was enhanced by the combination administration.

[0235] Figure 76 (2). In each single-administration group, tumors remained and the drug efficacy was limited (gray solid line and thin dashed line, respectively), whereas in the group administered NYZ-1010 in combination with anti-mouse CTLA-4 antibody, the estimated tumor volume was 0 mm in one of 10 cases. ３ The CT26 NY-SCT cells were re-transplanted into the completely regressed mice and into naive mice (female, 7-9 weeks old). ... ７The medium was adjusted to 100 cells / mL and 0.1 mL was implanted subcutaneously (Day 59). Tumors took root and grew in naive mice (rough dashed line), but in completely regressed mice, tumors did not take root and complete regression was maintained with NYZ-1010 and anti-mouse CTLA-4 antibody (black circles, solid line), indicating the establishment of immunological memory against the tumor cells.

[0236] Example 12 In vivo combined effect of Fc-tagged anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule (NYZ-1010) and anti-mouse TIGIT antibody using human CD3ε knock-in mice. CT26 NY-SCT prepared in Example 8 was diluted to 1 x 10 with PBS (Wako). ７ The medium was adjusted to give a concentration of 1000 cells / mL, and 0.1 mL of the medium was subcutaneously implanted (Day 0) into BALB / C human CD3ε knock-in mice (female, 7 to 9 weeks old) prepared in Example 7. From Day 8, the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the following formula.

[0237] Estimated Tumor Volume (mm ３ ) = average estimated tumor volume of each individual: estimated tumor volume of each individual (mm ３ ) = 1 / 2 x [tumor long diameter] x [tumor short diameter] x [tumor short diameter]

[0238] On Day 8, mice were divided into groups based on tumor volume (n = 5-10 per group), and an anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule (NYZ-1010, 5 mg / kg) was administered intravenously to the tail vein. Anti-mouse TIGIT antibody (clone 1B4, Absolute Antibody, 5 mg / kg) was also administered intraperitoneally. Administration was performed on Day 8. Tumor growth inhibition (%) was calculated using the following formula:

[0239] Tumor growth inhibition (%) = 100 - (Estimated tumor volume of each group / Estimated tumor volume of vehicle control × 100)

[0240] The results of administration of NYZ-1010 alone, anti-mouse TIGIT antibody alone, and combined administration of NYZ-1010 and anti-mouse TIGIT antibody are shown in Figure 77 (1). Twenty-four days after transplantation, the TGI was 55.5% in the NYZ-1010 alone administration group, -1.2% in the anti-mouse TIGIT antibody alone administration group, and 78.3% in the NYZ-1010 and anti-mouse TIGIT antibody combined administration group, demonstrating that the antitumor effect was enhanced by the combined administration.

[0241] Figure 77 (2). In each single-dose group, tumors remained and the efficacy was limited (gray solid line and thin dashed line, respectively), whereas the estimated tumor volume in 2 out of 10 cases in the combined group was 0 mm ３ The CT26 NY-SCT cells were re-transplanted into the completely regressed mice and into naive mice (female, 15-17 weeks old). ... ７ The medium was adjusted to 100 cells / mL and 0.1 mL was implanted subcutaneously (Day 58). Tumors took root and grew in naive mice (rough dashed line), but in mice with complete regression, tumors did not take root and complete regression was maintained with NYZ-1010 and anti-mouse TIGIT antibody (black circles, solid line), indicating the establishment of immunological memory against the tumor cells.

[0242] Example 13 In vivo effect of combined use of Fc-tagged anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule (NYZ-1010) and carboplatin on human squamous cell lung cancer cell lines. Human squamous cell lung cancer cell line NCI-H1703 (ATCC) was diluted to 6 x 10 with PBS containing 50% Matrigel (CORNING). ７ The medium was adjusted to 0.1 mL / mL and subcutaneously transplanted into NSG mice (female, 4-6 weeks old) at 0.1 mL on Day 0. Starting approximately one week later (Day 6-7), the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the following formula.

[0243] Estimated Tumor Volume (mm3) = Mean value of estimated tumor volume for each individual: Estimated tumor volume (mm ３) = 1 / 2 x [tumor long diameter] x [tumor short diameter] x [tumor short diameter]

[0244] On Day 14, the mice were divided into groups based on tumor volume (n = 5 per group), and T cells were stimulated and expanded using human PBMCs with CD3 / CD28 Dynabeads (ThermoFisher) at 5 × 10 in PBS. ７ The cells were adjusted to a concentration of 0.2 mL / mL, and 0.2 mL was transplanted into the tail vein. Three to four hours later, an anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule (NYZ-1010, 0.02 mg / kg) was administered into the tail vein. Carboplatin (TCI, 50 mg / kg) was administered intraperitoneally. Administration was performed on Day 14 and Day 21. Tumor growth inhibition (%) and change from baseline (%) were calculated using the following formula.

[0245] Tumor growth inhibition (%) = 100 - (Estimated tumor volume of each group / Estimated tumor volume of vehicle control × 100)

[0246] The results of NYZ-1010 administration alone, Carboplatin administration alone, and the combined administration of NYZ-1010 and Carboplatin are shown in Figure 78. The TGI 34 days after transplantation was 47.2% in the NYZ-1010 alone administration group, 15.0% in the Carboplatin alone administration group, and 61.6% in the NYZ-1010 and Carboplatin combined administration group, demonstrating that the antitumor effect was enhanced by the combined use.

[0247] Example 14 In vivo combined effect of Fc-tagged anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule (NYZ-1010) and sacituzumab govitecan-hziy on human esophageal cancer cell lines. Human esophageal cancer cell line OE-19 (DSMZ) was diluted at 5x10 with 50% Matrigel (Corning) in PBS. ７The medium was adjusted to 0.1 mL / mL and subcutaneously transplanted into NSG mice (female, 4-6 weeks old) at 0.1 mL on Day 0. Starting approximately one week later (Day 6-7), the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the following formula.

[0248] Estimated Tumor Volume (mm3) = Mean value of estimated tumor volume for each individual: Estimated tumor volume (mm ３ ) = 1 / 2 x [tumor long diameter] x [tumor short diameter] x [tumor short diameter]

[0249] On Day 6, the mice were divided into groups based on tumor volume (n = 5 per group), and sacituzumab govitecan-hziy (25 mg / kg) was administered intravenously into the tail vein. On Day 7, human PBMCs were stimulated and expanded with CD3 / CD28 Dynabeads (ThermoFisher), and T cells were diluted to 5 × 10 in PBS. ７ The solution was adjusted to 100 cells / mL, and 0.2 mL was transplanted into the tail vein. Three to four hours later, an anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule (NYZ-1010, 0.01 mg / kg) was administered into the tail vein. Administration was performed on Day 7 (NYZ-1010) or on Day 6 and Day 13 (sacituzumab govitecan-hziy). Tumor growth inhibition (%) and change from baseline (%) were calculated using the following formula.

[0250] Tumor growth inhibition (%) = 100 - (Estimated tumor volume of each group / Estimated tumor volume of vehicle control × 100)

[0251] The results of NYZ-1010 administration alone, sacituzumab govitecan-hziy administration alone, and the combination of NYZ-1010 and sacituzumab govitecan-hziy are shown in Figure 79(1). TGI 21 days after transplantation was 79.9% in the NYZ-1010 administration alone group, 35.4% in the sacituzumab govitecan-hziy administration alone group, and 96.7% in the NYZ-1010 and sacituzumab govitecan-hziy combination group, demonstrating that the antitumor effect was enhanced by the combination. The percentage change in tumor volume from Day 6 to Day 21 for each individual (Change from baseline (%)) was calculated using the formula below and shown in Figure 79 (2). The mean Change from baseline (%) for each group is shown in Table 5. The combined use of NYZ-1010 and sacituzumab govitecan-hziy demonstrated a tumor regression effect.

[0252] Change from baseline (%) = (Estimated tumor volume on Day 21 - Estimated tumor volume on Day 6) / Estimated tumor volume on Day 6 × 100

[0253]

[0254] Example 15. In vivo combined effect of Fc-tagged anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule (NYZ-1010) and doxorubicin on human lung adenocarcinoma cell lines. Human synovial sarcoma cell line SW982 (ATCC) was diluted at 5 x 10 with PBS containing 50% Matrigel (CORNING). ７ The medium was adjusted to 0.1 mL / mL and subcutaneously transplanted into NSG mice (female, 4-6 weeks old) at 0.1 mL on Day 0. Starting approximately one week later (Day 6-7), the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the following formula.

[0255] Estimated Tumor Volume (mm3) = Mean value of estimated tumor volume for each individual: Estimated tumor volume (mm ３ ) = 1 / 2 x [tumor long diameter] x [tumor short diameter] x [tumor short diameter]

[0256] On Day 40, the mice were divided into groups based on tumor volume (n = 5 per group), and Doxorubicin (Sandoz, 2.5 mg / kg) was administered intravenously to the tail vein. On Day 41, human PBMCs were stimulated and expanded with CD3 / CD28 Dynabeads (ThermoFisher), and T cells were diluted to 5 × 10 in PBS. ７ The solution was adjusted to 0.2 mL / mL, and 0.2 mL was transplanted into the tail vein. Three to four hours later, an anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule (NYZ-1010, 0.01 mg / kg) was administered into the tail vein. Administration was performed on Day 41 (NYZ-1010) or on Day 40 and Day 47 (Doxorubicin). Tumor growth inhibition (%) and change from baseline (%) were calculated using the following formula.

[0257] Tumor Growth Inhibition (%) = 100 - (Estimated Tumor Volume of each group / Estimated Tumor Volume of Vehicle Control × 100) The results of NYZ-1010 administration alone, Doxorubicin administration alone, and NYZ-1010 and Doxorubicin combination administration are shown in Figure 80 (1). The TGI 55 days after transplantation was 71.8% in the NYZ-1010 alone administration group, 25.6% in the Doxorubicin alone administration group, and 90.2% in the NYZ-1010 and Doxorubicin combination group, demonstrating that the antitumor effect was enhanced by the combination. The percentage change in tumor volume from Day 40 to Day 55 for each individual (Change from baseline (%)) was calculated using the formula shown below and shown in Figure 80 (2). The mean Change from baseline (%) for each group is shown in Table 6. The combined use of NYZ-1010 and doxorubicin demonstrated a tumor regression effect.

[0258] Change from baseline (%) = (Estimated tumor volume on Day 55 - Estimated tumor volume on Day 40) / Estimated tumor volume on Day 40 × 100

[0259]

[0260] Example 16 In vivo Combination Effect of Fc-Tagged Anti-HLA-A2 / NY-ESO-Anti-CD3 Bispecific Molecule (NYZ-1010) and Decitabine on Human Squamous Cell Lung Cancer Cell Lines Human squamous cell lung cancer cell line NCI-H1703 (ATCC) was cultured at 6 x 10 ng / mL in PBS containing 50% Matrigel (CORNING). ７ The medium was adjusted to 0.1 mL / mL and subcutaneously transplanted into NSG mice (female, 4-6 weeks old) at 0.1 mL on Day 0. Starting approximately one week later (Day 6-7), the major axis (mm) and minor axis (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the following formula.

[0261] Estimated Tumor Volume (mm3) = Mean value of estimated tumor volume for each individual: Estimated tumor volume (mm ３ ) = 1 / 2 x [tumor long diameter] x [tumor short diameter] x [tumor short diameter]

[0262] On Day 7, mice were divided into groups based on tumor volume (n = 5 per group), and Decitabine (Tokyo Chemical Industry Co., Ltd., 3 mg / kg) was administered intravenously to the tail vein. On Day 14, human PBMCs were stimulated and expanded with CD3 / CD28 Dynabeads (ThermoFisher), and the T cells were diluted to 5 × 10 in PBS. ７The solution was adjusted to 100 cells / mL, and 0.2 mL was transplanted into the tail vein. Three to four hours later, an anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule (NYZ-1010, 0.02 mg / kg) was administered into the tail vein. Administration was performed on Day 14 and Day 21 (NYZ-1010) or on Day 7, Day 9, and Day 11 (Decitabine). Tumor growth inhibition (%) and change from baseline (%) were calculated using the following formula.

[0263] Tumor growth inhibition (%) = 100 - (Estimated tumor volume of each group / Estimated tumor volume of vehicle control × 100) Change from baseline (%) = (Estimated tumor volume on Day 32 - Estimated tumor volume on Day 7) / Estimated tumor volume on Day 7 × 100

[0264] The results of NYZ-1010 administration alone, Decitabine administration alone, and NYZ-1010 and Decitabine combination administration are shown in Figure 81 (1). The TGI at 32 days after implantation was 68.0% in the NYZ-1010 alone administration group and 46.7% in the Decitabine alone administration group, while it was 93.0% in the NYZ-1010 and Decitabine combination group, demonstrating that the antitumor effect was enhanced by the combination. Furthermore, the percent change in tumor volume from Day 7 to Day 32 for each individual (Change from baseline (%)) was calculated using the formula shown below and is shown in Figure 81 (2). The mean Change from baseline (%) for each group is also shown in Table 7.

[0265]

[0266] Combining the antibodies provided by the present invention with other drugs makes it possible to treat or prevent various cancers.

[0267] SEQ ID NO: 1: Amino acid sequence of CDRH1 contained in NYA-0001 SEQ ID NO: 2: Amino acid sequence of CDRH2 contained in NYA-0001 SEQ ID NO: 3: Missing number (SEQ ID NO: 3 of Patent Application No. 2022-041253 filed on March 16, 2022 was the amino acid sequence of CDRH3 contained in NYA-0001) SEQ ID NO: 4: Amino acid sequence of CDRL1 contained in NYA-0001 SEQ ID NO: 5: Amino acid sequence of CDRL2 contained in NYA-0001 SEQ ID NO: 6: Amino acid sequence of CDRL3 contained in NYA-0001 SEQ ID NO: 7: Amino acid sequence of heavy chain CDR1 of C3E-7085 SEQ ID NO: 8: Amino acid sequence of heavy chain CDR3 of C3E-7085 SEQ ID NO: 9: Amino acid sequence of heavy chain CDR4 of C3E-7085 SEQ ID NO: 10: Amino acid sequence of light chain CDR1 of C3E-7085 SEQ ID NO: 11: Missing number (SEQ ID NO: 11 of the priority application dated March 16, 2022 was the amino acid sequence of the light chain CDR2 of C3E-7085) SEQ ID NO: 12: Amino acid sequence of the light chain CDR3 of C3E-7085 SEQ ID NO: 13: Amino acid sequence of the heavy chain variable region of NYA-0001 SEQ ID NO: 14: Amino acid sequence of the light chain variable region of NYA-0001 SEQ ID NO: 15: Amino acid sequence of the heavy chain variable region of NYA-0082 SEQ ID NO: 16: Amino acid sequence of the light chain variable region of NYA-0082 SEQ ID NO: 17: Amino acid sequence of the full length of NYA-1163 SEQ ID NO: 18: Amino acid sequence of the full length of NYA-2023 SEQ ID NO: 19: Amino acid sequence of the full length of NYA-2027 SEQ ID NO: 20: Amino acid sequence of the full length of NYA-1143 SEQ ID NO: 21: Amino acid sequence of the full length of NYA-1143 SEQ ID NO: 22: Amino acid sequence of full length NYA-2143 SEQ ID NO: 23: Amino acid sequence of NYA-1143-VL01 SEQ ID NO: 24: Amino acid sequence of full length NYA-2044 SEQ ID NO: 25: Amino acid sequence of full length NYA-2045 SEQ ID NO: 26: Amino acid sequence of full length NYA-2047 SEQ ID NO: 27: Amino acid sequence of full length NYA-2048 SEQ ID NO: 28: Amino acid sequence of full length NYA-2060 SEQ ID NO: 29: Amino acid sequence of full length NYA-2061 SEQ ID NO: 30: Amino acid sequence of full length NYA-0001 SEQ ID NO: 31: Amino acid sequence of full length HC1 SEQ ID NO: 32: Amino acid sequence of full length NYF-0016-HC2 SEQ ID NO: 33: Amino acid sequence of full length NYF-0019-HC2SEQ ID NO: 34: Amino acid sequence of NYF-0022-HC2 full length SEQ ID NO: 35: Amino acid sequence of NYF-0023-HC2 full length SEQ ID NO: 36: Amino acid sequence of NYF-0027-HC2 full length SEQ ID NO: 37: Amino acid sequence of NYF-0035-HC2 full length SEQ ID NO: 38: Amino acid sequence of NYF-0044-HC2 full length SEQ ID NO: 39: Amino acid sequence of NYF-0045-HC2 full length SEQ ID NO: 40: Amino acid sequence of NYF-0047-HC2 full length SEQ ID NO: 41: Amino acid sequence of NYF-0048-HC2 full length SEQ ID NO: 42: Amino acid sequence of NYF-0060-HC2 full length SEQ ID NO: 43: Amino acid sequence of NYF-0061-HC2 full length SEQ ID NO: 44: Amino acid sequence of NYA-0001-Fab-HC1-κ delete full length SEQ ID NO: 45: Amino acid sequence of NYA-0001-LC full length SEQ ID NO: 46: Amino acid sequence of C3E-7034 SEQ ID NO: 47: Amino acid sequence of C3E-7036 SEQ ID NO: 48: Amino acid sequence of C3E-7085 SEQ ID NO: 49: Amino acid sequence of C3E-7088 SEQ ID NO: 50: Amino acid sequence of C3E-7093 SEQ ID NO: 51: Amino acid sequence of C3E-7078 SEQ ID NO: 52: Amino acid sequence of NYF-0014-HC2 full length SEQ ID NO: 53: Amino acid sequence of NYF-0082-HC2 full length SEQ ID NO: 54: Amino acid sequence of NYF-0083-HC2 full length SEQ ID NO: 55: Amino acid sequence of NYZ-0082-HC2 full length SEQ ID NO: 56: Amino acid sequence of NYZ-0083-HC2 full length SEQ ID NO: 57: Amino acid sequence of NYZ-1010-HC2 full length SEQ ID NO: 58: Amino acid sequence of full-length C3E-7085-LC SEQ ID NO: 59: Full-length amino acid sequence of C3E-7085 scFv SEQ ID NO: 60: Amino acid sequence of full-length NYZ-1007-HC2 SEQ ID NO: 61: Amino acid sequence of full-length NYZ-1017-HC2 SEQ ID NO: 62: Full-length amino acid sequence of C3E-7096 SEQ ID NO: 63: Full-length amino acid sequence of C3E-7097 SEQ ID NO: 64: Full-length amino acid sequence of C3E-7098 SEQ ID NO: 65: Full-length amino acid sequence of C3E-7099 SEQ ID NO: 66: Amino acid sequence of NY-ESO SEQ ID NO: 67: Amino acid sequence of truncated form of HLA-A*0201 (GenBank: ASA47534.1) SEQ ID NO: 68: Amino acid sequence of β2-microglobulinSEQ ID NO: 69: Amino acid sequence of NYA-1143-VH02 SEQ ID NO: 70: Amino acid sequence of NYA-1143-VH03 SEQ ID NO: 71: Amino acid sequence of full-length NYA-1154

Claims

1. A pharmaceutical composition for the prevention and / or treatment of cancer comprising a multispecific antibody [i], wherein the pharmaceutical composition comprising the multispecific antibody [i] is administered simultaneously with, separately from, or in succession to, compound [ii]; The multispecific antibody [i] is An antibody or antigen-binding fragment thereof that binds to HLA / NY-ESO, and Antibodies that bind to CD3 or their antigen-binding fragments. Including; The aforementioned compound [ii] is Compounds that inhibit immune checkpoint molecules, or chemotherapeutic agents A pharmaceutical composition containing the following:

2. A pharmaceutical composition for the prevention and / or treatment of cancer comprising compound [ii], wherein the pharmaceutical composition comprising compound [ii] is administered simultaneously, separately, or consecutively with a multispecific antibody [i]; The multispecific antibody [i] is An antibody or antigen-binding fragment thereof that binds to HLA / NY-ESO, and Antibodies that bind to CD3 or their antigen-binding fragments. Including; The aforementioned compound [ii] is Compounds that inhibit immune checkpoint molecules, or chemotherapeutic agents A pharmaceutical composition containing the following:

3. A pharmaceutical composition for the prevention and / or treatment of cancer comprising a multispecific antibody [i] and a compound [ii], wherein the multispecific antibody [i] and the compound [ii] are administered simultaneously, separately, or sequentially; The multispecific antibody [i] is An antibody or antigen-binding fragment thereof that binds to HLA / NY-ESO, and Antibodies that bind to CD3 or their antigen-binding fragments. Including; The aforementioned compound [ii] is Compounds that inhibit immune checkpoint molecules, or chemotherapeutic agents A pharmaceutical composition containing the following:

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the multispecific antibody [i] is bispecific.

5. The multispecific antibody [i] The heavy chain CDRH1 consists of the amino acid sequence represented by Sequence ID No.

1. The heavy chain CDRH2 consists of the amino acid sequence represented by Sequence ID No.

2. The heavy chain CDRH3 consists of the amino acid sequence represented by Sequence ID No.

3. In the amino acid sequence represented by Sequence ID No. 4, the light chain CDRL1 consists of an amino acid sequence in which the 7th amino acid is W. A light chain CDRL2 consisting of the amino acid sequence represented by Asp Asn Asn (the fifth sequence from the top in Figure 11: Sequence ID No. 5 in Figure 82), and Light chain CDRL3 consisting of the amino acid sequence represented by Sequence ID No. 6 An antibody or antigen-binding fragment thereof that binds to HLA / NY-ESO, and, The heavy chain CDRH1 consists of the amino acid sequence represented by Sequence ID No.

7. The heavy chain CDRH2 consists of the amino acid sequence represented by Sequence ID No.

8. The heavy chain CDRH3 consists of the amino acid sequence represented by Sequence ID No.

9. The light chain CDRL1 consists of the amino acid sequence represented by Sequence ID No.

10. The light chain CDRL2 consists of the amino acid sequence represented by Arg Asp Asp (the fifth sequence from the top in Figure 12: Sequence ID No. 11 in Figure 82), and Light chain CDRL3 consisting of the amino acid sequence represented by Sequence ID No. 12 An antibody that binds to CD3 or an antigen-binding fragment thereof, A pharmaceutical composition according to any one of claims 1 to 3, comprising:

6. A pharmaceutical composition according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof that binds to HLA / NY-ESO comprises the following set of CDRH1 to CDRH3 and CDRL1 to CDRL3: The heavy chain CDRH1 consists of the amino acid sequence represented by Sequence ID No.

1. The heavy chain CDRH2 consists of the amino acid sequence represented by Sequence ID No.

2. The heavy chain CDRH3 consists of the amino acid sequence represented by Sequence ID No.

3. In the amino acid sequence represented by Sequence ID No. 4, the light chain CDRL1 consists of an amino acid sequence in which the 7th amino acid is W. The light chain CDRL2 consists of the amino acid sequence represented by Asp Asn Asn (the fifth sequence from the top in Figure 11: Sequence ID No. 5 in Figure 82), and A light chain CDRL3 consisting of the amino acid sequence represented by Sequence ID No.

6.

7. An antibody or antigen-binding fragment thereof that binds to HLA / NY-ESO, The heavy chain variable region corresponding to amino acid positions 21-140 of the amino acid sequence represented by SEQ ID NO: 57 and the light chain variable region corresponding to amino acid positions 161-271 of the amino acid sequence represented by SEQ ID NO: 57 A pharmaceutical composition according to any one of claims 1 to 3, comprising:

8. The pharmaceutical composition according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof that binds to HLA / NY-ESO is scFv.

9. The pharmaceutical composition according to claim 8, wherein the antibody or antigen-binding fragment thereof that binds to HLA / NY-ESO is an scFv consisting of the amino acid sequence from the 21st to the 271st amino acid sequence of the amino acid sequence represented by Sequence ID No.

57.

10. A pharmaceutical composition according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof that binds to CD3 comprises the following set of CDRH1 to CDRH3 and CDRL1 to CDRL3: The heavy chain CDRH1 consists of the amino acid sequence represented by Sequence ID No.

7. The heavy chain CDRH2 consists of the amino acid sequence represented by Sequence ID No.

8. The heavy chain CDRH3 consists of the amino acid sequence represented by Sequence ID No.

9. The light chain CDRL1 consists of the amino acid sequence represented by Sequence ID No.

10. The light chain CDRL2 consists of the amino acid sequence represented by Arg Asp Asp (the fifth sequence from the top in Figure 12: Sequence ID No. 11 in Figure 82), and A light chain CDRL3 consisting of the amino acid sequence represented by Sequence ID No.

12.

11. An antibody or its antigen-binding fragment that binds to CD3, Heavy chain variable region consisting of amino acid sequences 2 to 119 of the amino acid sequence represented by SEQ ID NO: 48 and light chain variable region consisting of amino acid sequences 135 to 241 of the amino acid sequence represented by SEQ ID NO: 48 A pharmaceutical composition according to claim 10, comprising:

12. The pharmaceutical composition according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof that binds to CD3 is scFv.

13. An antibody or its antigen-binding fragment that binds to CD3, scFv consisting of amino acid sequences 2 through 241 of the amino acid sequence represented by SEQ ID NO: 48 The pharmaceutical composition according to claim 12.

14. The multispecific antibody [i] From the N-terminus towards the C-terminus, A first polypeptide comprising, in that order, an antibody that specifically binds to HLA / NY-ESO which is svFv or an antigen-binding fragment thereof, the heavy chain variable region and constant region CH1 of the antibody that binds to CD3, and the immunoglobulin Fc region (i), A second polypeptide comprising the hinge region and Fc region (ii) of an immunoglobulin, and Third polypeptide comprising a variable region and a constant region, containing an antibody light chain that binds to CD3. Includes, Preferably, the first polypeptide and the second polypeptide are associated in Fc region (i) and Fc region (ii), and the first polypeptide is associated with the third polypeptide in the variable region and constant region CH1 of the antibody heavy chain, according to claim 10.

15. The pharmaceutical composition according to claim 14, wherein the first polypeptide comprises the amino acid sequence from the 20th to the 724th amino acid sequence of the amino acid sequence represented by Sequence ID No.

57.

16. The pharmaceutical composition according to claim 14, wherein the second polypeptide comprises the amino acid sequence from the 20th to the 246th amino acid sequence shown in SEQ ID NO:

31.

17. The pharmaceutical composition according to claim 14, wherein the third polypeptide comprises the amino acid sequence from the 21st to the 233rd amino acid sequence of the amino acid sequence represented by Sequence ID No.

58.

18. The first polypeptide consists of the amino acid sequence from the 20th to the 724th amino acid sequence of the amino acid sequence represented by Sequence ID No. 57; The second polypeptide consists of amino acid sequences 20 through 246 of the amino acid sequence represented by Sequence ID No. 31; The third polypeptide consists of amino acid sequences 21 through 233 of the amino acid sequence represented by Sequence ID No. 58; The pharmaceutical composition according to claim 14, wherein, optionally, one or more amino acids are deleted from the carboxyl terminus of the amino acid sequence of one or more polypeptides contained in the multispecific antibody [i].

19. Cancers include kidney cancer, melanoma, squamous cell carcinoma, basal cell carcinoma, conjunctival cancer, oral cancer, laryngeal cancer, pharyngeal cancer, thyroid cancer, lung cancer (non-small cell lung cancer (adenocarcinoma, squamous cell carcinoma, large cell carcinoma), small cell lung cancer), breast cancer, esophageal cancer, stomach cancer, duodenal cancer, small intestine cancer, colorectal cancer, rectal cancer, appendiceal cancer, anal cancer, liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, adrenal cancer, bladder cancer, prostate cancer, uterine cancer, vaginal cancer, liposarcoma, angiosarcoma, chondrosarcoma, and rhabdomyosarcoma. A pharmaceutical composition according to any one of claims 1 to 3, wherein one or more are selected from the group consisting of tumor, Ewing's sarcoma, osteosarcoma, undifferentiated pleomorphic sarcoma, myxoid fibrosarcoma, malignant peripheral schwannoma, retroperitoneal sarcoma, synovial sarcoma, uterine sarcoma, gastrointestinal stromal tumor, leiomyosarcoma, epithelioid sarcoma, B-cell lymphoma, T-NK cell lymphoma, Hodgkin lymphoma, myeloid leukemia, lymphocytic leukemia, myeloproliferative disorders, myelodysplastic syndromes, multiple myeloma, testicular cancer, and ovarian cancer.

20. The pharmaceutical composition according to any one of claims 1 to 3, wherein the multispecific antibody is administered after a compound or chemotherapeutic agent that inhibits an immune checkpoint molecule.

21. The pharmaceutical composition according to any one of claims 1 to 3, wherein the multispecific antibody is administered before a compound or chemotherapeutic agent that inhibits an immune checkpoint molecule.

22. The pharmaceutical composition according to any one of claims 1 to 3, wherein the multispecific antibody is administered simultaneously with a compound or chemotherapeutic agent that inhibits an immune checkpoint molecule.

23. A pharmaceutical composition according to any one of claims 1 to 3, to be used in combination with one or more further pharmaceuticals and / or treatments.

24. The pharmaceutical composition according to any one of claims 1 to 3, wherein compound [ii] is a compound that inhibits an immune checkpoint molecule.

25. The pharmaceutical composition according to any one of claims 1 to 3, wherein the immune checkpoint molecule is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, and TIGIT.

26. The pharmaceutical composition according to claim 25, wherein the immune checkpoint molecule is PD-1.

27. The pharmaceutical composition according to claim 26, wherein the compound that inhibits an immune checkpoint molecule is nivolumab or pembrolizumab, its antigen-binding fragment, or a compound containing its antigen-binding fragment.

28. The pharmaceutical composition according to claim 25, wherein the immune checkpoint molecule is PD-L1.

29. The pharmaceutical composition according to claim 28, wherein the compound that inhibits an immune checkpoint molecule is atezolizumab, durvalumab, or avelumab, its antigen-binding fragment, or a compound containing its antigen-binding fragment.

30. The pharmaceutical composition according to claim 25, wherein the immune checkpoint molecule is CTLA-4.

31. The pharmaceutical composition according to claim 30, wherein the compound that inhibits an immune checkpoint molecule is ipilimumab, tremelimumab, spartalizumab, or semiprimab, its antigen-binding fragment, or a compound containing its antigen-binding fragment.

32. The pharmaceutical composition according to claim 25, wherein the immune checkpoint molecule is TIGIT.

33. The pharmaceutical composition according to claim 32, wherein the compound that inhibits an immune checkpoint molecule is tilagolmab or vivostrimab, its antigen-binding fragment, or a compound containing its antigen-binding fragment.

34. The pharmaceutical composition according to any one of claims 1 to 3, wherein the compound that inhibits immune checkpoint molecules is pembrolizumab, nivolumab, spartalizumab, semiprimab, avelumab, atezolizumab, durvalumab, ipilimumab, or tremelimumab.

35. The pharmaceutical composition according to any one of claims 1 to 3, wherein the compound that inhibits an immune checkpoint molecule is pembrolizumab or an antigen-binding fragment of pembrolizumab, or a compound containing the antigen-binding fragment.

36. The pharmaceutical composition according to any one of claims 1 to 3, wherein the compound [ii] is a chemotherapeutic agent.

37. The pharmaceutical composition according to claim 36, wherein the chemotherapeutic agent is a microtubule formation inhibitor.

38. The pharmaceutical composition according to claim 37, wherein the chemotherapeutic agent is a taxane-based microtubule formation inhibitor.

39. The pharmaceutical composition according to claim 36, wherein the chemotherapeutic agent is selected from the group consisting of paclitaxel, docetaxel, vincristine, vinblastine, vindesine, eribulin, vinorelbine, albumin-bound paclitaxel oxaliplatin, carboplatin, cisplatin, nedaplatin, azacitidine, decitabine, doxorubicin, bleomycin, liposomal doxorubicin, ifosfamide, cyclophosphamide, and dacarbazine.

40. The pharmaceutical composition according to claim 39, wherein the chemotherapeutic agent is paclitaxel or albumin-suspended paclitaxel.

41. The pharmaceutical composition according to any one of claims 1 to 3, which is further administered in combination with a multi-kinase inhibitor.

42. The pharmaceutical composition according to claim 41, wherein the multikinase inhibitor is one or more selected from the group consisting of sorafenib, sunitinib, pazopanib, regorafenib, and lenvatinib.

43. The pharmaceutical composition according to claim 41, wherein the multikinase inhibitor is lenvatinib.

44. The pharmaceutical composition according to claim 41, wherein the compound [ii] is a compound that inhibits an immune checkpoint molecule, preferably pembrolizumab or an antigen-binding fragment of pembrolizumab, or a compound containing the antigen-binding fragment.

45. The pharmaceutical composition according to claim 41, wherein the compound that inhibits immune checkpoint molecules is pembrolizumab or an antigen-binding fragment of pembrolizumab, or a compound containing the antigen-binding fragment, and the multikinase inhibitor is lenvatinib.

46. The pharmaceutical composition according to any one of claims 1 to 3, wherein the chemotherapeutic agent is a taxane-based microtubule formation inhibitor selected from the group consisting of paclitaxel, docetaxel, vincristine, vinblastine, vindesine, eribulin, vinorelbine, and albumin-bound paclitaxel.

47. A pharmaceutical composition comprising a multispecific antibody [i], wherein the pharmaceutical composition comprising the multispecific antibody [i] is administered in combination with compound [ii], and the pharmaceutical composition comprising the multispecific antibody [i] is administered simultaneously with, separately from, or consecutively with compound [ii]. The multispecific antibody [i] is An antibody or antigen-binding fragment thereof that binds to HLA / NY-ESO, and Antibodies that bind to CD3 or their antigen-binding fragments. Including; The aforementioned compound [ii] is Compounds that inhibit immune checkpoint molecules, or chemotherapeutic agents A pharmaceutical composition containing the following:

48. A pharmaceutical composition comprising compound [ii], wherein the pharmaceutical composition comprising compound [ii] is administered in combination with a multispecific antibody [i], and the pharmaceutical composition comprising compound [ii] is administered simultaneously, separately, or consecutively with the multispecific antibody [i]. The multispecific antibody [i] is An antibody or antigen-binding fragment thereof that binds to HLA / NY-ESO, and Antibodies that bind to CD3 or their antigen-binding fragments. Including; The aforementioned compound [ii] is Compounds that inhibit immune checkpoint molecules, or chemotherapeutic agents A pharmaceutical composition containing the following:

49. A pharmaceutical composition comprising a multispecific antibody [i] and a compound [ii], The multispecific antibody [i] is An antibody or antigen-binding fragment thereof that binds to HLA / NY-ESO, and Antibodies that bind to CD3 or their antigen-binding fragments. Including; The aforementioned compound [ii] is Compounds that inhibit immune checkpoint molecules, or chemotherapeutic agents A pharmaceutical composition containing the following:

50. The pharmaceutical composition according to any one of claims 47 to 49, further comprising a multikinase inhibitor or administered in combination with a multikinase inhibitor.

51. The pharmaceutical composition according to claim 50, wherein the multikinase inhibitor is one or more selected from the group consisting of sorafenib, sunitinib, pazopanib, regorafenib, and lenvatinib.

52. The pharmaceutical composition according to claim 51, wherein the multikinase inhibitor is lenvatinib.