bispecific antibodies

Abstract

A novel antibody that can be used as an antitumor agent, and an antitumor agent comprising a molecule including said antibody, etc. as an active ingredient. An antibody or a binding fragment thereof comprising a heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO. 54, a heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO. 55, a heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO. 56, a light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO. 57, or a light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO. 57 in which the 7th amino acid is W and / or the 8th amino acid is K, a light chain CDRL2 consisting of the amino acid sequence represented by SEQ ID NO. 58, a light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO. 59, or a light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO. 57 in which the 2nd amino acid is A or S.

Description

Technology Field

[0001] The present invention relates to antibodies useful for cancer treatment, bispecific antibodies, etc. Background Technology

[0002] NY-ESO-1 is a molecule identified in esophageal cancer by the Serological Analysis of Recombinant cDNA Expression Libraries (SEREX) method (Non-patent Literature 1), and LAGE-1, also known as the alias NY-ESO-2, is a molecule identified by the Representational Difference Analysis of Tumor cDNA Libraries (Non-patent Literature 2). It is known that in normal tissues, the expression of both molecules is localized to the testes, but their functions are unknown. The association of NY-ESO-1 and LAGE-1 with cancer is suggested by the fact that expression has been reported in a wide range of cancers, including melanoma, lung cancer, bladder cancer, ovarian cancer, soft tissue sarcoma, and multiple myeloma (Non-patent Literature 3). In addition, regarding the correlation with malignancy, there are reports that NY-ESO-1 is expressed more in metastatic sites than in primary sites of melanoma (Non-patent Literature 4), that NY-ESO-1 and LAGE-1 are expressed more in advanced cancer compared to early cancer in urothelial carcinoma (Non-patent Literature 5), and that NY-ESO-1 is expressed more in myeloma of high-risk chromosomal abnormalities than in myeloma of normal chromosomes (Non-patent Literature 6). Based on this information, NY-ESO-1 and LAGE-1 are attracting attention as molecules with high cancer specificity, and although much research and development is being conducted as new drug targets for cancer vaccine therapy, no drugs have been approved to date.

[0003] It is known that SLLMWITQC, a 9-mer NY-ESO peptide at positions 157 through 165 of NY-ESO-1 and LAGE-1, forms a complex (HLA / NY-ESO peptide complex) with HLA (Histocompatibility Leukocyte Antigen)-A2 and is presented extracellularly (Non-patent Literature 7). As described above, given that the expression of NY-ESO-1 and LAGE-1 is cancer-specific, it is suggested that the HLA / NY-ESO peptide complex is a cancer-specific therapeutic target that exists only on cancer cells that are HLA-A2 positive and also NY-ESO-1 or LAGE-1 positive (Non-patent Literature 8). Furthermore, there are reports of TCRs (Patent Literatures 1 and 2) or antibodies (Patent Literatures 3 and 8) as molecules that bind to the HLA / NY-ESO peptide complex.

[0004] One application of binding molecules to cancer target molecules is CD3 bispecific antibodies that utilize T-cell redirection as a mechanism to induce T-cells into cancer cells and exhibit antitumor effects through cell damage (Non-patent Literature 9, 10). An example of a currently available CD3 bispecific antibody drug is Blinatumomab, a BiTE (bispecific T-cell engager) targeting CD19. It has been approved for acute lymphoblastic leukemia (ALL), and clinical trials targeting other blood cancers are underway. However, the bispecific antibody format is a tandem scFv (taFv) that lacks an Fc region, and compared to IgG antibodies commonly used as therapeutic antibodies, its blood half-life is very short when administered to patients (Non-patent Literature 11).

[0005] Research and clinical trials are underway for CD3 bispecific antibodies using various antibody formats, such as knobs-into-holes, CrossMAb, DuoBody (registered trademark) (Patent Documents 4, 5, 6, 7), as bispecific antibodies having a heterodimer Fc region with a blood half-life similar to that of IgG type antibodies, and CD3 bispecific antibodies using antibodies against HLA / NY-ESO peptide complexes have been reported (Non-patent Document 12). Prior art literature

[0006] WO2005 / 113595WO2017 / 109496WO2010 / 106431WO1998 / 050431WO2006 / 106905WO2011 / 028952WO2011 / 131746WO2021 / 003357

[0007] Proc Natl Acad Sci USA. ; 94 (5): 1914-8 (1997). Int J Cancer. ; 76 (6): 903-8 (1998). Immunol Cell Biol. ; 84 (3):303-17 (2006).J Surg Res. ; 98 (2): 76-80 (2001). Cancer Res. ; 61 (12): 4671-4 (2001). Blood. ; 105 (10):3939-44 (2005).J Exp Med. ; 187 (2):265-70 (1998).J Immunol. ; 176(12):7308-16 (2006).Nature; 314 (6012) ; 628-31 (1985).Int Rev Immunol. ; 4 (2) ; 159-73 (1989).Drug Des Devel Ther. ; 10 ; 757-765 (2016).Abstracts of the 21st Korean Society of Cancer Immunology ; O13-4 The problem to be solved

[0008] The present invention aims to provide a novel anti-HLA-A2 / NY-ESO antibody that can be used as an antitumor agent, and an antitumor agent containing a molecule that binds to HLA-A2 / NY-ESO containing the antibodies as an active ingredient. means of solving the problem

[0009] The inventors of the present invention have completed the present invention by conducting diligent research to solve the above problem and creating a novel anti-HLA-A2 / NY-ESO antibody and a molecule that binds to HLA-A2 / NY-ESO including the antibody.

[0010] That is, the present invention includes the following inventions.

[0011] [1] Heavy chain CDRH1 consisting of the amino acid sequence represented by sequence number 54,

[0012] Heavy chain CDRH2 consisting of the amino acid sequence represented by sequence number 55,

[0013] Heavy chain CDRH3 consisting of the amino acid sequence represented by sequence number 56,

[0014] A light chain CDRL1 comprising the amino acid sequence represented by SEQ No. 57, or a light chain CDRL1 comprising the amino acid sequence represented by SEQ No. 57, wherein the 7th amino acid is W and / or the 8th amino acid is K.

[0015] Light chain CDRL2 consisting of the amino acid sequence represented by SEQ ID No. 58, and

[0016] An antibody or binding fragment that specifically binds to human HLA / NY-ESO, comprising a light chain CDRL3 consisting of the amino acid sequence represented by SEQ No. 59, or a light chain CDRL3 consisting of the amino acid sequence represented by SEQ No. 59, wherein the second amino acid is A or S.

[0017] [2] (i) Heavy chain CDRH1 consisting of the amino acid sequence represented by sequence number 54,

[0018] Heavy chain CDRH2 consisting of the amino acid sequence represented by sequence number 55,

[0019] Heavy chain CDRH3 consisting of the amino acid sequence represented by sequence number 56,

[0020] Light chain CDRL1, consisting of the amino acid sequence represented by sequence number 57,

[0021] Light chain CDRL2 consisting of the amino acid sequence represented by SEQ ID No. 58, and,

[0022] Light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID No. 59

[0023] (ii) heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO. 54,

[0024] Heavy chain CDRH2 consisting of the amino acid sequence represented by sequence number 55,

[0025] Heavy chain CDRH3 consisting of the amino acid sequence represented by sequence number 56,

[0026] In the amino acid sequence represented by SEQ No. 57, a light chain CDRL1 consisting of an amino acid sequence in which the 7th amino acid is W,

[0027] Light chain CDRL2 consisting of the amino acid sequence represented by SEQ ID No. 58, and,

[0028] Light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID No. 59

[0029] (iii) heavy chain CDRH1 consisting of the amino acid sequence represented by sequence number 54,

[0030] Heavy chain CDRH2 consisting of the amino acid sequence represented by sequence number 55,

[0031] Heavy chain CDRH3 consisting of the amino acid sequence represented by sequence number 56,

[0032] Light chain CDRL1, consisting of the amino acid sequence represented by sequence number 57,

[0033] Light chain CDRL2 consisting of the amino acid sequence represented by SEQ ID No. 58, and,

[0034] In the amino acid sequence represented by SEQ No. 59, a light chain CDRL3 consisting of an amino acid sequence in which the second amino acid is A,

[0035] (iv) heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO. 54,

[0036] Heavy chain CDRH2 consisting of the amino acid sequence represented by sequence number 55,

[0037] Heavy chain CDRH3 consisting of the amino acid sequence represented by sequence number 56,

[0038] Light chain CDRL1, consisting of the amino acid sequence represented by sequence number 57,

[0039] Light chain CDRL2 consisting of the amino acid sequence represented by SEQ ID No. 58, and,

[0040] A light chain CDRL3 consisting of an amino acid sequence in which the second amino acid is S, as represented by the amino acid sequence of SEQ ID NO. 59, and,

[0041] (v) Heavy chain CDRH1 consisting of the amino acid sequence represented by sequence number 54,

[0042] Heavy chain CDRH2 consisting of the amino acid sequence represented by sequence number 55,

[0043] Heavy chain CDRH3 consisting of the amino acid sequence represented by sequence number 56,

[0044] A light chain CDRL1, which consists of an amino acid sequence represented by SEQ No. 57, wherein the 7th amino acid is W and the 8th amino acid is K,

[0045] Light chain CDRL2 consisting of the amino acid sequence represented by SEQ ID No. 58, and,

[0046] An antibody of [1] or a binding fragment thereof comprising one or more groups of CDRH1 to CDRH3 and CDRL1 to CDRL3 selected from the group consisting of (i) to (v) of the light chain CDRL3 having the amino acid sequence represented by SEQ ID NO. 59.

[0047] [3] An antibody of [1] or a binding fragment thereof comprising a medium chain variable region having 95% or more sequence identity with the amino acid sequence of amino acid numbers 21 to 140 of the amino acid sequence represented by sequence number 27, or the amino acid sequence represented by 38 or 39, and a light chain variable region having 95% or more sequence identity with the amino acid sequence of amino acid numbers 156 to 266 of the amino acid sequence represented by sequence number 27 or sequence number 52, or the amino acid sequence represented by sequence number 40.

[0048] [4] (H1) A medium-chain variable region consisting of the amino acid sequence represented by sequence number 6,

[0049] (H2) A medium chain variable region consisting of the amino acid sequence represented by sequence number 18,

[0050] (H3) A medium chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by sequence number 29,

[0051] (H4) A medium chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by sequence number 26,

[0052] (H5) A medium chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by sequence number 27,

[0053] (H6) A medium chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by sequence number 28,

[0054] (H7) A medium chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by sequence number 36,

[0055] (H8) A medium chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by sequence number 47,

[0056] (H9) A medium chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by sequence number 48,

[0057] (H10) A medium chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by sequence number 50,

[0058] (H11) A medium chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by sequence number 51,

[0059] (H12) A medium chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by sequence number 52,

[0060] (H13) A medium chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by sequence number 53,

[0061] (H14) A medium chain variable region consisting of the 21st to 140th amino acid sequences of the amino acid sequence represented by SEQ No. 30, or,

[0062] (H15) A medium chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 156, and,

[0063] (L1) A diagonal variable region consisting of the amino acid sequence represented by sequence number 8,

[0064] (L2) A diagonal variable region consisting of the amino acid sequence represented by sequence number 20,

[0065] (L3) A diagonal chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by sequence number 29,

[0066] (L4) A variable region of the light chain consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by sequence number 26,

[0067] (L5) A diagonal chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by sequence number 27,

[0068] (L6) A diagonal chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by sequence number 28,

[0069] (L7) A diagonal variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by sequence number 36,

[0070] (L8) A diagonal chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by sequence number 47,

[0071] (L9) A diagonal chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by sequence number 48,

[0072] (L10) A diagonal chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by sequence number 50,

[0073] (L11) A diagonal chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by sequence number 51,

[0074] (L12) A diagonal chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by sequence number 52,

[0075] (L13) A diagonal variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by sequence number 53,

[0076] (L14) A variable region of the light chain consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ No. 30, or,

[0077] (L15) An antibody of [1] or its binding fragment comprising a light chain variable region consisting of the 161st to 271st amino acid sequence of the amino acid sequence represented by sequence number 156.

[0078] [5] (H1L1) A medium chain variable region consisting of the amino acid sequence represented by sequence number 6 and a light chain variable region consisting of the amino acid sequence represented by sequence number 8,

[0079] (H2L2) A heavy chain variable region consisting of the amino acid sequence represented by sequence number 18 and a light chain variable region consisting of the amino acid sequence represented by sequence number 20,

[0080] (H3L3) A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ No. 29 and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ No. 29,

[0081] (H4L4) A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ No. 26 and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ No. 26,

[0082] (H5L5) A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ No. 27 and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ No. 27,

[0083] (H6L6) A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ No. 28 and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ No. 28,

[0084] (H7L7) A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ No. 36 and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ No. 36,

[0085] (H8L8) A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ No. 47 and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ No. 47,

[0086] (H9L9) A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ No. 48 and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ No. 48,

[0087] (H10L10) A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ No. 50 and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ No. 50,

[0088] (H11L11) A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ No. 51 and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ No. 51,

[0089] (H12L12) A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ No. 52 and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ No. 52,

[0090] (H13L13) A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ No. 53 and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ No. 53,

[0091] (H14L14) A heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ No. 30 and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ No. 30, or,

[0092] (H15L15) An antibody of [4] or a binding fragment thereof comprising a medium chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by sequence number 156 and a light chain variable region consisting of the 161st to 271st amino acid sequence of the amino acid sequence represented by sequence number 156.

[0093] [6] An antibody or its binding fragment of any one of [1] to [5], which is a scFv.

[0094] [7] (s1) scFv consisting of the 21st to 266th amino acid sequences of the amino acid sequence represented by sequence number 70,

[0095] (s2) scFv comprising a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO. 18 and a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO. 20,

[0096] (s3) scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by SEQ ID No. 29,

[0097] (s4) scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by sequence number 26,

[0098] (s5) scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by sequence number 27,

[0099] (s6) scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by sequence number 28,

[0100] (s7) scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by sequence number 36,

[0101] (s8) scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by sequence number 47,

[0102] (s9) scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by SEQ ID No. 48,

[0103] (s10) scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by sequence number 50,

[0104] (s11) scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by sequence number 51,

[0105] (s12) scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by sequence number 52,

[0106] (s13) scFv consisting of the amino acid sequence from the 21st to the 266th of the amino acid sequence represented by sequence number 53,

[0107] (s14) A scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 30, or

[0108] (s15) An antibody or its binding fragment that is a scFv consisting of the 21st to 271st amino acid sequence of the amino acid sequence represented by sequence number 156, [6].

[0109] [8] A polynucleotide encoding any one of the antibodies or their binding fragments from [1] to [7].

[0110] [9] [8] Vector containing polynucleotides.

[0111]

[10] [8] A host cell containing a polynucleotide or [9] a vector.

[0112]

[11] A method for producing an antibody or its binding fragment that specifically binds to human HLA / NY-ESO, comprising (i) a process of culturing host cells of

[10] , and (ii) a process of purifying the antibody or its binding fragment from the culture obtained in process (i).

[0113]

[12]

[11] An antibody or its binding fragment that specifically binds to human HLA / NY-ESO obtained by the method.

[0114]

[13] An antibody or its binding fragment that binds to HLA-A2 / NY-ESO having the properties described in (i) or (ii) below:

[0115] (i) Binds to a site on HLA-A2 / NY-ESO recognized by the antibody or its binding fragment described in [7];

[0116] (ii) Compete with the antibody or its binding fragment described in [7] for binding to human HLA-A2 / NY-ESO.

[0117]

[14] A pharmaceutical composition comprising as an active ingredient any one of the antibodies or their binding fragments of [1] ~ [7],

[12] and

[13] , the polynucleotide of [8], the vector of [9], or the cell of

[10] .

[0118]

[15] A molecule that specifically binds to human HLA / NY-ESO, comprising any one of the antibodies or their binding fragments from [1] to [7],

[12] and

[13] .

[0119]

[16] A molecule of

[15] a multispecific antibody.

[0120]

[17] A molecule of

[15] a double-specific antibody.

[0121]

[18] Any one of the molecules

[15] to

[17] containing an antibody that specifically binds to CD3 or a binding fragment thereof.

[0122]

[19] An antibody or its binding fragment that specifically binds to CD3,

[0123] (CCH1) Heavy chain CDRH1 consisting of the amino acid sequence represented by sequence number 141,

[0124] (CCH2) A heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ No. 142, or a heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ No. 142, wherein the third amino acid is N or S.

[0125] (CCH3) Heavy chain CDRH3 consisting of the amino acid sequence represented by sequence number 143,

[0126] (CCL1) Light chain CDRL1 consisting of the amino acid sequence represented by sequence number 144,

[0127] (CCL2) A light chain CDRL2 consisting of the amino acid sequence represented by SEQ No. 145, or a light chain CDRL2 consisting of the amino acid sequence represented by SEQ No. 145, wherein the second amino acid is N; and

[0128] A molecule of

[18] that is an antibody or its binding fragment that specifically binds to CD3, comprising a light chain CDRL3 consisting of the amino acid sequence represented by sequence number 146 (CCL3).

[0129]

[20] An antibody or its binding fragment that specifically binds to CD3,

[0130] (CH1) A medium chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by sequence number 136,

[0131] (CH2) A medium chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by sequence number 137,

[0132] (CH3) A medium chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by sequence number 147,

[0133] (CH4) A medium chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by sequence number 138,

[0134] (CH5) A medium chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by sequence number 139,

[0135] (CH6) A medium chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by sequence number 140,

[0136] (CH7) A medium chain variable region consisting of the 272nd to 389th amino acid sequence of the amino acid sequence represented by sequence number 155,

[0137] (CH8) A medium chain variable region consisting of the 277th to 394th amino acid sequence of the amino acid sequence represented by SEQ No. 156, or

[0138] (CH9) A medium chain variable region consisting of the 277th to 394th amino acid sequence of the amino acid sequence represented by SEQ No. 157, and,

[0139] (CL1) A diagonal chain variable region consisting of the 135th to 243rd amino acid sequence of the amino acid sequence represented by sequence number 136,

[0140] (CL2) A diagonal chain variable region consisting of the 135th to 241st amino acid sequence of the amino acid sequence represented by sequence number 137,

[0141] (CL3) A diagonal chain variable region consisting of the 135th to 243rd amino acid sequence of the amino acid sequence represented by sequence number 147,

[0142] (CL4) A diagonal chain variable region consisting of the 135th to 241st amino acid sequence of the amino acid sequence represented by sequence number 138,

[0143] (CL5) A diagonal chain variable region consisting of the 135th to 243rd amino acid sequence of the amino acid sequence represented by sequence number 139,

[0144] (CL6) A diagonal chain variable region consisting of the 135th to 243rd amino acid sequence of the amino acid sequence represented by sequence number 140,

[0145] (CL7) A diagonal chain variable region consisting of the 405th to 511th amino acid sequence of the amino acid sequence represented by sequence number 155,

[0146] (CL8) A light chain variable region consisting of the 410th to 516th amino acid sequence of the amino acid sequence represented by SEQ ID No. 156, or

[0147] (CL9) A molecule of

[19] comprising a diagonal chain variable region consisting of the 410th to 516th amino acid sequence of the amino acid sequence represented by sequence number 157.

[0148]

[21] An antibody or its binding fragment that specifically binds to CD3,

[0149] (CH1CL1) A heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by sequence number 136, and a light chain variable region consisting of the 135th to 243rd amino acid sequence of the amino acid sequence represented by sequence number 136,

[0150] (CH2CL2) A heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by sequence number 137, and a light chain variable region consisting of the 135th to 241st amino acid sequence of the amino acid sequence represented by sequence number 137,

[0151] (CH3CL3) A heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by sequence number 147, and a light chain variable region consisting of the 135th to 243rd amino acid sequence of the amino acid sequence represented by sequence number 147,

[0152] (CH4CL4) A heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by sequence number 138, and a light chain variable region consisting of the 135th to 241st amino acid sequence of the amino acid sequence represented by sequence number 138,

[0153] (CH5CL5) A heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by sequence number 139, and a light chain variable region consisting of the 135th to 243rd amino acid sequence of the amino acid sequence represented by sequence number 139,

[0154] (CH6CL6) A heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by sequence number 140, and a light chain variable region consisting of the 135th to 243rd amino acid sequence of the amino acid sequence represented by sequence number 140,

[0155] (CH7CL7) A heavy chain variable region consisting of the 272nd to 389th amino acid sequence of the amino acid sequence represented by SEQ No. 155, and a light chain variable region consisting of the 272nd to 389th amino acid sequence of the amino acid sequence represented by SEQ No. 155,

[0156] (CH8CL8) A heavy chain variable region consisting of the amino acid sequences from the 277th to the 394th of the amino acid sequence represented by SEQ No. 156, and a light chain variable region consisting of the amino acid sequences from the 410th to the 516th of the amino acid sequence represented by SEQ No. 156, or,

[0157] A molecule of

[20] comprising a medium chain variable region consisting of the 277th to 394th amino acid sequence of the (CH9CL9) sequence number 157 and a light chain variable region consisting of the 410th to 516th amino acid sequence of the amino acid sequence number 157.

[0158]

[22] Any one of the molecules of

[18] to

[21] that is an antibody or its binding fragment that specifically binds to CD3 and is scFv.

[0159]

[23] scFv a,

[0160] (CS1) scFv consisting of the 2nd to 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO. 136,

[0161] (CS2) scFv consisting of the 2nd to 241st amino acid sequence of the amino acid sequence represented by sequence number 137,

[0162] (CS3) scFv consisting of the 2nd to 243rd amino acid sequence of the amino acid sequence represented by sequence number 147,

[0163] (CS4) scFv consisting of the 2nd to 241st amino acid sequence of the amino acid sequence represented by SEQ ID NO. 138,

[0164] (CS5) scFv consisting of the 2nd to 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO. 139,

[0165] (CS6) scFv consisting of the 2nd to 243rd amino acid sequence of the amino acid sequence represented by sequence number 140,

[0166] (CS7) scFv consisting of the amino acid sequence from the 272nd to the 511th of the amino acid sequence represented by SEQ ID NO. 155,

[0167] (CS8) A scFv consisting of the amino acid sequence from the 277th to the 516th of the amino acid sequence represented by SEQ ID No. 156, or

[0168] (CS9) A molecule of

[22] comprising scFv consisting of amino acid sequences from the 277th to the 516th of the amino acid sequence represented by sequence number 157.

[0169]

[24] A first polypeptide comprising, in order from the N-terminus toward the C-terminus, a scFv that specifically binds to human HLA / NY-ESO, a scFv that specifically binds to CD3, and an Fc region (i); and,

[0170] A molecule comprising a second polypeptide including an Fc region (ii), preferably formed by association at the Fc region (i) and the Fc region (ii),

[18] to

[23] .

[0171]

[25] A scFv that specifically binds to human HLA / NY-ESO, a molecule of which is an antibody or its binding fragment that specifically binds to human HLA / NY-ESO, which is a scFv [1].

[24]

[0172]

[26] A scFv that specifically binds to human HLA / NY-ESO, a molecule of which is an antibody or its binding fragment that specifically binds to human HLA / NY-ESO, which is a scFv [2].

[24]

[0173]

[27] A scFv that specifically binds to human HLA / NY-ESO, a molecule of which is an antibody or its binding fragment that specifically binds to human HLA / NY-ESO, which is a scFv [3].

[24]

[0174]

[28] A scFv that specifically binds to human HLA / NY-ESO, a molecule of which is an antibody or its binding fragment that specifically binds to human HLA / NY-ESO, which is a scFv [4].

[24]

[0175]

[29] A scFv that specifically binds to human HLA / NY-ESO, a molecule of which is an antibody or its binding fragment that specifically binds to human HLA / NY-ESO, which is a scFv [5].

[24]

[0176]

[30] Any one of the molecules of scFv that specifically binds to CD3, which is an antibody or its binding fragment that specifically binds to CD3 of scFv

[18] ,

[19] to

[24] .

[0177]

[31] Any one of the molecules

[19] to

[24] which is an antibody or its binding fragment that specifically binds to CD3, which is a scFv that specifically binds to CD3 of

[19] scFv.

[0178]

[32] Any one of the molecules

[19] to

[24] which is an antibody or its binding fragment that specifically binds to CD3, or an scFv that specifically binds to CD3 of scFv

[20] or

[21] .

[0179]

[33] Any one of the molecules of scFv that specifically binds to CD3, which is an antibody or its binding fragment that specifically binds to CD3 of scFv

[23] ,

[19] to

[24] .

[0180]

[34] The 21st to 511th amino acid sequence of the amino acid sequence represented by sequence number 85, the 21st to 511th amino acid sequence of the amino acid sequence represented by sequence number 87, the 21st to 511th amino acid sequence of the amino acid sequence represented by sequence number 88, the 21st to 511th amino acid sequence of the amino acid sequence represented by sequence number 89, the 21st to 511th amino acid sequence of the amino acid sequence represented by sequence number 90, the 21st to 511th amino acid sequence of the amino acid sequence represented by sequence number 91, the 21st to 511th amino acid sequence of the amino acid sequence represented by sequence number 92, the 21st to 511th amino acid sequence of the amino acid sequence represented by sequence number 93, the 21st to 511th amino acid sequence of the amino acid sequence represented by sequence number 94, the amino acid sequence represented by sequence number 95 Any one of

[24] to

[33] , comprising an amino acid sequence selected from the group consisting of the 21st to 511th amino acid sequence of the amino acid sequence represented by sequence number 96, the 21st to 511th amino acid sequence of the amino acid sequence represented by sequence number 86, the 21st to 511th amino acid sequence of the amino acid sequence represented by sequence number 149, and the 21st to 511th amino acid sequence of the amino acid sequence represented by sequence number 150.

[0181]

[35] Any one of

[24] to

[33] , comprising an amino acid sequence selected from the group consisting of the 21st to 511th amino acid sequence of the amino acid sequence represented by sequence number 155, the 21st to 516th amino acid sequence of the amino acid sequence represented by sequence number 156, and the 21st to 516th amino acid sequence of the amino acid sequence represented by sequence number 157.

[0182]

[36] Any one of

[24] to

[34] , wherein the first polypeptide comprises the 529th to 745th amino acid sequence of the amino acid sequence represented by sequence number 85, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 86, 149 or 150.

[0183]

[37] Any one of

[24] to

[33] and

[35] , wherein the first polypeptide comprises the amino acid sequence of the 529th to 745th amino acid sequence of the amino acid sequence represented by sequence number 155, the amino acid sequence of the 534th to 750th amino acid sequence of the amino acid sequence represented by sequence number 156, or the amino acid sequence of the 534th to 750th amino acid sequence represented by sequence number 157.

[0184]

[38] The first polypeptide is the amino acid sequence from the 20th to the 745th of the amino acid sequence represented by sequence number 85, the amino acid sequence from the 20th to the 745th of the amino acid sequence represented by sequence number 87, the amino acid sequence from the 20th to the 745th of the amino acid sequence represented by sequence number 88, the amino acid sequence from the 20th to the 745th of the amino acid sequence represented by sequence number 89, the amino acid sequence from the 20th to the 745th of the amino acid sequence represented by sequence number 90, the amino acid sequence from the 20th to the 745th of the amino acid sequence represented by sequence number 91, the amino acid sequence from the 20th to the 745th of the amino acid sequence represented by sequence number 92, the amino acid sequence from the 20th to the 745th of the amino acid sequence represented by sequence number 93, the amino acid sequence from the 20th to the 745th of the amino acid sequence represented by sequence number 94, A molecule of

[34] or

[36] consisting of an amino acid sequence selected from the group consisting of the amino acid sequence from the 20th to the 745th of the amino acid sequence represented by sequence number 95, the amino acid sequence from the 20th to the 745th of the amino acid sequence represented by sequence number 96, the amino acid sequence from the 20th to the 745th of the amino acid sequence represented by sequence number 86, the amino acid sequence from the 20th to the 745th of the amino acid sequence represented by sequence number 149, and the amino acid sequence from the 20th to the 745th of the amino acid sequence represented by sequence number 150.

[0185]

[39] A molecule of

[35] or

[37] in which the first polypeptide is composed of an amino acid sequence selected from the group consisting of the 20th to 745th amino acid sequences of the amino acid sequence represented by sequence number 155, an amino acid sequence selected from the group consisting of the 20th to 750th amino acid sequences of the amino acid sequence represented by sequence number 156, or an amino acid sequence selected from the group consisting of the 20th to 750th amino acid sequences of the amino acid sequence represented by sequence number 157.

[0186]

[40] Any one of

[24] to

[39] , wherein the second polypeptide comprises the 20th to 246th amino acid sequence of the amino acid sequence represented by sequence number 84.

[0187]

[41] A first polypeptide, a second polypeptide and a third polypeptide, comprising,

[0188] The first polypeptide comprises, in order from the N-terminus toward the C-terminus, a scFv that specifically binds to human HLA / NY-ESO, a scFv that specifically binds to CD3, and an Fc region (i).

[0189] The second polypeptide is composed of an immunoglobulin heavy chain containing an Fc region (ii), and

[0190] The third polypeptide consists of an immunoglobulin light chain, and

[0191] Preferably, the second polypeptide and the third polypeptide are associated, and

[0192] Any one of the molecules

[18] to

[23] in which the first polypeptide and the second polypeptide are associated in their respective Fc regions.

[0193]

[42] A scFv that specifically binds to human HLA / NY-ESO, a molecule of which is an antibody or its binding fragment that specifically binds to human HLA / NY-ESO, which is a scFv [1].

[41]

[0194]

[43] A scFv that specifically binds to human HLA / NY-ESO, a molecule of which is an antibody or its binding fragment that specifically binds to human HLA / NY-ESO, which is a scFv [2].

[41]

[0195]

[44] A scFv that specifically binds to human HLA / NY-ESO, a molecule of which is an antibody or its binding fragment that specifically binds to human HLA / NY-ESO, which is a scFv [3].

[41]

[0196]

[45] A scFv that specifically binds to human HLA / NY-ESO, a molecule of which is an antibody or its binding fragment that specifically binds to human HLA / NY-ESO, which is a scFv [4].

[41]

[0197]

[46] A scFv that specifically binds to human HLA / NY-ESO, a molecule of which is an antibody or its binding fragment that specifically binds to human HLA / NY-ESO, which is a scFv [5].

[41]

[0198]

[47] Any one of the molecules of scFv that specifically binds to CD3, or an antibody or its binding fragment that specifically binds to CD3 of scFv

[18] ,

[42] to

[46] .

[0199]

[48] ​​Any one of the molecules of scFv that specifically binds to CD3, or an antibody or its binding fragment that specifically binds to CD3 of scFv

[19] ,

[42] to

[46] .

[0200]

[49] Any one of the molecules

[42] to

[46] which is an antibody or its binding fragment that specifically binds to CD3, which is a scFv that specifically binds to CD3 as described in

[20] or

[21] .

[0201]

[50] Any one of the molecules of scFv that specifically binds to CD3, or an antibody or its binding fragment that specifically binds to CD3 of scFv

[23] ,

[42] to

[46] .

[0202]

[51] Any one of

[41] to

[50] , wherein the second polypeptide comprises the amino acid sequence of amino acid numbers 20 to 242 of the amino acid sequence represented by sequence number 99.

[0203]

[52] Any one of

[41] to

[51] , wherein the third polypeptide comprises the amino acid sequence represented by sequence number 100.

[0204]

[53] The first polypeptide,

[0205] The amino acid sequence from the 21st to the 511th of the amino acid sequence represented by SEQ ID NO. 85, the amino acid sequence from the 21st to the 511th of the amino acid sequence represented by SEQ ID NO. 87, the amino acid sequence from the 21st to the 511th of the amino acid sequence represented by SEQ ID NO. 88, the amino acid sequence from the 21st to the 511th of the amino acid sequence represented by SEQ ID NO. 89, the amino acid sequence from the 21st to the 511th of the amino acid sequence represented by SEQ ID NO. 90, the amino acid sequence from the 21st to the 511th of the amino acid sequence represented by SEQ ID NO. 91, the amino acid sequence from the 21st to the 511th of the amino acid sequence represented by SEQ ID NO. 92, the amino acid sequence from the 21st to the 511th of the amino acid sequence represented by SEQ ID NO. 93, the amino acid sequence from the 21st to the 511th of the amino acid sequence represented by SEQ ID NO. 94, and the amino acid sequence represented by SEQ ID NO. 95 Any one of

[41] to

[52] comprising an amino acid sequence selected from the group consisting of the 21st to 511th amino acid sequence, the 21st to 511th amino acid sequence of the amino acid sequence represented by sequence number 96, the 21st to 511th amino acid sequence of the amino acid sequence represented by sequence number 86, the 21st to 511th amino acid sequence of the amino acid sequence represented by sequence number 149, and the 21st to 511th amino acid sequence of the amino acid sequence represented by sequence number 150.

[0206]

[54] The first polypeptide is the amino acid sequence from the 20th to the 745th of the amino acid sequence represented by sequence number 85, the amino acid sequence from the 20th to the 745th of the amino acid sequence represented by sequence number 87, the amino acid sequence from the 20th to the 745th of the amino acid sequence represented by sequence number 88, the amino acid sequence from the 20th to the 745th of the amino acid sequence represented by sequence number 89, the amino acid sequence from the 20th to the 745th of the amino acid sequence represented by sequence number 90, the amino acid sequence from the 20th to the 745th of the amino acid sequence represented by sequence number 91, the amino acid sequence from the 20th to the 745th of the amino acid sequence represented by sequence number 92, the amino acid sequence from the 20th to the 745th of the amino acid sequence represented by sequence number 93, the amino acid sequence from the 20th to the 745th of the amino acid sequence represented by sequence number 94, Any one of

[41] to

[53] , consisting of an amino acid sequence selected from the group consisting of the 20th to 745th amino acid sequence of the amino acid sequence represented by sequence number 95, the 20th to 745th amino acid sequence of the amino acid sequence represented by sequence number 96, the 20th to 745th amino acid sequence of the amino acid sequence represented by sequence number 86, the 20th to 745th amino acid sequence of the amino acid sequence represented by sequence number 149, and the 20th to 745th amino acid sequence of the amino acid sequence represented by sequence number 150.

[0207]

[55] A molecule described in any one of

[18] to

[23] comprising a first polypeptide comprising, in order from the N end toward the C end, a scFv that specifically binds to human HLA / NY-ESO, a variable region and a normal region CH1 of an antibody heavy chain that specifically binds to CD3, and an immunoglobulin Fc region (i); a second polypeptide comprising a hinge region and an Fc region (ii) of an immunoglobulin; and a third polypeptide comprising an antibody light chain comprising a variable region and a normal region, preferably wherein the first polypeptide and the second polypeptide associate in the Fc region (i) and the Fc region (ii), and the first polypeptide associates with the third polypeptide in the variable region and normal region CH1 of the antibody heavy chain.

[0208]

[56] A scFv that specifically binds to human HLA / NY-ESO, a molecule of which is an antibody or its binding fragment that specifically binds to human HLA / NY-ESO, which is a scFv [1].

[55]

[0209]

[57] A scFv that specifically binds to human HLA / NY-ESO, a molecule of which is an antibody or its binding fragment that specifically binds to human HLA / NY-ESO, which is a scFv [2].

[55]

[0210]

[58] A scFv that specifically binds to human HLA / NY-ESO, a molecule of which is an antibody or its binding fragment that specifically binds to human HLA / NY-ESO, which is a scFv [3].

[55]

[0211]

[59] A scFv that specifically binds to human HLA / NY-ESO, a molecule of which is an antibody or its binding fragment that specifically binds to human HLA / NY-ESO, which is a scFv [4].

[55]

[0212]

[60] A scFv that specifically binds to human HLA / NY-ESO, a molecule of which is an antibody or its binding fragment that specifically binds to human HLA / NY-ESO, [5] which is a scFv. [5]

[0213]

[61] Any one of the molecules of an antibody or its binding fragment that specifically binds to CD3, which is Fab,

[18] to

[21] .

[0214]

[62] Any one of the molecules of Fab that specifically binds to CD3, an antibody that specifically binds to the CD3 of Fab

[18] or a binding fragment thereof,

[56] to

[61] .

[0215]

[63] Any one of the molecules of Fab that specifically binds to CD3, an antibody that specifically binds to the CD3 of Fab

[19] or its binding fragment,

[56] to

[61] .

[0216]

[64] Any one of the molecules

[56] to

[61] that is an antibody or its binding fragment that specifically binds to CD3, or an antibody that specifically binds to CD3 of Fab,

[20] or

[21] .

[0217]

[65] Any one of

[55] to

[64] , wherein the first polypeptide comprises the 21st to 394th amino acid sequence of the amino acid sequence represented by sequence number 160.

[0218]

[66] Any one of

[55] to

[65] , wherein the first polypeptide comprises an amino acid sequence described in any one of (i) to (iii) below:

[0219] (i) The amino acid sequence from the 20th to the 724th of the amino acid sequence represented by sequence number 160;

[0220] (ii) The amino acid sequence from the 20th to the 719th of the amino acid sequence represented by SEQ ID NO. 197;

[0221] (iii) The amino acid sequence from the 20th to the 719th position of the amino acid sequence represented by sequence number 198.

[0222]

[67] Any one of

[55] to

[66] , wherein the second polypeptide comprises the 20th to 246th amino acid sequence of the amino acid sequence represented by sequence number 84.

[0223]

[68] Any one of

[56] to

[67] , wherein the third polypeptide comprises the 21st to 127th amino acid sequences of the amino acid sequence represented by sequence number 161.

[0224]

[69] Any one of

[56] to

[68] , wherein the third polypeptide comprises the 21st to 233rd amino acid sequence of the amino acid sequence represented by sequence number 161.

[0225]

[70]

[15] ~

[69] Any one of the molecules, wherein in the amino acid sequence of one or more polypeptides included in the molecule, one or two amino acids are deleted from the carboxyl terminus of the sequence.

[0226]

[71]

[15] ~

[70] A polynucleotide containing a nucleotide sequence that codes for an amino acid sequence included in any one of the molecules.

[0227]

[72]

[71] A vector containing polynucleotides.

[0228]

[73] Host cell containing the polynucleotide of

[71] or the vector of

[72] .

[0229]

[74] A method for producing a molecule that specifically binds to human HLA / NY-ESO and human CD3, comprising (i) a process of culturing host cells of

[73] , and (ii) a process of purifying an antibody or its binding fragment from the culture obtained in process (i).

[0230]

[75]

[74] A molecule that specifically binds to human HLA / NY-ESO and human CD3 obtained by the method.

[0231]

[76] A pharmaceutical composition comprising any one of the molecules of

[15] ~

[70] and

[75] , the polynucleotide of

[71] , the vector of

[72] , or the host cell of

[73] as an active ingredient.

[0232]

[77] A pharmaceutical composition of an anticancer agent,

[14] or

[76] .

[0233]

[78] Cancer, 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, 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, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, undifferentiated polymorphic sarcoma, myxoid fibrosarcoma, malignant peripheral schwannoma, retroperitoneal sarcoma, synovial sarcoma, uterine sarcoma, gastrointestinal stromal tumor, leiomyosarcoma, epithelial sarcoma, B-cell lymphoma, T-NK cell lymphoma, Hodgkin lymphoma, myeloid leukemia, lymphoid A pharmaceutical composition comprising one or more selected from the group consisting of leukemia, myeloproliferative disorders, myelodysplastic syndrome, multiple myeloma, testicular cancer and ovarian cancer, etc.

[77]

[0234]

[79] A pharmaceutical composition of any one of

[76] to

[78] used in combination with other drugs.

[0235] This specification includes the disclosure of Japanese Patent Application No. 2020-061476, which forms the basis of the priority of this application. Effects of the invention

[0236] According to the present invention, an antibody that binds to HLA-A2 / NY-ESO and a novel bispecific antibody (bispecific molecule) that binds to HLA-A2 / NY-ESO and also binds to CD3 are obtained. In addition, a novel pharmaceutical composition containing such an antibody (molecule) as an active ingredient is obtained. The antibody or the molecule, etc., has cell damage activity and is useful as a therapeutic agent or preventive agent for cancer, etc. Brief explanation of the drawing

[0237] Figure 1 is a table showing the standardized gMFI for T2 cells treated with various point mutation peptides of anti-HLA / NY-ESO scFv NYA-0001, 1143, 1154, 1163, 2023, 2027, 2035, 2044, 2045, 2047, 2048, 2060, 2061, 2143, NYC-0003, and 0004. * / Underlines indicate values ​​less than half of the standardized gMFI for T2 cells treated with NY-ESO peptides of each scFv. Figure 2a is a table showing information on selected homologous peptides. Binding affinity to HLA-A0201 was predicted by Net MHC Pa n2.8, and the 50% inhibition concentration (IC10) 50Figure 2b is a table showing the standardized gMFI for T2 cells with various homologous peptides added to anti-HLA / NY-ESO scFv NYA-0001, 1143, 1154, 1163, 2023, 2027, 2035, 2044, 2045, 2047, 2048, 2060, 2061, 2143, NYC-0003, 0004. * / Underline indicates a larger value compared to the standardized gMFI for DMSO-added T2 cells of each scFv. Figure 3 is a diagram showing the format of the antibody shown in this example. (a) scFv: A format in which the antibody H chain variable region (VH, described later) and the antibody L chain variable region (VL, described later) (all white) are connected by a linker. In this example, anti-HLA-A2 / NY-ESO and CD3 scFvs were used for evaluation. (b) Fab: A format consisting of VH (white) and the normal region of the antibody H chain (CH1: checkered pattern) and VL (white) and the normal region of the antibody L chain (horizontal line). In this example, anti-HLA-A2 / NY-ESO Fab, etc., were used for evaluation. (c) taFv: A format in which two types of scFv (white and upper right diagonal line) are connected by a linker. In this example, taFv containing anti-HLA-A2 / NY-ESO scFv and anti-CD3 scFv was used for evaluation. (d) taFv-heterodimer Fc form: An Fc (upper left diagonal line) containing a mutation forming a heterodimer at the C-terminal side of taFv is added (also called the first polypeptide),It is a format hetero-associated with another Fc (blackened: also called the second polypeptide). In this example, taFv-heterodimer Fc containing anti-HLA-A2 / NY-ESO scFv and anti-CD3scFv was used for evaluation. (e) taFv-Fab-heterodimer Fc type: A format in which Fab is added to the above taFv-heterodimer Fc type. In this example, taFv-Fab-heterodimer Fc using taFv containing anti-HLA-A2 / NY-ESO scFv and anti-CD3scFv and HLA-A2 / NY-ESO Fab was used for evaluation. (f) Shows the first polypeptide common to both the taFv-heterodimer Fc type and the taFv-Fab-heterodimer Fc type. (g) Represents a second polypeptide of the taFv-heterodimer Fc type. The second polypeptide comprises a hinge region and an Fc region (ii) in that order, extending from the N-terminus toward the C-terminus. (h) Represents a second polypeptide of the taFv-heterodimer Fc type. The second polypeptide comprises a hinge region and an Fc region (ii). (h) Represents a second polypeptide of the taFv-Fab-heterodimer Fc type. The second polypeptide comprises an immunoglobulin heavy chain comprising a hinge region and an Fc region (ii). (i) Represents a third polypeptide of the taFv-Fab-heterodimer Fc type. The third polypeptide comprises an immunoglobulin light chain. Fig. 4a shows Fc addition-type anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules, NYF-0016, NYF-0019, NYF-0022, NYF-0023, NYF-0027, NYF-0035, NYF-0044, and NYF-0047.Figure 4b shows that endogenous human NY-ESO-expressing U266B1 cells exhibit cell damage activity in the presence of human PBMCs. Error bars in the figure represent the standard deviation (n = 3). Figure 4b shows that Fc-addition type anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules, NYF-0016 and NYF-0058, exhibit cell damage activity in endogenous human NY-ESO-expressing U266B1 cells in the presence of human PBMCs. The error bars in the figure represent the standard deviation (n = 3). Figure 4c is a figure showing that Fc-addition type anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules, NYF-0023, NYF-0045, NYF-0048, NYF-0060, and NYF-0061, have cell damage activity against endogenous human NY-ESO-expressing U266B1 cells in the presence of human PBMCs. The error bars in the figure represent the standard deviation (n = 3). Figure 4d is a figure showing that Fc addition-type anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules, NYF-0016, NYF-0019, NYF-0022, NYF-0023, NYF-0027, NYF-0035, NYF-0044, and NYF-0047 have cell damage activity against endogenous human NY-ESO-expressing NCI-H1703 cells in the presence of human PBMCs. The error bars in the figure represent the standard deviation (n = 3). Figure 4e shows that Fc-addition type anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules, NYF-0016 and NYF-0058, exhibit cell damage activity against endogenous human NY-ESO-expressing NCI-H1703 cells in the presence of human PBMCs. The error bars in the figure represent the standard deviation (n = 3). Figure 4f shows Fc-addition type anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules, NYF-0023, NYF-0045, NYF-0048, and NYF-0060.Figure 4g shows that NYF-0061 exhibits cell damage activity against endogenous human NY-ESO-expressing NCI-H1703 cells in the presence of human PBMCs. Error bars in the figure represent the standard deviation (n = 3). Figure 4g shows that Fc-addition type anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules, NYF-0016, NYF-0019, NYF-0022, NYF-0023, NYF-0027, NYF-0035, NYF-0044, and NYF-0047 do not exhibit cell damage activity against endogenous human NY-ESO-non-expressing AGS cells in the presence of human PBMCs. The error bars in the figure represent the standard deviation (n = 3). Figure 4h is a figure showing that Fc-addition type anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules, NYF-0016 and NYF-0058, do not exhibit cell damage activity against endogenous human NY-ESO-non-expressing AGS cells in the presence of human PBMCs. The error bars in the figure represent the standard deviation (n = 3). Figure 4i is a figure showing that Fc-addition type anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules, NYF-0023, NYF-0045, NYF-0048, NYF-0060 and NYF-0061, do not exhibit cell damage activity against endogenous human NY-ESO-non-expressing AGS cells in the presence of human PBMCs. The error bars in the figure represent the standard deviation (n = 3). Fig. 4j shows the Fc addition-type HLA-A2 / NY-ESO-type CD3 bispecific molecules, NYF-0016, NYF-0019, NYF-0022, NYF-0023, NYF-0027, NYF-0035, NYF-0044, and NYF-0047.Figure 4k shows that endogenous human NY-ESO non-expressing CFPAC-1 cells do not exhibit cell damage activity in the presence of human PBMCs. Error bars in the figure represent the standard deviation (n = 3). Figure 4k shows that Fc-addition type anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules, NYF-0016 and NYF-0058, do not exhibit cell damage activity in endogenous human NY-ESO non-expressing CFPAC-1 cells in the presence of human PBMCs. The error bars in the figure represent the standard deviation (n = 3). Figure 41 is a figure showing that Fc-addition type anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules, NYF-0023, NYF-0045, NYF-0048, NYF-0060, and NYF-0061, do not exhibit cell damage activity against endogenous human NY-ESO-non-expressing CFPAC-1 cells in the presence of human PBMCs. The error bars in the figure represent the standard deviation (n = 3). Figure 5a is a figure showing that Fc-addition type anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules, NYF-0016, NYF-0019, NYF-0044, and NYF-0047, have antitumor activity in a human PBMC influx model. The error bars in the figure represent the standard error (n = 4 for NYF-0044 Day 32 only, n = 5 for others). Figure 5b is a figure showing that Fc addition-type anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules, NYF-0016, NYF-0022, NYF-0023, NYF-0027, NYF-0035, and NYF-0058 possess antitumor activity in a human PBMC influx model. The error bars in the figure represent the standard error (n = 5, n = 6 only for the Vehicle control group). Figure 5c shows Fc addition-type anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules, NYF-0016, NYF-0045, and NYF-0048.This figure shows that NYF-0060 and NYF-0061 possess antitumor activity in a human PBMC influencing model. The error bars in the figure represent the standard error (n = 5). Figure 6a is a figure showing the format of the antibody presented in this example. (a) Hybrid type: A format in which two Fc molecules are associated, with an Fc (slashed and blacked out) containing a mutation forming a heterodimer on the C-terminal side of each Fab and scFv. In this example, a hybrid type containing anti-HLA-A2 / NY-ESO Fab and anti-CD3scFv was used for evaluation. (b) Dual type: A format in which two Fc molecules are associated, with an Fc (slashed and blacked out) containing a mutation forming a heterodimer on the C-terminal side of each of two different types of scFv. In this embodiment, a dual form containing anti-HLA-A2 / NY-ESO scFv and anti-CD3scFv was used for evaluation. (c) scFv-Fab-heterodimer Fc form: This is a format in which an Fc (top left diagonal line) containing a mutation forming a heterodimer on the C-terminal side, which connects scFv and Fab with a linker, is added and associated with another Fc (black treatment). In this embodiment, a scFv-Fab-heterodimer Fc form containing anti-CD3 scFv (top right diagonal line) and anti-HLA-A2 / NY-ESO Fab was used for evaluation. Additionally, a scFv-Fab-heterodimer Fc form containing anti-HLA-A2 / NY-ESO scFv (top right diagonal line) and anti-CD3 Fab was also used for evaluation. Fig. 6b is a diagram showing the format of the antibody presented in this embodiment. (a) taFv-heterodimer Fc type: Same as Fig. 3(d). An Fc (top left diagonal line) with a mutation forming a heterodimer is added to the C-terminal side of taFv, andThis is a format hetero-associated with another Fc (blackened). In this example, a taFv-heterodimer Fc form consisting of anti-HLA-A2 / NY-ESO scFv and anti-CD3 scFv was used for evaluation. (b) taFv(inversed)-heterodimer Fc form: The order of the two scFvs in the above taFv-heterodimer Fc form is reversed. In this example, a taFv(inversed)-heterodimer Fc form consisting of anti-CD3scFv and anti-HLA-A2 / NY-ESO scFv was used for evaluation. (c) Shows the first polypeptide of the taFv(inversed)-heterodimer Fc form. The first polypeptide comprises, in order from the N-terminus toward the C-terminus, a scFv that specifically binds to CD3, a scFv that specifically binds to human HLA / NY-ESO, and an Fc region (i). (d) shows the second polypeptide of the taFv(inversed)-heterodimer Fc type. The second polypeptide comprises a hinge region and an Fc region (ii). Fig. 7a is a diagram showing the cell injury activity of various anti-HLA-A2 / NY-ESO-anti-CD3 dual-specific molecules, Hybrid (NYG-3143), Dual (NYG-2143), and taFv-heterodimer Fc (NYF-0011) on endogenous human NY-ESO-expressing U266B1 cells in the presence of human PBMCs. The error bars in the figure represent the standard deviation (n = 3). Fig. 7b shows the various anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules, scFv-Fab-heterodimer Fc complex (NYF-0003), taFv-heterodimer Fc complex (NYF-0010), and taFv(inversed)-heterodimer Fc complex (NYF-0004).This is a diagram showing the cell damage activity against endogenous human NY-ESO-expressing U266B1 cells in the presence of human PBMCs. The error bars in the figure represent the standard deviation (n = 3). Fig. 8 is the amino acid sequence of a peptide in NY-ESO (Sequence No. 1). Fig. 9 is the amino acid sequence of a peptide in MAGEC-1 (Sequence No. 2). Fig. 10 is primer 1 for scFv sequence analysis (Sequence No. 3). Fig. 11 is primer 2 for scFv sequence analysis (Sequence No. 4). Fig. 12 is the nucleotide sequence of the heavy chain variable region of NYA-0001 (Sequence No. 5). Fig. 13 is the amino acid sequence of the heavy chain variable region of NYA-0001 (Sequence No. 6). Fig. 14 is the nucleotide sequence of the light chain variable region of NYA-0001 (Sequence No. 7). Fig. 15 is the amino acid sequence of the light chain variable region of NYA-0001 (Sequence No. 8). Fig. 16 is Fig. 17 shows the nucleotide sequence of the heavy chain variable region of NYA-0060 (Sequence No. 9), Fig. 18 shows the amino acid sequence of the heavy chain variable region of NYA-0060 (Sequence No. 10), Fig. 19 shows the nucleotide sequence of the light chain variable region of NYA-0060 (Sequence No. 11), Fig. 20 shows the amino acid sequence of the light chain variable region of NYA-0060 (Sequence No. 12), Fig. 21 shows the nucleotide sequence of the heavy chain variable region of NYA-0068 (Sequence No. 13), Fig. 22 shows the amino acid sequence of the heavy chain variable region of NYA-0068 (Sequence No. 14), Fig. 23 shows the nucleotide sequence of the light chain variable region of NYA-0068 (Sequence No. 15), and Fig. 23 shows the amino acid sequence of the light chain variable region of NYA-0068. (Sequence No. 16) FIG. 24 is the nucleotide sequence of the medium chain variable region of NYA-0082 (Sequence No. 17) FIG. 25 is the amino acid sequence of the medium chain variable region of NYA-0082 (Sequence No. 18) FIG. 26 is,Fig. 27 shows the nucleotide sequence of the variable light chain region of NYA-0082 (Sequence No. 19), Fig. 28 shows the amino acid sequence of the variable light chain region of NYA-0082 (Sequence No. 20), Fig. 29 shows the nucleotide sequence of the NYA-1163 tag adduct (Sequence No. 21), Fig. 30 shows the nucleotide sequence of the NYA-2023 tag adduct (Sequence No. 22), Fig. 31 shows the nucleotide sequence of the NYA-2027 tag adduct (Sequence No. 23), Fig. 32 shows the nucleotide sequence of the NYA-1143 tag adduct (Sequence No. 24), Fig. 33 shows the nucleotide sequence of the NYA-2143 tag adduct (Sequence No. 25), and Fig. 36 shows the amino acid sequence of the NYA-1163 tag adduct. NYA-1163 is amino acid Figures 21–266, Fig. 34 is the amino acid sequence of the NYA-2023 tag adduct (Sequence No. 27), NYA-2023 is the amino acid sequence of the NYA-2027 tag adduct (Sequence No. 28), NYA-2027 is the amino acid sequence of the NYA-1143 tag adduct (Sequence No. 29), NYA-1143 is the amino acid sequence of the NYA-2143 tag adduct (Sequence No. 30), NYA-2143 is the amino acid sequence of the NYA-1154 tag adduct (Sequence No. 31), Fig. 39 is, The amino acid sequence of the NYA-1154 tag adduct (Sequence No. 32), NYA-1154 with amino acid numbers 21–266, Fig. 40, the amino acid sequence of the truncate of HLA-A*0201 (GenBank: ASA47534.1) (Sequence No. 33), Fig. 41, the amino acid sequence of β2-microglobulin (Sequence No. 34), Fig. 42, and the nucleotide sequence of the NYA-2035 tag adduct (Sequence No. 35), Fig. 43,Amino acid sequence of NYA-2035 tag adduct (Sequence No. 36), NYA-2035 amino acid numbers 21–266, Fig. 44, amino acid sequence of NYA-1143-VH01 (Sequence No. 37), Fig. 45, amino acid sequence of NYA-1143-VH02 (Sequence No. 38), Fig. 46, amino acid sequence of NYA-1143-VH03 (Sequence No. 39), Fig. 47, amino acid sequence of NYA-1143-VL01 (Sequence No. 40), Fig. 48, nucleotide sequence of NYA-2044 tag adduct (Sequence No. 41), Fig. 49, nucleotide sequence of NYA-2045 tag adduct (Sequence No. 42), Fig. 50, NYA-2047 tag Fig. 51 shows the nucleotide sequence of the adduct (SEQ No. 43), Fig. 52 shows the nucleotide sequence of the NYA-2048 tag adduct (SEQ No. 44), Fig. 53 shows the nucleotide sequence of the NYA-2060 tag adduct (SEQ No. 45), Fig. 54 shows the nucleotide sequence of the NYA-2061 tag adduct (SEQ No. 46), Fig. 55 shows the amino acid sequence of the NYA-2044 tag adduct (SEQ No. 47), where NYA-2044 has amino acid numbers 21–266, Fig. 56 shows the amino acid sequence of the NYA-2045 tag adduct (SEQ No. 48), where NYA-2045 has amino acid numbers 21–266, Fig. 57 shows the amino acid sequence of NYA-0082 (SEQ No. 49), and NYA-2047 tag Amino acid sequence of the adduct (Sequence No. 50), NYA-2047, amino acid numbers 21–266, 58; amino acid sequence of the NYA-2048 tag adduct (Sequence No. 51), NYA-2048, amino acid numbers 21–266, 59; amino acid sequence of the NYA-2060 tag adduct (Sequence No. 52), NYA-2060, amino acid numbers 21–266, 60; amino acid sequence of the NYA-2061 tag adduct (Sequence No. 53), NYA-2061, amino acid numbers 21–266, 61;Fig. 62 shows the amino acid sequences of CDRH1–CDRH3 of the heavy chain and CDRL1–L3 of the light chain of NYA-0001 (Sequence Nos. 54–59); Fig. 63 shows the amino acid sequence of CDRL1 of NYA-2023 (Sequence No. 60); Fig. 64 shows the amino acid sequence of CDRL3 of NYA-2027 (Sequence No. 61); Fig. 65 shows the amino acid sequences of CDRH3 and CDRL3 of NYA-1154 (Sequence Nos. 62 and 63); Fig. 66 shows the amino acid sequence of CDRL1 of NYA-0035 (Sequence No. 64); Fig. 67 shows the nucleotide sequence of the tag adduct of NYC-0003 (Sequence No. 65); and Fig. 66 shows the nucleotide sequence of the tag adduct of NYC-0004 (Sequence No. 66). 68 is the amino acid sequence of the NYC-0003 tag adduct (Sequence No. 67), NYC-0003 is amino acid numbers 21–263. 69 is the amino acid sequence of the NYC-0004 tag adduct (Sequence No. 68), NYC-0004 is amino acid numbers 21–263. 70 is the nucleotide sequence of the NYA-0001 tag adduct (Sequence No. 69). 71 is the amino acid sequence of the NYA-0001 tag adduct (Sequence No. 70), NYA-0001 is amino acid numbers 21–266. 72 is the nucleotide sequence of HC1 (Sequence No. 71). 73 is the nucleotide sequence of NYF-0016-HC2 (Sequence No. 72). 74 is, Figure 75, the nucleotide sequence of NYF-0019-HC2 (Sequence No. 73); Figure 76, the nucleotide sequence of NYF-0022-HC2 (Sequence No. 74); Figure 77, the nucleotide sequence of NYF-0023-HC2 (Sequence No. 75); Figure 78, the nucleotide sequence of NYF-0027-HC2 (Sequence No. 76); Figure 79, the nucleotide sequence of NYF-0035-HC2 (Sequence No. 77); and Figure 80, the nucleotide sequence of NYF-0044-HC2 (Sequence No. 78).Figure 81 is the nucleotide sequence of NYF-0045-HC2 (Sequence No. 79), Figure 82 is the nucleotide sequence of NYF-0047-HC2 (Sequence No. 80), Figure 83 is the nucleotide sequence of NYF-0048-HC2 (Sequence No. 81), Figure 84 is the nucleotide sequence of NYF-0060-HC2 (Sequence No. 82), Figure 85 is the nucleotide sequence of NYF-0061-HC2 (Sequence No. 83), Figure 86 is the amino acid sequence of HC1 (Sequence No. 84), Figure 85 is the amino acid sequence of NYF-0016-HC2 (Sequence No. 85), NYA-1143 has amino acid numbers 21–266, and C3E-7085 has amino acid numbers 272– 511D 87 is the amino acid sequence of NYF-0019-HC2 (sequence number 86), NYA-2143 is amino acid numbers 21–266, C3E-7085 is amino acid number 272–511D 88 is the amino acid sequence of NYF-0022-HC2 (sequence number 87), NYA-1163 is amino acid numbers 21–266, C3E-7085 is amino acid number 272–511D 89 is the amino acid sequence of NYF-0023-HC2 (sequence number 88), NYA-2023 is amino acid numbers 21–266, C3E-7085 is amino acid number 272–511D 90 is of NYF-0027-HC2 Amino acid sequence (Sequence No. 89), NYA-2027 is amino acid numbers 21–266, C3E-7085 is amino acid numbers 272–511, 91 is the amino acid sequence of NYF-0035-HC2 (Sequence No. 90), NYA-2035 is amino acid numbers 21–266, C3E-7085 is amino acid numbers 272–511, 92 is the amino acid sequence of NYF-0044-HC2 (Sequence No. 91), NYA-2044 is amino acid numbers 21–266, C3E-7085 is amino acid numbers 272–511, 93 is the amino acid sequence of NYF-0045-HC2 (Sequence No. 92),NYA-2045 is amino acid numbers 21–266, C3E-7085 is amino acid numbers 272–511, and 94 is the amino acid sequence of NYF-0047-HC2 (sequence number 93), NYA-2047 is amino acid numbers 21–266, C3E-7085 is amino acid numbers 272–511, and 95 is the amino acid sequence of NYF-0048-HC2 (sequence number 94), NYA-2048 is amino acid numbers 21–266, C3E-7085 is amino acid numbers 272–511, and 96 is the amino acid sequence of NYF-0060-HC2 (sequence number 95), and NYA-2060 is amino acid numbers 21–266, C3E-7085 is amino acid numbers 272–511, Figure 97 is the amino acid sequence of NYF-0061-HC2 (Sequence No. 96), NYA-2061 is amino acid numbers 21–266, C3E-7085 is amino acid numbers 272–511, Figure 98 is the nucleotide sequence of NYA-0001-Fab-HC1-k delete (Sequence No. 97), Figure 99 is the nucleotide sequence of NYA-0001-LC (Sequence No. 98), Figure 100 is the amino acid sequence of NYA-0001-Fab-HC1-k delete (Sequence No. 99), the heavy chain variable region of NYA-0001 is amino acid numbers 20–139, Figure 101 is, The amino acid sequence of NYA-0001-LC (Sequence No. 100), the light chain variable region of NYA-0001 is amino acid numbers 21–131, Figure 102, the nucleotide sequence of NYA-1143-Fab-HC1-k delete (Sequence No. 101), Figure 103, the nucleotide sequence of NYA-1143-LC (Sequence No. 102), Figure 104, the nucleotide sequence of C3E-7085-HC2-k deleteC (Sequence No. 103), Figure 105, the amino acid sequence of NYA-1143-Fab-HC1-k delete (Sequence No. 104), the heavy chain variable region of NYA-1143 is amino acid numbers 20–139, Figure 106,Amino acid sequence of NYA-1143-LC (Sequence No. 105), the variable region of the light chain of NYA-1143 is amino acid numbers 21–131, Figure 107 is the amino acid sequence of C3E-7085-HC2-k delete (Sequence No. 106), C3E-7085 is amino acid numbers 21–260, Figure 108 is the nucleotide sequence of NYA-1143-HC1-k delete (Sequence No. 107), Figure 109 is the amino acid sequence of NYA-1143-HC1-k delete (Sequence No. 108), NYA-1143 is amino acid numbers 21–266, Figure 110 is the nucleotide sequence of C3E-7085-NYA-1154-Fab-HC2-k delete (Sequence No. 109) Fig. 111 is the nucleotide sequence of NYA-1154-LC (Sequence No. 110). Fig. 112 is the nucleotide sequence of OAA-HC1-k delete (Sequence No. 111). Fig. 113 is the amino acid sequence of C3E-7085-NYA-1154-Fab-HC2-k delete (Sequence No. 112), C3E-7085 is amino acid numbers 21–260, and the heavy chain variable region of NYA-1154 is amino acid numbers 266–285. Fig. 114 is the amino acid sequence of NYA-1154-LC (Sequence No. 113), and the light chain variable region of NYA-1154 is amino acid numbers 21–131. Fig. 115 is the amino acid sequence of OAA-HC1-k delete (Sequence No. Figures 114), 116 are the nucleotide sequence of NYF-0010-HC2-k delete (sequence number 115), Figure 117 is the nucleotide sequence of NYF-0004-HC2-k delete (sequence number 116), Figure 118 is the nucleotide sequence of NYF-0011-HC2-k delete (sequence number 117), Figure 119 is the amino acid sequence of NYF-0010-HC2-k delete (sequence number 18), NYA-1154 is amino acid numbers 21–266,C3E-7085 is amino acid numbers 272–511, Figure 120 is the amino acid sequence of NYF-0004-HC2-k delete (Sequence No. 119), C3E-7085 is amino acid numbers 21–260, NYA-1154 is amino acid numbers 272–511, Figure 121 is the amino acid sequence of NYF-0011-HC2-k delete (Sequence No. 120), NYA-1143 is amino acid numbers 21–266, C3E-7085 is amino acid numbers 272–511, Figure 122 is the amino acid sequence of point variant NY-ESO peptide 1F (Sequence No. 121), Figure 123 is the amino acid sequence of point variant NY-ESO peptide 2M (Sequence No. 122) Fig. 124 is the amino acid sequence of the point variant NY-ESO peptide 3A (Sequence No. 123); Fig. 125 is the amino acid sequence of the point variant NY-ESO peptide 4A (Sequence No. 124); Fig. 126 is the amino acid sequence of the point variant NY-ESO peptide 5A (Sequence No. 125); Fig. 127 is the amino acid sequence of the point variant NY-ESO peptide 6L (Sequence No. 126); Fig. 128 is the amino acid sequence of the point variant NY-ESO peptide 7F (Sequence No. 127); Fig. 129 is the amino acid sequence of the point variant NY-ESO peptide 8A (Sequence No. 128); Fig. 130 is the amino acid sequence of the point variant NY-ESO peptide 9A (Sequence No. 129); Fig. 131 is of the gp100 peptide Amino acid sequence (Sequence No. 130) Fig. 132, amino acid sequence of homologous peptide DOLPP1 (Sequence No. 131) Fig. 133, amino acid sequence of homologous peptide IL20RB (Sequence No. 132) Fig. 134, amino acid sequence of homologous peptide PRKD2 (Sequence No. 133) Fig. 135, amino acid sequence of homologous peptide CD163 (Sequence No. 134) Fig. 136, amino acid sequence of homologous peptide P2RY8 (Sequence No. 135) Fig. 137,Fig. 138 shows the amino acid sequence of C3E-7034 (Sequence No. 136), Fig. 139 shows the amino acid sequence of C3E-7036 (Sequence No. 137), Fig. 140 shows the amino acid sequence of C3E-7085 (Sequence No. 138), Fig. 141 shows the amino acid sequence of C3E-7088 (Sequence No. 139), Fig. 142 shows the amino acid sequence of C3E-7093 (Sequence No. 140), Fig. 143 shows the amino acid sequences of CDRH1–CDRH3 of the heavy chain and CDRL1–CDRL3 of the light chain of C3E-7085 (Sequence Nos. 141–146), Fig. 144 shows the amino acid sequence of C3E-7078 (Sequence No. 147), NYF-0014-HC2 Fig. 145 is the nucleotide sequence of NYF-0014-HC2 (SEQ No. 148), the amino acid sequence of NYF-0014-HC2 (SEQ No. 149), NYA-0001 is amino acid numbers 21–266, and C3E-7085 is amino acid numbers 272–511. Fig. 146 is the amino acid sequence of NYF-0082-HC2 (SEQ No. 150), NYA-0082 is amino acid numbers 21–266, and C3E-7085 is amino acid numbers 272–511. Fig. 147 is the amino acid sequence of human CD3ε (SEQ No. 151). Fig. 148 is the full-length nucleotide sequence of NYZ-0038-HC2 (SEQ No. 152). Fig. 149 is NYZ-0082-HC2 Fig. 150 is the full-length nucleotide sequence (Sequence No. 153) of NYZ-0083-HC2, Fig. 154 is the full-length nucleotide sequence of NYZ-0038-HC2, Fig. 151 is the full-length amino acid sequence (Sequence No. 155), NYA-2061 contains amino acid numbers 21–266, C3E-7096 contains amino acid numbers 272–511, Fig. 152 is the full-length amino acid sequence of NYZ-0082-HC2 (Sequence No. 156), NYA-3061 contains amino acid numbers 21–271, C3E-7096 contains amino acid numbers 277–516, Fig. 153 is,NYZ-0083-HC2 full-length amino acid sequence (Sequence No. 157), NYA-3061 amino acid numbers 21–271, C3E-7097 amino acid numbers 277–516, Fig. 154, NYZ-1010-HC2 full-length nucleotide sequence (Sequence No. 158), Fig. 155, C3E-7085-LC full-length nucleotide sequence (Sequence No. 159), Fig. 156, NYZ-1010-HC2 full-length amino acid sequence (Sequence No. 160), NYA-3061 amino acid numbers 21–271, C3E-7085 medium chain variable region amino acid numbers 277–394, Fig. 157, C3E-7085-LC full-length amino acid sequence (Sequence No 161) Figure 158a is a table showing the standardized gMFI for CD3e knockout T2 cells with various point mutation peptides of Fc addition type anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules NYZ-0038, 0082, 0083, and 1010. * / Underlined indicates less than half compared to the standardized gMFI for each Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule in CD3e knockout T2 cells with NY-ESO peptide added, * Bold indicates less than one-fourth. Figure 158b is a table showing the standardized gMFI for CD3e knockout T2 cells with various homologous peptides of the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules NYZ-0038, 0082, 0083, and 1010. * / Underlined indicates a large value compared to the standardized gMFI for DMSO-added CD3e knockout T2 cells for each Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule. Figure 159a shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules, NYZ-0038, 0082, and 0083,Figure 159b shows that the Fc-addition type anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, NYZ-1010, has cell damage activity in endogenous human NY-ESO-expressing U266B1 cells in the presence of human PBMCs. The error bars in the figure represent the standard deviation (n = 3). Figure 159c shows that the Fc-addition type anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, NYZ-0082, 1010, has cell damage activity in endogenous human NY-ESO-expressing NCI-H1703 cells in the presence of human PBMCs. The error bars in the figure represent the standard deviation (n = 3). Figure 159d is a figure showing that the Fc-addition type anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules, NYZ-0038, 0083, exhibit cell damage activity against endogenous human NY-ESO-expressing NCI-H1703 cells in the presence of human PBMCs. The error bars in the figure represent the standard deviation (n = 3). Figure 159e is a figure showing that the Fc-addition type anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules, NYZ-0038, 0082, 0083, do not exhibit cell damage activity against endogenous human NY-ESO-non-expressing AGS cells in the presence of human PBMCs. The error bars in the figure represent the standard deviation (n = 3). Figure 159f is a figure showing that the Fc-addition type anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, NYZ-1010, does not exhibit cell damage activity against endogenous human NY-ESO-non-expressing AGS cells in the coexistence of human PBMCs. The error bars in the figure represent the standard deviation (n = 3). Figure 159g is,Figure 159h is a figure showing that the Fc-addition type anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, NYZ-0082, 1010, does not exhibit cell damage activity against endogenous human NY-ESO-non-expressing CFPAC-1 cells in the presence of human PBMCs. The error bars in the figure represent the standard deviation (n = 3). Figure 159h is a figure showing that the Fc-addition type anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, NYZ-0038, 0083, does not exhibit cell damage activity against endogenous human NY-ESO-non-expressing CFPAC-1 cells in the presence of human PBMCs. The error bars in the figure represent the standard deviation (n = 3). Figure 160a is a figure showing that the Fc-addition type anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, NYZ-0038, has antitumor activity in a human PBMC influx model. The error bars in the figure represent the standard error (n = 5). Figure 160b is a figure showing that the Fc-addition type anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, NYZ-0082, has antitumor activity in a human PBMC influx model. The error bars in the figure represent the standard error (n = 5). Figure 160c is a figure showing that the Fc-addition type anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, NYZ-0083, has antitumor activity in a human PBMC influx model. The error bars in the figure represent the standard error (n = 5). Fig. 160d is a figure showing that the Fc addition-type anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, NYZ-1010, possesses antitumor activity in a human PBMC influx model. The error bars in the figure represent the standard error (n = 5). Fig. 161 is the amino acid sequence of the peptide linker (Sequence No. 162). Fig. 162 is,This is a figure showing the content (%) of the multimer when various Fc addition-type anti-HLA / NY-ESO-anti-CD3 bispecific molecules are acid-treated. Fig. 163 is a figure showing the results of the solution stability evaluation of the anti-HLA / NY-ESO scFv-heterodimer Fc. Fig. 164 is the full-length nucleotide sequence of NYA-3061 (Sequence No. 163), Fig. 165 is the full-length amino acid sequence of NYA-3061 (Sequence No. 164), Fig. 166 is the full-length nucleotide sequence of NYC-0005 (Sequence No. 165), Fig. 167 is the full-length amino acid sequence of NYC-0005 (Sequence No. 166), Fig. 168 is the full-length nucleotide sequence of NYC-0006 (Sequence No. 167), and Fig. 169 is Fig. 170 shows the full-length amino acid sequence of NYC-0006 (Sequence No. 168), Fig. 171 shows the full-length nucleotide sequence of NYC-0007 (Sequence No. 169), Fig. 172 shows the full-length amino acid sequence of NYC-0007 (Sequence No. 170), Fig. 173 shows the full-length nucleotide sequence of NYC-0008 (Sequence No. 171), Fig. 174 shows the full-length amino acid sequence of NYC-0008 (Sequence No. 172), Fig. 175 shows the full-length nucleotide sequence of NYC-0009 (Sequence No. 173), Fig. 176 shows the full-length amino acid sequence of NYC-0009 (Sequence No. 174), and Fig. 177 shows the full-length nucleotide sequence of NYC-0010 (Sequence No. 175). Fig. 178, the full-length amino acid sequence of NYC-0010 (Sequence No. 176); Fig. 179, the full-length nucleotide sequence of HC-h (Sequence No. 177); Fig. 180, the full-length amino acid sequence of HC-h (Sequence No. 178); Fig. 181, the full-length nucleotide sequence of NYD-2047-HC-k (Sequence No. 179); Fig. 182, the full-length amino acid sequence of NYD-2047-HC-k (Sequence No. 180).Fig. 183, the full-length nucleotide sequence of NYD-2061-HC-k (Sequence No. 181); Fig. 184, the full-length amino acid sequence of NYD-2061-HC-k (Sequence No. 182); Fig. 185, the full-length nucleotide sequence of NYD-3061-HC-k (Sequence No. 183); Fig. 186, the full-length amino acid sequence of NYD-3061-HC-k (Sequence No. 184); Fig. 187, the full-length nucleotide sequence of NYC-0011-HC-k (Sequence No. 185); Fig. 188, the full-length amino acid sequence of NYC-0011-HC-k (Sequence No. 186); and Fig. 189, the full-length nucleotide sequence of NYC-0012-HC-k (Sequence No. 187). Fig. 190, the full-length amino acid sequence of NYC-0012-HC-k (Sequence No. 188); Fig. 191, the full-length nucleotide sequence of NYC-0013-HC-k (Sequence No. 189); Fig. 192, the full-length amino acid sequence of NYC-0013-HC-k (Sequence No. 190); Fig. 193, the full-length nucleotide sequence of NYC-0014-HC-k (Sequence No. 191); Fig. 194, the full-length amino acid sequence of NYC-0014-HC-k (Sequence No. 192); Fig. 195, the full-length nucleotide sequence of NYC-0015-HC-k (Sequence No. 193); Fig. 196, the full-length amino acid sequence of NYC-0015-HC-k (Sequence No. 194). Fig. 197 shows the full-length nucleotide sequence of NYC-0016-HC-k (Sequence No. 195), Fig. 198 shows the full-length amino acid sequence of NYC-0016-HC-k (Sequence No. 196), Fig. 197 shows the full-length amino acid sequence of NYZ-1007-HC2 (Sequence No. 197), NYA-2061 is amino acid numbers 21–266, and the C3E-7085 medium chain variable region is amino acid numbers 272–389. Fig. 199 shows the full-length amino acid sequence of NYZ-1017-HC2 (Sequence No. 198).NYA-2047 has amino acid numbers 21–266, and the C3E-7085 medium chain variable region has amino acid numbers 277–389. Specific details for implementing the invention

[0238] The present invention will be described in detail below.

[0239] 1. Definition

[0240] In the present invention, "gene" means a nucleotide chain containing a base sequence encoding an amino acid of a protein or a complementary chain thereof, and for example, polynucleotides, oligonucleotides, DNA, mRNA, cDNA, cRNA, etc., which are nucleotide chains containing a base sequence encoding an amino acid of a protein or a complementary chain thereof, are included in the meaning of "gene." Such genes are nucleotides in a single strand, a double strand, or three or more strands, and aggregates of DNA strands and RNA strands, a mixture of ribonucleotides (RNA) and deoxyribonucleotides (DNA) on a single nucleotide chain, and two or more nucleotide chains containing such nucleotide chains are also included in the meaning of "gene." In the present invention, base sequence and nucleotide sequence have the same meaning.

[0241] In the present invention, “polynucleotide,” “nucleotide chain,” “nucleic acid,” and “nucleic acid molecule” have the same meaning, and for example, DNA, RNA, probe, oligonucleotide, primer, etc. are also included in the meaning of “polynucleotide.” Such polynucleotides are polynucleotides composed of a single chain, a two-chain, or three or more chains, and aggregates of DNA chains and RNA chains, ribonucleotides (RNA) and deoxyribonucleotides (DNA) mixed on a single polynucleotide chain, and aggregates of two or more chains containing such polynucleotide chains are also included in the meaning of “polynucleotide.”

[0242] In the present invention, "polypeptide," "peptide," and "protein" have the same meaning.

[0243] In the present invention, "antigen" may be used in the sense of "immunogen."

[0244] In the present invention, "cell" includes various cells derived from animal individuals, serially cultured cells, primary cultured cells, cell lines, recombinant cells, and microorganisms, etc.

[0245] In the present invention, "antibody" has the same meaning as "immunoglobulin." However, in the case of the anti-HLA / NY-ESO antibody of the present invention, the term "antibody" is used to mean an immunoglobulin having a normal region and a variable region. The antibody is not particularly limited, whether it is a natural one or an immunoglobulin produced by partial or complete synthesis. The anti-HLA / NY-ESO antibody of the present invention is included in the "molecule" described below.

[0246] In the present invention, "NY-ESO peptide" means a peptide (SLLMWITQC: Sequence No. 1) composed of 9 amino acids from the 157th to the 165th of NY-ESO-1 and LAGE-1.

[0247] 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 denoted as “HLA / NY-ESO”.

[0248] In the present invention, "anti-HLA-A2 / NY-ESO antibody" means an antibody that binds to HLA-A2 / NY-ESO, in other words, an antibody that recognizes HLA-A2 / NY-ESO. Likewise, "anti-HLA-A2 / NY-ESO scFv" means an scFv that binds to HLA / NY-ESO, in other words, recognizes HLA-A2 / NY-ESO. "Anti-HLA-A2 / NY-ESO antibody" and "anti-HLA-A2 / NY-ESO scFv" are also denoted as "anti-HLA / NY-ESO antibody" and "anti-HLA / NY-ESO scFv," respectively.

[0249] The structure of a basic 4-chain antibody consists of two identical light chains (L chains) and two identical heavy chains (H chains). The light chains are bound to the heavy chains by one covalent disulfide bond. The two heavy chains are bound to each other by one or more disulfide bonds, depending on the isotype of the heavy chain. Each light and heavy chain has intra-chain disulfide bonds at regular intervals. The heavy and light chains contain a normal region exhibiting very high amino acid sequence similarity and a variable region with low amino acid sequence similarity. The light chains have a variable region (VH) at the amino terminus followed by a normal region (CL). The heavy chains have a variable region (VH) at the amino terminus followed by three normal regions (CH1 / CH2 / CH3). VL and VH are paired, and CL runs parallel to the first normal region (CH1) of the heavy chain. VL and VH pair up and form a single antigen-binding site.

[0250] The normal region of the antibody of the present invention is not particularly limited, but preferably, a human antibody is used as the antibody of the present invention for treating or preventing human diseases. Examples of the heavy chain normal region of the human antibody include Cγ1, Cγ2, Cγ3, Cγ4, Cμ, Cδ, Cα1, Cα2, Cε, etc. Examples of the light chain normal region of the human antibody include Cκ, Cλ, etc.

[0251] Fab consists of the heavy chain VH and the CH1 following it, and the light chain VL and the CL following it. VH and VL include a complementarity determining region (CDR).

[0252] Fc (also referred to as the Fc region) is a carboxyl terminal region of the normal region of the heavy chain and is a dimer containing CH2 and CH3. The Fc of the present invention may be a natural sequence Fc or a variant Fc in which a mutation has occurred in the natural sequence (referred to as "variant Fc"), and in the multispecificity molecule and bispecificity molecule of the present invention, the preferred Fc region is the variant Fc, and more preferably, a set of Fc capable of forming a heterodimer. Examples of a set of Fc include a combination of Fc (i) included in the first polypeptide and Fc (ii) included in the second polypeptide, which will be described later, but are not limited to those as long as the set of Fc can associate (form a heterodimer).

[0253] Variant Fc includes: a modified Fc region (including a heterodimeric Fc region) contained in a heteromultimer having enhanced stability disclosed in WO2013 / 063702; Fc comprising a CH3 region of an immunoglobulin derived from an IgG antibody having "protrusions" and "voids" contained in a heteromultimer disclosed in WO1996 / 27011; Fc comprising a CH3 domain contained in an electrostatically advantageous heterodimer by substituting one or more amino acid residues with charged amino acids disclosed in WO2009 / 089004; a heterodimeric Fc region contained in a heterodimer using stereochemical variations and / or pI (isoelectric point) variations disclosed in WO2014 / 110601; and a variant disclosed in WO2010 / 151792 that loses or reduces binding to Protein A. Examples include heterodimers such as Fc containing a CH3 domain including a modification, but are not limited thereto.

[0254] The variable region consists of a region of extreme variability called the hypervariable region (HVR) and a relatively invariable region called the framework region (FR) separated by that region. The variable regions of the natural heavy and light chains contain four FRs connected by three hypervariable regions, and the hypervariable region of each chain is maintained near the pole together with the hypervariable region of another chain by the FR, contributing to the formation of the antigen-binding site of the antibody.

[0255] It is known that the heavy chain and light chain of an antibody molecule each have three complementarity determining regions (CDRs). The complementarity determining region, also called the supervariable region, is a region within the variable region of the antibody's heavy chain and light chain that exhibits particularly high variability in the primary structure, and is typically divided into three points on the primary structure of the polypeptide chains of the heavy chain and light chain. In the present invention, regarding the complementarity determining regions of the antibody, the complementarity determining region of the heavy chain is denoted as CDRH1, CDRH2, and CDRH3 from the amino-terminal side of the heavy chain amino acid sequence, and the complementarity determining region of the light chain is denoted as CDRL1, CDRL2, and CDRL3 from the amino-terminal side of the light chain amino acid sequence. These regions are close to each other in terms of stereostructure and determine specificity for the antigen to which they bind.

[0256] In the present invention, the position and length of the CDR were determined by the definition of IMGT (Developmental and Comparative Immunology 27 (2003) 55-77).

[0257] FR is a variable region other than the CDR residue. The variable region generally has four FRs: FR1, FR2, FR3, and FR4.

[0258] CDR and FR included in the heavy chain and light chain are arranged in the order of FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4 and FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4, respectively, from the amino terminus toward the carboxyl terminus.

[0259] The positions of CDR and FR can be determined by various well-known definitions in the field of the art, for example, in addition to IMGT, definitions such as Kabat, Chothia, AbM, and contact.

[0260] In the present invention, "antigen-binding fragment of an antibody" refers to a partial fragment of an antibody having binding activity with an antigen, comprising a medium-chain variable region and a light-chain variable region. Examples of "antigen-binding fragments of an antibody" include, but are not limited to, antigen-binding fragments such as Fab, F(ab')2, scFv, Fab', Fv, and single-domain antibody (sdAb). In addition to being obtained by treating the full-length molecule of an antibody protein with an enzyme such as papain or pepsin, such an antigen-binding fragment of an antibody may also be a recombinant protein produced in a suitable host cell using a recombinant gene. In the present invention, "binding fragment of an antibody" has the same meaning as "antigen-binding fragment of an antibody."

[0261] In the present invention, the "site" to which the antibody binds, i.e., the "site" recognized by the antibody, refers to a partial peptide or partial higher-order structure on the antigen to which the antibody binds or recognizes.

[0262] In the present invention, such a region is also referred to as an epitope or a binding site of an antibody. In the present invention, "variant antibody" refers to a polypeptide having an amino acid sequence formed by substituting, deleting, or adding (addition includes insertion) amino acids to the amino acid sequence of the original antibody (hereinafter collectively referred to as "variant"), and which also binds to the HLA / NY-ESO of the present invention. The number of variant amino acids in such a variant antibody is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, or 50. Such a variant antibody is also included in the "antibody" of the present invention.

[0263] In the present invention, "number of" in "1 or a number of" refers to 2 to 10.

[0264] In this specification, "molecule" is a molecule comprising the antibody described above or an antigen-binding fragment of the antibody, and also includes a multispecific molecule formed from the antibody or a plurality of antigen-binding fragments derived therefrom.

[0265] In this specification, “multispecific molecule,” “multispecific(of) molecule,” and “multispecific molecule” have the same meaning and are not particularly limited to molecules capable of binding to multiple different epitopes on one molecule and / or to different epitopes on two or more molecules. Multispecific molecules include antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL). Such multispecific molecules include, but are not limited to, full-length antibody molecules having two or more different heavy chains and light chains, i.e., IgG-type multispecific molecules, and molecules composed of antigen-binding fragments having two or more types of VL and VH, i.e., Fab, Fab', Fv, scFv, sdAb, etc., molecules derived by combining tandem scFv, diabodies, single-chain diabodies, tribodies, etc. In addition, molecules generated by genetically or chemically linking a protein having antigen-binding ability without an immunoglobulin backbone to an antigen-binding fragment are also included as multispecific molecules. In the present invention, such multispecific molecules may also be referred to as "multispecific antibodies," "multispecific antibodies," etc., except in cases where they do not contain antibodies or their antigen-binding fragments.

[0266] The activities and properties obtained by the anti-HLA / NY-ESO antibody of the present invention, the antigen-binding fragment of the antibody, or the molecule of the present invention may include, for example, biological activity, physicochemical properties (also called physical properties), and specifically, various biological activities such as cell damage activity, ADCC activity, and antitumor activity (all described later), binding activity to antigens or epitopes, stability during manufacturing or storage, and thermal stability.

[0267] In the present invention, "hybridize under strict conditions" means to perform hybridization at 65°C in a solution containing 5 × SSC, and then perform hybridization under conditions or equivalent conditions, such as washing at 65°C for 20 minutes in an aqueous solution containing 2 × SSC and 0.1% SDS, at 65°C for 20 minutes in an aqueous solution containing 0.5 × SSC and 0.1% SDS, and at 65°C for 20 minutes in an aqueous solution containing 0.2 × SSC and 0.1% SDS, respectively. SSC is an aqueous solution of 150 mM NaCl and 15 mM sodium citrate, and n × SSC means SSC at n times the concentration.

[0268] In the present invention, "cell injury" refers to causing pathological changes in a cell in any form, and does not stop at direct trauma, but means damage to the structure or function of the cell, such as DNA cutting, formation of base dimers, chromosome cutting, damage to cell division machinery, and reduction of various enzyme activities. In the present invention, "cell injury activity" means causing the above-mentioned cell injury.

[0269] In the present invention, "antibody-dependent cell damage activity" refers to "antibody-dependent cellular cytotoxicity (ADCC) activity," and means the activity of NK cells inflicting damage on target cells, such as tumor cells, through the intermediary of antibodies.

[0270] In the present invention, "cell injury activity by T cell redirection" means causing said cell injury by interposing a multispecific molecule comprising an anti-tumor antigen antibody and an anti-HLA / NY-ESO antibody. That is, it means that the anti-tumor antigen antibody binds to a target tumor cell and the anti-HLA / NY-ESO antibody binds to a T cell, thereby bringing the distance between the target tumor cell and the T cell closer and inducing cell injury through T cell activation. The molecule may be contained in a pharmaceutical composition.

[0271] 2. Antigen

[0272] 2-1. HLA / NY-ESO Antigen

[0273] In the present invention, "HLA / NY-ESO" is used with the same meaning as HLA / NY-ESO protein.

[0274] HLA / NY-ESO is a triadic complex of HLA-A2, β2-microglobulin, and NY-ESO peptide. HLA-A2 is a type of HLA peptide and is known to be the most frequent peptide in Caucasians. HLA forms a triadic complex of β2-microglobulin and a peptide fragment of its own protein within the cell's endoplasmic reticulum, presents it extracellularly, and is recognized by the T cell receptor (TCR) of T cells. The NY-ESO peptide (SLLMWITQC: sequence number 1, Fig. 8) is a peptide consisting of 9 amino acids from the 157th to the 165th position of NY-ESO-1 and LAGE-1, and it has been reported to be presented to HLA-A2.

[0275] 2-2. CD3 Antigen

[0276] In the present invention, "CD3" is used with the same meaning as CD3 protein.

[0277] CD3 is expressed on T cells as part of a multimolecular T cell receptor complex and is a complex of five types of polypeptides (molecular weights of 25,000-28,000, 21,000, 20,000, 16,000, and 22,000, respectively) of γ chain, δ chain, ε chain, ζ chain, and η chain.

[0278] Examples of CD3 complex chains include γ, δ, ε, ζ, and η. These are also referred to as subunits. By binding to T cells, anti-CD3 antibodies induce cell damage through T cell activation. Most anti-CD3 antibodies bind to CD3ε.

[0279] The nucleotide sequence of the cDNA encoding human CD3ε is registered in NCBI / GenBank as accession number: NM_000733 (NM_000733.3), and the amino acid sequence of human CD3ε is registered in NCBI / GenPept as accession number: NP_000724 (NM_000724.1). The nucleotide sequence of the cDNA encoding crab-eating monkey CD3 is registered in GenBank as accession number: NM_001283615.1. The amino acid sequence of human CD3ε is listed in sequence number 151 of the sequence table (Fig. 147).

[0280] 2-3. Preparation of Antigens

[0281] The antigen protein described above used in the present invention; HLA / NY-ESO, CD3 (hereinafter collectively referred to as the antigen protein) can be prepared by purification and isolation from animal tissue (including body fluid), cells derived from the tissue, or cell cultures thereof, genetic recombination, in vitro translation, chemical synthesis, etc.

[0282] The cDNA of the antigen protein can be obtained by the so-called PCR method, for example, by using a cDNA library of an organ expressing the mRNA of the antigen protein as a template and performing a polymerase chain reaction (hereinafter referred to as “PCR”) (Saiki, RK, et al., Science (1988) 239, 487-49) using primers that specifically amplify the cDNA of the antigen protein.

[0283] A polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence encoding the antigen protein expressed in humans or rats is hybridized under stringent conditions, and a polynucleotide encoding a protein having biological activity equivalent to that antigen protein is also included in the cDNA of the antigen protein.

[0284] In addition, a splicing variant transcribed from the antigen protein locus expressed in humans or rats, or a polynucleotide hybridized under conditions stringent thereto, and a polynucleotide encoding a protein having biological activity equivalent to that of the antigen protein, are also included in the cDNA of the antigen protein.

[0285] In the amino acid sequence of the antigen protein of a human or rat, or the amino acid sequence from which the signal sequence is excluded, a nucleotide sequence encoding a protein having biological activity equivalent to that of the antigen protein, wherein one to several amino acids are substituted, deleted, or added to the amino acid sequence, is also included in the nucleotide sequence of the antigen protein gene.

[0286] An amino acid sequence encoded in a splicing variant transcribed from a locus of the antigen protein of a human or rat, or in the amino acid sequence, one or more amino acids are substituted, deleted, or added to the amino acid sequence, and a protein having biological activity equivalent to that of the antigen protein is also included in the antigen protein.

[0287] 2-4. Binding specificity to antigen proteins

[0288] The anti-HLA / NY-ESO antibody and its antigen-binding fragment, etc. of the present invention recognize HLA / NY-ESO. That is, they bind to the HLA / NY-ESO antigen. The presence of HLA / NY-ESO in non-human animals such as mice, rats, and crab-eating monkeys is not known.

[0289] The anti-CD3 antibody and its binding fragment, etc., included in the multispecific molecule of the present invention recognize, i.e., bind to, the CD3 antigen. These anti-CD3 antibodies, etc., preferably bind to human CD3, monkey CD3, etc., and more preferably bind to human CD3 and crab-eating monkey CD3. Meanwhile, these preferred anti-CD3 antibodies do not bind to rat and / or mouse CD3.

[0290] The antitumor activity of the multispecific molecule of the present invention can be evaluated by, for example, (i) transplanting human cancer cells, human cancer tissues, etc. into a non-human animal transplanted with human peripheral hemolymphocyte cells, preferably a rat or mouse, more preferably a rat or mouse with impaired endogenous effector function (e.g., an immunocompromised rat or mouse), or (ii) transplanting mouse cancer cells, etc., into which HLA and NY-ESO have been introduced into a non-human animal transplanted with the human CD3 gene, preferably a rat or mouse. By performing evaluations using such immunocompromised or transplanted animals, various assays, immunohistochemistry, etc., can be performed using the in vivo of mice and / or rats, which is advantageous for the development of drugs, nonclinical studies, etc., containing the multispecific molecule of the present invention.

[0291] In the present invention, "recognition," i.e., "binding," refers to binding rather than non-specific adsorption. As a criterion for determining whether recognition is occurring, i.e., whether binding is taking place, for example, the dissociation constant (hereinafter referred to as "KD"). The KD value for HLA / NY-ESO or CD3 of the preferred antibody of the present invention is 1 × 10⁻⁶ -5 M or less, 5 × 10 -6 M or less, 2 × 10 -6 M or less, 1 × 10 -6 M or less, and the desirable 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 × 10 -9 M or less, and more preferably 1 × 10-9 It is M or less. NYA-1143, NYA-2023, NYA-2143, NYA-2044, NYA-2045, NYA-2060, NYA-2061, and NYA-3061 can be exemplified as the anti-HLA / NY-ESO scFv of the present invention having excellent antigen-binding activity, and the KD value for HLA / NY-ESO such as NYA-1143, NYA-2044, NYA-2045, and NYA-2143 is 1 × 10 -9 M is less than or equal to (Example 4, etc.).

[0292] The binding of antigens and antibodies in the present invention can be measured or determined by a biomolecular interaction analysis system such as the SPR method or BLI method, or by the ELISA method, RIA method, etc. The binding of antigens and antibodies expressed on the cell surface can be measured by the flow cytometry method, etc.

[0293] The SPR method (Surface Plasmon Resonance analysis method) is used as an analytical method to determine the dissociation constant (KD value), which is an indicator of affinity, by measuring the binding rate constant (Ka value) and the dissociation rate constant (Kd value) through reaction kinetic analysis. Examples of instruments used for SPR analysis include Biacore (trademark) (manufactured by GE Healthcare), ProteOn (trademark) (manufactured by BioRad), SPR-Navi (trademark) (manufactured by BioNavis), Spreeta (trademark) (manufactured by Texas Instruments), SPRi-PlexII (trademark) (manufactured by Horiba), and Autolab SPR (trademark) (manufactured by Metrohm).

[0294] BLI (BioLayer Interferometry) is a method for measuring interactions between biomolecules using biolayer interference. Examples of instruments used for analyzing interactions using BLI include the Octet system (manufactured by Pall ForteBio).

[0295] ELISA (Enzyme-linked ImmunoSorbent Assay) is a method for detecting and quantifying a target antigen or antibody contained in a sample solution by capturing it with a specific antibody or antigen and using an enzyme reaction. An enzyme-labeled antigen or antibody is introduced into a reaction system, and enzyme activity is detected. For the detection of enzyme activity, a substrate whose absorption spectrum changes upon reaction is used, and the result is quantified by measuring absorbance.

[0296] Cell-ELISA is a method that captures the target on the cell surface in each cell and detects and quantifies it using an enzymatic reaction.

[0297] In the RIA method (Radio Immunoassay method), antibodies can be quantified by labeling them with a radioactive material and measuring the radioactivity from the antibodies.

[0298] Flow cytometry is a method of optically analyzing individual cells by dispersing them in a fluid and flowing the fluid in a fine stream. Antibodies labeled with fluorescent dyes bind to antigens on the cell surface through an antigen-antibody reaction, and the antigen-binding ability of antibodies is quantified by measuring the fluorescence intensity produced by the antibodies bound to the cells.

[0299] Among the anti-HLA / NY-ESO antibodies of the present invention, 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, which are anti-HLA / NY-ESO scFvs having excellent antigen-binding specificity, can be exemplified (Example 6, etc.).

[0300] 3. Antibody or binding fragment that specifically binds to HLA / NY-ESO

[0301] 3-1. Anti-HLA / NY-ESO or its binding fragment

[0302] The present invention provides an antibody that recognizes and binds to HLA / NY-ESO or a binding fragment thereof.

[0303] As previously mentioned, HLA / NY-ESO is a complex comprising HLA-A2 and a 9-mer NY-ESO peptide (SLLMWITQC: SEQ No. 1). The NY-ESO peptide is an intracellular protein and is a peptide derived from NY-ESO-1 or LAGE-1, which are Cancer-Testis antigens. HLA / NY-ESO is presented on the surface of cancer cells.

[0304] The anti-HLA / NY-ESO antibody of the present invention and the antigen-binding fragment of the antibody (hereinafter also referred to as the antibody of the present invention) may be either a monoclonal antibody or a polyclonal antibody. The isotype of the monoclonal antibody of the present invention is not particularly limited and may include, for example, IgG such as IgG1, IgG2, IgG3, IgG4, IgM, IgA such as IgA1, IgA2, IgD, Ig, etc. The isotype and subclass of the monoclonal antibody may be determined, for example, by the octerone method, ELISA method, RIA method, etc. 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"), humanized antibodies, etc., and preferably, human antibodies may be used. The scope of the antibody of the present invention includes variants of the antibody (the "variant antibody" described below), and for example, the scope of the human antibody includes human variant antibodies.

[0305] Examples of non-human animal antibodies include antibodies derived from vertebrates such as mammals and birds. Examples of mammalian-derived antibodies include mouse antibodies and rodent-derived antibodies such as rat antibodies. Examples of bird-derived antibodies include chicken antibodies.

[0306] Examples of chimeric antibodies include antibodies formed by combining a variable region derived from a non-human animal antibody with a normal region of a human antibody (human immunoglobulin), but are not limited to these.

[0307] Examples of humanized antibodies include those in which the CDR of the variable region of a non-human animal antibody is transplanted into a human antibody (variable region of human immunoglobulin), those in which the sequence of the framework region of a non-human animal antibody is also transplanted into a human antibody in addition to the CDR, and those in which one or more amino acids derived from any of the non-human animal antibodies are substituted with human-type amino acids, but are not limited to these.

[0308] Antibodies can be produced by various known methods. Known methods include producing them using hybridomas or cellular immunoassays, or by genetic recombination. Additionally, methods for obtaining human antibodies derived from phage displays selected from a human antibody library are also known. For example, a phage display method can be used to select phages that bind to antigens by expressing the variable region of a human antibody as scFv on the surface of a phage. By analyzing the gene of the phage selected by binding to the antigen, the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined. When the DNA sequence of an scFv that binds to an antigen is determined, a human antibody can be obtained by constructing an expression vector having the sequence and introducing it into a suitable host for expression (WO92 / 01047, WO92 / 20791, WO93 / 06213, WO93 / 11236, WO93 / 19172, WO95 / 01438, WO95 / 15388, Annu. Rev. Immunol (1994) 12, 433-455).

[0309] The antibody with high activity obtained in this way can be used as a lead antibody, and the gene encoding the lead antibody can be mutated to produce a variant with even higher activity (the “variant antibody” described below).

[0310] The CDRH1 to CDRH3 included in the heavy chain of the anti-HLA / NY-ESO antibody of the present invention or the antigen-binding fragment thereof preferably include a combination of CDRH1 consisting of the amino acid sequence represented by SEQ ID NO. 54 (Fig. 61), CDRH2 consisting of the amino acid sequence represented by SEQ ID NO. 55 (Fig. 61), and the amino acid sequence represented by SEQ ID NO. 56 (Fig. 61), or the heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO. 56 (Fig. 61) in which the 6th amino acid is N (Asn). More preferably, a NYA-0001 heavy chain variable region consisting of the amino acid sequence represented by SEQ No. 6 (Fig. 13), a NYA-0082 heavy chain variable region consisting of the amino acid sequence represented by SEQ No. 18 (Fig. 25), a NYA-2023 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence represented by SEQ No. 27 (Fig. 34), a NYA-2027 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence represented by SEQ No. 28 (Fig. 35), a NYA-1143 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence represented by SEQ No. 29 (Fig. 36), a NYA-1163 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence represented by SEQ No. 26 (Fig. 33), and a sequence NYA-2023 medium-chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence represented by number 27 (Fig. 34), NYA-2027 medium-chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence represented by number 28 (Fig. 35), NYA-2035 medium-chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence represented by number 36 (Fig. 43),NYA-2044 heavy chain variable region consisting of amino acid numbers 21–140 of the amino acid sequence represented by sequence number 47 (Fig. 54), NYA-2045 heavy chain variable region consisting of amino acid numbers 21–140 of the amino acid sequence represented by sequence number 48 (Fig. 55), NYA-2047 heavy chain variable region consisting of amino acid numbers 21–140 of the amino acid sequence represented by sequence number 50 (Fig. 57), NYA-2048 heavy chain variable region consisting of amino acid numbers 21–140 of the amino acid sequence represented by sequence number 51 (Fig. 58), NYA-2060 heavy chain variable region consisting of amino acid numbers 21–140 of the amino acid sequence represented by sequence number 52 (Fig. 59), and the amino acid number of the amino acid sequence represented by sequence number 53 (Fig. 60). Examples include a combination of CDRH1 to CDRH3 included in the NYA-2061 medium-chain variable region consisting of amino acid numbers 21 to 140, or the NYA-2143 medium-chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence represented by sequence number 30 (Fig. 37).

[0311] In addition, a combination of CDRH1 to CDRH3 included in the medium chain variable region of NYA-3061 consisting of amino acid numbers 21 to 140 of the amino acid sequence represented by sequence number 156 (Fig. 152) can be cited.

[0312] CDRL1 to CDRL3 included in the light chain of the anti-HLA / NY-ESO antibody of the present invention or the antigen-binding fragment thereof may preferably be a combination of CDRL1, which is composed of the amino acid sequence represented by SEQ ID NO. 57 (Fig. 61), or the amino acid sequence represented by SEQ ID NO. 57 (Fig. 61), in which the 7th amino acid is W (Trp) or the 8th amino acid is K (Lys); CDRL2, which is composed of the amino acid sequence represented by SEQ ID NO. 58 (Fig. 61); and CDRL3, which is composed of the amino acid sequence represented by SEQ ID NO. 59 (Fig. 61), or the amino acid sequence represented by SEQ ID NO. 59 (Fig. 61), in which the 2nd amino acid is A (Ala) or S (Ser).

[0313] More preferably, a NYA-0001 light chain variable region consisting of the amino acid sequence represented by SEQ ID NO. 8 (Fig. 15), a NYA-0082 light chain variable region consisting of the amino acid sequence represented by SEQ ID NO. 20 (Fig. 27), a NYA-1143 light chain variable region consisting of amino acid numbers 156 to 266 of the amino acid sequence represented by SEQ ID NO. 29 (Fig. 36), a NYA-1163 light chain variable region consisting of amino acid numbers 156 to 266 of the amino acid sequence represented by SEQ ID NO. 26 (Fig. 33), a NYA-2023 light chain variable region consisting of amino acid numbers 156 to 266 of the amino acid sequence represented by SEQ ID NO. 27 (Fig. 34), a NYA-2027 light chain variable region consisting of amino acid numbers 156 to 266 of the amino acid sequence represented by SEQ ID NO. 28 (Fig. 35), and SEQ ID NO. NYA-2035 light chain variable region consisting of amino acid numbers 156–266 of the amino acid sequence represented by 36 (Fig. 43), NYA-2044 light chain variable region consisting of amino acid numbers 156–266 of the amino acid sequence represented by sequence number 47 (Fig. 54), NYA-2045 light chain variable region consisting of amino acid numbers 156–266 of the amino acid sequence represented by sequence number 48 (Fig. 55), NYA-2047 light chain variable region consisting of amino acid numbers 156–266 of the amino acid sequence represented by sequence number 50 (Fig. 57), NYA-2048 light chain variable region consisting of amino acid numbers 156–266 of the amino acid sequence represented by sequence number 51 (Fig. 58), and amino acid number 156 of the amino acid sequence represented by sequence number 52 (Fig. 59). NYA-2060 light chain variable region consisting of ~266, NYA-2061 light chain variable region consisting of amino acid numbers 156 ~266 of the amino acid sequence represented by sequence number 53 (Fig. 60),Alternatively, a combination of CDRL1 to CDRL3 included in the NYA-2143 light chain variable region consisting of amino acid numbers 156 to 266 of the amino acid sequence represented by sequence number 30 (Fig. 37) may be cited.

[0314] In addition, a combination of CDRL1 to CDRL3 included in the NYA-3061 light chain variable region consisting of amino acid numbers 161 to 271 of the amino acid sequence represented by sequence number 156 (Fig. 152)) can be cited.

[0315] The CDRH1 to CDRH3 included in the heavy chain and CDRL1 to CDRL3 included in the light chain of the anti-HLA / NY-ESO antibody or its antigen-binding fragment of the present invention are preferably CDRH1 composed of the amino acid sequence represented by SEQ ID NO. 54 (Fig. 61), CDRH2 composed of the amino acid sequence represented by SEQ ID NO. 55 (Fig. 61), heavy chain CDRH3 composed of the amino acid sequence represented by SEQ ID NO. 56 (Fig. 61), or an amino acid sequence in which the 6th amino acid in the amino acid sequence represented by SEQ ID NO. 56 (Fig. 61) is N (Asn), CDRL1 composed of the amino acid sequence represented by SEQ ID NO. 57 (Fig. 61), or an amino acid sequence in which the 7th amino acid in the amino acid sequence represented by SEQ ID NO. 57 (Fig. 61) is N (Asn) and / or the 8th amino acid is K (Lys), and CDRL1 composed of the amino acid sequence represented by SEQ ID NO. 58 (Fig. 61). Examples include a combination of CDRL2 and, the amino acid sequence represented by SEQ ID NO. 59 (Fig. 61), or CDRL3, which is an amino acid sequence in which the second amino acid in the amino acid sequence represented by SEQ ID NO. 59 is A (Ala) or S (Ser). More preferably, the NYA-0001 heavy chain variable region and light chain variable region formed by the amino acid sequences represented by SEQ ID NOs 6 and 8, respectively; the NYA-1143 heavy chain variable region and light chain variable region formed by amino acid numbers 21 to 140 and 156 to 266 of the amino acid sequence represented by SEQ ID NO. 29 (Fig. 36); and the NYA-1163 heavy chain variable region and light chain variable region formed by amino acid numbers 21 to 140 and 156 to 266 of the amino acid sequence represented by SEQ ID NO. 26 (Fig. 33).NYA-2023 heavy chain variable region and light chain variable region consisting of amino acid numbers 21–140 and 156–266 of the amino acid sequence represented by SEQ No. 27 (Fig. 34); NYA-2027 heavy chain variable region and light chain variable region consisting of amino acid numbers 21–140 and 156–266 of the amino acid sequence represented by SEQ No. 28 (Fig. 35); NYA-2035 heavy chain variable region and light chain variable region consisting of amino acid numbers 21–140 and 156–266 of the amino acid sequence represented by SEQ No. 36 (Fig. 43); NYA-2044 heavy chain variable region and light chain variable region consisting of amino acid numbers 21–140 and 156–266 of the amino acid sequence represented by SEQ No. 47 (Fig. 54); SEQ No. NYA-2045 heavy chain variable region and light chain variable region consisting of amino acid numbers 21–140 and 156–266 of the amino acid sequence represented by 48 (Fig. 55); NYA-2047 heavy chain variable region and light chain variable region consisting of amino acid numbers 21–140 and 156–266 of the amino acid sequence represented by sequence number 50 (Fig. 57); NYA-2048 heavy chain variable region and light chain variable region consisting of amino acid numbers 21–140 and 156–266 of the amino acid sequence represented by sequence number 51 (Fig. 58); NYA-2060 heavy chain variable region and light chain variable region consisting of amino acid numbers 21–140 and 156–266 of the amino acid sequence represented by sequence number 52 (Fig. 59); and sequence number NYA-2061 medium-chain variable region and light-chain variable region consisting of amino acid numbers 21–140 and 156–266 of the amino acid sequence shown in 53 (Fig. 60), or,Examples include combinations of CDRH1–CDRH3 and CDRL1–CDRL3, respectively, contained in the NYA-2143 medium chain variable region and light chain variable region, which consist of amino acid numbers 21–140 and 156–266 of the amino acid sequence represented by sequence number 30 (Fig. 37).

[0316] In addition, combinations of CDRH1 to CDRH3 and CDRL1 to CDRL3 included in the NYA-3061 medium 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 sequence number 156 (Fig. 152).

[0317] Preferably, the heavy chain variable region of the anti-HLA / NY-ESO antibody of the present invention or its antigen-binding fragment may include the heavy chain CDR or a combination thereof, and more preferably, 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, NYA-2027 heavy chain variable region, 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, and, The NYA-3061 medium chain variable region can be exemplified. The amino acid sequence of each medium chain variable region is as described above.

[0318] Preferably, the light chain variable region of the anti-HLA / NY-ESO antibody of the present invention or its antigen-binding fragment may include the light chain CDR or a combination thereof, and more preferably, 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, NYA-2027 light chain variable region, NYA-2035 light chain variable region, NYA-2044 light chain variable region, NYA-2045 light chain variable region, NYA-2047 light chain variable region, NYA-2048 light chain variable region, NYA-2060 light chain variable region, NYA-2061 light chain variable region, NYA-2143 light chain variable region, and NYA-3061 light chain variable region may be exemplified. The amino acid sequence of each diagonal chain variable region is as above.

[0319] Preferably, the heavy chain variable region and light chain variable region of the anti-HLA / NY-ESO antibody of the present invention or its antigen-binding fragment may include the CDR of said heavy chain and light chain or a combination thereof, and more preferably, 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, NYA-2035 heavy chain variable region and light chain variable region, NYA-2044 heavy chain variable region and light chain variable region, NYA-2045 heavy chain variable region and light chain variable region, NYA-2047 heavy chain variable region and light chain variable region. Examples include NYA-2048 medium chain variable region and light chain variable region, NYA-2060 medium chain variable region and light chain variable region, NYA-2061 medium chain variable region and light chain variable region, and, combinations of NYA-2143 medium chain variable region and light chain variable region, and, combinations of NYA-3061 medium chain variable region and light chain variable region.

[0320] As a preferred example, the heavy chain of the anti-HLA / NY-ESO antibody of the present invention or its antigen-binding fragment may include the preferred or more preferred heavy chain variable region, and more preferably, 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, and NYA-3061 heavy chain may be exemplified.

[0321] As a preferred example, the oblique chain of the anti-HLA / NY-ESO antibody of the present invention or its antigen-binding fragment may include the preferred or more preferred oblique chain variable region, and more preferably, NYA-0001 oblique chain, NYA-0082 oblique chain, NYA-1143 oblique chain, NYA-1163 oblique chain, NYA-2023 oblique chain, NYA-2027 oblique chain, NYA-2035 oblique chain, NYA-2044 oblique chain, NYA-2045 oblique chain, NYA-2047 oblique chain, NYA-2048 oblique chain, NYA-2060 oblique chain, NYA-2061 oblique chain, NYA-2143 oblique chain, and NYA-3061 oblique chain may be exemplified.

[0322] As preferred examples, the heavy chain and light chain of the anti-HLA / NY-ESO antibody of the present invention or its antigen-binding fragment may each include the preferred or more preferred heavy chain variable region and light chain variable region, and more preferably, combinations of the heavy chain and light chain 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, and NYA-3061 may be exemplified.

[0323] An antigen-binding fragment of an antibody refers to a fragment or a modification thereof that maintains at least antigen-binding ability among the functions of the antibody. Generally, such functions of the antibody include antigen-binding activity, activity that regulates antigen activity, antibody-dependent cell damage activity, and complement-dependent cell damage activity. Functions when the antibody of the present invention and a multispecific molecule containing the antibody of the present invention are made include, for example, T cell redirection, T cell activation, and cell damage activity of cancer cells by activating T cells.

[0324] The antigen-binding fragment of an antibody is not particularly limited as long as it is a fragment of the antibody that maintains at least antigen-binding ability among the activities of the antibody. Examples include Fab, Fab', F(ab')2, Fv, a single-chain Fv (scFv) formed by connecting a heavy chain and a light chain Fv with a suitable linker, and a single-domain antibody (sdAb), but are not limited thereto. Molecules containing a portion other than the antigen-binding fragment of the antibody of the present invention, such as scFv having a linker portion, are also included in the meaning of the antigen-binding fragment of the antibody of the present invention.

[0325] A molecule formed by deleting one or more amino acids at the amino-terminal and / or carboxyl-terminal ends of an antibody protein, and which retains at least some of the functions of the antibody, is also included in the meaning of an antigen-binding fragment of an antibody. A modification of such an antigen-binding fragment of an antibody is also included in the antibody of the present invention, the antigen-binding fragment thereof, or the modification thereof (described below).

[0326] One embodiment of the antibody or its antigen-binding fragment according to the present invention is a scFv. A scFv is obtained by connecting a medium-chain variable region and a light-chain variable region of an antibody with a polypeptide linker (Pluckthun A. The Pharmacology of Monoclonal Antibodies 113, Rosenburg and Moore eds., Springer Verlag, New York, 269-315 (1994), Nature Biotechnology (2005), 23, 1126-1136). In addition, a tandem scFv produced by combining two scFvs with a polypeptide linker can also be used as a bispecific molecule. Furthermore, a triabodiment composed of three or more scFvs can also be used as a multispecific molecule.

[0327] As scFvs specific to HLA / NY-ESO (also called "anti-HLA / NY-ESO scFvs"), preferably include CDRH1 to CDRH3 and CDRL1 to CDRL3, more preferably include the heavy chain variable region and light chain variable region, and even more preferably NYA-0001 (amino acid numbers 21 to 266 of the amino acid sequence represented by SEQ ID NO. 70 (Fig. 71)), NYA-0082 (including the amino acid sequence represented by SEQ ID NO. 18 (Fig. 25) and the amino acid sequence represented by SEQ ID NO. 20 (Fig. 27)), NYA-1143 (amino acid numbers 21 to 266 of the amino acid sequence represented by SEQ ID NO. 29 (Fig. 36)), NYA-1163 (amino acid numbers 21 to 266 of the amino acid sequence represented by SEQ ID NO. 26 (Fig. 33)), NYA-2023 (amino acid numbers 21–266 of the amino acid sequence represented by sequence number 27 (Fig. 34)), NYA-2027 (amino acid numbers 21–266 of the amino acid sequence represented by sequence number 28 (Fig. 35)), NYA-2035 (amino acid numbers 21–266 of the amino acid sequence represented by sequence number 36 (Fig. 43)), NYA-2044 (amino acid numbers 21–266 of the amino acid sequence represented by sequence number 47 (Fig. 54)), NYA-2045 (amino acid numbers 21–266 of the amino acid sequence represented by sequence number 48 (Fig. 55)), NYA-2047 (amino acid numbers 21–266 of the amino acid sequence represented by sequence number 50 (Fig. 57)), NYA-2048 (sequence number amino acid numbers 21–266 of the amino acid sequence represented by 51 (Fig. 58), NYA-2060 (amino acid numbers 21–266 of the amino acid sequence represented by sequence number 52 (Fig. 59)), NYA-2061 (amino acid numbers 21–266 of the amino acid sequence represented by sequence number 53 (Fig. 60)), and,NYA-2143 (amino acid numbers 21 to 266 of the amino acid sequence represented by sequence number 30 (Fig. 37)) can be exemplified. Additionally, NYA-3061 (amino acid numbers 21 to 271 of the amino acid sequence represented by sequence number 156 (Fig. 152)) can be exemplified.

[0328] A preferred embodiment of anti-HLA / NY-ESO scFv includes a FLAG-His tag fused to the carboxyl terminal side (also simply called a "tag adduct"). Preferred tag adducts include NYA-0001 tag adduct (amino acid numbers 20–292 of sequence no. 70 (Fig. 71)), NYA-1143 tag adduct (amino acid numbers 20–292 of sequence no. 29 (Fig. 36)), NYA-1163 tag adduct (amino acid numbers 20–292 of sequence no. 26 (Fig. 33)), NYA-2023 tag adduct (amino acid numbers 20–292 of sequence no. 27 (Fig. 34)), NYA-2027 tag adduct (amino acid numbers 20–292 of sequence no. 28 (Fig. 35)), NYA-2035 tag adduct (amino acid numbers 20–292 of sequence no. 36 (Fig. 43)), and NYA-2044 tag adduct. (amino acid numbers 20–292 of sequence number 47 (Fig. 54), NYA-2045 tag adduct (amino acid numbers 20–292 of sequence number 48 (Fig. 55)), NYA-2047 tag adduct (amino acid numbers 20–292 of sequence number 50 (Fig. 57)), NYA-2048 tag adduct (amino acid numbers 20–292 of sequence number 51 (Fig. 58)), NYA-2060 tag adduct (amino acid numbers 20–292 of sequence number 52 (Fig. 59)), NYA-2061 tag adduct (amino acid numbers 20–292 of sequence number 53 (Fig. 60)), and, NYA-2143 tag adduct (amino acid of sequence number 30 (Fig. 37) Numbers 20 to 292) can be cited.

[0329] Among them, NYA-2023 and its tag adduct, NYA-2047 and its tag adduct, NYA-2048 and its tag adduct, NYA-2060 and its tag adduct, and NYA-2061 and its tag adduct are more preferable as Fc addition type bispecific molecules, having excellent biological activity, physical properties, etc.

[0330] In addition, when anti-HLA / NY-ESO scFv and its tag adduct are expressed in a host cell, a signal peptide can be added to the amino terminus. Examples of amino acid sequences of anti-HLA / NY-ESO scFv tag adducts with added signal peptides include sequence numbers 70, 29, 26 to 28, 36, 47, 48, 50 to 53, and 30 (Fig. 71, 36, 33 to 35, 43, 54, 55, 57 to 60, and 37).

[0331] scFv can be obtained by a phage display method (Nature Biotechnology (2005), 23, (9), p.1105-1116) in which a variable region of an antibody is expressed on the surface of a phage as a one-chain antibody (scFv) and a phage that binds to an antigen is selected. By analyzing the gene of the phage selected by binding to the antigen, the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined. When the DNA sequence of an scFv that binds to an antigen is determined, a human antibody can be obtained by constructing an expression vector having the sequence and introducing it into a suitable host for expression (WO92 / 01047, WO92 / 20791, WO93 / 06213, WO93 / 11236, WO93 / 19172, WO95 / 01438, WO95 / 15388, Annu. Rev. Immunol (1994) 12, p.433-455, Nature Biotechnology (2005) 23 (9), p.1105-1116).

[0332] The antibody of the present invention may be an antibody having a single medium-chain variable region and not having a light-chain sequence. Such antibodies are referred to as single-domain antibodies (sdAb) or nanobodies, and it has been reported that antigen-binding ability is maintained (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 included in the meaning of the antigen-binding fragment of the antibody in the present invention.

[0333] In addition, the present invention includes a single-chain immunoglobulin in which the entire length sequences of the heavy chain and light chain of an antibody are linked using a suitable linker (Lee, HS, et. al., Molecular Immunology (1999) 36, 61-71; Shirrmann, T. et. al., kbbs (2010), 2 (1), 1-4). By dimerizing such a single-chain immunoglobulin, it is possible to maintain a structure and activity similar to that of an antibody, which is originally a tetramer. The anti-HLA / NY-ESO antibody of the present invention may be a single-chain immunoglobulin.

[0334] In the scFv of the present invention, the medium chain variable region and the light chain variable region may be disulfide bonded.

[0335] The anti-HLA / NY-ESO antibody of the present invention may be an antibody composed of portions derived from a plurality of different antibodies when it binds to HLA / NY-ESO, for example, a case in which the heavy chain and / or light chain are exchanged between a plurality of different antibodies, a case in which the entire length of the heavy chain and / or light chain is exchanged, a case in which only the variable region or only the normal region is exchanged, or a case in which only all or part of the CDR is exchanged, etc. The heavy chain variable region and the light chain variable region of the chimeric antibody may be derived from different anti-HLA / NY-ESO antibodies of the present invention. The heavy chain CDR1 to heavy chain CDR3 and the light chain CDR1 to light chain CDR3 among the variable regions of the heavy chain and light chain of the humanized antibody may be derived from two or more types of anti-HLA / NY-ESO antibodies of the present invention. The heavy chain CDR1 to heavy chain CDR3 and the light chain CDR1 to light chain CDR3 among the variable regions of the heavy chain and light chain of the human antibody may be a combination of CDRs having two or more of the anti-HLA / NY-ESO antibodies of the present invention.

[0336] The anti-HLA / NY-ESO antibody of the present invention includes an amino acid sequence encoded by a nucleotide sequence included in a polynucleotide that hybridizes under stringent conditions with a complementary chain of a polynucleotide containing a nucleotide sequence encoding an amino acid sequence included in the anti-HLA / NY-ESO antibody of the present invention, and also includes an antibody that binds to HLA / NY-ESO.

[0337] An amino acid sequence included in the medium chain variable region of the anti-HLA / NY-ESO antibody of the present invention or the antigen-binding fragment thereof (preferably the amino acid sequence of amino acid numbers 21 to 140 of the amino acid sequence represented by SEQ ID NO. 27, the amino acid sequence represented by SEQ ID NO. 38, the amino acid sequence represented by SEQ ID NO. 39, or, the amino acid sequence of amino acid numbers 21 to 140 of the amino acid sequence represented by SEQ ID NO. 160, SEQ ID NO. 197, or SEQ ID NO. 198), and / or an amino acid sequence included in the light chain variable region (preferably the amino acid sequence of amino acid numbers 156 to 266 of the amino acid sequence represented by SEQ ID NO. 27, the amino acid sequence of amino acid numbers 156 to 266 of the amino acid sequence represented by SEQ ID NO. 52, the amino acid sequence represented by SEQ ID NO. 40, the amino acid sequence of amino acid numbers 161 to 271 of the amino acid sequence represented by SEQ ID NO. 160, or, SEQ ID NO. 197 or It may also be an antibody or its antigen-binding fragment that binds to HLA / NY-ESO and contains an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to the amino acid sequence represented by sequence number 198 (amino acid sequence number 156 to 266).

[0338] When the position and length of the light chain variable region are determined using a definition different from IMGT (e.g., Kabat, Chothia, AbM, contact, etc.) compared to the case where they are determined by the definition of IMGT, there may be additionally one or more amino acids, for example, arginine or glycine, at the carboxyl terminus of the amino acid sequence of the light chain variable region determined by the definition of IMGT. An antibody having such a light chain variable region or a binding fragment thereof is also included in the antibody or binding fragment thereof of the present invention.

[0339] The antibody of the present invention may be one in which a mutation is introduced into the binding fragment of the anti-HLA / NY-ESO antibody of the present invention to optimize the binding ability to HLA / NY-ESO, particularly human and / or crab-eating monkey HLA / NY-ESO. Specific methods for introducing the mutation include random mutagenesis using the erra-front PCR method, introduction of site-specific amino acid mutations using an NNK library, introduction of site-specific mutations using structural information, and combinations thereof.

[0340] 3-2. Variants of Anti-HLA / NY-ESO Antibodies (Variant Antibodies)

[0341] The variant antibodies of the anti-HLA / NY-ESO antibody of the present invention may preferably have reduced susceptibility to protein degradation or oxidation, maintained or improved biological activity or function, inhibited reduction or change, improved or regulated antigen binding ability, or conferred physicochemical or functional properties. It is known that the function or activity of a protein changes when specific amino acid side chains on its surface are altered, and such examples include deamidation of the asparagine side chain and isomerization of the aspartic acid side chain. Substitutions made with a different amino acid to prevent such changes in the amino acid side chain are also included within the scope of the variant antibodies of the present invention.

[0342] As an example of a variant antibody of the present invention, an antibody having an amino acid sequence formed by conservative amino acid substitution in the amino acid sequence of the antibody may be cited. Conservative amino acid substitution is a substitution that occurs within an amino acid group associated with an amino acid side chain.

[0343] 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 non-polar family = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Other preferred amino acid groups are as follows: aliphatic hydroxyl group = serine and threonine; amide-containing group = asparagine and glutamine; aliphatic group = alanine, valine, leucine and isoleucine; and aromatic group = phenylalanine, tryptophan and tyrosine. In such variant antibodies, amino acid substitution is preferably performed within a range that does not reduce the antigen-binding activity of the original antibody.

[0344] Antibodies of the present invention such as NYA-2023 having an amino acid sequence in which a conservative amino acid substitution and / or other variation is performed, and also a variant antibody that binds to HLA / NY-ESO, the antigen-binding fragments thereof, molecules containing the same, etc.; chimeric antibodies, humanized antibodies, human antibodies, the antigen-binding fragments thereof, molecules containing the same, which have an amino acid sequence in which a conservative amino acid substitution and / or other variation is performed in any of CDRH1 to CDRH3 and CDRL1 to CDRL3 derived from antibodies of the present invention including NYA-2023, etc., and also bind to HLA / NY-ESO, are also included in the anti-HLA / NY-ESO antibody of the present invention, the antigen-binding fragment thereof, the variant thereof (variant antibody or the antigen-binding fragment thereof), or the molecules of the present invention.

[0345] 3-3. Binding fragment of anti-HLA / NY-ESO antibody

[0346] As one aspect of the present invention, an antigen-binding fragment of the anti-HLA / NY-ESO antibody of the present invention (hereinafter simply referred to as "binding fragment") is provided. The binding fragment of the anti-HLA / NY-ESO antibody of the present invention includes a binding fragment of a chimeric antibody, a humanized antibody, or a human antibody. A binding fragment of an antibody means a fragment or a modification thereof that maintains at least antigen-binding ability among the functions of the antibody. Generally, the functions of such an antibody include antigen-binding activity, activity that regulates the activity of an antigen (e.g., agonist activity), activity that internalizes an antigen into a cell, and activity that inhibits or promotes the interaction of a substance interacting with an antigen.

[0347] The binding fragment of an antibody is not particularly limited as long as it is a fragment of the antibody that maintains at least antigen-binding activity among the activities of the antibody. Examples of such binding fragments of an antibody include Fab, Fab', F(ab')2, a single-chain Fab (scFab) in which the carboxyl terminus of the Fab light chain and the amino terminus of the Fab heavy chain are connected by a suitable linker, Fv, a single-chain Fv (scFv) in which the heavy chain and light chain Fv are connected by a suitable linker, a single-domain antibody (sdAb) having a single heavy chain variable region and not a light chain sequence, or a nanobody. (Muyldemans S. et. al., Protein Eng., (1994) 7 (9), 1129-35; Hamers-Casterman C. et. al., Nature (1993) 363 (6428), 446-448), etc., but are not limited to these. Molecules containing a portion other than the binding fragment of the antibody of the present invention, such as scFab and scFv having a linker portion, are also included in the meaning of the binding fragment of the antibody of the present invention.

[0348] 3-4. Modifiers or complexes of anti-HLA / NY-ESO antibodies or their binding fragments

[0349] The present invention provides a modification of an antibody or its binding fragment. A modification of an antibody or its binding fragment according to the present invention means that the antibody or its binding fragment according to the present invention is formed by performing chemical or biological modification. Chemical modifications include the binding of a chemical portion to an amino acid backbone, chemical modifications of N-bond or O-bond carbohydrate chains, etc. Biological modifications include those that have undergone post-translational modification (e.g., addition of a sugar chain to an N-bond or O-bond, processing of an amino-terminal region or a carboxyl-terminal region, deamidation, isomerization of aspartic acid, oxidation of methionine), and those in which a methionine residue is added to the amino-terminus by expressing using a prokaryotic host cell. Furthermore, labels that enable the detection or isolation of the antibody or antigen according to the present invention, such as enzyme labels, fluorescent labels, and affinity labels, are also included in the meaning of such modifications. The present invention’s antibody or its binding fragment is useful for improving the stability and retention of the original antibody or its binding fragment in the blood, reducing antigenicity, and detecting or isolating such antibody or antigen.

[0350] Examples of chemical parts included in the chemical modification include water-soluble polymers such as polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymer, carboxymethylcellulose, dextran, and polyvinyl alcohol.

[0351] Examples of biological modified plants include those modified by enzyme treatment or cell treatment, fusion products to which other peptides such as tags are added by genetic recombination, and those prepared using cells expressing endogenous or exogenous sugar chain modifying enzymes as hosts.

[0352] Such modifications may be performed at any location or desired location on the antibody or its binding fragment, and the same or two or more different modifications may be performed at one or more locations.

[0353] However, deletion of these heavy chain sequences, or modification of heavy or light chain sequences, does not significantly affect the antigen binding ability and effector function of the antibody (such as complement activation or antibody-dependent cell damage).

[0354] Accordingly, the present invention also includes antibodies that have undergone such deletion or modification. Examples include a deletion form in which one or two amino acids are deleted at the medium chain carboxyl terminus (Journal of Chromatography A; 705; 129-134 (1995)), a deletion form in which two amino acid residues of glycine and lysine at the medium chain carboxyl terminus are deleted and a proline residue newly located at the carboxyl terminus is amidated (Analytical Biochemistry, 360: 75-83 (2007)), and an antibody in which a glutamine or glutamic acid residue at the amino terminus of the medium or light chain of the antibody is modified by pyroglutamylation (International Patent Publication WO2013 / 147153), etc. (these are collectively referred to as “deletion forms”). However, as long as antigen binding ability and effector function are maintained, the deletions of the carboxyl ends of the heavy chains and light chains of the antibody related to the present invention are not limited to the types described above. When the antibody related to the present invention comprises two or more chains (e.g., heavy chains), said 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 deletions described above, or a combination of any two types. Although the ratio of the quantity or number of molecules of each deletion may be influenced by the type of mammalian culture cell and culture conditions producing the antibody related to the present invention, the main component of the antibody related to the present invention may be a case in which one amino acid residue of the carboxyl end is deleted from both of the two heavy chains.

[0355] In addition, if the desired antigen-binding activity is maintained even when one to several amino acids derived from an expression vector and / or signal sequence are added to the amino terminus and / or carboxyl terminus of the antibody or its antigen-binding fragment of the present invention (including the molecule, multispecific molecule, bispecific molecule, etc. of the present invention) (and some or all thereof are modified as described above), the antibody or its antigen-binding fragment of the present invention is included in the scope of the modification of the antibody or its antigen-binding fragment of the present invention, and a molecule containing such a modification of the antibody or its antigen-binding fragment is also included in the scope of the molecule of the present invention.

[0356] In the present invention, "antibody or its binding fragment" includes "modifiers of the antibody or its antigen-binding fragment" in its meaning. Furthermore, "antibody or its antigen-binding fragment" included in the molecules, multispecific molecules, bispecific molecules, etc., of the present invention includes such "modifiers of the antibody or its antigen-binding fragment" in its meaning.

[0357] In addition, it is possible to enhance antibody-dependent cell injury activity by controlling the modification of the sugar chain bound to the antibody of the present invention (glycosylation, defucosation, etc.). Techniques for controlling the modification of the sugar chain of the antibody are known, such as International Patent Publications WO99 / 54342, WO00 / 61739, WO02 / 31140, but are not limited thereto.

[0358] The present invention also includes a complex (Immunoconjugate) in which the antibody described above is linked to another molecule by a linker. An example of an antibody-drug complex in which the antibody is bound to a radioactive substance or a compound (drug) having pharmacological action is an Antibody-Drug Conjugate (ADC) ((Methods Mol Biol. (2013) 1045 : 1-27 ; Nature Biotechnology (2005) 23, p.1137-1146).

[0359] In addition, the present invention also includes a complex in which these antibodies are linked to other functional polypeptides. An example of such an antibody-peptide complex is a complex in which the antibody is combined with an albumin-binding polypeptide (Protein Eng Des Sel. (2012) (2) : 81-8).

[0360] The antibody modification, the glycosylated antibody modification, and the complex described above are included in the antibody of the present invention, and the binding fragment of the antibody modification, the glycosylated antibody modification, and the complex described above is included in the binding fragment of the antibody of the present invention.

[0361] 4. Method of Antibody Production

[0362] The antibody of the present invention can be produced in cells as a recombinant antibody by, for example, inserting DNA encoding a medium-chain variable region or DNA encoding a light-chain variable region into an expression vector, transforming a host cell for expression into the vector, and culturing the host cell.

[0363] DNA encoding antibodies is obtained by linking DNA encoding a heavy chain variable region and DNA encoding a heavy chain normal region to obtain DNA encoding a heavy chain, and also by linking DNA encoding a light chain variable region and DNA encoding a light chain normal region to obtain DNA encoding a light chain.

[0364] 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. At this time, the DNA encoding the heavy chain and the DNA encoding the light chain may be introduced into the same expression vector and the host cell may be transformed using the vector, or the DNA encoding the heavy chain and the DNA encoding the light chain may be inserted into different vectors and the host cell may be transformed using two vectors. At this time, the DNA encoding the heavy chain variable region and the light chain variable region may be introduced into a vector in which the DNA encoding the heavy chain normal region and the DNA encoding the light chain normal region have been introduced beforehand. In addition, the vector may contain DNA encoding a signal peptide that promotes the secretion of antibodies from the host cell, and in this case, the DNA encoding the signal peptide and the DNA encoding the antibody are linked in-frame. By removing the signal peptide after the antibody is produced, the antibody can be obtained as a mature protein.

[0365] At this time, DNA encoding a medium-strand variable region, DNA encoding a light-strand variable region, DNA formed by linking the DNA encoding the medium-strand variable region and the DNA encoding the medium-strand normal region, and DNA formed by linking the DNA encoding the light-strand variable region and the DNA encoding the light-strand normal region may be functionally linked with elements such as promoters, enhancers, and polyadenylation signals. Here, functional linkage means linking so that the elements perform their functions.

[0366] The expression vector is not particularly limited as long as it is capable of replication among hosts such as animal cells, bacteria, and yeast; examples include known plasmids and phages. Examples of vectors used to construct the expression vector include pcDNA (trademark) (ThermoFissher SCIENTIFIC), Flexi (registered trademark) vector (Promega), pUC19, pUEX2 (manufactured by Armersham), pGEX-4T, pKK233-2 (manufactured by Pharmacia), and pMAM-neo (manufactured by CloneTech). As for the host cell, prokaryotic cells such as E. coli and Bacillus subtilis, as well as eukaryotic cells such as yeast and animal cells, may be used, but eukaryotic cells are preferred. For example, as animal cells, human fetal kidney cell line HEK293 cells or Chinese hamster ovary (CHO) cells may be used. The expression vector is introduced into the host cell by a known method, and the host cell is transformed. Examples include electroporation, calcium phosphate precipitation, and DEAE-dextran transfection. The generated antibodies can be purified using separation and purification methods commonly used for proteins. For instance, affinity chromatography, other chromatographs, filtration, ultrafiltration, salting out, dialysis, etc., can be appropriately selected and combined.

[0367] 5. Molecules that bind to HLA / NY-ESO

[0368] The molecule of the present invention comprises the anti-HLA / NY-ESO antibody of the present invention or its antigen-binding fragment. The molecule of the present invention is preferably a multispecific molecule having two or more antigen-binding sites. That is, it is a molecule capable of binding to two or more different epitopes on one molecule or to two or more different epitopes on two or more molecules, and surrounds a plurality of different antigen-binding fragments. Such a multispecific molecule includes, but is not limited to, IgG-type multispecific molecules, multispecific molecules having two or more types of variable regions, for example, tandem scFv (taFv), antibody fragments such as single-chain diabodies, diabodies and tribodies, and antibody fragments connected by covalent or non-covalent bonds. The multispecific molecule may include Fc.

[0369] The multispecific molecule of the present invention may comprise, in addition to the anti-HLA / NY-ESO antibody of the present invention or its antigen-binding fragment, one or more additional antibodies or antigen-binding fragments of the antibodies. Examples of additional antibody antigen-binding fragments include Fab, F(ab)', Fv, scFv, and sdAb.

[0370] A preferred multispecific molecule of the present invention further comprises an anti-CD3 antibody or its antigen-binding fragment and specifically binds to CD3 as well.

[0371] The anti-CD3 antibody or its antigen-binding fragment included in the multispecific molecule of the present invention is not particularly limited to being an antibody or its binding fragment that binds to human CD3, but preferably also binds to CD3 of non-human primates such as crab-eating monkeys. More preferred examples of the anti-CD3 antibody or its antigen-binding fragment may include an antibody or its antigen-binding fragment comprising a medium-chain variable region CDRH1 formed by the amino acid sequence represented by SEQ ID NO. 141, a medium-chain variable region CDRH2 formed by the amino acid sequence represented by SEQ ID NO. 142, a medium-chain variable region CDRH3 formed by the amino acid sequence represented by SEQ ID NO. 143, a light-chain variable region CDRL1 formed by the amino acid sequence represented by SEQ ID NO. 144, a light-chain variable region CDRL2 formed by the amino acid sequence represented by SEQ ID NO. 145, and a light-chain variable region CDRL3 formed by the amino acid sequence represented by SEQ ID NO. 146 (all above, FIG. 142).

[0372] A more preferred antibody or antigen-binding fragment comprising the CDRH1 to CDRH3 and CDRL1 to CDRL3 comprises: a C3E-7034 medium-chain variable region consisting of amino acid sequences 2 to 119 of the amino acid sequence represented by SEQ ID NO. 136 (Fig. 137); a C3E-7036 medium-chain variable region consisting of amino acid sequences 2 to 119 of the amino acid sequence represented by SEQ ID NO. 137 (Fig. 138); a C3E-7085 medium-chain variable region consisting of amino acid sequences 2 to 119 of the amino acid sequence represented by SEQ ID NO. 138 (Fig. 139); a C3E-7088 medium-chain variable region consisting of amino acid sequences 2 to 119 of the amino acid sequence represented by SEQ ID NO. 139 (Fig. 140); and an amino acid sequence represented by SEQ ID NO. 140 (Fig. 141). An antibody or its antigen-binding fragment may be exemplified by comprising a C3E-7093 medium chain variable region consisting of amino acid sequences of amino acid numbers 2 to 119, a C3E-7096 medium chain variable region consisting of amino acid sequences of amino acid numbers 272 to 389 of the amino acid sequence represented by sequence number 155 (Fig. 151), a C3E-7096 medium chain variable region consisting of amino acid sequences of amino acid numbers 277 to 394 of the amino acid sequence represented by sequence number 156 (Fig. 152), and a C3E-7097 medium chain variable region consisting of amino acid sequences of amino acid numbers 277 to 394 of the amino acid sequence represented by sequence number 157 (Fig. 153).

[0373] In addition, a more preferred antibody or antigen-binding fragment comprising the CDRH1 to CDRH3 and CDRL1 to CDRL3 comprises: a C3E-7034 light chain variable region consisting of amino acid sequences of amino acid numbers 135 to 243 of the amino acid sequence represented by SEQ ID NO. 136 (Fig. 137); a C3E-7036 light chain variable region consisting of amino acid sequences of amino acid numbers 135 to 241 of the amino acid sequence represented by SEQ ID NO. 137 (Fig. 138); a C3E-7085 light chain variable region consisting of amino acid sequences of amino acid numbers 135 to 241 of the amino acid sequence represented by SEQ ID NO. 138 (Fig. 139); a C3E-7088 light chain variable region consisting of amino acid sequences of amino acid numbers 135 to 243 of the amino acid sequence represented by SEQ ID NO. 139 (Fig. 140); and SEQ ID NO. 140 (Fig. 141). An antibody or its antigen-binding fragment may be exemplified by comprising a C3E-7093 light chain variable region consisting of amino acid sequences of amino acid numbers 135 to 243 of the amino acid sequence represented by sequence number 155 (Fig. 151), a C3E-7096 light chain variable region consisting of amino acid sequences of amino acid numbers 405 to 511 of the amino acid sequence represented by sequence number 155 (Fig. 151), a C3E-7096 light chain variable region consisting of amino acid sequences of amino acid numbers 410 to 516 of the amino acid sequence represented by sequence number 156 (Fig. 152), and a C3E-7097 light chain variable region consisting of amino acid sequences of amino acid numbers 410 to 516 of the amino acid sequence represented by sequence number 157 (Fig. 153).

[0374] In addition, a more preferred antibody or antigen-binding fragment comprising the CDRH1 to CDRH3 and CDRL1 to CDRL3 includes a combination of a C3E-7034 heavy chain variable region and a light chain variable region consisting of amino acids 2 to 119 and 135 to 243 represented by SEQ ID NO. 136 (FIG. 137); a combination of a C3E-7036 heavy chain variable region and a light chain variable region consisting of amino acids 2 to 119 and 135 to 241 represented by SEQ ID NO. 137 (FIG. 138); a combination of a C3E-7078 heavy chain variable region and a light chain variable region consisting of amino acids 2 to 119 and 135 to 243 represented by SEQ ID NO. 147 (FIG. 143); and amino acids 2 to 119 and 135 to 243 represented by SEQ ID NO. 138 (FIG. 139). A combination of a C3E-7085 heavy chain variable region and a light chain variable region consisting of 241; a combination of a C3E-7088 heavy chain variable region and a light chain variable region consisting of amino acids 2–119 and 135–243 represented by sequence number 139 (Fig. 140); a combination of a C3E-7093 heavy chain variable region and a light chain variable region consisting of amino acids 2–119 and 135–243 represented by sequence number 140 (Fig. 141); a combination of a C3E-7096 heavy chain variable region and a light chain variable region consisting of amino acids 272–389 and 405–511 represented by sequence number 155 (Fig. 151); and amino acids 277–394 and 410– An antibody or its antigen-binding fragment may be exemplified by comprising a combination of a C3E-7096 medium chain variable region and a light chain variable region consisting of 516, and a combination of a C3E-7097 medium chain variable region and a light chain variable region consisting of amino acid numbers 277 to 394 and 410 to 516 represented by sequence number 157 (Fig. 153).

[0375] In addition, a more preferred antibody or antigen-binding fragment comprising the CDRH1 to CDRH3 and CDRL1 to CDRL3 includes C3E-7034 scFv consisting of the amino acid sequence of amino acid numbers 2 to 243 of the amino acid sequence represented by SEQ ID NO. 136 (Fig. 137), C3E-7036 scFv consisting of the amino acid sequence of amino acid numbers 2 to 241 of the amino acid sequence represented by SEQ ID NO. 137 (Fig. 138), C3E-7078 scFv consisting of the amino acid sequence of amino acid numbers 2 to 243 of the amino acid sequence represented by SEQ ID NO. 147 (Fig. 143), C3E-7085 scFv consisting of the amino acid sequence of amino acid numbers 2 to 241 of the amino acid sequence represented by SEQ ID NO. 138 (Fig. 139), and amino acid numbers 2 to Examples may include C3E-7088scFv consisting of an amino acid sequence of 243, C3E-7093scFv consisting of an amino acid sequence of amino acid numbers 2 to 243 of the amino acid sequence represented by sequence number 140 (Fig. 141), and an antibody or a binding fragment of an antibody comprising any one of these scFvs. A preferred embodiment of a scFv specific to CD3 (also called an "anti-CD3 scFv") includes one in which a FLAG-His tag is added to the carboxyl terminal side (also simply called a "tag adduct"). Preferred tag adducts may be exemplified by C3E-7034 (Sequence No. 136: FIG. 137), C3E-7036 (Sequence No. 137: FIG. 138), C3E-7085 (Sequence No. 138: FIG. 139), C3E-7088 (Sequence No. 139: FIG. 140), and C3E-7093 (Sequence No. 140: FIG. 141), and more preferably C3E-7085.

[0376] As a preferred example of a multispecific molecule of the present invention, a bispecific molecule may be cited. "Bispecificity" means the ability to bind to two different epitopes of the same molecule or to different epitopes on two molecules, and includes an antibody or antigen-binding fragment having such bispecificity. The bispecific molecule of the present invention binds to HLA / NY-ESO and also binds to CD3.

[0377] Examples of the dual-specificity molecules of the present invention may include those having the following structure (format).

[0378] In dual scFv type bispecific molecules, two types of scFvs binding to different epitopes are each linked to one side of the dimeric Fc by a linker or directly without a linker. Alternatively, two types of scFvs binding to different epitopes are each linked to CH and CL by a linker, and also each linked to one side of the dimeric Fc by a linker. The bispecific molecule is a format in which Fc is hetero-associated with a mutation that forms a heterodimer downstream of the two different scFv groups. Dual scFv type bispecific molecules are called dual-type bispecific molecules, or simply dual-type (Fig. 6a(b)).

[0379] In the present invention, for example, a dual-type bispecific molecule consisting of anti-HLA-A2 / NY-ESO scFv and anti-CD3scFv may be used.

[0380] Alternatively, as the bispecific molecule of the present invention, the Fab and scFv that bind to different epitopes may be a bispecific molecule in which the Fab of the first antibody is attached to one side of the dimer Fc and the scFv of the second antibody is attached to the other side via a linker. The bispecific molecule is in a format in which Fc, having a mutation forming a heterodimer downstream of each of Fab and scFv, is hetero-associated. Such a bispecific molecule is called a hybrid bispecific molecule or hybrid type (Fig. 6a(a)). In the present invention, for example, a hybrid type consisting of anti-HLA-A2 / NY-ESO Fab and anti-CD3scFv may be used.

[0381] In addition, it may be a bispecific molecule in which the Fab of the first antibody and the scFv of the second antibody are attached to one side of the dimer Fc via a linker. In this case, Fab may be attached to Fc and scFv may be attached to the Fab, or scFv may be attached to Fc and Fab may be attached to the scFv. Preferably, Fab is attached and scFv is attached to the Fab. The binding of Fab and scFv can be achieved by attaching scFv to the variable region of Fab via a linker. The bispecific molecule is in a format in which Fc, having a mutation forming a heterodimer, is attached downstream of the linkage between scFv and Fab via a linker. Such a bispecific molecule is called a scFv-Fab-heterodimer Fc type bispecific molecule or scFv-Fab-heterodimer Fc type (Fig. 6a(c)).

[0382] In the present invention, for example, a scFv-Fab-heterodimer Fc type consisting of anti-CD3scFv and anti-HLA-A2 / NY-ESO Fab may be used.

[0383] In addition, taFv (Fig. 3(c)), having a format in which two types of scFv, a first antibody and a second antibody, are linked by a linker, may be directly attached to one of the dimeric Fc with or without a linker. Such a bispecific molecule is called a taFv-heterodimer Fc type bispecific molecule or a taFv-heterodimer Fc type (Fig. 3(d)). The bispecific molecule is a format in which Fc, having a mutation forming a heterodimer downstream of taFv, is hetero-associated. Although the linkage order of the first antibody and the second antibody in taFv is not limited, when the linkage order of the first antibody and the second antibody in taFv is reversed, the first bispecific molecule is called the taFv-heterodimer Fc type, and is called the taFv(inversed)-heterodimer Fc type (also called the taFv(inversed)-Fc type).

[0384] Figure 6a(a) shows the structure of a hybrid type bispecific molecule, Figure 6a(b) shows the structure of a dual type bispecific molecule, and Figure 6a(c) shows the structure of a scFv-Fab-heterodimer Fc type bispecific molecule. Also, Figure 3(a) shows the structure of scFv, Figure 3(b) shows the structure of Fab, Figure 3(c) shows the structure of taFv, Figure 3(d) shows the structure of a taFv-heterodimer Fc type bispecific molecule, and Figure 3(e) shows the structure of a taFv-Fab-heterodimer Fc type bispecific molecule. In addition, Figure 6b(a) shows the structure of a taFv-heterodimer Fc type bispecific molecule (identical to Figure 3(d)), Figure 6b(b) shows the structure of a taFv(inversed)-heterodimer Fc type bispecific molecule, Figure 6b(c) shows the structure of a first polypeptide included in a taFv(inversed)-heterodimer Fc type bispecific molecule, and Figure 6(d) shows the structure of a second polypeptide included in a taFv(inversed)-heterodimer Fc type bispecific molecule.

[0385] The dual-specific molecule of the present invention has a structure in which a plurality of polypeptides are associated.

[0386] In the present invention, for example, as taFv, anti-HLA-A2 / NY-ESO scFv and anti-CD3 scFv taFv may be used. The taFv-heterodimer Fc-type bispecific molecule preferably comprises (a) a first polypeptide comprising, in order from the N-terminus toward the C-terminus, a scFv specifically binding to HLA / NY-ESO, a scFv specifically binding to CD3, and an immunoglobulin Fc region (i), and a second polypeptide comprising an immunoglobulin hinge region and an Fc region (ii), and more preferably (b) the first polypeptide and the second polypeptide are associated at the Fc region (i) and the Fc region (ii). The Fc regions of the first polypeptide and the second polypeptide may include mutations for forming a heterodimer. An example of a taFv-heterodimer Fc-type bispecific molecule is shown in FIG. 3(d). As shown in FIG. 3(d), the Fc region (i) of the first polypeptide and the Fc region (ii) of the second polypeptide, indicated by black treatment, are combined, so that the first polypeptide and the second polypeptide are associated. FIG. 3(f) shows the first polypeptide, and FIG. 3(g) shows the second polypeptide. For example, in FIG. 3(d), the scFv shown in white is anti-HLA-A2 / NY-ESO scFv, and the scFv shown by the upper right diagonal line is anti-CD3scFv.

[0387] A first polypeptide included in a more preferred taFv-heterodimer Fc-type bispecific molecule of the present invention comprises the amino acid sequence of the 21st to 511th positions of the amino acid sequence represented by SEQ ID NO. 85, the amino acid sequence of the 21st to 511th positions of the amino acid sequence represented by SEQ ID NO. 87, the amino acid sequence of the 21st to 511th positions of the amino acid sequence represented by SEQ ID NO. 88, the amino acid sequence of the 21st to 511th positions of the amino acid sequence represented by SEQ ID NO. 89, the amino acid sequence of the 21st to 511th positions of the amino acid sequence represented by SEQ ID NO. 90, the amino acid sequence of the 21st to 511th positions of the amino acid sequence represented by SEQ ID NO. 91, the amino acid sequence of the 21st to 511th positions of the amino acid sequence represented by SEQ ID NO. 92, the amino acid sequence of the 21st to 511th positions of the amino acid sequence represented by SEQ ID NO. 93, and SEQ ID NO. 94 The amino acid sequence of the 21st to 511th positions of the amino acid sequence represented by [Name], the amino acid sequence of the 21st to 511th positions of the amino acid sequence represented by [Name], the amino acid sequence of the 21st to 511th positions of the amino acid sequence represented by [Name], the amino acid sequence of the 21st to 511th positions of the amino acid sequence represented by [Name], the amino acid sequence of the 21st to 511th positions of the amino acid sequence represented by [Name], the amino acid sequence of the 21st to 511th positions of the amino acid sequence represented by [Name], the amino acid sequence of the 21st to 511th positions of the amino acid sequence represented by [Name], the amino acid sequence of the 20th to 511th positions of the amino acid sequence represented by [Name], the amino acid sequence of the 20th to 516th positions of the amino acid sequence represented by [Name], or the amino acid sequence of the 20th to 516th positions of the amino acid sequence represented by [Name], and is further preferred. The first polypeptide included in the taFv-heterodimer Fc-type bispecific molecule is sequence numbers 85, 87, 88,The first polypeptide included in a more preferred taFv-heterodimer Fc-type bispecific molecule comprises the amino acid sequences from the 529th to the 745th of the amino acid sequence represented by 89, 90, 91, 92, 93, 94, 95, 96, 86, 149, 150, or 155, or the amino acid sequences from the 534th to the 750th of the amino acid sequence represented by SEQ ID NO. 156 or 157, and further comprises the amino acid sequences from the 21st to the 745th of the amino acid sequence represented by SEQ ID NO. 85, the amino acid sequences from the 21st to the 745th of the amino acid sequence represented by SEQ ID NO. 87, the amino acid sequences from the 21st to the 745th of the amino acid sequence represented by SEQ ID NO. 88, the amino acid sequences from the 21st to the 745th of the amino acid sequence represented by SEQ ID NO. 89, and the sequence The amino acid sequence from the 21st to the 745th of the amino acid sequence represented by number 90, the amino acid sequence from the 21st to the 745th of the amino acid sequence represented by sequence number 91, the amino acid sequence from the 21st to the 745th of the amino acid sequence represented by sequence number 92, the amino acid sequence from the 21st to the 745th of the amino acid sequence represented by sequence number 93, the amino acid sequence from the 21st to the 745th of the amino acid sequence represented by sequence number 94, the amino acid sequence from the 21st to the 745th of the amino acid sequence represented by sequence number 95, the amino acid sequence from the 21st to the 745th of the amino acid sequence represented by sequence number 96, the amino acid sequence from the 21st to the 745th of the amino acid sequence represented by sequence number 86, the amino acid sequence from the 21st to the 745th of the amino acid sequence represented by sequence number 149, and the amino acid sequence represented by sequence number 150 It consists of the amino acid sequence from the 21st to the 745th, or the amino acid sequence from the 20th to the 745th of the amino acid sequence represented by SEQ ID NO. 155. Or,It consists of the 20th to 750th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 156 or the 20th to 750th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 157.

[0388] A second polypeptide included in the preferred taFv-heterodimer Fc-type bispecific molecule of the present invention comprises a hinge region derived from a human antibody and a variant Fc, and a second polypeptide included in a more preferred taFv-heterodimer Fc-type bispecific molecule comprises the 20th to 246th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 84.

[0389] Among these, NYF-0016, formed by assembling a first polypeptide consisting of amino acid sequences from the 21st to the 745th of the amino acid sequence represented by SEQ ID NO. 85 and a second polypeptide consisting of amino acid sequences from the 21st to the 246th of the amino acid sequence represented by SEQ ID NO. 84; NYF-0022, formed by assembling a first polypeptide consisting of amino acid sequences from the 21st to the 745th of the amino acid sequence represented by SEQ ID NO. 87 and a second polypeptide consisting of amino acid sequences from the 21st to the 246th of the amino acid sequence represented by SEQ ID NO. 84; NYF-0023, formed by assembling a first polypeptide consisting of amino acid sequences from the 21st to the 745th of the amino acid sequence represented by SEQ ID NO. 88 and a second polypeptide consisting of amino acid sequences from the 21st to the 246th of the amino acid sequence represented by SEQ ID NO. 84; and the amino acid sequence represented by SEQ ID NO. 89 NYF-0027 formed by assembling a first polypeptide consisting of amino acid sequences from the 21st to the 745th and a second polypeptide consisting of amino acid sequences from the 21st to the 246th of the amino acid sequence represented by SEQ No. 84; NYF-0035 formed by assembling a first polypeptide consisting of amino acid sequences from the 21st to the 745th of the amino acid sequence represented by SEQ No. 90 and a second polypeptide consisting of amino acid sequences from the 21st to the 246th of the amino acid sequence represented by SEQ No. 84; NYF-0044 formed by assembling a first polypeptide consisting of amino acid sequences from the 21st to the 745th of the amino acid sequence represented by SEQ No. 91 and a second polypeptide consisting of amino acid sequences from the 21st to the 246th of the amino acid sequence represented by SEQ No. 84.NYF-0045 formed by assembling a first polypeptide consisting of amino acid sequences from the 21st to the 745th of the amino acid sequence represented by SEQ ID NO. 92 and a second polypeptide consisting of amino acid sequences from the 21st to the 246th of the amino acid sequence represented by SEQ ID NO. 84; NYF-0047 formed by assembling a first polypeptide consisting of amino acid sequences from the 21st to the 745th of the amino acid sequence represented by SEQ ID NO. 93 and a second polypeptide consisting of amino acid sequences from the 21st to the 246th of the amino acid sequence represented by SEQ ID NO. 84; NYF-0048 formed by assembling a first polypeptide consisting of amino acid sequences from the 21st to the 745th of the amino acid sequence represented by SEQ ID NO. 94 and a second polypeptide consisting of amino acid sequences from the 21st to the 246th of the amino acid sequence represented by SEQ ID NO. 84; and the 21st of the amino acid sequence represented by SEQ ID NO. 95 NYF-0060 formed by assembling a first polypeptide consisting of amino acid sequences from the 21st to the 246th position of the amino acid sequence represented by SEQ ID NO. 84; NYF-0061 formed by assembling a first polypeptide consisting of amino acid sequences from the 21st to the 745th position of the amino acid sequence represented by SEQ ID NO. 96 and a second polypeptide consisting of amino acid sequences from the 21st to the 246th position of the amino acid sequence represented by SEQ ID NO. 84; NYF-0019 formed by assembling a first polypeptide consisting of amino acid sequences from the 21st to the 745th position of the amino acid sequence represented by SEQ ID NO. 86 and a second polypeptide consisting of amino acid sequences from the 21st to the 246th position of the amino acid sequence represented by SEQ ID NO. 84.NYF-0014, formed by associating a first polypeptide consisting of amino acid sequences from the 21st to the 745th of the amino acid sequence represented by SEQ ID NO. 149 and a second polypeptide consisting of amino acid sequences from the 21st to the 246th of the amino acid sequence represented by SEQ ID NO. 84, and NYF-0082, formed by associating a first polypeptide consisting of amino acid sequences from the 21st to the 745th of the amino acid sequence represented by SEQ ID NO. 150 and a second polypeptide consisting of amino acid sequences from the 21st to the 246th of the amino acid sequence represented by SEQ ID NO. 84, can be exemplified as a preferred taFv-heterodimer Fc-type bispecific molecule of the present invention.

[0390] In addition, NYZ-0038, which is formed by assembling a first polypeptide consisting of amino acid sequences from the 20th to the 745th of the amino acid sequence represented by SEQ ID NO. 155 and a second polypeptide consisting of amino acid sequences from the 20th to the 246th of the amino acid sequence represented by SEQ ID NO. 84; NYZ-0082, which is formed by assembling a first polypeptide consisting of amino acid sequences from the 20th to the 750th of the amino acid sequence represented by SEQ ID NO. 156 and a second polypeptide consisting of amino acid sequences from the 20th to the 246th of the amino acid sequence represented by SEQ ID NO. 84; and NYZ-0083, which is formed by assembling a first polypeptide consisting of amino acid sequences from the 20th to the 750th of the amino acid sequence represented by SEQ ID NO. 157 and a second polypeptide consisting of amino acid sequences from the 20th to the 246th of the amino acid sequence represented by SEQ ID NO. 84, are preferred embodiments of the present invention. It can be exemplified as a taFv-heterodimer Fc-type bispecific molecule.

[0391] Among them, NYF-0023, NYF-0047, NYF-0048, NYF-0060, NYF-0061, NYZ-0038, NYZ-0082, and NYZ-0083 are particularly desirable as they possess excellent biological activity, physical properties, etc.

[0392] In addition, taFv of the first antibody may be directly bound to one side of the Fc of the dimer, either via a linker or without a linker, and Fab of the first antibody or the second antibody may be directly bound to the other side of the Fc, either via a linker or without a linker. The bispecific molecule is a format in which Fab is added upstream of the Fc region (ii) (blackened) side of the second polypeptide of the taFv-heterodimer Fc type. Such a bispecific molecule is called a taFv-Fab-heterodimer Fc type bispecific molecule or a taFv-Fab-heterodimer Fc type (Fig. 3).

[0393] In the present invention, for the taFv included in the taFv-Fab-heterodimer Fc type bispecific molecule, for example, anti-HLA-A2 / NY-ESO scFv and anti-CD3scFv taFv can be used, and for the Fab, for example, HLA / NY-ESO Fab can be used.

[0394] The taFv-Fab-heterodimer Fc type bispecific molecule preferably comprises (a) a first polypeptide comprising, in order from the N-terminus toward the C-terminus, a scFv that specifically binds to human HLA / NY-ESO, a scFv that specifically binds to CD3, and an immunoglobulin Fc region (i); a second polypeptide comprising an immunoglobulin heavy chain comprising an Fc region (ii); and a third polypeptide comprising an immunoglobulin light chain, more preferably (b) the second polypeptide and the third polypeptide are associated, and (c) the first polypeptide and the second polypeptide are associated in the Fc region (i) and the Fc region (ii). An example of a taFv-Fab-heterodimer Fc type bispecific molecule is shown in FIG. 3(e), a first polypeptide is shown in FIG. 3(f), a second polypeptide is shown in FIG. 3(h), and a third polypeptide is shown in FIG. 3(i). As shown in FIG. 3(e), a second polypeptide consisting of an immunoglobulin heavy chain including the Fc region (i) of the first polypeptide and the Fc region (ii) indicated by black treatment is bound to the Fc region (i) of the first polypeptide and the Fc region (ii) of the second polypeptide, and an immunoglobulin light chain is bound to the second polypeptide. Such a preferred taFv-Fab-heterodimer Fc-type bispecific molecule may be formed by binding Fab to a taFv-heterodimer Fc-type bispecific molecule comprising taFv and the Fc region of immunoglobulin. The amino acid sequence included in the first polypeptide included in the preferred taFv-Fab-heterodimer Fc-type bispecific molecule is the amino acid sequence from the 21st to the 511th of the amino acid sequence represented by SEQ ID NO. 85, the amino acid sequence from the 21st to the 511th of the amino acid sequence represented by SEQ ID NO. 87,The amino acid sequence from the 21st to the 511th of the amino acid sequence represented by SEQ ID NO. 88, the amino acid sequence from the 21st to the 511th of the amino acid sequence represented by SEQ ID NO. 89, the amino acid sequence from the 21st to the 511th of the amino acid sequence represented by SEQ ID NO. 90, the amino acid sequence from the 21st to the 511th of the amino acid sequence represented by SEQ ID NO. 91, the amino acid sequence from the 21st to the 511th of the amino acid sequence represented by SEQ ID NO. 92, the amino acid sequence from the 21st to the 511th of the amino acid sequence represented by SEQ ID NO. 93, the amino acid sequence from the 21st to the 511th of the amino acid sequence represented by SEQ ID NO. 94, the amino acid sequence from the 21st to the 511th of the amino acid sequence represented by SEQ ID NO. 95, the amino acid sequence from the 21st to the 511th of the amino acid sequence represented by SEQ ID NO. 96, and the amino acid sequence represented by SEQ ID NO. 86 Examples include the amino acid sequences of the 21st to 511th positions, the amino acid sequences of the 21st to 511th positions of the amino acid sequence represented by SEQ ID NO. 149, and the amino acid sequences of the 21st to 511th positions of the amino acid sequence represented by SEQ ID NO. 150; furthermore, the first polypeptide included in a preferred taFv-Fab heterodimer Fc-type bispecific molecule comprises the amino acid sequences of the 21st to 745th positions of the amino acid sequence represented by SEQ ID NO. 85, the amino acid sequences of the 21st to 745th positions of the amino acid sequence represented by SEQ ID NO. 87, the amino acid sequences of the 21st to 745th positions of the amino acid sequence represented by SEQ ID NO. 88, the amino acid sequences of the 21st to 745th positions of the amino acid sequence represented by SEQ ID NO. 89, the amino acid sequences of the 21st to 745th positions of the amino acid sequence represented by SEQ ID NO. 90, and the amino acid sequence represented by SEQ ID NO. 91. The amino acid sequence of the 21st to 745th positions of the sequence,It consists of the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 92, the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 93, the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 94, the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 95, the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 96, the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 86, the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 149, or the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 150.

[0395] A second polypeptide included in the preferred taFv-Fab-heterodimer Fc type bispecific molecule of the present invention comprises a variable region, a CH1 region, and a hinge region of a human antibody or humanized antibody heavy chain, and a variant Fc, and a more preferred second polypeptide included in the taFv-Fab-heterodimer Fc type bispecific molecule comprises the 20th to 242nd amino acid sequence of the amino acid sequence represented by SEQ ID NO. 99.

[0396] A third polypeptide included in the preferred taFv-Fab-heterodimer Fc type bispecific molecule of the present invention comprises a variable region and a normal region of a human antibody or humanized antibody light chain, and a more preferred third polypeptide included in the taFv-Fab-heterodimer Fc type bispecific molecule comprises the 21st to 131st amino acid sequence represented by SEQ ID NO. 100.

[0397] The variable region and CH1 region of the second polypeptide included in this preferred taFv-Fab-heterodimer Fc type bispecific molecule and the third polypeptide constitute Fab, and the preferred Fab is the Fab of the anti-HLA / NY-ESO antibody, for example, the Fab of NYA-0001.

[0398] In the present invention, for the scFv included in the scFv-Fab-heterodimer Fc type bispecific molecule, for example, anti-HLA-A2 / NY-ESO scFv or anti-CD3scFv may be used, and for the Fab, for example, HLA / NY-ESO Fab or anti-CD3 Fab may be used.

[0399] A scFv-Fab-heterodimer Fc type bispecific molecule preferably comprises (a) a first polypeptide comprising, in order from the N-terminus toward the C-terminus, a scFv that specifically binds to human HLA / NY-ESO, a variable region and normal region CH1 of an antibody heavy chain that specifically binds to CD3, and an immunoglobulin Fc region (i); a second polypeptide comprising a hinge region and an Fc region (ii) of an immunoglobulin; and a third polypeptide comprising an antibody light chain comprising a variable region and a normal region, and more preferably (b) the first polypeptide and the second polypeptide associate in the Fc region (i) and the Fc region (ii), and the first polypeptide associate with the third polypeptide (antibody light chain) in the variable region and normal region CH1 of the antibody heavy chain. The Fc regions of the first polypeptide and the second polypeptide may be wild-type or may contain mutations that form a heterodimer. An example of a scFv-Fab-heterodimer Fc-type bispecific molecule is shown in FIG. 6a(c). The right half of FIG. 6a(c) consists of the first polypeptide and the third polypeptide, and the left half consists of the second polypeptide. As shown in FIG. 6a(c), the Fc region (i) of the first polypeptide and the Fc region (ii) of the second polypeptide, indicated by black treatment, are associated, and the first polypeptide and the third polypeptide are associated. For example, in FIG. 6a(c), the scFv indicated by the upper right diagonal line is the HLA-A2 / NY-ESO scFv, and the Fab indicated by white, checkered, and horizontal lines is the CD3Fab.

[0400] As amino acid sequences included in the first polypeptide included in the preferred scFv-Fab-heterodimer Fc type bispecific molecule, the amino acid sequences from the 21st to the 394th of the amino acid sequence represented by SEQ ID NO. 160, and more preferably from the 20th to the 724th, can be exemplified.

[0401] In addition, other amino acid sequences included in the first polypeptide included in the preferred scFv-Fab-heterodimer Fc type bispecific molecule may be exemplified by the amino acid sequences from the 21st to the 389th, and more preferably from the 20th to the 719th.

[0402] In addition, other amino acid sequences included in the first polypeptide included in the preferred scFv-Fab-heterodimer Fc type bispecific molecule may be exemplified by the amino acid sequences from the 21st to the 389th, and more preferably from the 20th to the 719th, of the amino acid sequence represented by SEQ ID NO. 198.

[0403] A second polypeptide included in the preferred scFv-Fab-heterodimer Fc-type bispecific molecule of the present invention comprises a hinge region derived from a human antibody and a variant Fc, and a more preferred second polypeptide included in the scFv-Fab-heterodimer Fc-type bispecific molecule comprises the 20th to 246th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 84.

[0404] A third polypeptide included in the preferred scFv-Fab-heterodimer Fc type bispecific molecule of the present invention comprises a light chain derived from a human antibody, and a third polypeptide included in the more preferred scFv-Fab-heterodimer Fc type bispecific molecule may be exemplified by the amino acid sequence of the 21st to 127th amino acid sequence represented by SEQ ID NO. 161, and more preferably the amino acid sequence of the 21st to 233rd amino acid sequence.

[0405] Among these, NYZ-1010, which is formed by associating a first polypeptide consisting of the 20th to 724th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 160, a second polypeptide consisting of the 20th to 246th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 84, and a third polypeptide consisting of the 21st to 233rd amino acid sequence of the amino acid sequence represented by SEQ ID NO. 161, can be exemplified as a preferred scFv-Fab-heterodimer Fc type bispecific molecule of the present invention.

[0406] In addition, NYZ-1007, which is formed by associating a first polypeptide consisting of the 20th to 719th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 197, a second polypeptide consisting of the 20th to 246th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 84, and a third polypeptide consisting of the 21st to 233rd amino acid sequence of the amino acid sequence represented by SEQ ID NO. 161, can also be exemplified as a preferred scFv-Fab-heterodimer Fc type bispecific molecule of the present invention.

[0407] In addition, NYZ-1017, which is formed by associating a first polypeptide consisting of amino acid sequences from the 20th to the 719th of the amino acid sequence represented by SEQ ID NO. 198, a second polypeptide consisting of amino acid sequences from the 20th to the 246th of the amino acid sequence represented by SEQ ID NO. 84, and a third polypeptide consisting of amino acid sequences from the 21st to the 233rd of the amino acid sequence represented by SEQ ID NO. 161, can also be exemplified as a preferred scFv-Fab-heterodimer Fc type bispecific molecule of the present invention.

[0408] One, two, or three or more peptides included in the bispecific molecule of the present invention may be the aforementioned "deleted bodies," that is, in the carboxyl terminus, particularly the carboxyl terminus derived from the antibody heavy chain, one or two (or more) amino acids may be modified (including deletions). For example, the carboxyl terminus of the amino acid sequence of the first polypeptide included in NYZ-1010, one of the preferred scFv-Fab-heterodimer Fc type bispecific molecules of the present invention, may be any of Lys (724th) of SEQ ID NO. 160, Gly (723rd) formed by the deletion of one amino acid, or a mixture containing the same. Likewise, the carboxyl terminus of the amino acid sequence of the second polypeptide included in the preferred scFv-Fab-heterodimer Fc type bispecific molecule of the present invention may be any of the 246th Lys of SEQ ID NO. 84, the 245th Gly formed by the deletion of one amino acid, or a mixture comprising the same.

[0409] scFv and Fab included in the dual-specificity molecule of the present invention are preferably scFv and Fab of a humanized antibody or human antibody, and Fc is preferably Fc of a human antibody.

[0410] In the variable regions included in the dual-specificity molecule of the present invention, the medium-chain variable region and the light-chain variable region may be bound in that order from the amino-terminal side of the antibody, or the light-chain variable region and the medium-chain variable region may be bound in that order. A linker may be present between the two variable regions (optional). Additionally, a glycine residue may be present at the amino-terminal side of the variable region (optional). In the tandem scFv type dual-specificity molecule, a linker, a FLAG tag, and / or a His tag may be bound to the carboxyl-terminal side of the variable region (optional). As one preferred embodiment, a configuration in which the medium-chain variable region, the first linker, the light-chain variable region, the second linker, the FLAG tag, and the His tag are bound in that order from the amino-terminal side may be exemplified.

[0411] The linker includes a one-piece chain polypeptide or a one-piece chain oligopeptide, or synthetic products such as PEG, nucleotide, sugar chain, or compound. In addition, as long as it combines two polypeptides, it is not particularly limited and it is possible to use a known linker.

[0412] The length of the linker is, for example, 5 to 30 amino acids in the case of a peptide linker. When a double-specific molecule contains multiple linkers, peptide linkers of the same length may all be used, or peptide linkers of different lengths may be used.

[0413] As peptide linkers, for example, a repetition of (Gly·Gly·Gly·Gly·Ser) (Sequence No. 161) is exemplified, but one to several amino acid residues different from Gly and Ser may be added to these.

[0414] Among the structures (formats) that can be employed by the multispecific antibody of the present invention, particularly the bispecific antibody as described above, the preferred ones are the taFv-heterodimer Fc type, the taFv-Fab-heterodimer Fc type, and the scFv-Fab-heterodimer Fc type, and the more preferred one is the taFv-heterodimer Fc type, and the type in which anti-HLA / NY-ESO scFv and anti-CD3 scFv are positioned in order from the N-terminus toward the C-terminus (taFv-heterodimer Fc type) is more preferred than the type in which they are positioned in the opposite order (taFv(inversed)-heterodimer Fc type) (Example 11, etc.). In addition, the scFv-Fab-heterodimer Fc type is another more preferred one.

[0415] The present invention also includes a molecule comprising a polynucleotide comprising a nucleotide sequence encoding an amino acid sequence included in the molecule of the present invention and a polynucleotide that hybridizes under stringent conditions with a complementary chain of a polynucleotide comprising a nucleotide sequence encoding an amino acid sequence included in the molecule of the present invention, and also a molecule that binds to HLA / NY-ESO and, preferably, additionally binds to CD3.

[0416] The present invention also includes a molecule that has an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to the amino acid sequence included in the molecule of the present invention, and also binds to HLA / NY-ESO, and preferably additionally binds to CD3.

[0417] The antibodies of the present invention, their binding fragments, and multispecific antibodies containing them possess properties such as excellent biological activity, physicochemical properties (hereinafter referred to as "physical properties"), safety, and pharmacokinetics. (a) Examples of biological activity or indicators thereof include antigen binding activity, in vitro cell damage activity, and in vivo antitumor activity. 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, more preferably 5 nM or less. Also, for example, the EC of cell damage activity exerted using human peripheral blood mononuclear cells as effector cells against endogenous human NY-ESO expressing cell lines U266B1 and / or NCI-H1703 50 The value is 20 nM or less, preferably 10 nM or less, more preferably 5 nM or less (as for the measurement and calculation of in vitro cell injury activity, the method described in Example 8 may be exemplified, but is not limited thereto). In addition, for example, 6 × 10 of the human squamous cell lung cancer cell line NCI-H1703 7 0.1 mL of the cell / mL suspension was implanted subcutaneously into NOG mice, and after 4 days, 3.75 × 10⁶ human peripheral blood mononuclear cells 7When 0.2 ml of the cell / ml suspension is transplanted into the tail vein and the tumor volume is measured after administering the antibody three times once a week starting 14 days later, the tumor proliferation inhibitory activity compared to the control group administered the solvent is 50% or more, preferably 75% or more, more preferably 90% or more (the method described in Example 9 may be exemplified for the measurement and calculation of in vivo anti-tumor activity, but is not limited thereto). (b) Since impurities contained in biopharmaceuticals affect the safety of the drug, it is required to manage them by appropriately setting standards for whether they increase during manufacturing and storage. Among these, HMWS (aggregates) is one of the major impurities and is an item that must be managed particularly strictly because it is involved in immunogenicity risks or a decrease in efficacy. The management of impurities needs to be evaluated not only at the time of manufacturing but also including stability over time (presence or absence of increase) during the manufacturing process and after manufacturing. Since the shelf life of a drug is determined based on the results of long-term stability tests, a longer shelf life can be established for antibodies that are stable over time. Therefore, physicochemical properties in the present invention that serve as indicators when selecting a desirable antibody for biopharmaceuticals include acid resistance (such as inhibition of HMWS formation) and solution stability (such as inhibition of HMWS formation). Other indicators include a large quantity and high yield in a culture of recombinant cells obtained by introducing a gene encoding the amino acid sequence contained therein into host cells suitable for producing the antibody of the present invention, its binding fragment, or molecules containing them, such as Expi293F cells.The preferred antibodies of the present invention having the physicochemical properties, the antigen-binding fragments thereof, and the multispecific antibodies containing them: make it possible to expose their solutions to acidic conditions, and thus enable or facilitate the manufacture of them, for example, chromatography such as protein A, ion exchange, and virus inactivation; since the generation of HMWS is suppressed to a low level even when they are in solution, it enables or facilitates the manufacture, formulation, distribution, and preservation of pharmaceuticals containing them; and it makes it possible to produce them efficiently. Regarding acid resistance, for example, the content of generated HMWS calculated by holding 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 (the method described in Example 19-1 may be exemplified for measuring and calculating the HMWS content for acid resistance evaluation, but is not limited thereto). Regarding solution stability, for example, the solution is dissolved to a concentration of 25 mg / mL in pH 6.0 consisting of 25 mM histidine and 5% sorbitol, stored at 25°C for 6 days, and then the HMWS content calculated by measuring HMWS by size exclusion chromatography is 20% or less, preferably 10% or less (as a method for measuring and calculating the HMWS content for evaluating solution stability, the method described in Example 19-2 may be exemplified, but is not limited thereto). As a method for measuring and calculating production efficiency or yield, the method described in Examples 20 and 21 may be exemplified, but is not limited thereto. (c) As for safety or its indicators, antigen recognition characteristics, findings upon administration, etc. may be exemplified.For example, it recognizes multiple amino acids on the wild-type NY-ESO peptide and does not bind to homologous peptides having amino acid sequences similar to but not identical to the wild-type NY-ESO peptide, resulting in a low risk of side effects caused by off-target effects. In addition, it can be predicted that it has low immunogenicity in ISPRI web-based immunogenicity screening (EpiVax, Inc) and a low risk of side effects such as cytokine production caused by anti-antibodies. Furthermore, for example, when NYF-0023, NYF-0045, NYF-0047, NYF-0048, NYF-0060, NYF-0061, NYZ-0082, or NYZ-1010 included in the bispecific antibodies of the present invention were administered to Balb / c mice, no problematic findings regarding blood half-life were observed, nor were weight loss or other significant toxic findings observed. In addition, when NYZ-0082 or NYZ-1010 was administered as a single dose to crab-eating monkeys, no problematic findings regarding the blood half-life were observed, and no changes attributable to administration were observed regarding general condition, body weight, intake amount, body temperature, and plasma cytokine levels. (d) Examples of kinetics or indicators thereof include the blood half-life. For example, when several bispecific antibodies obtained in the present invention were administered to Balb / c mice or crab-eating monkeys, no problematic findings regarding the blood half-life were observed. The antibodies, their binding fragments, and molecules of the present invention, which possess such excellent biological activity, physicochemical properties, safety, and pharmacokinetics, can be preferably included in a pharmaceutical composition.Preferred antibodies of the present invention or antigen-binding fragments having the antigen-binding activity described in (a) and the properties described in (b) may 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. In addition, the preferred multiple-specific 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, NYF00058, NYF-0060, NYF-0061, NYZ-0038, NYZ-0082, NYZ-0088, and NYZ-1010, more preferably NYF-0061, NYZ-0038, NYZ-0082, NYZ-0088, NYZ-1007, NYZ-1010, and NYZ-1017. Each can be exemplified, but is not limited to them.

[0418] In the present invention, the "site" to which the antibody binds, that is, the "site" recognized by the antibody, refers to a partial peptide or partial higher-order structure on the antigen to which the antibody binds or recognizes. In the present invention, such a site is also referred to as an epitope or a binding site of the antibody. Examples of sites on the HLA / NY-ESO to which the anti-HLA / NY-ESO antibody of the present invention binds or recognizes include multiple amino acids in the HLA / NY-ESO peptide, partial higher-order structures, etc.

[0419] An antibody or binding fragment of the present invention and an “antibody or its binding fragment that binds to the same site” are also included in the present invention. An “antibody that binds to the same site” as any antibody refers to another antibody that binds to a site on an antigen molecule recognized by that antibody. If a second antibody binds to a partial peptide or partial stereostructure on an antigen molecule to which a first antibody binds, it can be determined that the first antibody and the second antibody bind to the same site. If the 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 the same activity, and such a second antibody is also included in the present invention. Furthermore, an antibody that competes with the first antibody of the present invention for binding to HLA / NY-ESO is also included in the present invention if it has the antigen-binding activity described in (a). An antibody that binds to a site on the HLA / NY-ESO recognized by such a monoclonal antibody of the present invention, an antibody that competes with the monoclonal antibody of the present invention for binding to the HLA / NY-ESO, and their binding fragments preferably have one or more of the in vitro cell injury activity and in vivo antitumor activity described in (a), more preferably three or more of the properties described in (b) to (d), and most preferably all of them.

[0420] The binding site of an antibody can be determined by methods known to those skilled in the art, such as immunoassays. For example, a series of peptides formed by appropriately cutting the amino acid sequence of an antigen from the C-terminus or N-terminus, and the reactivity of antibodies against them can be examined to determine a rough recognition site. Subsequently, the binding site can be determined by synthesizing shorter peptides and examining the reactivity of antibodies against those peptides. Alternatively, for example, the binding site can be determined by deleting a specific region or area from the amino acid sequence of an antigen or an antigen fragment peptide, substituting it with a different amino acid sequence, or introducing a mutation into the amino acid sequence, and examining the reactivity of antibodies against those peptides. Antigen fragment peptides can be prepared using techniques such as genetic recombination or peptide synthesis.

[0421] When an antibody binds to or recognizes a partial higher-order structure of an antigen, the binding site of such 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 having an interaction distance with the antibody can be identified by crystallizing the antibody or its fragment and 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 having such an interaction distance with the antibody may constitute the antigen binding site (epitope) of the antibody. If there are two or more such amino acid residues, each amino acid does not need to be adjacent to each other in the primary sequence.

[0422] The anti-HLA / NY-ESO antibody or its binding fragment of the present invention specifically recognizes a plurality of amino acids present in the amino acid sequence of HLA / NY-ESO. An antibody or its binding fragment that recognizes such a plurality of amino acids, competes with the antibody or its binding fragment of the present invention for binding to HLA / NY-ESO, or has an interaction distance with such a plurality of amino acids is also included in the present invention. Furthermore, a multispecific antibody comprising such an antibody or its binding fragment is also included in the present invention.

[0423] 6. Pharmaceutical composition

[0424] The present invention includes an anticancer agent comprising, as an active ingredient, an anti-HLA / NY-ESO antibody of the present invention or a multispecific molecule that binds to HLA / NY-ESO.

[0425] The anticancer agent of the present invention can be used for one or more types selected from carcinoma, sarcoma, lymphoma, leukemia, myeloma, germ cell tumor, brain tumor, carcinoid, neuroblastoma, retinoblastoma, and neoblastoma. Specifically, carcinomas include renal 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, etc. Sarcomas include liposarcoma, angiosarcoma, chondrosarcoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, undifferentiated pleomorphic sarcoma, myxoid fibrosarcoma, malignant peripheral schwannoma, retroperitoneal sarcoma, synovial sarcoma, uterine sarcoma, gastrointestinal stromal tumor, leiomyosarcoma, epithelial sarcoma, etc. Lymphomas include B-cell lymphoma, T-NK cell lymphoma, Examples include Hodgkin lymphoma, leukemias include myeloid leukemia, lymphocytic leukemia, myeloproliferative disorders, and myelodysplastic syndrome, myelomas include multiple myeloma, germ cell tumors include testicular cancer and ovarian cancer, and brain tumors include glioma and meningioma.

[0426] The anticancer agent of the present invention may comprise an effective amount of anti-HLA / NY-ESO antibody or a multispecific molecule that binds to HLA / NY-ESO, a pharmaceutically acceptable carrier, a diluent, a solubilizer, an emulsifier, a preservative, an adjuvant, etc. The "pharmaceutically acceptable carrier," etc., can be appropriately selected from a wide range depending on the type of target disease or the form of administration of the drug. The method of administration of the anticancer agent of the present invention can be appropriately selected, for example, by injection, local injection, intraperitoneal injection, selective intravenous injection, intravenous injection, subcutaneous injection, or organ perfusion fluid infusion. In addition, the solution for injection may be formulated using a carrier consisting of a salt solution, a glucose solution, a mixture of saline and glucose solutions, various buffer solutions, etc. Alternatively, it may be formulated in a powder state and mixed with the liquid carrier at the time of use to prepare the injection solution.

[0427] Other methods of administration can also be appropriately selected along with the development of the formulation. For example, in the case of oral administration, oral liquid preparations, powders, pills, capsules, and tablets may be applied. In the case of oral liquid preparations, they may be manufactured using water, sugars such as sucrose, sorbitol, and fructose, glycols such as PEG, oils such as sesame oil and soybean oil, preservatives such as alkyl parahydroxybenzoate, and flavors such as strawberry and peppermint, as oral liquid formulations such as suspensions and syrups. Powders, pills, capsules, and tablets can be formulated using excipients such as lactose, glucose, sucrose, and mannitol; disintegrants such as starch and sodium arginate; lubricants such as magnesium stearate and talc; binders such as polyvinyl alcohol, hydroxypropyl cellulose, and gelatin; surfactants such as fatty acid esters; and plasticizers such as glycerin. Tablets and capsules are preferred unit dosage forms in the composition of the present invention in that they facilitate administration. When manufacturing tablets or capsules, a solid manufacturing carrier is used.

[0428] The amount of anti-HLA / NY-ESO antibody or multispecific molecule binding to HLA / NY-ESO effective for therapeutic use may be varied depending on the nature of the condition being treated, the patient's age or condition, and ultimately determined by the physician. For example, it is 0.0001 mg to 100 mg per kg of body weight per dose. The prescribed dosage may be administered once every 1 to 180 days, or divided into 2, 3, 4, or more doses per day at appropriate intervals.

[0429] In addition, the present invention includes a polynucleotide encoding an amino acid sequence included in the anti-HLA / NY-ESO antibody of the present invention or a multispecific molecule binding to HLA / NY-ESO, a vector comprising said polynucleotide, a cell comprising said polynucleotide or the vector, and a pharmaceutical composition comprising any of said polynucleotide, said vector, and said cell as an active ingredient. Such pharmaceutical compositions are preferably anticancer agents.

[0430] In addition, the pharmaceutical composition of the present invention may be used in combination with other drugs. Other drugs are not specifically limited to chemotherapy agents, radiation therapy agents, biopharmaceuticals, etc., and may be administered or applied as a single preparation containing the pharmaceutical composition of the present invention or as two or more different preparations or therapies, either simultaneously with or at a different schedule than the pharmaceutical composition of the present invention.

[0431] [Example]

[0432] The present invention will be described in more detail below in the embodiments, but the present invention is not limited thereto.

[0433] In addition, in the following examples, unless otherwise specified, each operation regarding gene manipulation was performed by the method described in *Molecular Cloning* (by Sambrook, J., Fritsch, EF and Maniatis, T., published by Cold Spring Harbor Laboratory Press in 1989) and other methods described in laboratory manuals used by those skilled in the art, or, if commercially available reagents or kits were used, by following the instructions of the commercially available products. The synthesis of primers required for gene synthesis or vector construction was outsourced as necessary (FASMAC, Inc., Thermo Fisher Scientific, and Eurofin Genomics).

[0434] (Example 1) Acquisition of anti-HLA / NY-ESO antibody derived from human antibody phage library

[0435] 1) Preparation of 1HLA / NY-ESO antigen protein

[0436] Each inclusion body prepared with E. coli (BL21 (DE3), Agilent Technologies) expressing the truncate of HLA-A*0201 (GenBank: ASA47534.1) (biotin ligase recognition sequence addition: sequence no. 33) and β2-microglobulin (UniProtKB-P61769: sequence no. 34), respectively, and NY-ESO peptide: SLLMWITQC (sequence no. 1 of the sequence table) were refolded by heavy dilution, and then an HLA-A*0201 / β2-microglobulin / NY-ESO peptide complex (hereinafter referred to as HLA / NY-ESO) was prepared using a gel filtration column (Superdex 200 10 / 300, GE Healthcare).

[0437] As a negative control antigen, the HLA-A*0201 / β2-microglobulin / MAGEC-1 peptide complex (hereinafter referred to as HLA / MC1) was prepared by refolding in the same manner using MAGEC-1 peptide: ILFGISLREV (sequence number 2 of the sequence table). In addition, the prepared HLA / NY-ESO and HLA / MC1 were biotinylated with E. coli biotin ligase and then fractionated using a gel filtration column (Superdex 200 10 / 300, GE Healthcare) to prepare each biotinylated protein.

[0438] 1)-2 Isolation of scFv having binding ability to HLA / NY-ESO

[0439] scFvs binding to HLA / NY-ESO were isolated from a human antibody phage library. First, the phages were added to Dynabeads Streptavidin M-280 (Thermo Fisher Scientific), which solidified biotinylated HLA / MC1, and non-binding phages were recovered. Next, Dynabeads Streptavidin M-280Ag, which solidified biotinylated HLA / NY-ESO, was added, and non-binding phages were removed by a washing operation using a magnet stand (DynaMag-2, Thermo Fisher Scientific).

[0440] After that, phages bound to HLA / NY-ESO were infected into Escherichia coli (XL-1Blue, Agilent Technologies), and phages bound to HLA / NY-ESO were recovered and amplified. After a total of three panning cycles, the polyclonal phagemid was transferred to an expression vector for Escherichia coli that added a FLAG tag and a His tag to the carboxyl terminus of scFv, and then Escherichia coli were transformed and scFv was expressed in the presence of IPTG (Isopropyl-β-D-thiogalactopyranoside) (Sigma-Aldrich) and provided for screening by ELISA.

[0441] 1)-3 Screening of HLA / NY-ESO-binding scFv by ELISA

[0442] 50 µl of NeutrAvidin (Life Technologies), diluted to 1 µg / ml in PBS (0.01 M phosphate-buffered physiological saline containing 0.138 M sodium chloride and 0.0027 M potassium chloride, pH 7.4, Sigma-Aldrich), was added to a 384-well Maxi-sorp plate (Black, Nunc), and solidified by standing overnight at 4°C. After washing three times with PBS (ELISA buffer) containing 0.05% Tween-20 (Bio-Rad), biotinylated HLA / NY-ESO, also used in Examples 1-2 and diluted to 1 µg / ml in PBS, was added and shaken at room temperature for 1 hour. After washing three times with ELISA buffer, the mixture was blocked with Blocker Casein (Thermo Fisher Scientific) and washed three times with ELISA buffer. Subsequently, a culture of E. coli expressing scFv was added, and the mixture was reacted at room temperature for 2 hours. After washing three times with ELISA buffer, 50 µl of a Horseradish peroxidase (HRP) labeled FLAG antibody (Sigma-Aldrich) diluted 5000-fold with ELISA buffer was added, and the mixture was reacted at room temperature for 1 hour. After washing five times with ELISA buffer, SuperSignal Pico ELISA Chemiluminescent substrate (Thermo Fisher Scientific) was added, and chemiluminescence after 10 minutes was measured using a plate reader (Envision 2104 Multilabel Reader, Perkin Elmer) to isolate ELISA-positive clones binding to HLA / NY-ESO.

[0443] 1) Determination of nucleotide and amino acid sequences of ELISA-positive clone NY-R119

[0444] Among the ELISA-positive clones obtained in 1)-3, NY-R119 was selected as a scFv with strong binding affinity to HLA / NY-ESO and excellent recognition specificity. The nucleotide sequences of the heavy chain and light chain variable regions of NY-R119 were analyzed using the Dye Terminator method (BigDye (registered trademark) Terminator v3.1, Thermo Fisher Scientific). The primer sequences used for sequence analysis are as follows.

[0445] PrimerA : 5'-CTCTTCGCTATTACGCCAGCTGGCGA-3' (Sequence number 3 in the sequence table (Fig. 10))

[0446] PrimerB : 5'-ATAACAATTTCACACAGGAAACAGCTATGA-3' (Sequence number 4 in the sequence table (Fig. 11))

[0447] The nucleotide sequence of cDNA encoding the variable region of the determined heavy chain of NY-R119 is shown in sequence number 5 (Fig. 12), and the amino acid sequence is shown in sequence number 6 (Fig. 13).

[0448] The nucleotide sequence of the cDNA encoding the variable region of the determined NY-R119 light chain is shown in sequence number 7 (Fig. 14), and the amino acid sequence is shown in sequence number 8 (Fig. 15).

[0449] The CDR sequences of NY-R119 in the definition of CDR in IMGT are respectively CDRH1 at sequence number 54 (Fig. 61), CDRH2 at sequence number 55 (Fig. 61), CDRH3 at sequence number 56 (Fig. 61), CDRL1 at sequence number 57 (Fig. 61), CDRL2 at sequence number 58 (Fig. 61), and CDRL3 at sequence number 59 (Fig. 61).

[0450] 1)-5 Production of NYA-0001

[0451] 1)-5-1 Construction of the NYA-0001 expression vector

[0452] To prepare various evaluation samples, a mammalian culture expression vector of NY-R119 was constructed. Additionally, NY-R119 expressed in mammalian cultured cells was named NYA-0001. The scFv portions, variable regions of the heavy and light chains, and CDRH1–3 and CDRL1–3 of NY-R119 and NYA-0001 consist of identical amino acid sequences. A DNA fragment encoding NYA-0001 was inserted into a mammalian cell expression vector backed by pcDNA3.3 (Thermo Fisher Scientific) using the In-Fusion HD Cloning Kit (CLONTECH) to construct the NYA-0001 expression vector.

[0453] The nucleotide sequence of the constructed NYA-0001 expression vector was reanalyzed, and it was confirmed that the full-length nucleotide sequence of NYA-0001 is the nucleotide sequence shown in sequence number 69 of the sequence table (Fig. 70). Furthermore, from the said nucleotide sequence, the full-length amino acid sequence of NYA-0001 encoded by said sequence was the amino acid sequence shown in sequence number 70 (Fig. 71). In said sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 266th is NYA-0001, and the amino acid sequence from the 267th to the 292nd is the Flag-His tag. Also, the amino acid sequence from the 46th to the 53rd is CDRH1, the amino acid sequence from the 71st to the 78th is CDRH2, and the amino acid sequence from the 117th to the 129th is CDRH3. Also, the amino acid sequence from the 181st to the 188th is CDRL1, the amino acid sequence from the 206th to the 208th is CDRL2, and the amino acid sequence from the 245th to the 256th is CDRL3.

[0454] 1)-5-2 NYA-0001 Expression·Purification

[0455] Expi293F cells (Thermo Fisher Scientific) were subcultured according to the manual. 2.5 × 10⁶ of the Expi293F cell culture medium in the logarithmic growth phase 6 The solution was diluted with Expi293 Expression medium (Thermo Fisher Scientific) to a cell / mL ratio and used for the production of NYA-0001. 0.3 mg of NYA-0001 expression vector and 0.9 mg of Polyethyleneimine (Polyscience #24765) were added to 20 mL of Opti-Pro SFM medium (Thermo Fisher Scientific), stirred slowly, and then incubated for 5 minutes before being added to Expi293F cells. The culture supernatant obtained after shaking at 135 rpm for 6 days in a 37°C, 8% CO2 incubator was filtered through a 0.2 µm filter (Millipore) to obtain the NYA-0001 culture supernatant. Purification was performed by eluting with Ni Sepharose excel (GE Healthcare), concentrating, and then purifying using a gel filtration column (Superdex 200 Increase, GE Healthcare) equilibrated with 25 mM Histidine, 300 mM NaCl, 5% sorbitol, and pH 6.0. The purified protein samples were fed into analytical size exclusion chromatography (SEC) to determine purity and concentration, after which they were used for various evaluations.

[0456] (Example 2) Production of NYA-0001 variant

[0457] 2)-1 Acquisition of NYA-0001 Variant

[0458] A phage library was constructed using a method of introducing mutations using PCR with the NYA-0001 gene as a template (Error-prone-based library), or by synthesizing oligomers randomly mutated into 20 types of amino acids for each residue of the entire CDR and constructing a library (oligo-based library), screening of high-binding-ability clones was performed, and NYA-0060, NYA-0068, and NYA-0082 were obtained as high-binding-ability variants, and their nucleotide sequences were determined.

[0459] The nucleotide sequence of the cDNA encoding the variable region of the heavy chain of the acquired NYA-0060 is shown in sequence number 9 (Fig. 16), and the amino acid sequence is shown in sequence number 10 (Fig. 17).

[0460] The nucleotide sequence of the cDNA encoding the variable region of the acquired NYA-0060 light chain is shown in sequence number 11 (Fig. 18), and the amino acid sequence is shown in sequence number 12 (Fig. 19).

[0461] The nucleotide sequence of the cDNA encoding the variable region of the heavy chain of the acquired NYA-0068 is shown in sequence number 13 (Fig. 20), and the amino acid sequence is shown in sequence number 14 (Fig. 21).

[0462] The nucleotide sequence of the cDNA encoding the variable region of the acquired NYA-0068 light chain is shown in sequence number 15 (Fig. 22), and the amino acid sequence is shown in sequence number 16 (Fig. 23).

[0463] The nucleotide sequence of the cDNA encoding the variable region of the heavy chain of the acquired NYA-0082 is shown in sequence number 17 (Fig. 24), and the amino acid sequence is shown in sequence number 18 (Fig. 25).

[0464] The nucleotide sequence of the cDNA encoding the variable region of the acquired NYA-0082 light chain is shown in sequence number 19 (Fig. 26), and the amino acid sequence is shown in sequence number 20 (Fig. 27).

[0465] 2)-2 Production of high-binding-ability variants by identified variant sites and combinations from NYA-0060, NYA-0068, and NYA-0082

[0466] DNA fragments encoding NYA-1163 and NYA-2023, each having the mutation sites NYA-0060 and NYA-0068 respectively, were inserted into a mammalian cell expression vector with pcDNA3.3 (Thermo Fisher Scientific) as a scaffold using the In-Fusion HD Cloning Kit (CLONTECH), and a mammalian cell scFv expression vector was constructed.

[0467] In addition, NYA-2027, NYA-1143, and NYA-2143 were designed using variant sites identified from NYA-0060, NYA-0068, and NYA-0082 and combinations thereof. Each mammalian cell scFv expression vector was constructed by introducing a site-specific variant into the NYA-0001 expression vector constructed in 1)-5, or by inserting a DNA fragment encoding the target scFv into a mammalian cell expression vector with pcDNA3.3 (Thermo Fisher Scientific) as a backbone using the In-Fusion HD Cloning Kit (CLONTECH).

[0468] The nucleotide sequence of the constructed scFv expression vector was reanalyzed and it was confirmed that the nucleotide sequence of the full length of NYA-1163 is the nucleotide sequence shown in sequence number 21 of the sequence table (Fig. 28).

[0469] It was confirmed that the full-length nucleotide sequence of NYA-2023 is the nucleotide sequence shown in sequence number 22 of the sequence table (Fig. 29).

[0470] It was confirmed that the full length nucleotide sequence of NYA-2027 is the nucleotide sequence shown in sequence number 23 of the sequence table (Fig. 30).

[0471] It was confirmed that the full-length nucleotide sequence of NYA-1143 is the nucleotide sequence shown in sequence number 24 of the sequence table (Fig. 31).

[0472] It was confirmed that the full-length nucleotide sequence of NYA-2143 is the nucleotide sequence shown in sequence number 25 of the sequence table (Fig. 32).

[0473] In addition, from the above nucleotide sequence, the full-length amino acid sequences of NYA-1163, NYA-2023, NYA-2027, NYA-1143, and NYA-2143 encoded by the said sequence were identified.

[0474] The full-length amino acid sequence of NYA-1163 is the amino acid sequence shown in sequence number 26 of the sequence table (Fig. 33). In the said sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 266th is NYA-1163, and the amino acid sequence from the 267th to the 292nd is the Flag-His tag. Also, the amino acid sequence from the 46th to the 53rd is CDRH1, the amino acid sequence from the 71st to the 78th is CDRH2, and the amino acid sequence from the 117th to the 129th is CDRH3. Also, amino acid numbers 181 to 188 are CDRL1, amino acid numbers 206 to 208 are CDRL2, and amino acid numbers 245 to 256 are CDRL3.

[0475] The full-length amino acid sequence of NYA-2023 is the amino acid sequence shown in sequence number 27 of the sequence table (Fig. 34). In the said sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 266th is NYA-2023, and the amino acid sequence from the 267th to the 292nd is the Flag-His tag. Also, the amino acid sequence from the 46th to the 53rd is CDRH1, the amino acid sequence from the 71st to the 78th is CDRH2, and the amino acid sequence from the 117th to the 129th is CDRH3. Also, amino acid numbers 181 to 188 are CDRL1, amino acid numbers 206 to 208 are CDRL2, and amino acid numbers 245 to 256 are CDRL3.

[0476] The full-length amino acid sequence of NYA-2027 is the amino acid sequence shown in sequence number 28 of the sequence table (Fig. 35). In the said sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 266th is NYA-2027, and the amino acid sequence from the 267th to the 292nd is the Flag-His tag. Also, the amino acid sequence from the 46th to the 53rd is CDRH1, the amino acid sequence from the 71st to the 78th is CDRH2, and the amino acid sequence from the 117th to the 129th is CDRH3. Also, amino acid numbers 181 to 188 are CDRL1, amino acid numbers 206 to 208 are CDRL2, and amino acid numbers 245 to 256 are CDRL3.

[0477] The full-length amino acid sequence of NYA-1143 is the amino acid sequence shown in sequence number 29 of the sequence table (Fig. 36). In the said sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 266th is NYA-1143, and the amino acid sequence from the 267th to the 292nd is the Flag-His tag. Also, the amino acid sequence from the 46th to the 53rd is CDRH1, the amino acid sequence from the 71st to the 78th is CDRH2, and the amino acid sequence from the 117th to the 129th is CDRH3. Also, amino acid numbers 181 to 188 are CDRL1, amino acid numbers 206 to 208 are CDRL2, and amino acid numbers 245 to 256 are CDRL3.

[0478] The full-length amino acid sequence of NYA-2143 is the amino acid sequence shown in sequence number 30 of the sequence table (Fig. 37). In the said sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 266th is NYA-2143, and the amino acid sequence from the 267th to the 292nd is the Flag-His tag. Also, the amino acid sequence from the 46th to the 53rd is CDRH1, the amino acid sequence from the 71st to the 78th is CDRH2, and the amino acid sequence from the 117th to the 129th is CDRH3. Also, amino acid numbers 181 to 188 are CDRL1, amino acid numbers 206 to 208 are CDRL2, and amino acid numbers 245 to 256 are CDRL3.

[0479] In addition, the CDR sequences of NYA-1163 are all identical to the CDR sequences of NYA-0001. Also, the CDR sequences of NYA-1143, NYA-2143, and NYA-2023 are identical, and CDRH1 is shown in sequence number 54 (Fig. 61), CDRH2 in sequence number 55 (Fig. 61), CDRH3 in sequence number 56 (Fig. 61), CDRL1 in sequence number 60 (Fig. 62), CDRL2 in sequence number 58 (Fig. 61), and CDRL3 in sequence number 59 (Fig. 61). In addition, the CDR sequences of NYA-2027 are shown as follows: CDRH1 at sequence number 54 (Fig. 61), CDRH2 at sequence number 55 (Fig. 61), CDRH3 at sequence number 56 (Fig. 61), CDRL1 at sequence number 57 (Fig. 61), CDRL2 at sequence number 58 (Fig. 61), and CDRL3 at sequence number 61 (Fig. 63).

[0480] In addition, NYA-1154 was designed by combining the binding variant sites observed during the screening of high-binding-ability clones, and a NYA-1154 expression vector with pcDNA3.3 (Thermo Fisher Scientific) as a backbone was constructed by introducing site-specific variants into the NYA-0001 expression vector. The nucleotide sequence of the constructed scFv expression vector was reanalyzed and confirmed to be the nucleotide sequence shown in Sequence No. 31 (Fig. 38). From the above nucleotide sequence, the full-length amino acid sequence of NYA-1154 encoded by the said sequence was identified (Sequence No. 32 (Fig. 39)). In the said sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 266th is NYA-1154, and the amino acid sequence from the 267th to the 292nd is the Flag-His tag. Also, the amino acid sequence from the 46th to the 53rd is CDRH1, the amino acid sequence from the 71st to the 78th is CDRH2, and the amino acid sequence from the 117th to the 129th is CDRH3. Also, amino acid numbers 181 to 188 are CDRL1, amino acid numbers 206 to 208 are CDRL2, and amino acid numbers 245 to 256 are CDRL3.

[0481] The CDR sequences of NYA-1154 are shown as follows: CDRH1 at sequence number 54 (Fig. 61), CDRH2 at sequence number 55 (Fig. 61), CDRH3 at sequence number 62 (Fig. 64), CDRL1 at sequence number 57 (Fig. 61), CDRL2 at sequence number 58 (Fig. 61), and CDRL3 at sequence number 63 (Fig. 64).

[0482] NYA-1163, NYA-2023, NYA-2027, NYA-1143, NYA-2143, and NYA-1154 were expressed in the same manner as in 1)-5-2, and each target scFv was purified. The purified protein samples were provided to an analytical SEC to determine purity and concentration, and then used in various assays.

[0483] 2)-3 NYA-1143 Variant Creation

[0484] 2)-3-1 Production of NYA-2035

[0485] NYA-2035 was designed as a variant of NYA-1143, and a NYA-2035 expression vector was constructed by introducing a site-specific variant into a NYA-1143 expression vector for mammalian cells. The nucleotide sequence of the constructed scFv expression vector was reanalyzed and confirmed to be the nucleotide sequence shown in Sequence No. 35 (Fig. 42). From the above nucleotide sequence, the full-length amino acid sequence of NYA-2035 encoded by the said sequence was determined (Sequence No. 36 (Fig. 43)). In the said sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 266th is NYA-2035, and the amino acid sequence from the 267th to the 292nd is the Flag-His tag. Also, the amino acid sequence from the 46th to the 53rd is CDRH1, the amino acid sequence from the 71st to the 78th is CDRH2, and the amino acid sequence from the 117th to the 129th is CDRH3. Also, amino acid numbers 181 to 188 are CDRL1, amino acid numbers 206 to 208 are CDRL2, and amino acid numbers 245 to 256 are CDRL3. The CDR sequences of NYA-2035 in the definition of CDR in IMGT are shown as follows: CDRH1 at sequence number 54 (Fig. 61), CDRH2 at sequence number 55 (Fig. 61), CDRH3 at sequence number 56 (Fig. 61), CDRL1 at sequence number 64 (Fig. 65), CDRL2 at sequence number 58 (Fig. 61), and CDRL3 at sequence number 59 (Fig. 61).

[0486] 2)-3-2 Production of the NYA-1143CDR Graft Variant

[0487] A graft variant of NYA-1143CDR was designed with the goal of improving physical properties. The sequences of the framework regions of VH and VL of NYA-1143 were compared with the framework regions of human subgroup consensus sequences or germline sequences established in KABAT et al. (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service National Institutes of Health, Bethesda, MD. (1991)). As a result, the human γ chain subgroup 3 consensus sequence IGHV3_30*15 was selected as the human germline sequence for VH, and the human germline sequence IGLV1-44*01 was selected as the human germline sequence IGLV1-44*01 for VL, as acceptors with high sequence identity in their framework regions. Next, the amino acid residues of the framework region of each acceptor were aligned with the amino acid residues of NYA-1143 to identify residues in which different amino acids were used.

[0488] Next, using a three-dimensional model of NYA-1143, framework residues to be incorporated into the acceptor were selected with reference to criteria provided by Queen et al. (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)). By the above method, NYA-1143-VH01, NYA-1143-VH02, and NYA-1143-VH03 were designed as CDR graft variant amino acid sequences for the VH portion of NYA-1143. The respective amino acid sequences are sequence numbers 37 (Fig. 44), 38 (Fig. 45), and 39 (Fig. 46). NYA-1143-VL01 was designed as a CDR graft variant amino acid sequence for the VL portion of NYA-1143. Each corresponding amino acid sequence is sequence number 40 (Fig. 47).

[0489] Each designed VH and VL sequence was combined to design various scFvs as shown below. A scFv in which the VH portion of NYA-1143 was substituted with the amino acid sequence of NYA-1143-VH01 was named NYA-2044. Additionally, a scFv in which the VL portion of NYA-2044 was substituted with the amino acid sequence of NYA-1143-VL01 was named NYA-2045.

[0490] scFv in which the VH portion of NYA-1143 was substituted with the amino acid sequence of NYA-1143-VH02 was named NYA-2047. Also, scFv in which the VL portion of NYA-2047 was substituted with the amino acid sequence of NYA-1143-VL01 was named NYA-2048.

[0491] scFv in which the VH portion of NYA-1143 was substituted with the amino acid sequence of NYA-1143-VH03 was named NYA-2060. Also, scFv in which the VL portion of NYA-2060 was substituted with the amino acid sequence of NYA-1143-VL01 was named NYA-2061.

[0492] Various designed NYA-1143CDR graft variants were synthesized entirely from DNA fragments (Pharmmark) and combined with the DNA fragments using the In-Fusion HD Cloning Kit (CLONTECH) to construct scFv expression vectors for mammalian cells based on pcDNA3.3 (Thermo Fisher Scientific).

[0493] The nucleotide sequence of the constructed scFv expression vector was reanalyzed and it was confirmed that the nucleotide sequence of the full length of NYA-2044 is the nucleotide sequence shown in sequence number 41 of the sequence table (Fig. 48).

[0494] It was confirmed that the full-length nucleotide sequence of NYA-2045 is the nucleotide sequence shown in sequence number 42 of the sequence table (Fig. 49).

[0495] It was confirmed that the full length nucleotide sequence of NYA-2047 is the nucleotide sequence shown in sequence number 43 of the sequence table (Fig. 50).

[0496] It was confirmed that the full length nucleotide sequence of NYA-2048 is the nucleotide sequence shown in sequence number 44 of the sequence table (Fig. 51).

[0497] It was confirmed that the full length nucleotide sequence of NYA-2060 is the nucleotide sequence shown in sequence number 45 of the sequence table (Fig. 52).

[0498] It was confirmed that the full-length nucleotide sequence of NYA-2061 is the nucleotide sequence shown in sequence number 46 of the sequence table (Fig. 53).

[0499] In addition, from the above nucleotide sequence, the full-length amino acid sequences of NYA-2044, NYA-2045, NYA-2047, NYA-2048, NYA-2060, and NYA-2061 encoded by the said sequence were determined. Also, the CDR sequence of NYA-2044, NYA-2045, NYA-2047, NYA-2048, NYA-2060, and NYA-2061 is the same as NYA-1143.

[0500] The full-length amino acid sequence of NYA-2044 is the amino acid sequence shown in sequence number 47 of the sequence table (Fig. 54). In the said sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 266th is NYA-2044, and the amino acid sequence from the 267th to the 292nd is the Flag-His tag. Also, the amino acid sequence from the 46th to the 53rd is CDRH1, the amino acid sequence from the 71st to the 78th is CDRH2, and the amino acid sequence from the 117th to the 129th is CDRH3. Also, amino acid numbers 181 to 188 are CDRL1, amino acid numbers 206 to 208 are CDRL2, and amino acid numbers 245 to 256 are CDRL3.

[0501] The full-length amino acid sequence of NYA-2045 is the amino acid sequence shown in sequence number 48 of the sequence table (Fig. 55). In the said sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 266th is NYA-2045, and the amino acid sequence from the 267th to the 292nd is the Flag-His tag. Also, the amino acid sequence from the 46th to the 53rd is CDRH1, the amino acid sequence from the 71st to the 78th is CDRH2, and the amino acid sequence from the 117th to the 129th is CDRH3. Also, amino acid numbers 181 to 188 are CDRL1, amino acid numbers 206 to 208 are CDRL2, and amino acid numbers 245 to 256 are CDRL3.

[0502] The full-length amino acid sequence of NYA-2047 is the amino acid sequence shown in sequence number 50 of the sequence table (Fig. 57). In the said sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 266th is NYA-2047, and the amino acid sequence from the 267th to the 292nd is the Flag-His tag. Also, the amino acid sequence from the 46th to the 53rd is CDRH1, the amino acid sequence from the 71st to the 78th is CDRH2, and the amino acid sequence from the 117th to the 129th is CDRH3. Also, amino acid numbers 181 to 188 are CDRL1, amino acid numbers 206 to 208 are CDRL2, and amino acid numbers 245 to 256 are CDRL3.

[0503] The full-length amino acid sequence of NYA-2048 is the amino acid sequence shown in sequence number 51 of the sequence table (Fig. 58). In the said sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 266th is NYA-2048, and the amino acid sequence from the 267th to the 292nd is the Flag-His tag. Also, the amino acid sequence from the 46th to the 53rd is CDRH1, the amino acid sequence from the 71st to the 78th is CDRH2, and the amino acid sequence from the 117th to the 129th is CDRH3. Also, amino acid numbers 181 to 188 are CDRL1, amino acid numbers 206 to 208 are CDRL2, and amino acid numbers 245 to 256 are CDRL3.

[0504] The full-length amino acid sequence of NYA-2060 is the amino acid sequence shown in sequence number 52 of the sequence table (Fig. 59). In the said sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 266th is NYA-2060, and the amino acid sequence from the 267th to the 292nd is the Flag-His tag. Also, the amino acid sequence from the 46th to the 53rd is CDRH1, the amino acid sequence from the 71st to the 78th is CDRH2, and the amino acid sequence from the 117th to the 129th is CDRH3. Also, amino acid numbers 181 to 188 are CDRL1, amino acid numbers 206 to 208 are CDRL2, and amino acid numbers 245 to 256 are CDRL3.

[0505] The full-length amino acid sequence of NYA-2061 is the amino acid sequence shown in sequence number 53 of the sequence table (Fig. 60). In the said sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 266th is NYA-2061, and the amino acid sequence from the 267th to the 292nd is the Flag-His tag. Also, the amino acid sequence from the 46th to the 53rd is CDRH1, the amino acid sequence from the 71st to the 78th is CDRH2, and the amino acid sequence from the 117th to the 129th is CDRH3. Also, amino acid numbers 181 to 188 are CDRL1, amino acid numbers 206 to 208 are CDRL2, and amino acid numbers 245 to 256 are CDRL3.

[0506] 2)-3-3 Expression and Purification of NYA-1143 Variant

[0507] NYA-2035, NYA-2044, NYA-2045, NYA-2047, NYA-2048, NYA-2060, and NYA-2061 produced in 2)-3-1 and 2)-3-2 were expressed in the same manner as in 1)-5-2, and each target scFv was purified. The purified protein samples were provided to an analytical SEC to determine their purity and concentration, and then used in various assays.

[0508] (Example 3) Production of scFv of anti-HLA / NY-ESO reference antibody

[0509] scFvs of antibodies 3M4E5 and T1 (WO2010 / 106431) with high binding ability to HLA / NY-ESO were designed, and the scFv of 3M4E5 was named NYC-0003 and the scFv of T1 was named NYC-0004.

[0510] All DNA fragments of NYC-0003 and NYC-0004 were synthesized (Thermo Fisher Scientific), and a scFv expression vector for mammalian cells with pcDNA3.3 (Thermo Fisher Scientific) as a backbone was constructed using the In-Fusion HD Cloning Kit (CLONTECH).

[0511] The nucleotide sequences of the constructed scFv expression vector were reanalyzed, and it was confirmed that the full-length nucleotide sequences of NYC-0003 and NYC-0004 are the nucleotide sequences shown in sequence number 65 (Fig. 66) and sequence number 66 (Fig. 67) of the sequence table. In addition, from the said nucleotide sequences, the full-length amino acid sequence of NYC-0003 encoded by the said sequence is sequence number 67 (Fig. 68), and the full-length amino acid sequence of NYC-0004 is the amino acid sequence shown in sequence number 68 (Fig. 69).

[0512] In the same manner as in 1)-5-2, NYC-0003 and NYC-0004 were expressed to purify each target scFv. Each purified protein sample was provided to an analytical SEC to determine its purity and concentration, after which it was used in various assays.

[0513] (Example 4) Evaluation of binding ability to HLA / NY-ESO by Biacore

[0514] Using a Biacore T200, anti-HLA / NY-ESO scFv was captured as a ligand on an immobilized anti-His antibody, and the antigen was measured using an analyzer. The HLA / NY-ESO prepared in 1)-1 was used as the antigen. The anti-His antibody (His Capture kit, GE Healthcare) was immobilized on the Sensor Chip CM5 (GE Healthcare) according to the kit instructions. Each anti-HLA / NY-ESO scFv to be evaluated, diluted to 0.5 µg / ml with HBS-EP+ (GE Healthcare), was immobilized by contacting it at a flow rate of 10 µl / min for 60 seconds. Subsequently, multiple concentrations of HLA / NY-ESO diluted with HBS-EP+ were added to each sample as an analyzer at a flow rate of 30 µl / min for 120 seconds, and K was measured by single-cycle kinetic analysis by measuring the discrepancy over 600 seconds. D was calculated, and the results are listed in Table 1. Compared to the affinity antibody NYA-0001, all variants were found to have stronger binding to HLA / NY-ESO. Furthermore, compared to NYC-0004, NYA-1143, NYA-2023, NYA-2143, NYA-2044, NYA-2045, NYA-2060, and NYA-2061 showed stronger binding, and among these, NYA-1143, NYA-2044, NYA-2045, and NYA-2143 K D It showed less than 1 nM.

[0515]

[0516] (Example 5) Recognition of recognition amino acids in NY-ESO peptide of anti-HLA / NY-ESO scFv

[0517] Human lymphoblast fusion cell line T2 (ATCC) cells were prepared in AIM-V medium containing 20% ​​FBS (Thermo Fisher Scientific) at appropriate concentrations, and NY-ESO peptide (SEQ No. 1), point variant NY-ESO peptides 1F, 2M, 3A, 4A, 5A, 6L, 7F, 8A, 9A (SEQ No. 121 (Fig. 122), 122 (Fig. 123), 123 (Fig. 124), 124 (Fig. 125), 125 (Fig. 126), 126 (Fig. 127), 127 (Fig. 128), 128 (Fig. 129), 129 (Fig. 130)), and gp100 peptide (SEQ No. 130 (Fig. 131)) (all Sigma Genosys) were diluted in 5 mM DMSO. The solution dissolved in [the liquid] was incubated at 37°C for 4 hours with the addition of 1 / 100 volume of DMSO to a final concentration of 50 μM. After washing twice with AIM-V medium containing 20% ​​FBS, the solution was adjusted to an appropriate concentration with PBS containing 5% FBS, LIVE / DEAD Fixable Dead Cell Stain Kit (Thermo Fisher Scientific) was added, and the mixture was incubated at 4°C for 30 minutes. After washing twice with PBS containing 5% FBS, the cells were divided into two groups and 10 [units of volume] was added with PBS containing 5% FBS. 5Cells were seeded into a 96-well U-bottom microplate, and the supernatant was removed after centrifugation. For one set of cells, 25 µl / well of anti-HLA / NY-ESO scFv diluted to 100 nM in PBS containing 5% FBS was added, and the mixture was incubated at 4°C for 30 minutes. After washing twice with PBS containing 5% FBS, 25 µl / well of Penta-His Alexa Fluor 488 (QIAGEN) diluted in PBS containing 5% FBS was added, and the mixture was incubated at 4°C for 30 minutes. After washing twice with PBS containing 5% FBS, 25 µl / well of Anti-mouse-IgG Alexa Fluor 488 (Thermo Fisher Scientific) diluted in PBS containing 5% FBS was added, and the mixture was incubated at 4°C for 30 minutes. After washing twice with PBS containing 5% FBS, the cells were fixed overnight with Mild Form 10 N (Fujifilm Wako Pure Chemicals) and resuspended in PBS containing 5% FBS. To standardize the amount of HLA / peptide complex, 25 µl / well of HLA-A2 antibody BB7.2-Alexa Fluor 488 or mouse IgG2b-Alexa Fluor 488, diluted to 10 µg / ml in PBS containing 5% FBS, was added to the other group of cells and incubated at 4°C for 30 minutes. After washing twice with PBS containing 5% FBS, the cells were fixed overnight with Mild Form 10 N (Fujifilm Wako Pure Chemicals) and resuspended in PBS containing 5% FBS. These cell suspensions were detected using a fluxometer (Canto II, Becton Dickinson). Data analysis was performed using Flowjo (Treestar) to measure the geometric mean fluorescence intensity (gMFI) of Alexa Fluor 488 in the T2 cell necrolysis fraction.The standardized gMFI, a value representing the binding affinity of each scFv standardized by the amount of HLA / peptide complex on T2 cells, was calculated using the following formula.

[0518] Standardized gMFI = (A / B) / ((C / D) / (E / F))

[0519] ㆍA / B = Relative gMFI

[0520] A: gMFI of antibody-added DMSO or each peptide-added T2 cell

[0521] B: gMFI of antibody-free DMSO or T2 cells with each peptide added

[0522] ㆍ(C / D) / (E / F) = Corrected value for HLA / peptide complex amount in T2 cells treated with DMSO or each peptide

[0523] C: gMFI of DMSO with HLA-A2 antibody or each peptide-added T2 cells

[0524] D: gMFI of T2 cells in DMSO with mouse IgG2b antibody or each peptide added

[0525] E: gMFI of DMSO-added T2 cells with HLA-A2 antibody

[0526] F: gMFI of DMSO-added T2 cells with mouse IgG2b antibody

[0527] As shown in Figure 1, anti-HLA / NY-ESO scFv NYA-0001, 1143, 2044, 2045, 2047, 2048, 2060, 2061 were shown to recognize the 1st, 4th, 5th, and 7th amino acids of the NY-ESO peptide, as the binding affinity to peptide addition T2, which introduces point mutations to the 1st, 4th, 5th, and 7th amino acids compared to the wild-type NY-ESO peptide, was reduced to less than half. Likewise, it was suggested that NYA-1154 recognizes the 1st and 5th, NYA-1163 recognizes the 1st, 3rd, 4th, 5th, 6th, and 7th, NYA-2023, 2027, and 2035 recognize the 1st, 4th, and 5th, and NYA-2143 recognizes the 1st, 5th, and 7th. Meanwhile, it was suggested that NYC-0003 and 0004 recognize the 4th and 5th.

[0528] (Example 6) Evaluation of Antigen Binding Specificity of Anti-HLA / NY-ESO scFv

[0529] In order to search for human peptides (hereinafter referred to as "homologous peptides") that have amino acid sequences similar but not identical to the NY-ESO peptide: SLLMWITQC (Sequence No. 1) among the human proteome (Swiss-Prot) that are likely to bind, we searched for 9-mer peptides with matching 1st, 4th, and 5th amino acids, which are suggested to be recognized by most of the antibodies. Regarding the searched 9-mer peptides, the 9-mer peptide shown in Figure 2a, which predicted binding affinity to HLA-A0201 using Net MHC Pa n2.8 and had an IC50 of 500 nM or less, was selected as the homologous peptide for evaluating the binding specificity of anti-HLA / NY-ESO scFv. T2 cells were prepared in AIM-V medium containing 20% ​​FBS (Thermo Fisher Scientific) at an appropriate concentration, and a solution was prepared by dissolving NY-ESO peptide (SEQ No. 1), homologous peptides DOLPP1, IL20RB, PRKD2, CD163, P2RY8 (SEQ No. 131 (Fig. 132), 132 (Fig. 133), 133 (Fig. 134), 134 (Fig. 135), 135 (Fig. 136)), and gp100 peptide (SEQ No. 130 (Fig. 131)) (all from Sigma Genosys) in DMSO to 5 mM, and DMSO was added in an amount of 1 / 100 to make the final concentration 50 μM, and the binding ability of each antibody was evaluated in the same manner as in Example 5. The standardized gMFI, a value representing the binding affinity of each scFv standardized by the amount of HLA / peptide complex on T2 cells, was calculated using the following formula.

[0530] Standardized gMFI = (A / B) / ((C / D) / (E / F))

[0531] ㆍA / B = Relative gMFI

[0532] A: gMFI of antibody-added DMSO or each peptide-added T2 cell

[0533] B: gMFI of antibody-free DMSO or T2 cells with each peptide added

[0534] ㆍ(C / D) / (E / F) = Corrected value for HLA / peptide complex amount in T2 cells treated with DMSO or each peptide

[0535] C: gMFI of DMSO with HLA-A2 antibody or each peptide-added T2 cells

[0536] D: gMFI of T2 cells in DMSO with mouse IgG2b antibody or each peptide added

[0537] E: gMFI of DMSO-added T2 cells with HLA-A2 antibody

[0538] F: gMFI of DMSO-added T2 cells with mouse IgG2b antibody

[0539] As shown in Figure 2b, anti-HLA / NY-ESO scFv NYA-0001, 1143, 1163, 2023, 2027, 2035, 2044, 2045, 2047, 2048, 2060, 2061, and 2143 did not bind to any homologous peptide-added cells, suggesting high specificity. On the other hand, NYA-1154, NYC-0003, and 0004 showed binding to some homologous peptide-added T2 cells.

[0540] (Example 7) Preparation of Fc addition-type anti-HLA / NY-ESO-anti-CD3 bispecific molecules

[0541] 7)-1 Fc Addition-Type Anti-HLA / NY-ESO-Anti-CD3 Bispecific Molecular Expression Vector Construction

[0542] 7)-1-1 taFv-heterodimer Fc-type bispecific molecular expression vector construction

[0543] To evaluate taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecules, expression vectors for each molecule were designed. For the anti-HLA / NY-ESO antibodies, NYA-1143, NYA-2143, NYA-1163, NYA-2023, NYA-2027, NYA-2035, NYA-2044, NYA-2045, NYA-2047, NYA-2048, NYA-2060, and NYA-2061 were used. For the anti-CD3 antibody, the humanized anti-CD3scFv C3E-7085 (WO2018 / 117237) was used. For the heterodimer Fc sequence, an Fc sequence (WO2014 / 190441) was used that introduced a mutation that degrades the effector function and also forms a heteromer.

[0544] A DNA fragment encoding Fc (HC1 or HC2) with a mutation that degrades effector function and also forms a heteromer was synthesized (Pharmmark), and an expression vector for mammalian cells with pcDNA3.3 (Thermo Fisher Scientific) as a backbone was constructed using the In-Fusion HD Cloning Kit (CLONTECH) and named "p_HC1".

[0545] An expression vector for mammalian cells was constructed by inserting a DNA fragment encoding HC2 into the carboxyl terminus of taFv, which is formed by linking NYA-1143 and C3E-7085 with a GGGGS linker, and was named "p_NYF-0016-HC2".

[0546] An expression vector for mammalian cells in which a DNA fragment encoding HC2 is inserted into the carboxyl terminus of taFv, in which NYA-2143 and C3E-7085 are linked by a GGGGS linker, was constructed by introducing a site-specific mutation into p_NYF-0016-HC2 and named "p_NYF-0019-HC2".

[0547] An expression vector for mammalian cells, in which a DNA fragment encoding HC2 is inserted into the carboxyl terminus of taFv, in which NYA-1163 and C3E-7085 are linked by a GGGGS linker, was constructed by replacing the nucleotide sequence portion encoding NYA-1143 of p_NYF-0016-HC2 with a DNA fragment encoding NYA-1163 using the In-Fusion HD Cloning Kit (CLONTECH), and was named "p_NYF-0022-HC2".

[0548] An expression vector for mammalian cells in which a DNA fragment encoding HC2 is inserted into the carboxyl terminus of taFv, in which NYA-2023 and C3E-7085 are linked by a GGGGS linker, was constructed by introducing a site-specific mutation into p_NYF-0016-HC2 and named "p_NYF-0023-HC2".

[0549] A mammalian cell expression vector in which a DNA fragment encoding HC2 is inserted into the carboxyl terminus of taFv, in which NYA-2027 and C3E-7085 are linked by a GGGGS linker, was constructed by introducing a site-specific mutation into a mammalian cell expression vector in which a DNA fragment encoding HC2 is inserted into the carboxyl terminus of taFv, in which NYA-0001 and C3E-7085 are linked by a GGGGS linker, and was named "p_NYF-0027-HC2".

[0550] An expression vector for mammalian cells in which a DNA fragment encoding HC2 is inserted into the carboxyl terminus of taFv, in which NYA-2035 and C3E-7085 are linked by a GGGGS linker, was constructed by introducing a site-specific mutation into p_NYF-0016-HC2 and named "p_NYF-0035-HC2".

[0551] An expression vector for mammalian cells, in which a DNA fragment encoding HC2 is inserted into the carboxyl terminus of taFv, which is formed by linking NYA-2044 and C3E-7085 with a GGGGS linker, was constructed by replacing the nucleotide sequence portion encoding NYA-1143 of p_NYF-0016-HC2 with a DNA fragment encoding NYA-2044 using the In-Fusion HD Cloning Kit (CLONTECH), and was named "p_NYF-0044-HC2".

[0552] An expression vector for mammalian cells, in which a DNA fragment encoding HC2 is inserted into the carboxyl terminus of taFv, which is formed by linking NYA-2045 and C3E-7085 with a GGGGS linker, was constructed by replacing the nucleotide sequence portion encoding NYA-1143 of p_NYF-0016-HC2 with a DNA fragment encoding NYA-2045 using the In-Fusion HD Cloning Kit (CLONTECH), and was named "p_NYF-0045-HC2".

[0553] An expression vector for mammalian cells, in which a DNA fragment encoding HC2 is inserted into the carboxyl terminus of taFv, in which NYA-2047 and C3E-7085 are linked by a GGGGS linker, was constructed by replacing the nucleotide sequence portion encoding NYA-1143 of p_NYF-0016-HC2 with a DNA fragment encoding NYA-2047 using the In-Fusion HD Cloning Kit (CLONTECH), and was named "p_NYF-0047-HC2".

[0554] An expression vector for mammalian cells, in which a DNA fragment encoding HC2 is inserted into the carboxyl terminus of taFv, which is formed by linking NYA-2048 and C3E-7085 with a GGGGS linker, was constructed by replacing the nucleotide sequence portion encoding NYA-1143 of p_NYF-0016-HC2 with a DNA fragment encoding NYA-2048 using the In-Fusion HD Cloning Kit (CLONTECH), and was named "p_NYF-0048-HC2".

[0555] An expression vector for mammalian cells in which a DNA fragment encoding HC2 is inserted into the carboxyl terminus of taFv, in which C3E-7085 is linked to NYA-2060 via a GGGGS linker, was constructed by introducing a site-specific mutation into p_NYF-0044-HC2 and named "p_NYF-0060-HC2".

[0556] An expression vector for mammalian cells in which a DNA fragment encoding HC2 is inserted into the carboxyl terminus of taFv, in which NYA-2061 and C3E-7085 are linked by a GGGGS linker, was constructed by introducing a site-specific mutation into p_NYF-0045-HC2 and named "p_NYF-0061-HC2".

[0557] The nucleotide sequence of p_HC1 was reanalyzed and it was confirmed that the nucleotide sequence of the entire length of HC1 is the nucleotide sequence shown in sequence number 71 of the sequence table (Fig. 72).

[0558] The nucleotide sequence of p_NYF-0016-HC2 was reanalyzed and it was confirmed that the nucleotide sequence of the full length of NYF-0016-HC2 is the nucleotide sequence shown in sequence number 72 of the sequence table (Fig. 73).

[0559] The nucleotide sequence of p_NYF-0019-HC2 was reanalyzed and it was confirmed that the nucleotide sequence of the full length of NYF-0019-HC2 is the nucleotide sequence shown in sequence number 73 of the sequence table (Fig. 74).

[0560] The nucleotide sequence of p_NYF-0022-HC2 was reanalyzed and it was confirmed that the nucleotide sequence of the full length of NYF-0022-HC2 is the nucleotide sequence shown in sequence number 74 of the sequence table (Fig. 75).

[0561] The nucleotide sequence of p_NYF-0023-HC2 was reanalyzed and it was confirmed that the nucleotide sequence of the full length of NYF-0023-HC2 is the nucleotide sequence shown in sequence number 75 of the sequence table (Fig. 76).

[0562] The nucleotide sequence of p_NYF-0027-HC2 was reanalyzed and it was confirmed that the nucleotide sequence of the full length of NYF-0027-HC2 is the nucleotide sequence shown in sequence number 76 of the sequence table (Fig. 77).

[0563] The nucleotide sequence of p_NYF-0035-HC2 was reanalyzed and it was confirmed that the nucleotide sequence of the full length of NYF-0035-HC2 is the nucleotide sequence shown in sequence number 77 of the sequence table (Fig. 78).

[0564] The nucleotide sequence of p_NYF-0044-HC2 was reanalyzed and it was confirmed that the nucleotide sequence of the full length of NYF-0044-HC2 is the nucleotide sequence shown in sequence number 78 of the sequence table (Fig. 79).

[0565] The nucleotide sequence of p_NYF-0045-HC2 was reanalyzed and it was confirmed that the nucleotide sequence of the full length of NYF-0045-HC2 is the nucleotide sequence shown in sequence number 79 of the sequence table (Fig. 80).

[0566] The nucleotide sequence of p_NYF-0047-HC2 was reanalyzed and it was confirmed that the nucleotide sequence of the full length of NYF-0047-HC2 is the nucleotide sequence shown in sequence number 80 of the sequence table (Fig. 81).

[0567] The nucleotide sequence of p_NYF-0048-HC2 was reanalyzed and it was confirmed that the nucleotide sequence of the full length of NYF-0048-HC2 is the nucleotide sequence shown in sequence number 81 of the sequence table (Fig. 82).

[0568] The nucleotide sequence of p_NYF-0060-HC2 was reanalyzed and it was confirmed that the nucleotide sequence of the full length of NYF-0060-HC2 is the nucleotide sequence shown in sequence number 82 of the sequence table (Fig. 83).

[0569] The nucleotide sequence of p_NYF-0061-HC2 was reanalyzed and it was confirmed that the nucleotide sequence of the full length of NYF-0061-HC2 is the nucleotide sequence shown in sequence number 83 of the sequence table (Fig. 84).

[0570] In addition, from the above nucleotide sequence, the amino acid sequences of the full length of HC1, NYF-0016-HC2, NYF-0019-HC2, NYF-0022-HC2, NYF-0023-HC2, NYF-0027-HC2, NYF-0035-HC2, NYF-0044-HC2, NYF-0045-HC2, NYF-0047-HC2, NYF-0048-HC2, NYF-0060-HC2, and NYF-0061-HC2 encoded by the said sequence were confirmed.

[0571] The amino acid sequence of the full length of HC1 is the amino acid sequence shown in sequence number 84 of the sequence table (Fig. 85). In the said sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, and the amino acid sequence from the 20th to the 246th is HC.

[0572] The full length amino acid sequence of NYF-0016-HC2 is the amino acid sequence shown in sequence number 85 of the sequence table (Fig. 86). In the said sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 511th is NYA-1143-C3E-7085taFv, the amino acid sequence from the 512th to the 513th is the linker, and the amino acid sequence from the 514th to the 745th is HC2.

[0573] The full length amino acid sequence of NYF-0019-HC2 is the amino acid sequence shown in sequence number 86 of the sequence table (Fig. 87). In the said sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 511th is NYA-2143-C3E-7085taFv, the amino acid sequence from the 512th to the 513th is the linker, and the amino acid sequence from the 514th to the 745th is HC2.

[0574] The full length amino acid sequence of NYF-0022-HC2 is the amino acid sequence shown in sequence number 87 of the sequence table (Fig. 88). In the said sequence, the 1st to 19th amino acid sequence is the signal sequence, the 21st to 511th amino acid sequence is NYA-1163-C3E-7085taFv, the 512th to 513th amino acid sequence is the linker, and the 514th to 745th amino acid sequence is HC2.

[0575] The full length amino acid sequence of NYF-0023-HC2 is the amino acid sequence shown in sequence number 88 of the sequence table (Fig. 89). In the said sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 511th is NYA-2023-C3E-7085taFv, the amino acid sequence from the 512th to the 513th is the linker, and the amino acid sequence from the 514th to the 745th is HC2.

[0576] The full length amino acid sequence of NYF-0027-HC2 is the amino acid sequence shown in sequence number 89 of the sequence table (Fig. 90). In the said sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 511th is NYA-2027-C3E-7085taFv, the amino acid sequence from the 512th to the 513th is the linker, and the amino acid sequence from the 514th to the 745th is HC2.

[0577] The full length amino acid sequence of NYF-0035-HC2 is the amino acid sequence shown in sequence number 90 of the sequence table (Fig. 91). In the said sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 511th is NYA-2035-C3E-7085taFv, the amino acid sequence from the 512th to the 513th is the linker, and the amino acid sequence from the 514th to the 745th is HC2.

[0578] The full length amino acid sequence of NYF-0044-HC2 is the amino acid sequence shown in sequence number 91 of the sequence table (Fig. 92). In the said sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 511th is NYA-2044-C3E-7085taFv, the amino acid sequence from the 512th to the 513th is the linker, and the amino acid sequence from the 514th to the 745th is HC2.

[0579] The full length amino acid sequence of NYF-0045-HC2 is the amino acid sequence shown in sequence number 92 of the sequence table (Fig. 93). In the said sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 511th is NYA-2045-C3E-7085taFv, the amino acid sequence from the 512th to the 513th is the linker, and the amino acid sequence from the 514th to the 745th is HC2.

[0580] The full length amino acid sequence of NYF-0047-HC2 is the amino acid sequence shown in sequence number 93 of the sequence table (Fig. 94). In the said sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 511th is NYA-2047-C3E-7085taFv, the amino acid sequence from the 512th to the 513th is the linker, and the amino acid sequence from the 514th to the 745th is HC2.

[0581] The full length amino acid sequence of NYF-0048-HC2 is the amino acid sequence shown in sequence number 94 of the sequence table (Fig. 95). In the said sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 511th is NYA-2048-C3E-7085taFv, the amino acid sequence from the 512th to the 513th is the linker, and the amino acid sequence from the 514th to the 745th is HC2.

[0582] The full length amino acid sequence of NYF-0060-HC2 is the amino acid sequence shown in sequence number 95 of the sequence table (Fig. 96). In the said sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 511th is NYA-2060-C3E-7085taFv, the amino acid sequence from the 512th to the 513th is the linker, and the amino acid sequence from the 514th to the 745th is HC2.

[0583] The full length amino acid sequence of NYF-0061-HC2 is the amino acid sequence shown in sequence number 96 of the sequence table (Fig. 97). In the said sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 511th is NYA-2061-C3E-7085taFv, the amino acid sequence from the 512th to the 513th is the linker, and the amino acid sequence from the 514th to the 745th is HC2.

[0584] 7)-1-2 taFv-Fab-heterodimer Fc-type bispecific molecular expression vector construction

[0585] A vector for the expression of taFv-Fab-heterodimer Fc type anti-HLA / NY-ESO-anti-CD3 bispecific molecules was designed. NYA-0001 was used as the anti-HLA / NY-ESO antibody. C3E-7085 (WO2018 / 117237), a humanized anti-CD3scFv antibody, was used as the anti-CD3 antibody. For the heterodimer Fc sequence, an Fc sequence (WO2014 / 190441) was used that introduced a mutation that degrades effector function and also forms a heteromer.

[0586] An expression vector for mammalian cells was constructed by inserting a DNA fragment encoding the heavy chain variable region of NYA-0001, the human IgG-derived CH1 region, and the HC1-k delete, which degrades effector function, and was named "p_NYA-0001-Fab-HC1-k delete". Additionally, an expression vector for mammalian cells was constructed by inserting a DNA fragment encoding the light chain variable region of NYA-0001 and the human IgG-derived CL region, and was named "p_NYA-0001-LC".

[0587] The nucleotide sequence of p_NYA-0001-Fab-HC1-k delete was reanalyzed, and it was confirmed that the nucleotide sequence of the full length of NYA-0001-Fab-HC1-k delete is the nucleotide sequence shown in sequence number 97 of the sequence table (Fig. 98).

[0588] The nucleotide sequence of p_NYA-0001-LC was reanalyzed and it was confirmed that the nucleotide sequence of the full length of NYA-0001-LC is the nucleotide sequence shown in sequence number 98 of the sequence table (Fig. 99).

[0589] In addition, from the above nucleotide sequence, the amino acid sequences of the full length of NYA-0001-Fab-HC1-k delete and NYA-0001-LC encoded by the said sequence were identified.

[0590] The amino acid sequence of the full length of NYA-0001-Fab-HC1-k delete is the amino acid sequence shown in sequence number 99 of the sequence table (Fig. 100). In the said sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 20th to the 139th is the variable region, and the amino acid sequence from the 140th to the 468th is the normal region. Also, the amino acid sequence from the 45th to the 52nd is CDRH1 (sequence number 54 (Fig. 61)), the amino acid sequence from the 70th to the 77th is CDRH2 (sequence number 55 (Fig. 61)), and the amino acid sequence from the 116th to the 128th is CDRH3 (sequence number 56 (Fig. 61)).

[0591] The amino acid sequence of the full length of NYA-0001-LC is the amino acid sequence shown in sequence number 100 of the sequence table (Fig. 101). The amino acid sequence from the 1st to the 20th is the signal sequence, the amino acid sequence from the 21st to the 131st is the variable region, and the amino acid sequence from the 132nd to the 237th is the normal region. Also, amino acid numbers 46 to 53 are CDRL1 (sequence number 57 (Fig. 61)), amino acid numbers 71 to 73 are CDRL2 (sequence number 58 (Fig. 61)), and amino acid numbers 110 to 121 are CDRL3 (sequence number 59 (Fig. 61)).

[0592] 7)-2 Fc addition-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule expression

[0593] 7)-2-1 Expression of taFv-heterodimer Fc-type bispecific molecules

[0594] Expi293F cells (Thermo Fisher Scientific) were subcultured according to the manual. 2.5 × 10⁶ of the Expi293F cell culture medium in the logarithmic growth phase 6The solution was diluted with Expi293 Expression medium (Thermo Fisher Scientific) to a cell / mL ratio and used for the production of various bispecific molecules. A vector mixture of vectors p_NYF-0016-HC2 and p_HC1 mixed in a ratio of 1:1.5, along with 0.3 mg and 0.9 mg of Polyethyleneimine (Polyscience #24765), was added to 20 mL of Opti-Pro SFM medium (Thermo Fisher Scientific), stirred slowly, and then added to Expi293F cells after standing for 5 minutes. The culture supernatant obtained by shaking culture at 135 rpm for 6 days in an 8% CO2 incubator at 37 ℃ was filtered through a 0.2 µm filter (Millopore) to obtain the culture supernatant of the taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0016). The amino acid sequences obtained by expressing each vector constituting NYF-0016 are shown in sequence numbers 85 (Fig. 86) and 84 (Fig. 85) of the sequence table.

[0595] In the same manner, the culture supernatant of the taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0019) was prepared for expression using p_NYF-0019-HC2 and p_HC1. The amino acid sequences obtained by expressing each vector constituting NYF-0019 are shown in sequence numbers 86 (Fig. 87) and 84 (Fig. 85) of the sequence table.

[0596] The culture supernatant of the taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0022) was prepared for expression using p_NYF-0022-HC2 and p_HC1. The amino acid sequences obtained by expressing each vector constituting NYF-0022 are shown in sequence numbers 87 (Fig. 88) and 84 (Fig. 85) of the sequence table.

[0597] The culture supernatant of the taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0023) was prepared for expression using p_NYF-0023-HC2 and p_HC1. The amino acid sequences obtained by expressing each vector constituting NYF-0023 are shown in sequence numbers 88 (Fig. 89) and 84 (Fig. 85) of the sequence table.

[0598] The culture supernatant of the taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0027) was prepared for expression using p_NYF-0027-HC2 and p_HC1. The amino acid sequences obtained by expressing each vector constituting NYF-0027 are shown in sequence numbers 89 (Fig. 90) and 84 (Fig. 85) of the sequence table.

[0599] The culture supernatant of the taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0035) was prepared for expression using p_NYF-0035-HC2 and p_HC1. The amino acid sequences obtained by expressing each vector constituting NYF-0035 are shown in sequence numbers 90 (Fig. 91) and 84 (Fig. 85) of the sequence table.

[0600] The culture supernatant of the taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0044) was prepared for expression using p_NYF-0044-HC2 and p_HC1. The amino acid sequences obtained by expressing each vector constituting NYF-0044 are shown in sequence numbers 91 (Fig. 92) and 84 (Fig. 85) of the sequence table.

[0601] The culture supernatant of the taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0045) was prepared for expression using p_NYF-0045-HC2 and p_HC1. The amino acid sequences obtained by expressing each vector constituting NYF-0045 are shown in sequence numbers 92 (Fig. 93) and 84 (Fig. 85) of the sequence table.

[0602] The culture supernatant of the taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0047) was prepared for expression using p_NYF-0047-HC2 and p_HC1. The amino acid sequences obtained by expressing each vector constituting NYF-0047 are shown in sequence numbers 93 (Fig. 94) and 84 (Fig. 85) of the sequence table.

[0603] The culture supernatant of the taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0048) was prepared for expression using p_NYF-0048-HC2 and p_HC1. The amino acid sequences obtained by expressing each vector constituting NYF-0048 are shown in sequence numbers 94 (Fig. 95) and 84 (Fig. 85) of the sequence table.

[0604] The culture supernatant of the taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0060) was prepared for expression using p_NYF-0060-HC2 and p_HC1. The amino acid sequences obtained by expressing each vector constituting NYF-0060 are shown in sequence numbers 95 (Fig. 96) and 84 (Fig. 85) of the sequence table.

[0605] The culture supernatant of the taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0061) was prepared for expression using p_NYF-0061-HC2 and p_HC1. The amino acid sequences obtained by expressing each vector constituting NYF-0061 are shown in sequence numbers 96 (Fig. 97) and 84 (Fig. 85) of the sequence table.

[0606] 7)-2-2 Expression of taFv-Fab-heterodimer Fc-type bispecific molecules

[0607] 7)-2-1 Using the same method as in 7)-2-1, a vector mixture was prepared by mixing p_NYF-0023-HC2, p_NYA-0001-Fab-HC1-k delete, and p_NYA-0001-LC in a ratio of 1:1:1.5 to prepare an expression supernatant of the taFv-Fab-heterodimer Fc-type bispecific molecule (NYF-0058). The amino acid sequences obtained by expressing each vector constituting NYF-0058 are shown in amino acid numbers 20 to 745 of sequence number 88 (Fig. 89), amino acid numbers 20 to 468 of sequence number 99 (Fig. 100), and amino acid numbers 21 to 237 of sequence number 100 (Fig. 101).

[0608] 7)-3 Fc Addition-Type Anti-HLA / NY-ESO-Anti-CD3 Bispecific Molecule Purification

[0609] Various bispecific molecules from the culture supernatant obtained in 7)-2 were purified in a two-step process of Protein A affinity chromatography and gel filtration chromatography.

[0610] The culture supernatant was applied to a MabSelectSuRe column (GE Healthcare Bioscience; also simply referred to as "GE Healthcare") equilibrated to PBS pH 7.4 to adsorb the target bispecific molecule. After removing the non-adsorbed component with PBS, the adsorbed component was eluted with acetic acid buffer pH 3.5. The eluted fraction was concentrated after adjusting the pH to neutral with Tris buffer pH 9.5, and then fed to a gel filtration column Superdex 200 10 / 300 (GE Healthcare Bioscience) equilibrated with 25 mM Histidine, 300 mM NaCl, 5% sorbitol, and pH 5.5. From the peak fractions obtained by gel filtration chromatography, the fraction corresponding to the target heterodimer was recovered, and the formation of the target anti-HLA / NY-ESO-anti-CD3 bispecific molecule was confirmed by SDS-polyacrylamide electrophoresis (SDS-PAGE). The purified protein sample was fed into an analytical SEC to determine its purity and concentration, and then used for various evaluations.

[0611] (Example 8) Evaluation of the cell injury activity of an Fc addition-type anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule

[0612] 8)-1 Preparation of Target Cells

[0613] Endogenous human NY-ESO expressing cell lines (U266B1, and NCI-H1703), and endogenous human NY-ESO non-expressing cell lines (AGS, and CFPAC-1) were prepared in RPMI1640 medium containing 10% FBS (Fujifilm Wako Pure Pharmaceutical Co., Ltd.) at a rate of 1 × 10⁶ 6The cells were adjusted to a concentration of cells / mL, 100 µL of Chromium-51 Radionuclide (Perkin Elmer) was added to 1 mL of each cell suspension, and the cells were incubated for 2 hours under conditions of 37°C and 5% CO2. After washing twice with RPMI1640 medium containing 10% FBS, the cells were resuspended in RPMI1640 medium containing 10% FBS to a concentration of 1 × 10⁵ cells / mL and used as target cells.

[0614] 8)-2 Preparation of Effector Cells

[0615] Thaw commercially available frozen human PBMC (Cellular Technology Limited) at 37°C, transfer to a solution of Anti-aggregateWash reagent (Cellular Technology Limited) added to RPMI1640 medium containing 10% FBS and wash twice, then transfer 1 × 10⁶ of RPMI1640 medium containing 10% FBS. 6 The solution was prepared to a cell / mL ratio and used as an effector cell.

[0616] 8)-3 Cell injury assay

[0617] Each target cell obtained in 8)-1 was added to a 96-well U-bottom microplate at a rate of 50 µl / well. Various Fc-addition type anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules prepared in Example 7, adjusted to various concentrations, were added at a rate of 50 µl / well. Additionally, 100 µl / well of effector cells prepared in Example 8)-2 were added. After centrifugation at 1000 rpm for 1 minute at room temperature, the cells were incubated for 20 to 24 hours under conditions of 37°C and 5% CO2. 50 µl of the supernatant was collected in a LumaPlate (Perkin Elmer), dried at 50°C for approximately 2 hours, and then measured using a plate reader (TopCount: Perkin Elmer). The test was performed in triplicate, and the cell lysis rate was calculated using the following formula.

[0618] Cell lysis rate (%) = (AB) / (CB) × 100

[0619] A: Count of sample wells.

[0620] B: Average count of background (wells without antibody addition) (n = 3). 50 µl of assay medium was added when antibodies were added. Otherwise, the same procedure as for the sample wells was performed. C: Average count of maximum release (wells with target cells lysed with surfactant) (n = 3). 50 µl of assay medium was added when antibodies were added. 100 µl of surfactant was added, and 50 µl was transferred to a LumaPlate for measurement, just as with the sample wells.

[0621] As shown in Figures 4a–f, the cytotoxic activity of various anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules was observed against endogenous human NY-ESO-expressing cell lines (U266B1 and NCI-H1703). EC was calculated using the Four Pa-rameter Logistics Curve formula of the analysis software (Sigmaplot, version 12.0).50 The results were calculated, and the results for the U266B1 cell line are listed in Table 2, and the results for the NCI-H1703 cell line are listed in Table 3. Not Applicable (NA) indicates that the correlation coefficient R value was not calculated and curve fitting could not be performed. Meanwhile, as shown in Figure 4g-l, no cell injury activity was confirmed against endogenous human NY-ESO non-expressing cell lines (AGS, and CFPAC-1).

[0622]

[0623]

[0624] (Example 9) Evaluation of in vivo activity of Fc addition-type anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule in a human PBMC influx model

[0625] 6 × 10⁶ human squamous cell lung cancer cell line NCI-H1703 (ATCC) in PBS containing 50% Matrigel (CORNING) 7 The solution was prepared to a cell / mL concentration, and 0.1 mL was implanted subcutaneously into NOG mice (magnetic, 6–7 weeks old) (Day 0). On Day 4, human PBMC was diluted with PBS at a concentration of 3.75 × 10⁶ 7 The solution was prepared to a cell / mL concentration and 0.2 mL was transplanted into the tail vein. Starting about 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 formula shown below.

[0626] Estimated Tumor Volume (mm³) = Average of the estimated tumor volumes for each individual

[0627] Estimated tumor volume for each individual = Major axis × Minor axis 2 / 2

[0628] On Day 14, groups were divided based on tumor volume so that n = 5 to 6 for each group, and various anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules were administered intratail vein (1 mg / kg, NYF-0058 at 1.5 mg / kg for comparison at the same molar amount). Administration was performed on Day 14, Day 21, and Day 28. Antitumor effects were confirmed in the groups administered various anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules (Figs. 5a to c). The tumor growth inhibition rate (%) on Days 31 to 32 was calculated using the formula shown below and is listed in Table 4.

[0629] Tumor growth inhibition rate (%) = 100 - (Estimated tumor volume of each group / Estimated tumor volume of vehicle control × 100)

[0630]

[0631] (Example 10) Preparation of various Fc addition-type format anti-HLA / NY-ESO-anti-CD3 bispecific molecules

[0632] 10)-1 Construction of various Fc addition format anti-HLA / NY-ESO-anti-CD3 bispecific molecular expression vectors

[0633] 10)-1-1 Hybrid type bispecific molecular expression vector construction

[0634] To evaluate hybrid anti-HLA / NY-ESO-anti-CD3 bispecific molecules, expression vectors for each molecule were designed. NYA-1143 was used as the anti-HLA / NY-ESO antibody. C3E-7085 (PCT / JP2017 / 046006), a humanized anti-CD3scFv, was used as the anti-CD3 antibody. For the heterodimer Fc sequence, an Fc sequence (WO2014 / 190441) was used that introduced a mutation that degrades effector function and also forms a heterodimer.

[0635] An expression vector for mammalian cells was constructed by inserting a DNA fragment encoding the heavy chain variable region of NYA-1143, the human IgG-derived CH1 region, and the Fc region, into which a mutation that degrades effector function and also forms heteromers was introduced, and was named "p_NYA-1143-Fab-HC1-k delete". In addition, an expression vector for mammalian cells was constructed by inserting a DNA fragment encoding the light chain variable region of NYA-1143 and the human IgG-derived CL region, and was named "p_NYA-1143-LC". In addition, an expression vector for mammalian cells "p_C3E-7085-HC2-k delete" was constructed by inserting a DNA fragment encoding the Fc region, into which a mutation that degrades effector function and forms heteromers was introduced at the carboxyl terminus of humanized anti-CD3scFv (C3E-7085).

[0636] The nucleotide sequence of p_NYA-1143-Fab-HC1-k delete was reanalyzed, and it was confirmed that the nucleotide sequence of the full length of NYA-1143-Fab-HC1-k delete is the nucleotide sequence shown in sequence number 101 of the sequence table (Fig. 102).

[0637] The nucleotide sequence of p_NYA-1143-LC was reanalyzed and it was confirmed that the nucleotide sequence of the full length of NYA-1143-LC is the nucleotide sequence shown in sequence number 102 of the sequence table (Fig. 103).

[0638] The nucleotide sequence of p_C3E-7085-HC2-k delete was reanalyzed, and it was confirmed that the nucleotide sequence of the full length of C3E-7085-HC2-k delete is the nucleotide sequence shown in sequence number 103 of the sequence table (Fig. 104).

[0639] In addition, from the above nucleotide sequence, the full-length amino acid sequences of NYA-1143-Fab-HC1-k delete, NYA-1143-LC, and C3E-7085-HC2-k delete encoded by the said sequence were identified.

[0640] The amino acid sequence of the full length of NYA-1143-Fab-HC1-k delete is the amino acid sequence shown in sequence number 104 of the sequence table (Fig. 105). In the said sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 139th is the variable region, and the amino acid sequence from the 140th to the 468th is the normal region. Also, the amino acid sequence from the 45th to the 52nd is CDRH1 (sequence number 54 (Fig. 61)), the amino acid sequence from the 70th to the 77th is CDRH2 (sequence number 55 (Fig. 61)), and the amino acid sequence from the 116th to the 128th is CDRH3 (sequence number 56 (Fig. 61)).

[0641] The amino acid sequence of the full length of NYA-1143-LC is the amino acid sequence shown in sequence number 105 of the sequence table (Fig. 106). The amino acid sequence from the 1st to the 20th is the signal sequence, the amino acid sequence from the 21st to the 131st is the variable region, and the amino acid sequence from the 132nd to the 237th is the normal region. Also, amino acid numbers 46 to 53 are CDRL1 (sequence number 60 (Fig. 62)), amino acid numbers 71 to 73 are CDRL2 (sequence number 58 (Fig. 61)), and amino acid numbers 110 to 121 are CDRL3 (sequence number 59 (Fig. 61)).

[0642] The full-length amino acid sequence of C3E-7085-HC2-k delete is the amino acid sequence shown in sequence number 106 of the sequence table (Fig. 107). The amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 260th is C3E-7085, the amino acid sequence from the 261st to the 262nd is the linker, and the amino acid sequence from the 263rd to the 493rd is Fc, which introduces a mutation that degrades the effector function and also forms a heterodimer.

[0643] 10)-1-2 Construction of Dual-Type Bispecific Molecular Expression Vectors

[0644] To evaluate dual-type anti-HLA / NY-ESO-anti-CD3 dual-specific molecules, the following expression vector was designed. An expression vector for mammalian cells, "p_NYA-1143-HC1-k delete," was constructed by inserting a DNA fragment encoding an Fc region into which a mutation was introduced at the carboxyl terminus of NYA-1143 to reduce effector function and form a heteromer.

[0645] The nucleotide sequence of p_NYA-1143-HC1-k delete was reanalyzed, and it was confirmed that the nucleotide sequence of the full length of NYA-1143-HC1-k delete is the nucleotide sequence shown in sequence number 107 of the sequence table (Fig. 108). In addition, from the said nucleotide sequence, the amino acid sequence of the full length of NYA-1143-HC1-k delete encoded by said sequence was confirmed. The amino acid sequence of the full length of NYA-1143-HC1-k delete is the amino acid sequence shown in sequence number 108 of the sequence table (Fig. 109). In the sequence, the 1st to 19th amino acid sequence is the signal sequence, the 21st to 266th amino acid sequence is NYA-1143, the 267th to 268th amino acid sequence is the linker, and the 269th to 499th amino acid sequence is Fc that introduces a mutation that reduces the effector function and also forms a heterodimer.

[0646] 10)-1-3 scFv-Fab-heterodimer Fc-type bispecific molecular expression vector construction

[0647] To evaluate scFv-Fab-heterodimer Fc type anti-HLA / NY-ESO-anti-CD3 bispecific molecules, expression vectors for each molecule were designed. NYA-1154 was used as the anti-HLA / NY-ESO antibody and C3E-7085 was used as the anti-CD3 antibody. For the heterodimer Fc sequence, an Fc sequence was used that introduced a mutation that degrades effector function and also forms a heteromer.

[0648] An expression vector for mammalian cells was constructed by inserting a DNA fragment encoding the NYA-1154 medium chain variable region, the human IgG-derived CH1 region, and the Fc region, which introduces a mutation that degrades effector function and also forms heteromers, into the carboxyl terminus of C3E-7085, and was named "p_C3E-7085-NYA-1154-Fab-HC2-k delete". Additionally, an expression vector for mammalian cells was constructed by inserting a DNA fragment encoding the NYA-1154 light chain variable region and the human IgG-derived CL region, and was named "p_NYA-1154-LC". Additionally, an expression vector for mammalian cells was constructed by inserting a DNA fragment encoding the HC1-k delete, and was named "p_OAA-HC1-k delete".

[0649] The nucleotide sequence of p_C3E-7085-NYA-1154-Fab-HC2-k delete was reanalyzed, and it was confirmed that the nucleotide sequence of the full length of C3E-7085-NYA-1154-Fab-HC2-k delete is the nucleotide sequence shown in sequence number 109 of the sequence table (Fig. 110).

[0650] The nucleotide sequence of p_NYA-1154-LC was reanalyzed and it was confirmed that the nucleotide sequence of the full length of NYA-1154-LC is the nucleotide sequence shown in sequence number 110 of the sequence table (Fig. 111).

[0651] The nucleotide sequence of p_OAA-HC1-k delete was reanalyzed and it was confirmed that the nucleotide sequence of the full length of OAA-HC1-k delete is the nucleotide sequence shown in sequence number 111 of the sequence table (Fig. 112).

[0652] In addition, from the above nucleotide sequence, the full-length amino acid sequences of C3E-7085-NYA-1154-Fab-HC2-k delete, NYA-1154-LC, and OAA-HC1-k delete encoded by the said sequence were identified.

[0653] The full-length amino acid sequence of C3E-7085-NYA-1154-Fab-HC2-k delete is the amino acid sequence shown in sequence number 112 of the sequence table (Fig. 113). The amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 260th is C3E-7085, the amino acid sequence from the 261st to the 385th is the linker, and the amino acid sequence from the 266th to the 385th is the NYA-1154-heavy chain variable region. Also, the amino acid sequence from amino acid number 386 to the 714th is HC2-k delete.

[0654] The amino acid sequence of the full length of NYA-1154-LC is the amino acid sequence shown in sequence number 113 of the sequence table (Fig. 114). The amino acid sequence from the 1st to the 20th is the signal sequence, the amino acid sequence from the 21st to the 131st is the variable region, and the amino acid sequence from the 132nd to the 237th is the normal region. Also, amino acid numbers 46 to 53 are CDRL1 (sequence number 57 (Fig. 61)), amino acid numbers 71 to 73 are CDRL2 (sequence number 58 (Fig. 61)), and amino acid numbers 110 to 121 are CDRL3 (sequence number 63 (Fig. 64)).

[0655] The full-length amino acid sequence of OAA-HC1-k delete is the amino acid sequence shown in sequence number 114 of the sequence table (Fig. 115). In the said sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, and the amino acid sequence from the 20th to the 245th is OAA-HC1-k delete.

[0656] 10)-1-4 Preparation of taFv-heterodimer Fc-type bispecific molecular expression vectors for evaluation of various formats

[0657] To evaluate taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecules for evaluation in various formats, expression vectors for each molecule were designed. NYA-1143 and NYA-1154 were used as anti-HLA / NY-ESO antibodies. C3E-7085, a humanized anti-CD3scFv, was used as the anti-CD3 antibody. For the heterodimer Fc sequence, an Fc sequence was used that introduced a mutation that degrades effector function and also forms a heteromer.

[0658] An expression vector for mammalian cells was constructed by inserting a DNA fragment encoding HC2-k delete into the carboxyl terminus of taFv, which is formed by linking NYA-1154 and C3E-7085 with a GGGGS linker, and was named "p_NYF-0010-HC2-k delete".

[0659] An expression vector for mammalian cells was constructed by inserting a DNA fragment encoding HC2-k delete into the carboxyl terminus of taFv, which was formed by linking NYA-1154 to C3E-7085 using a GGGGS linker, and was named "p_NYF-0004-HC2-k delete".

[0660] An expression vector for mammalian cells was constructed by inserting a DNA fragment encoding HC2-k delete into the carboxyl terminus of taFv, which is formed by linking NYA-1143 and C3E-7085 with a GGGGS linker, and was named "p_NYF-0011-HC2-k delete".

[0661] The nucleotide sequence of p_NYF-0010-HC2-k delete was reanalyzed and it was confirmed that the nucleotide sequence of the full length of NYF-0010-HC2-k delete is the nucleotide sequence shown in sequence number 115 of the sequence table (Fig. 116).

[0662] The nucleotide sequence of p_NYF-0004-HC2-k delete was reanalyzed and it was confirmed that the nucleotide sequence of the full length of NYF-0004-HC2-k delete is the nucleotide sequence shown in sequence number 116 of the sequence table (Fig. 117).

[0663] The nucleotide sequence of p_NYF-0011-HC2-k delete was reanalyzed and it was confirmed that the nucleotide sequence of the full length of NYF-0011-HC2-k delete is the nucleotide sequence shown in sequence number 117 of the sequence table (Fig. 118).

[0664] In addition, from the above nucleotide sequence, the amino acid sequences of the full length of NYF-0010-HC2-k delete, NYF-0004-HC2-k delete and NYF-0011-HC2-k delete encoded by the said sequence were identified.

[0665] The full-length amino acid sequence of NYF-0010-HC2-k delete is the amino acid sequence shown in sequence number 118 of the sequence table (Fig. 119). In the said sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 511th is NYA-1154-C3E-7085taFv, the amino acid sequence from the 512th to the 513th is the linker, and the amino acid sequence from the 514th to the 744th is HC2-k delete.

[0666] The full-length amino acid sequence of NYF-0004-HC2-k delete is the amino acid sequence shown in sequence number 119 of the sequence table (Fig. 120). In the said sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 511th is C3E-7085-NYA-1154taFv, the amino acid sequence...

Claims

Claim 1 An antibody or binding fragment that specifically binds to human HLA / NY-ESO, comprising a heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 53 and a light chain variable region consisting of the 156th to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 53, or a heavy chain variable region consisting of the 21st to 140th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 156 and a light chain variable region consisting of the 161st to 271st amino acid sequence of the amino acid sequence represented by SEQ ID NO.

156. Claim 2 In claim 1, an antibody or its binding fragment that is scFv. Claim 3 In claim 2, a scFv consisting of the 21st to 266th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 53, or a scFv consisting of the 21st to 271st amino acid sequence of the amino acid sequence represented by SEQ ID NO. 156, a phosphorus, an antibody, or a conjugated fragment thereof. Claim 4 A polynucleotide encoding the antibody or its binding fragment described in claim 1. Claim 5 A vector comprising the polynucleotide described in claim 4. Claim 6 An isolated host cell comprising the polynucleotide described in claim 4 or a vector comprising the polynucleotide described in claim 4. Claim 7 A method for producing an antibody or its binding fragment that specifically binds to human HLA / NY-ESO, comprising: (i) a process of culturing host cells as described in claim 6; and (ii) a process of purifying the antibody or its binding fragment from the culture obtained in process (i). Claim 8 An antibody or its binding fragment that specifically binds to human HLA / NY-ESO, obtained by the method described in claim 7. Claim 9 A pharmaceutical composition that is an anticancer agent, comprising as an active ingredient an antibody or its binding fragment described in any one of claims 1 to 3 and 8, a polynucleotide described in claim 4, a vector described in claim 5, or a cell described in claim 6. Claim 10 A molecule that is a bispecific antibody comprising the antibody or binding fragment described in claim 1 that specifically binds to human HLA / NY-ESO, and the antibody or binding fragment that specifically binds to CD3, wherein the antibody or binding fragment that specifically binds to CD3 comprises: (CCH1) a heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO. 141; (CCH2) a heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO. 142, or a heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO. 142, wherein the third amino acid is N or S; (CCH3) a heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO. 143; (CCL1) a light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO. 144; (CCL2) a light chain CDRL2 consisting of the amino acid sequence represented by SEQ ID NO. 145, or a light chain CDRL2 consisting of the amino acid sequence represented by SEQ ID NO. 145, wherein the second amino acid is N A molecule comprising a light chain CDRL2 consisting of an amino acid sequence, and a light chain CDRL3 consisting of an amino acid sequence represented by SEQ ID NO. 146, comprising an antibody or its binding fragment that specifically binds to CD3. Claim 11 In claim 10, the antibody or its binding fragment that specifically binds to CD3 comprises: (CH1) a medium-chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 136; (CH2) a medium-chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 137; (CH3) a medium-chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 147; (CH4) a medium-chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 138; (CH5) a medium-chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 139; and (CH6) a medium-chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO.

140. Region, (CH7) a medium chain variable region consisting of the amino acid sequences from the 272nd to the 389th of the amino acid sequence represented by SEQ ID NO. 155, (CH8) a medium chain variable region consisting of the amino acid sequences from the 277th to the 394th of the amino acid sequence represented by SEQ ID NO. 156, or (CH9) a medium chain variable region consisting of the amino acid sequences from the 277th to the 394th of the amino acid sequence represented by SEQ ID NO. 157, and, (CL1) a light chain variable region consisting of the amino acid sequences from the 135th to the 243rd of the amino acid sequence represented by SEQ ID NO. 136, (CL2) a light chain variable region consisting of the amino acid sequences from the 135th to the 241st of the amino acid sequence represented by SEQ ID NO. 137, (CL3) a light chain variable region consisting of the amino acid sequences from the 135th to the 243rd of the amino acid sequence represented by SEQ ID NO. 147,(CL4) a light chain variable region consisting of the 135th to 241st amino acid sequence of the amino acid sequence represented by SEQ No. 138, (CL5) a light chain variable region consisting of the 135th to 243rd amino acid sequence of the amino acid sequence represented by SEQ No. 139, (CL6) a light chain variable region consisting of the 135th to 243rd amino acid sequence of the amino acid sequence represented by SEQ No. 140, (CL7) a light chain variable region consisting of the 405th to 511th amino acid sequence of the amino acid sequence represented by SEQ No. 155, (CL8) a light chain variable region consisting of the 410th to 516th amino acid sequence of the amino acid sequence represented by SEQ No. 156, or (CL9) a light chain variable region consisting of the 410th to 516th amino acid sequence of the amino acid sequence represented by SEQ No. 157, comprising a molecule. Claim 12 In claim 11, the antibody or its binding fragment that specifically binds to CD3 comprises: (CH1CL1) a heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 136 and a light chain variable region consisting of the 135th to 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO. 136; (CH2CL2) a heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 137 and a light chain variable region consisting of the 135th to 241st amino acid sequence of the amino acid sequence represented by SEQ ID NO. 137; (CH3CL3) a heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 147 and a light chain variable region consisting of the 135th to 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO.

147. Region, (CH4CL4) a heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 138 and a light chain variable region consisting of the 135th to 241st amino acid sequence of the amino acid sequence represented by SEQ ID NO. 138, (CH5CL5) a heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 139 and a light chain variable region consisting of the 135th to 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO. 139, (CH6CL6) a heavy chain variable region consisting of the 2nd to 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 140 and a light chain variable region consisting of the 135th to 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO. 140,(CH7CL7) a heavy chain variable region consisting of the amino acid sequences from the 272nd to the 389th of the amino acid sequence represented by SEQ ID NO. 155 and a light chain variable region consisting of the amino acid sequences from the 405th to the 511th of the amino acid sequence represented by SEQ ID NO. 155; (CH8CL8) a heavy chain variable region consisting of the amino acid sequences from the 277th to the 394th of the amino acid sequence represented by SEQ ID NO. 156 and a light chain variable region consisting of the amino acid sequences from the 410th to the 516th of the amino acid sequence represented by SEQ ID NO. 156; or, (CH9CL9) a heavy chain variable region consisting of the amino acid sequences from the 277th to the 394th of the amino acid sequence represented by SEQ ID NO. 157 and a light chain variable region consisting of the amino acid sequences from the 410th to the 516th of the amino acid sequence represented by SEQ ID NO. 157, comprising a molecule. Claim 13 In claim 10, a molecule in which an antibody or its binding fragment that specifically binds to CD3 is scFv. Claim 14 In claim 13, scFv comprises: (CS1) a scFv consisting of the 2nd to 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO. 136; (CS2) a scFv consisting of the 2nd to 241st amino acid sequence of the amino acid sequence represented by SEQ ID NO. 137; (CS3) a scFv consisting of the 2nd to 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO. 147; (CS4) a scFv consisting of the 2nd to 241st amino acid sequence of the amino acid sequence represented by SEQ ID NO. 138; (CS5) a scFv consisting of the 2nd to 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO. 139; (CS6) a scFv consisting of the 2nd to 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO. 140; and (CS7) the 272nd to A molecule comprising a scFv consisting of the 511th amino acid sequence (CS8), a scFv consisting of the 277th to 516th amino acid sequence of the amino acid sequence represented by sequence number 156, or a scFv consisting of the 277th to 516th amino acid sequence of the amino acid sequence represented by sequence number 157 (CS9). Claim 15 A molecule comprising: a first polypeptide comprising, in order from the N-terminus toward the C-terminus, a scFv specifically binding to human HLA / NY-ESO, a linker, a scFv specifically binding to CD3, a linker, a hinge region, and an Fc region (i); and a second polypeptide comprising a hinge region and an Fc region (ii). Claim 16 In claim 15, the scFv that specifically binds to human HLA / NY-ESO is a molecule that is an antibody or a binding fragment thereof that specifically binds to the human HLA / NY-ESO described in claim 1, which is the scFv. Claim 17 In claim 15, the scFv that specifically binds to human HLA / NY-ESO is a molecule that is an antibody or a binding fragment thereof that specifically binds to the human HLA / NY-ESO described in claim 2, which is a scFv. Claim 18 In claim 15, the scFv that specifically binds to human HLA / NY-ESO is a molecule that is an antibody or a binding fragment thereof that specifically binds to human HLA / NY-ESO as described in claim 3, which is a scFv. Claim 19 In claim 15, the scFv that specifically binds to CD3 is a molecule that is an antibody or a binding fragment thereof that specifically binds to CD3 as described in claim 10, which is a scFv. Claim 20 In claim 15, the scFv that specifically binds to CD3 is a molecule that is an antibody or a binding fragment thereof that specifically binds to CD3 as described in claim 11, which is a scFv. Claim 21 In claim 15, the scFv that specifically binds to CD3 is a molecule that is an antibody or a binding fragment thereof that specifically binds to CD3 as described in claim 12, which is a scFv. Claim 22 In claim 15, the scFv that specifically binds to CD3 is a molecule that is an antibody or a binding fragment thereof that specifically binds to CD3 as described in claim 13 or 14, which is a scFv. Claim 23 A molecule according to claim 15, comprising the 21st to 511th amino acid sequence of the amino acid sequence represented by sequence number 96. Claim 24 A molecule according to claim 15, comprising an amino acid sequence selected from the group consisting of the 21st to 516th amino acid sequence of the amino acid sequence represented by sequence number 156 and the 21st to 516th amino acid sequence of the amino acid sequence represented by sequence number 157. Claim 25 In claim 15, the molecule wherein the first polypeptide comprises the 529th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO.

96. Claim 26 A molecule according to claim 15, wherein the first polypeptide comprises the 534th to 750th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 156, or the 534th to 750th amino acid sequence of the amino acid sequence represented by SEQ ID NO.

157. Claim 27 A molecule according to claim 23 or 25, wherein the first polypeptide consists of the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO.

96. Claim 28 A molecule according to claim 24 or 26, wherein the first polypeptide is composed of the 20th to 750th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 156, or the 20th to 750th amino acid sequence of the amino acid sequence represented by SEQ ID NO.

157. Claim 29 In claim 15, the molecule wherein the second polypeptide consists of the 20th to 246th amino acid sequence of the amino acid sequence represented by SEQ ID NO.

84. Claim 30 A molecule according to claim 10, comprising a first polypeptide consisting of, in the order of a scFv that specifically binds to human HLA / NY-ESO from the N-terminus toward the C-terminus, a linker, a variable region and a normal region CH1 of an antibody heavy chain that specifically binds to CD3, a hinge region of the immunoglobulin, and an Fc region (i) of the immunoglobulin; a second polypeptide consisting of a hinge region and an Fc region (ii) of the immunoglobulin; and a third polypeptide consisting of an antibody light chain that specifically binds to CD3, consisting of a variable region and a normal region. Claim 31 In claim 30, the scFv that specifically binds to human HLA / NY-ESO is a molecule that is an antibody or a binding fragment thereof that specifically binds to the human HLA / NY-ESO described in claim 1, which is the scFv. Claim 32 In claim 30, the scFv that specifically binds to human HLA / NY-ESO is a molecule that is an antibody or a binding fragment thereof that specifically binds to human HLA / NY-ESO as described in claim 2, which is a scFv. Claim 33 In claim 30, the scFv that specifically binds to human HLA / NY-ESO is a molecule that is an antibody or a binding fragment thereof that specifically binds to the human HLA / NY-ESO described in claim 3, which is the scFv. Claim 34 In claim 30, the antibody or binding fragment thereof that specifically binds to CD3 is a molecule in which Fab. Claim 35 In claim 34, the molecule in which Fab specifically binds to CD3 is the antibody or binding fragment thereof that specifically binds to CD3 as described in claim 10, which is Fab. Claim 36 In claim 34, the molecule in which Fab specifically binds to CD3 is the antibody or binding fragment thereof that specifically binds to CD3 as described in claim 11, which is Fab. Claim 37 In claim 34, the molecule in which Fab specifically binds to CD3 is an antibody or its binding fragment that specifically binds to CD3 as described in claim 12, which is Fab. Claim 38 In claim 30, the molecule wherein the first polypeptide comprises the 21st to 394th amino acid sequence of the amino acid sequence represented by SEQ ID NO.

160. Claim 39 In claim 30, the molecule wherein the first polypeptide is composed of the amino acid sequence described in (i) or (ii) below: (i) the 20th to 724th amino acid sequence of the amino acid sequence represented by SEQ ID NO. 160; (ii) the 20th to 719th amino acid sequence of the amino acid sequence represented by SEQ ID NO.

197. Claim 40 In claim 30, the molecule in which the second polypeptide consists of the 20th to 246th amino acid sequence of the amino acid sequence represented by SEQ ID NO.

84. Claim 41 In claim 30, the molecule wherein the third polypeptide comprises the 21st to 127th amino acid sequence of the amino acid sequence represented by SEQ ID NO.

161. Claim 42 In claim 30, the molecule wherein the third polypeptide consists of the 21st to 233rd amino acid sequence of the amino acid sequence represented by SEQ ID NO.

161. Claim 43 A molecule described in claim 10, wherein, in the amino acid sequence of one or more polypeptides included in the molecule, one or two amino acids are deleted from the carboxyl terminus of the sequence. Claim 44 A polynucleotide comprising a nucleotide sequence encoding the molecule described in claim 10. Claim 45 A vector comprising the polynucleotide described in claim 44. Claim 46 An isolated host cell comprising the polynucleotide described in claim 44 or a vector comprising the polynucleotide described in claim 44. Claim 47 A method for producing a molecule that specifically binds to human HLA / NY-ESO and human CD3, comprising: (i) a process of culturing host cells as described in claim 46; and (ii) a process of purifying an antibody or its binding fragment from a culture obtained in process (i). Claim 48 A molecule that specifically binds to human HLA / NY-ESO and human CD3, obtained by the method described in claim 47. Claim 49 A pharmaceutical composition that is an anticancer agent, comprising as an active ingredient a molecule described in any one of claims 10 to 15, 23 to 26, 29, 30, 34, 38 to 43, and 48, a polynucleotide described in claim 44, a vector described in claim 45, or a host cell described in claim 46. Claim 50 In paragraph 49, cancer is renal cancer, melanoma, acanthic cell carcinoma, basal cell carcinoma, conjunctival cancer, oral cancer, laryngeal cancer, pharyngeal cancer, thyroid cancer, lung cancer, breast cancer, esophageal cancer, gastric cancer, duodenal cancer, small intestine cancer, colorectal cancer, rectal cancer, appendiceal cancer, anal cancer, liver cancer, gallbladder cancer, cholangiocarcinoma, pancreatic cancer, adrenal cancer, bladder cancer, prostate cancer, uterine cancer, vaginal cancer, liposarcoma, angiosarcoma, chondrosarcoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, undifferentiated pleomorphic sarcoma, myxoid fibrosarcoma, malignant peripheral schwannoma, retroperitoneal sarcoma, synovial sarcoma, uterine sarcoma, gastrointestinal stromal tumor, leiomyosarcoma, epithelial sarcoma, B-cell lymphoma, T-NK cell lymphoma, Hodgkin lymphoma, myeloid leukemia, lymphoid leukemia, myelodysplastic syndrome, multiple myeloma, testicular cancer and A pharmaceutical composition comprising one or more selected from the group consisting of ovarian cancer. Claim 51 A pharmaceutical composition according to claim 50, wherein the lung cancer is one or two selected from the group consisting of non-small cell lung cancer and small cell lung cancer. Claim 52 A pharmaceutical composition according to claim 51, wherein the non-small cell lung cancer is one or more selected from the group consisting of adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. Claim 53 A first polypeptide comprising, in order from the N-terminus toward the C-terminus, a scFv specifically binding to human HLA / NY-ESO, a linker, a scFv specifically binding to CD3, a linker, a hinge region, and an Fc region (i); And, comprising a second polypeptide consisting of a hinge region and an Fc region (ii), formed by association in the Fc region (i) and the Fc region (ii), wherein the first polypeptide is composed of the amino acid sequence of the 20th to 745th position of the amino acid sequence represented by SEQ ID NO. 96, the amino acid sequence of the 20th to 745th position of the amino acid sequence represented by SEQ ID NO. 155, the amino acid sequence of the 20th to 750th position of the amino acid sequence represented by SEQ ID NO. 156, or the amino acid sequence of the 20th to 750th position of the amino acid sequence represented by SEQ ID NO. 157, or an amino acid sequence formed by deleting one or two amino acids from the carboxyl terminus of the amino acid sequence, and the second polypeptide is composed of the amino acid sequence of the 20th to 246th position of the amino acid sequence represented by SEQ ID NO. 84, or one or two amino acids from the carboxyl terminus of the amino acid sequence A bispecific molecule consisting of a deletion-based amino acid sequence that specifically binds to human HLA / NY-ESO and human CD3. Claim 54 A first polypeptide comprising, in the order from the N-terminus toward the C-terminus, a scFv that specifically binds to human HLA / NY-ESO, a linker, a variable region and normal region CH1 of an antibody heavy chain that specifically binds to CD3, a hinge region of the immunoglobulin, and an Fc region (i) of the immunoglobulin; a second polypeptide comprising a hinge region and an Fc region (ii) of the immunoglobulin; and a third polypeptide comprising an antibody light chain that specifically binds to CD3, comprising a variable region and a normal region, wherein the first polypeptide and the second polypeptide are associated in the Fc region (i) and the Fc region (ii), and the first polypeptide is formed by associating with the third polypeptide in the variable region and normal region CH1 of the antibody heavy chain, and the first polypeptide is formed such that the 20th to of the amino acid sequence represented by SEQ No. 160 A bispecific molecule that specifically binds to human HLA / NY-ESO and human CD3, comprising an amino acid sequence of the 724th position or an amino acid sequence of the 20th to 719th positions of the amino acid sequence represented by SEQ ID NO. 197, or an amino acid sequence formed by deleting one or two amino acids from the carboxyl terminus of the amino acid sequence, wherein a second polypeptide comprises an amino acid sequence of the 20th to 246th positions of the amino acid sequence represented by SEQ ID NO. 84, and a third polypeptide comprises an amino acid sequence of the 21st to 127th positions of the amino acid sequence represented by SEQ ID NO. 161, or an amino acid sequence of the 21st to 233rd positions of the amino acid sequence represented by SEQ ID NO.

161. Claim 55 A polynucleotide comprising a nucleotide sequence encoding a molecule described in claim 53 or 54. Claim 56 A vector comprising the polynucleotide described in claim 55. Claim 57 An isolated host cell comprising the polynucleotide described in claim 55 or a vector comprising the polynucleotide described in claim 55. Claim 58 A method for producing a bispecific molecule that specifically binds to human HLA / NY-ESO and human CD3, comprising: (i) a process of culturing host cells as described in claim 57; and (ii) a process of purifying an antibody or its binding fragment from the culture obtained in process (i). Claim 59 A bispecific molecule that specifically binds to human HLA / NY-ESO and human CD3, obtained by the method described in claim 58. Claim 60 A pharmaceutical composition that is an anticancer agent, comprising as an active ingredient a molecule described in claim 53 or 54; a polynucleotide comprising a nucleotide sequence encoding said molecule; a vector comprising said polynucleotide; or an isolated host cell comprising said polynucleotide or said polynucleotide. Claim 61 In paragraph 60, cancer is renal cancer, melanoma, acanthic cell carcinoma, basal cell carcinoma, conjunctival cancer, oral cancer, laryngeal cancer, pharyngeal cancer, thyroid cancer, lung cancer, breast cancer, esophageal cancer, gastric cancer, duodenal cancer, small intestine cancer, colorectal cancer, rectal cancer, appendiceal cancer, anal cancer, liver cancer, gallbladder cancer, cholangiocarcinoma, pancreatic cancer, adrenal cancer, bladder cancer, prostate cancer, uterine cancer, vaginal cancer, liposarcoma, angiosarcoma, chondrosarcoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, undifferentiated pleomorphic sarcoma, myxoid fibrosarcoma, malignant peripheral schwannoma, retroperitoneal sarcoma, synovial sarcoma, uterine sarcoma, gastrointestinal stromal tumor, leiomyosarcoma, epithelial sarcoma, B-cell lymphoma, T-NK cell lymphoma, Hodgkin lymphoma, myeloid leukemia, lymphoid leukemia, myelodysplastic syndrome, multiple myeloma, testicular cancer and A pharmaceutical composition comprising one or more selected from the group consisting of ovarian cancer. Claim 62 A pharmaceutical composition according to claim 61, wherein the lung cancer is one or two selected from the group consisting of non-small cell lung cancer and small cell lung cancer. Claim 63 A pharmaceutical composition according to claim 62, wherein the non-small cell lung cancer is one or more selected from the group consisting of adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. Claim 64 delete Claim 65 delete Claim 66 delete Claim 67 delete Claim 68 delete Claim 69 delete Claim 70 delete Claim 71 delete Claim 72 delete Claim 73 delete Claim 74 delete Claim 75 delete Claim 76 delete Claim 77 delete Claim 78 delete Claim 79 delete

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