Bispecific Antibodies

Abstract

The present invention aims to provide a novel antibody that can be used as an antitumor agent, and an antitumor agent containing a molecule including the antibody as an effective ingredient. The antibody or its binding fragment contains the following heavy and light chains: a heavy chain CDRH1 consisting of the amino acid sequence shown in SEQ ID NO:54; a heavy chain CDRH2 consisting of the amino acid sequence shown in SEQ ID NO:55; a heavy chain CDRH3 consisting of the amino acid sequence shown in SEQ ID NO:56; a light chain CDRL1 consisting of the amino acid sequence shown in SEQ ID NO:57, or a light chain CDRL1 consisting of an amino acid sequence in which the 7th amino acid is W and / or the 8th amino acid is K in the amino acid sequence shown in SEQ ID NO:57; a light chain CDRL2 consisting of an amino acid sequence shown in SEQ ID NO:58; and a light chain CDRL3 consisting of an amino acid sequence shown in SEQ ID NO:59, or a light chain CDRL3 consisting of an amino acid sequence in which the 2nd amino acid is A or S in the amino acid sequence shown in SEQ ID NO:57.

Description

Technical Field

[0001] The present invention relates to antibodies, bispecific antibodies, etc. that can be used to treat cancer. Background Art

[0002] NY-ESO-1 is a molecule identified from esophageal cancer by serological analysis of recombinant cDNA expression libraries (SEREX) (Non-Patent Document 1), and LAGE-1, also known as NY-ESO-2, is a molecule identified by representative difference analysis of tumor cDNA libraries (Non-Patent Document 2). Although it is known that these molecules are limited to expression in the testis in normal tissues, their functions are still unclear. There are reports that NY-ESO-1 and LAGE-1 are expressed in a wide range of cancers such as melanoma, lung cancer, bladder cancer, ovarian cancer, soft tissue sarcoma, and myeloma (Non-Patent Document 3), suggesting their association with cancer. In addition, regarding the association with malignancy, there are reports that NY-ESO-1 expression is higher in melanoma metastases than in primary lesions (Non-Patent Document 4); in urothelial carcinoma, NY-ESO-1 and LAGE-1 expression is higher in intermediate-stage cancers compared to early-stage cancers (Non-Patent Document 5); and NY-ESO-1 expression is higher in high-risk myeloma with chromosomal abnormalities than in myeloma with normal chromosomes (Non-Patent Document 6). These findings suggest that NY-ESO-1 and LAGE-1 are attracting attention as highly cancer-specific molecules, and are being extensively researched and developed as drug discovery targets for cancer vaccine therapy, but no approved drugs have yet been identified.

[0003] The 9-mer NY-ESO peptide SLLMWITQC, located at positions 157 to 165 of NY-ESO-1 and LAGE-1, is known to form a complex (HLA / NY-ESO peptide complex) with HLA (Histocompatibility Leukocyte Antigen)-A2 and be presented extracellularly (Non-Patent Document 7). As described above, since the expression of NY-ESO-1 and LAGE-1 is cancer-specific, the HLA / NY-ESO peptide complex is suggested to be a cancer-specific therapeutic target, present only on HLA-A2-positive and NY-ESO-1 or LAGE-1-positive cancer cells (Non-Patent Document 8). Furthermore, TCRs (Patent Documents 1 and 2) and antibodies (Patent Documents 3 and 8) have been reported as molecules that bind to the HLA / NY-ESO peptide complex.

[0004] As one of the purposes of binding molecules for cancer target molecules, there are CD3 bispecific (bispecific) antibodies (non-patent literature 9, 10), whose mechanism is to induce T cells to cancer cells, thereby showing T cell redirection of the anti-tumor effect caused by cytotoxicity. As a currently listed CD3 bispecific antibody drug, blinatumomab (BiTE) (bispecfic T-cell engager, bispecific T cell adapter) for CD19 can be cited. It is recognized in acute lymphoblastic leukemia (ALL), and clinical trials targeting other blood cancers are underway. However, compared with IgG type antibodies commonly used as therapeutic antibodies, the half-life in the blood when the tandem scFv (taFv) without Fc region as a bispecific antibody configuration is administered to the patient is very short (non-patent literature 11).

[0005] As bispecific antibodies having heterodimeric Fc regions with half-lives in the blood comparable to those of IgG antibodies, research and clinical trials are underway using various CD3 bispecific antibodies with antibody configurations such as knobs-into-holes, CrossMAb, and DuoBody (registered trademark) (Patent Documents 4, 5, 6, and 7). A CD3 bispecific antibody utilizing an antibody against the HLA / NY-ESO peptide complex has been reported (Non-Patent Document 12).

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: WO2005 / 113595

[0009] Patent Document 2: WO2017 / 109496

[0010] Patent Document 3: WO2010 / 106431

[0011] Patent Document 4: WO1998 / 050431

[0012] Patent Document 5: WO2006 / 106905

[0013] Patent Document 6: WO2011 / 028952

[0014] Patent Document 7: WO2011 / 131746

[0015] Patent Document 8: WO2021 / 003357

[0016] Non-patent literature

[0017] Non-patent literature 1: Proc Natl Acad Sci US A.; 94(5): 1914-8 (1997).

[0018] Non-patent document 2: Int J Cancer.; 76(6): 903-8 (1998).

[0019] Non-patent document 3: Immunol Cell Biol.; 84(3): 303-17 (2006).

[0020] Non-patent document 4: J Surg Res.; 98(2): 76-80 (2001).

[0021] Non-patent document 5: Cancer Res.; 61(12): 4671-4 (2001).

[0022] Non-patent literature 6: Blood.; 105(10): 3939-44 (2005).

[0023] Non-patent literature 7: J Exp Med.; 187(2): 265-70 (1998).

[0024] Non-patent literature 8: J Immunol.; 176(12): 7308-16 (2006).

[0025] Non-patent literature 9: Nature; 314(6012); 628-31(1985).

[0026] Non-patent literature 10: Int Rev Immunol.; 4(2); 159-73 (1989).

[0027] Non-patent literature 11: Drug Des Devel Ther.; 10; 757-765 (2016).

[0028] Non-patent literature 12: Transcripts of the 21st Chapter of the Society for Immunology; O13-4 Summary of the Invention

[0029] [Problems to be solved by the invention]

[0030] The present invention aims to provide novel anti-HLA-A2 / NY-ESO antibodies useful as antitumor agents, and antitumor agents containing molecules such as the antibodies that bind to HLA-A2 / NY-ESO as active ingredients.

[0031] Technical solutions to solve the problem

[0032] The present inventors have conducted intensive studies to solve the above-mentioned problems, created novel anti-HLA-A2 / NY-ESO antibodies, and molecules binding to HLA-A2 / NY-ESO including the antibodies, thereby completing the present invention.

[0033] That is, the present invention includes the following contents.

[0034] [1] An antibody or a binding fragment thereof that specifically binds to human HLA / NY-ESO, the antibody or the binding fragment thereof comprising:

[0035] a heavy chain CDRH1 consisting of the amino acid sequence shown in SEQ ID NO:54;

[0036] a heavy chain CDRH2 consisting of the amino acid sequence shown in SEQ ID NO:55;

[0037] a heavy chain CDRH3 consisting of the amino acid sequence shown in SEQ ID NO:56;

[0038] A light chain CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 57, or a light chain CDRL1 consisting of an amino acid sequence shown in SEQ ID NO: 57 in which amino acid position 7 is W and / or amino acid position 8 is K;

[0039] Light chain CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 58; and

[0040] A light chain CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 59, or a light chain CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 59 wherein the second amino acid is A or S.

[0041] [2] The antibody or binding fragment thereof according to [1], comprising one or more CDRH1 to CDRH3 and CDRL1 to CDRL3 selected from the group consisting of the following groups (i) to (v):

[0042] (i) a heavy chain CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 54;

[0043] a heavy chain CDRH2 consisting of the amino acid sequence shown in SEQ ID NO:55;

[0044] a heavy chain CDRH3 consisting of the amino acid sequence shown in SEQ ID NO:56;

[0045] Light chain CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 57;

[0046] Light chain CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 58; and

[0047] Light chain CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 59;

[0048] (ii) a heavy chain CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 54;

[0049] a heavy chain CDRH2 consisting of the amino acid sequence shown in SEQ ID NO:55;

[0050] a heavy chain CDRH3 consisting of the amino acid sequence shown in SEQ ID NO:56;

[0051] A light chain CDRL1 consisting of an amino acid sequence in which amino acid position 7 of the amino acid sequence shown in SEQ ID NO: 57 is W;

[0052] Light chain CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 58; and

[0053] Light chain CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 59;

[0054] (iii) a heavy chain CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 54;

[0055] a heavy chain CDRH2 consisting of the amino acid sequence shown in SEQ ID NO:55;

[0056] a heavy chain CDRH3 consisting of the amino acid sequence shown in SEQ ID NO:56;

[0057] Light chain CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 57;

[0058] Light chain CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 58; and

[0059] A light chain CDRL3 consisting of an amino acid sequence in which the second amino acid in the amino acid sequence of SEQ ID NO: 59 is A;

[0060] (iv) a heavy chain CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 54;

[0061] a heavy chain CDRH2 consisting of the amino acid sequence shown in SEQ ID NO:55;

[0062] a heavy chain CDRH3 consisting of the amino acid sequence shown in SEQ ID NO:56;

[0063] Light chain CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 57;

[0064] Light chain CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 58; and

[0065] A light chain CDRL3 consisting of an amino acid sequence represented by SEQ ID NO: 59, wherein the second amino acid is S; and

[0066] (v) a heavy chain CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 54;

[0067] a heavy chain CDRH2 consisting of the amino acid sequence shown in SEQ ID NO:55;

[0068] a heavy chain CDRH3 consisting of the amino acid sequence shown in SEQ ID NO:56;

[0069] A light chain CDRL1 consisting of an amino acid sequence in which amino acid position 7 is W and amino acid position 8 is K in the amino acid sequence shown in SEQ ID NO: 57;

[0070] Light chain CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 58; and

[0071] Light chain CDRL3 consisting of the amino acid sequence shown in SEQ ID NO:59.

[0072] [3] The antibody or binding fragment thereof according to [1], wherein the antibody or binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of an amino acid sequence of amino acids 21 to 140 of the amino acid sequence set forth in SEQ ID NO: 27, or an amino acid sequence having 95% or greater sequence identity with the amino acid sequence set forth in SEQ ID NO: 38 or SEQ ID NO: 39; and the light chain variable region consists of an amino acid sequence of amino acids 156 to 266 of the amino acid sequence set forth in SEQ ID NO: 27 or SEQ ID NO: 52, or an amino acid sequence having 95% or greater sequence identity with the amino acid sequence set forth in SEQ ID NO: 40.

[0073] [4] The antibody or binding fragment thereof according to [1], wherein the antibody or binding fragment thereof comprises the following heavy chain variable region and light chain variable region:

[0074] (H1) a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 6;

[0075] (H2) a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 18;

[0076] (H3) a heavy chain variable region consisting of the amino acid sequence from positions 21 to 140 of the amino acid sequence set forth in SEQ ID NO: 29;

[0077] (H4) a heavy chain variable region consisting of the amino acid sequence from positions 21 to 140 of the amino acid sequence set forth in SEQ ID NO: 26;

[0078] (H5) a heavy chain variable region consisting of the amino acid sequence from positions 21 to 140 of the amino acid sequence set forth in SEQ ID NO: 27;

[0079] (H6) a heavy chain variable region consisting of the amino acid sequence from positions 21 to 140 of the amino acid sequence set forth in SEQ ID NO: 28;

[0080] (H7) a heavy chain variable region consisting of the amino acid sequence from positions 21 to 140 of the amino acid sequence set forth in SEQ ID NO: 36;

[0081] (H8) a heavy chain variable region consisting of the amino acid sequence from positions 21 to 140 of the amino acid sequence shown in SEQ ID NO: 47;

[0082] (H9) a heavy chain variable region consisting of the amino acid sequence from positions 21 to 140 of the amino acid sequence set forth in SEQ ID NO: 48;

[0083] (H10) a heavy chain variable region consisting of the amino acid sequence from positions 21 to 140 of the amino acid sequence set forth in SEQ ID NO: 50;

[0084] (H11) a heavy chain variable region consisting of the amino acid sequence from positions 21 to 140 of the amino acid sequence shown in SEQ ID NO: 51;

[0085] (H12) a heavy chain variable region consisting of the amino acid sequence from positions 21 to 140 of the amino acid sequence shown in SEQ ID NO: 52;

[0086] (H13) a heavy chain variable region consisting of amino acids 21 to 140 of the amino acid sequence of SEQ ID NO: 53;

[0087] (H14) a heavy chain variable region consisting of the amino acid sequence from positions 21 to 140 of the amino acid sequence shown in SEQ ID NO: 30; or

[0088] (H15) a heavy chain variable region consisting of the amino acid sequence from positions 21 to 140 of the amino acid sequence shown in SEQ ID NO: 156; and

[0089] (L1) a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 8;

[0090] (L2) a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 20;

[0091] (L3) a light chain variable region consisting of the amino acid sequence from positions 156 to 266 of the amino acid sequence set forth in SEQ ID NO: 29;

[0092] (L4) a light chain variable region consisting of the amino acid sequence from positions 156 to 266 of the amino acid sequence set forth in SEQ ID NO: 26;

[0093] (L5) a light chain variable region consisting of the amino acid sequence from positions 156 to 266 of the amino acid sequence set forth in SEQ ID NO: 27;

[0094] (L6) a light chain variable region consisting of the amino acid sequence from positions 156 to 266 of the amino acid sequence set forth in SEQ ID NO: 28;

[0095] (L7) a light chain variable region consisting of the amino acid sequence from positions 156 to 266 of the amino acid sequence set forth in SEQ ID NO: 36;

[0096] (L8) a light chain variable region consisting of the amino acid sequence from positions 156 to 266 of the amino acid sequence set forth in SEQ ID NO: 47;

[0097] (L9) a light chain variable region consisting of the amino acid sequence from positions 156 to 266 of the amino acid sequence set forth in SEQ ID NO:48;

[0098] (L10) a light chain variable region consisting of the amino acid sequence from positions 156 to 266 of the amino acid sequence shown in SEQ ID NO: 50;

[0099] (L11) a light chain variable region consisting of the amino acid sequence from positions 156 to 266 of the amino acid sequence shown in SEQ ID NO: 51;

[0100] (L12) a light chain variable region consisting of the amino acid sequence from positions 156 to 266 of the amino acid sequence set forth in SEQ ID NO: 52;

[0101] (L13) a light chain variable region consisting of the amino acid sequence from positions 156 to 266 of the amino acid sequence shown in SEQ ID NO: 53;

[0102] (L14) a light chain variable region consisting of the amino acid sequence from positions 156 to 266 of the amino acid sequence shown in SEQ ID NO: 30; or

[0103] (L15) A light chain variable region consisting of the amino acid sequence from positions 161 to 271 of the amino acid sequence shown in SEQ ID NO: 156.

[0104] [5] The antibody or binding fragment thereof according to [4], wherein the antibody or binding fragment thereof comprises the following heavy chain variable region and light chain variable region:

[0105] (H1L1) a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO:6, and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO:8;

[0106] (H2L2) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 18, and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 20;

[0107] (H3L3) a heavy chain variable region consisting of amino acids 21 to 140 of the amino acid sequence set forth in SEQ ID NO: 29, and a light chain variable region consisting of amino acids 156 to 266 of the amino acid sequence set forth in SEQ ID NO: 29;

[0108] (H4L4) a heavy chain variable region consisting of amino acids 21 to 140 of the amino acid sequence of SEQ ID NO: 26, and a light chain variable region consisting of amino acids 156 to 266 of the amino acid sequence of SEQ ID NO: 26;

[0109] (H5L5) a heavy chain variable region consisting of amino acids 21 to 140 of the amino acid sequence of SEQ ID NO: 27, and a light chain variable region consisting of amino acids 156 to 266 of the amino acid sequence of SEQ ID NO: 27;

[0110] (H6L6) a heavy chain variable region consisting of amino acids 21 to 140 of SEQ ID NO: 28, and a light chain variable region consisting of amino acids 156 to 266 of SEQ ID NO: 28;

[0111] (H7L7) a heavy chain variable region consisting of amino acids 21 to 140 of SEQ ID NO: 36, and a light chain variable region consisting of amino acids 156 to 266 of SEQ ID NO: 36;

[0112] (H8L8) a heavy chain variable region consisting of amino acids 21 to 140 of SEQ ID NO:47, and a light chain variable region consisting of amino acids 156 to 266 of SEQ ID NO:47;

[0113] (H9L9) a heavy chain variable region consisting of amino acids 21 to 140 of SEQ ID NO:48, and a light chain variable region consisting of amino acids 156 to 266 of SEQ ID NO:48;

[0114] (H10L10) a heavy chain variable region consisting of amino acids 21 to 140 of the amino acid sequence set forth in SEQ ID NO:50, and a light chain variable region consisting of amino acids 156 to 266 of the amino acid sequence set forth in SEQ ID NO:50;

[0115] (H11L11) a heavy chain variable region consisting of amino acids 21 to 140 of the amino acid sequence of SEQ ID NO:51, and a light chain variable region consisting of amino acids 156 to 266 of the amino acid sequence of SEQ ID NO:51;

[0116] (H12L12) a heavy chain variable region consisting of amino acids 21 to 140 of SEQ ID NO: 52, and a light chain variable region consisting of amino acids 156 to 266 of SEQ ID NO: 52;

[0117] (H13L13) a heavy chain variable region consisting of amino acids 21 to 140 of SEQ ID NO: 53, and a light chain variable region consisting of amino acids 156 to 266 of SEQ ID NO: 53;

[0118] (H14L14) a heavy chain variable region consisting of the amino acid sequence from positions 21 to 140 of the amino acid sequence set forth in SEQ ID NO: 30, and a light chain variable region consisting of the amino acid sequence from positions 156 to 266 of the amino acid sequence set forth in SEQ ID NO: 30; or

[0119] (H15L15) A heavy chain variable region consisting of the amino acid sequence from positions 21 to 140 of the amino acid sequence shown in SEQ ID NO: 156, and a light chain variable region consisting of the amino acid sequence from positions 161 to 271 of the amino acid sequence shown in SEQ ID NO: 156.

[0120] [6] The antibody or binding fragment thereof according to any one of [1] to [5], wherein the antibody or binding fragment thereof is scFv.

[0121] [7] The antibody or binding fragment thereof according to [6], wherein the antibody or binding fragment thereof is the following scFv:

[0122] (s1) an scFv consisting of the amino acid sequence from positions 21 to 266 of the amino acid sequence shown in SEQ ID NO: 70;

[0123] (s2) an scFv comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 18 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 20;

[0124] (s3) an scFv consisting of the amino acid sequence from positions 21 to 266 of the amino acid sequence shown in SEQ ID NO: 29;

[0125] (s4) an scFv consisting of the amino acid sequence from positions 21 to 266 of the amino acid sequence shown in SEQ ID NO: 26;

[0126] (s5) an scFv consisting of the amino acid sequence from positions 21 to 266 of the amino acid sequence shown in SEQ ID NO: 27;

[0127] (s6) an scFv consisting of the amino acid sequence from positions 21 to 266 of the amino acid sequence shown in SEQ ID NO: 28;

[0128] (s7) an scFv consisting of the amino acid sequence from positions 21 to 266 of the amino acid sequence shown in SEQ ID NO: 36;

[0129] (s8) an scFv consisting of the amino acid sequence from positions 21 to 266 of the amino acid sequence shown in SEQ ID NO: 47;

[0130] (s9) an scFv consisting of the amino acid sequence from positions 21 to 266 of the amino acid sequence shown in SEQ ID NO: 48;

[0131] (s10) an scFv consisting of the amino acid sequence from positions 21 to 266 of the amino acid sequence shown in SEQ ID NO: 50;

[0132] (s11) an scFv consisting of the amino acid sequence from positions 21 to 266 of the amino acid sequence shown in SEQ ID NO: 51;

[0133] (s12) an scFv consisting of the amino acid sequence from positions 21 to 266 of the amino acid sequence shown in SEQ ID NO: 52;

[0134] (s13) an scFv consisting of the amino acid sequence from positions 21 to 266 of the amino acid sequence shown in SEQ ID NO: 53;

[0135] (s14) an scFv consisting of the amino acid sequence from positions 21 to 266 of the amino acid sequence shown in SEQ ID NO: 30; or

[0136] (s15) scFv consisting of the amino acid sequence from positions 21 to 271 of the amino acid sequence shown in SEQ ID NO: 156.

[0137] [8] A polynucleotide encoding the antibody or binding fragment thereof according to any one of [1] to [7].

[0138] [9] A vector comprising the polynucleotide described in [8].

[0139]

[10] A host cell containing the polynucleotide described in [8] or the vector described in [9].

[0140]

[11] A method for producing an antibody or a binding fragment thereof that specifically binds to human HLA / NY-ESO, the method comprising: (i) culturing the host cell described in

[10] ; and (ii) purifying the antibody or the binding fragment thereof from the culture obtained in step (i).

[0141]

[12] An antibody or a binding fragment thereof that specifically binds to human HLA / NY-ESO, wherein the antibody or the binding fragment thereof is obtained by the method described in

[11] .

[0142]

[13] An antibody or a binding fragment thereof that binds to HLA-A2 / NY-ESO, wherein the antibody or the binding fragment thereof has the property described in (i) or (ii) below:

[0143] (i) binds to the site on HLA-A2 / NY-ESO recognized by the antibody or binding fragment thereof described in [7];

[0144] (ii) competing with the antibody or binding fragment thereof described in [7] for binding to human HLA-A2 / NY-ESO.

[0145]

[14] A pharmaceutical composition comprising as an active ingredient: the antibody or binding fragment thereof described in any one of [1] to [7],

[12] and

[13] ; the polynucleotide described in [8]; the vector described in [9]; or the cell described in

[10] .

[0146]

[15] A molecule that specifically binds to human HLA / NY-ESO, comprising the antibody or binding fragment thereof according to any one of [1] to [7],

[12] and

[13] .

[0147]

[16] The molecule according to

[15] , which is a multispecific antibody.

[0148]

[17] The molecule according to

[15] , which is a bispecific antibody.

[0149]

[18] The molecule according to any one of

[15] to

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

[0150]

[19] The molecule according to

[18] , wherein the antibody or its binding fragment that specifically binds to CD3 is an antibody or its binding fragment that specifically binds to CD3 comprising the following chains:

[0151] (CCH1) heavy chain CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 141;

[0152] (CCH2) a heavy chain CDRH2 consisting of the amino acid sequence of SEQ ID NO: 142, or a heavy chain CDRH2 consisting of an amino acid sequence in which the third amino acid in the amino acid sequence of SEQ ID NO: 142 is N or S;

[0153] (CCH3) heavy chain CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 143;

[0154] (CCL1) light chain CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 144;

[0155] (CCL2) a light chain CDRL2 consisting of the amino acid sequence of SEQ ID NO: 145, or a light chain CDRL2 consisting of an amino acid sequence of SEQ ID NO: 145 wherein the second amino acid is N; and

[0156] (CCL3) Light chain CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 146.

[0157]

[20] The molecule according to

[19] , wherein the antibody or binding fragment thereof that specifically binds to CD3 comprises:

[0158] (CH1) a heavy chain variable region consisting of the amino acid sequence from positions 2 to 119 of the amino acid sequence set forth in SEQ ID NO: 136;

[0159] (CH2) a heavy chain variable region consisting of amino acids 2 to 119 of the amino acid sequence of SEQ ID NO: 137;

[0160] (CH3) a heavy chain variable region consisting of amino acids 2 to 119 of the amino acid sequence of SEQ ID NO: 147;

[0161] (CH4) a heavy chain variable region consisting of the amino acid sequence from positions 2 to 119 of the amino acid sequence of SEQ ID NO: 138;

[0162] (CH5) a heavy chain variable region consisting of the amino acid sequence from positions 2 to 119 of the amino acid sequence of SEQ ID NO: 139;

[0163] (CH6) a heavy chain variable region consisting of the amino acid sequence from positions 2 to 119 of the amino acid sequence of SEQ ID NO: 140;

[0164] (CH7) a heavy chain variable region consisting of the amino acid sequence from positions 272 to 389 of the amino acid sequence set forth in SEQ ID NO: 155;

[0165] (CH8) a heavy chain variable region consisting of the amino acid sequence from positions 277 to 394 of the amino acid sequence shown in SEQ ID NO: 156; or

[0166] (CH9) a heavy chain variable region consisting of the amino acid sequence from positions 277 to 394 of the amino acid sequence shown in SEQ ID NO: 157; and

[0167] (CL1) a light chain variable region consisting of the amino acid sequence from positions 135 to 243 of the amino acid sequence set forth in SEQ ID NO: 136;

[0168] (CL2) a light chain variable region consisting of the amino acid sequence from positions 135 to 241 of the amino acid sequence set forth in SEQ ID NO: 137;

[0169] (CL3) a light chain variable region consisting of the amino acid sequence from positions 135 to 243 of the amino acid sequence set forth in SEQ ID NO: 147;

[0170] (CL4) a light chain variable region consisting of the amino acid sequence from positions 135 to 241 of the amino acid sequence set forth in SEQ ID NO: 138;

[0171] (CL5) a light chain variable region consisting of the amino acid sequence from positions 135 to 243 of the amino acid sequence set forth in SEQ ID NO: 139;

[0172] (CL6) a light chain variable region consisting of the amino acid sequence from positions 135 to 243 of the amino acid sequence set forth in SEQ ID NO: 140;

[0173] (CL7) a light chain variable region consisting of the amino acid sequence from positions 405 to 511 of the amino acid sequence set forth in SEQ ID NO: 155;

[0174] (CL8) a light chain variable region consisting of the amino acid sequence from positions 410 to 516 of the amino acid sequence shown in SEQ ID NO: 156; or

[0175] (CL9) A light chain variable region consisting of the amino acid sequence from positions 410 to 516 of the amino acid sequence shown in SEQ ID NO: 157.

[0176]

[21] The molecule according to

[20] , wherein the antibody or binding fragment thereof that specifically binds to CD3 comprises:

[0177] (CH1CL1) a heavy chain variable region consisting of amino acids 2 to 119 of the amino acid sequence of SEQ ID NO: 136, and a light chain variable region consisting of amino acids 135 to 243 of the amino acid sequence of SEQ ID NO: 136;

[0178] (CH2CL2) a heavy chain variable region consisting of amino acids 2 to 119 of SEQ ID NO: 137, and a light chain variable region consisting of amino acids 135 to 241 of SEQ ID NO: 137;

[0179] (CH3CL3) a heavy chain variable region consisting of amino acids 2 to 119 of SEQ ID NO: 147, and a light chain variable region consisting of amino acids 135 to 243 of SEQ ID NO: 147;

[0180] (CH4CL4) a heavy chain variable region consisting of amino acids 2 to 119 of SEQ ID NO: 138, and a light chain variable region consisting of amino acids 135 to 241 of SEQ ID NO: 138;

[0181] (CH5CL5) a heavy chain variable region consisting of amino acids 2 to 119 of the amino acid sequence of SEQ ID NO: 139, and a light chain variable region consisting of amino acids 135 to 243 of the amino acid sequence of SEQ ID NO: 139;

[0182] (CH6CL6) a heavy chain variable region consisting of amino acids 2 to 119 of SEQ ID NO: 140, and a light chain variable region consisting of amino acids 135 to 243 of SEQ ID NO: 140;

[0183] (CH7CL7) a heavy chain variable region consisting of amino acids 272 to 389 of SEQ ID NO: 155, and a light chain variable region consisting of amino acids 272 to 389 of SEQ ID NO: 155;

[0184] (CH8CL8) a heavy chain variable region consisting of the amino acid sequence from positions 277 to 394 of the amino acid sequence set forth in SEQ ID NO: 156, and a light chain variable region consisting of the amino acid sequence from positions 410 to 516 of the amino acid sequence set forth in SEQ ID NO: 156; or

[0185] (CH9CL9) a heavy chain variable region consisting of the amino acid sequence from positions 277 to 394 of the amino acid sequence shown in SEQ ID NO: 157, and a light chain variable region consisting of the amino acid sequence from positions 410 to 516 of the amino acid sequence shown in SEQ ID NO: 157.

[0186]

[22] The molecule according to any one of

[18] to

[21] , wherein the antibody or binding fragment thereof that specifically binds to CD3 is a scFv.

[0187]

[23] The molecule of

[22] , wherein the scFv comprises:

[0188] (CS1) an scFv consisting of the amino acid sequence from positions 2 to 243 of the amino acid sequence of SEQ ID NO: 136;

[0189] (CS2) an scFv consisting of the amino acid sequence from positions 2 to 241 of the amino acid sequence of SEQ ID NO: 137;

[0190] (CS3) an scFv consisting of the amino acid sequence from positions 2 to 243 of the amino acid sequence of SEQ ID NO: 147;

[0191] (CS4) an scFv consisting of the amino acid sequence from positions 2 to 241 of the amino acid sequence of SEQ ID NO: 138;

[0192] (CS5) an scFv consisting of the amino acid sequence from positions 2 to 243 of the amino acid sequence of SEQ ID NO: 139;

[0193] (CS6) an scFv consisting of the amino acid sequence from positions 2 to 243 of the amino acid sequence of SEQ ID NO: 140;

[0194] (CS7) an scFv consisting of the amino acid sequence from positions 272 to 511 of the amino acid sequence of SEQ ID NO: 155;

[0195] (CS8) an scFv consisting of the amino acid sequence from positions 277 to 516 of the amino acid sequence shown in SEQ ID NO: 156; or

[0196] (CS9) scFv consisting of the amino acid sequence from positions 277 to 516 of the amino acid sequence shown in SEQ ID NO: 157.

[0197]

[24] The molecule according to any one of

[18] to

[23] , wherein the molecule comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises, from the N-terminus to the C-terminus, an scFv that specifically binds to human HLA / NY-ESO, an scFv that specifically binds to CD3, and an Fc region (i); the second polypeptide comprises an Fc region (ii); preferably, the molecule associates in the Fc region (i) and the Fc region (ii).

[0198]

[25] The molecule according to

[24] , wherein the scFv that specifically binds to human HLA / NY-ESO is the antibody or binding fragment thereof that specifically binds to human HLA / NY-ESO as described in [1] as an scFv.

[0199]

[26] The molecule according to

[24] , wherein the scFv that specifically binds to human HLA / NY-ESO is the antibody or binding fragment thereof that specifically binds to human HLA / NY-ESO as described in [2] as an scFv.

[0200]

[27] The molecule according to

[24] , wherein the scFv that specifically binds to human HLA / NY-ESO is the antibody or binding fragment thereof that specifically binds to human HLA / NY-ESO as described in [3] as an scFv.

[0201]

[28] The molecule according to

[24] , wherein the scFv that specifically binds to human HLA / NY-ESO is the antibody or binding fragment thereof that specifically binds to human HLA / NY-ESO as described in [4] as an scFv.

[0202]

[29] The molecule according to

[24] , wherein the scFv that specifically binds to human HLA / NY-ESO is the antibody or binding fragment thereof that specifically binds to human HLA / NY-ESO as described in [5] as an scFv.

[0203]

[30] The molecule according to any one of

[19] to

[24] , wherein the scFv that specifically binds to CD3 is the antibody or binding fragment thereof that specifically binds to CD3 according to

[18] as an scFv.

[0204]

[31] The molecule according to any one of

[19] to

[24] , wherein the scFv that specifically binds to CD3 is the antibody or binding fragment thereof that specifically binds to CD3 according to

[19] as an scFv.

[0205]

[32] The molecule according to any one of

[19] to

[24] , wherein the scFv that specifically binds to CD3 is the antibody or binding fragment thereof that specifically binds to CD3 as described in

[20] or

[21] as an scFv.

[0206]

[33] The molecule according to any one of

[19] to

[24] , wherein the scFv that specifically binds to CD3 is the antibody or binding fragment thereof that specifically binds to CD3 according to

[23] as an scFv.

[0207]

[34] The molecule according to any one of

[24] to

[33] , which contains an amino acid sequence selected from the group consisting of the amino acid sequence at positions 21 to 511 of the amino acid sequence of SEQ ID NO: 85, the amino acid sequence at positions 21 to 511 of the amino acid sequence of SEQ ID NO: 87, the amino acid sequence at positions 21 to 511 of the amino acid sequence of SEQ ID NO: 88, the amino acid sequence at positions 21 to 511 of the amino acid sequence of SEQ ID NO: 89, the amino acid sequence at positions 21 to 511 of the amino acid sequence of SEQ ID NO: 90, the amino acid sequence at positions 21 to 511 of the amino acid sequence of SEQ ID NO: 91, the amino acid sequence at positions 21 to 511 of the amino acid sequence of SEQ ID NO: 92, the amino acid sequence at positions 21 to 511 of the amino acid sequence of SEQ ID NO: 93, the amino acid sequence at positions 21 to 511 of the amino acid sequence of SEQ ID NO: 94, the amino acid sequence from positions 21 to 511 of the amino acid sequence shown in SEQ ID NO: 95, the amino acid sequence from positions 21 to 511 of the amino acid sequence shown in SEQ ID NO: 96, the amino acid sequence from positions 21 to 511 of the amino acid sequence shown in SEQ ID NO: 86, the amino acid sequence from positions 21 to 511 of the amino acid sequence shown in SEQ ID NO: 149, and the amino acid sequence from positions 21 to 511 of the amino acid sequence shown in SEQ ID NO: 150.

[0208]

[35] The molecule according to any one of

[24] to

[33] , which contains an amino acid sequence selected from the group consisting of the amino acid sequence at positions 21 to 511 of the amino acid sequence shown in SEQ ID NO: 155, the amino acid sequence at positions 21 to 516 of the amino acid sequence shown in SEQ ID NO: 156, and the amino acid sequence at positions 21 to 516 of the amino acid sequence shown in SEQ ID NO: 157.

[0209]

[36] The molecule according to any one of

[24] to

[34] , wherein the first polypeptide comprises the amino acid sequence from positions 529 to 745 of the amino acid sequence represented by SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:86, SEQ ID NO:149 or SEQ ID NO:150.

[0210]

[37] The molecule according to any one of

[24] to

[33] and

[35] , wherein the first polypeptide comprises: the amino acid sequence of positions 529 to 745 of the amino acid sequence shown in SEQ ID NO: 155, the amino acid sequence of positions 534 to 750 of the amino acid sequence shown in SEQ ID NO: 156, or the amino acid sequence of positions 534 to 750 of the amino acid sequence shown in SEQ ID NO: 157.

[0211]

[38] The molecule according to

[34] or

[36] , wherein the first polypeptide consists of an amino acid sequence selected from the group consisting of the amino acid sequence of positions 20 to 745 of the amino acid sequence of SEQ ID NO: 85, the amino acid sequence of positions 20 to 745 of the amino acid sequence of SEQ ID NO: 87, the amino acid sequence of positions 20 to 745 of the amino acid sequence of SEQ ID NO: 88, the amino acid sequence of positions 20 to 745 of the amino acid sequence of SEQ ID NO: 89, the amino acid sequence of positions 20 to 745 of the amino acid sequence of SEQ ID NO: 90, the amino acid sequence of positions 20 to 745 of the amino acid sequence of SEQ ID NO: 91, the amino acid sequence of positions 20 to 745 of the amino acid sequence of SEQ ID NO: 92, the amino acid sequence of positions 20 to 745 of the amino acid sequence of SEQ ID NO: 93, the amino acid sequence of positions 20 to 745 of the amino acid sequence of SEQ ID NO: 94, the amino acid sequence of positions 20 to 745 of the amino acid sequence of SEQ ID NO: 95, the amino acid sequence from positions 20 to 745 of the amino acid sequence shown in SEQ ID NO: 95, the amino acid sequence from positions 20 to 745 of the amino acid sequence shown in SEQ ID NO: 96, the amino acid sequence from positions 20 to 745 of the amino acid sequence shown in SEQ ID NO: 86, the amino acid sequence from positions 20 to 745 of the amino acid sequence shown in SEQ ID NO: 149, and the amino acid sequence from positions 20 to 745 of the amino acid sequence shown in SEQ ID NO: 150.

[0212]

[39] The molecule according to

[35] or

[37] , wherein the first polypeptide consists of an amino acid sequence selected from the group consisting of: the amino acid sequence at positions 20 to 745 of the amino acid sequence shown in SEQ ID NO: 155, the amino acid sequence at positions 20 to 750 of the amino acid sequence shown in SEQ ID NO: 156; or the amino acid sequence at positions 20 to 750 of the amino acid sequence shown in SEQ ID NO: 157.

[0213]

[40] The molecule according to any one of

[24] to

[39] , wherein the second polypeptide comprises the amino acid sequence from positions 20 to 246 of the amino acid sequence shown in SEQ ID NO: 84.

[0214]

[41] The molecule according to any one of

[18] to

[23] , comprising a first polypeptide, a second polypeptide and a third polypeptide,

[0215] The first polypeptide comprises, from the N-terminus to 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);

[0216] The second polypeptide consists of an immunoglobulin heavy chain containing an Fc region (ii);

[0217] The third polypeptide consists of an immunoglobulin light chain,

[0218] Preferably, the second polypeptide and the third polypeptide associate,

[0219] The first polypeptide and the second polypeptide are associated in their respective Fc regions.

[0220]

[42] The molecule according to

[41] , wherein the scFv that specifically binds to human HLA / NY-ESO is the antibody or binding fragment thereof that specifically binds to human HLA / NY-ESO as described in [1] as an scFv.

[0221]

[43] The molecule according to

[41] , wherein the scFv that specifically binds to human HLA / NY-ESO is the antibody or binding fragment thereof that specifically binds to human HLA / NY-ESO as described in [2] as an scFv.

[0222]

[44] The molecule according to

[41] , wherein the scFv that specifically binds to human HLA / NY-ESO is the antibody or binding fragment thereof that specifically binds to human HLA / NY-ESO as described in [3] as an scFv.

[0223]

[45] The molecule according to

[41] , wherein the scFv that specifically binds to human HLA / NY-ESO is the antibody or binding fragment thereof that specifically binds to human HLA / NY-ESO as described in [4] as an scFv.

[0224]

[46] The molecule according to

[41] , wherein the scFv that specifically binds to human HLA / NY-ESO is the antibody or binding fragment thereof that specifically binds to human HLA / NY-ESO as described in [5] as an scFv.

[0225]

[47] The molecule according to any one of

[42] to

[46] , wherein the scFv that specifically binds to CD3 is the antibody or binding fragment thereof that specifically binds to CD3 as described in

[18] as an scFv.

[0226]

[48] ​​The molecule according to any one of

[42] to

[46] , wherein the scFv that specifically binds to CD3 is the antibody or binding fragment thereof that specifically binds to CD3 as described in

[19] as an scFv.

[0227]

[49] The molecule according to any one of

[42] to

[46] , wherein the scFv that specifically binds to CD3 is the antibody or binding fragment thereof that specifically binds to CD3 as described in

[20] or

[21] as an scFv.

[0228]

[50] The molecule according to any one of

[42] to

[46] , wherein the scFv that specifically binds to CD3 is the antibody or binding fragment thereof that specifically binds to CD3 as described in

[23] as an scFv.

[0229]

[51] The molecule according to any one of

[41] to

[50] , wherein the second polypeptide comprises an amino acid sequence of amino acids 20 to 242 of the amino acid sequence shown in SEQ ID NO:99.

[0230]

[52] The molecule according to any one of

[41] to

[51] , wherein the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 100.

[0231]

[53] The molecule according to any one of

[41] to

[52] , wherein the first polypeptide comprises an amino acid sequence selected from the group consisting of the amino acid sequence at positions 21 to 511 of the amino acid sequence of SEQ ID NO: 85, the amino acid sequence at positions 21 to 511 of the amino acid sequence of SEQ ID NO: 87, the amino acid sequence at positions 21 to 511 of the amino acid sequence of SEQ ID NO: 88, the amino acid sequence at positions 21 to 511 of the amino acid sequence of SEQ ID NO: 89, the amino acid sequence at positions 21 to 511 of the amino acid sequence of SEQ ID NO: 90, the amino acid sequence at positions 21 to 511 of the amino acid sequence of SEQ ID NO: 91, the amino acid sequence at positions 21 to 511 of the amino acid sequence of SEQ ID NO: 92, the amino acid sequence at positions 21 to 511 of the amino acid sequence of SEQ ID NO: 93, the amino acid sequence at positions 21 to 511 of the amino acid sequence of SEQ ID NO: 94, the amino acid sequence at positions 21 to 511 of the amino acid sequence of SEQ ID NO: The amino acid sequence from positions 21 to 511 of the amino acid sequence shown in SEQ ID NO: 95, the amino acid sequence from positions 21 to 511 of the amino acid sequence shown in SEQ ID NO: 96, the amino acid sequence from positions 21 to 511 of the amino acid sequence shown in SEQ ID NO: 86, the amino acid sequence from positions 21 to 511 of the amino acid sequence shown in SEQ ID NO: 149, and the amino acid sequence from positions 21 to 511 of the amino acid sequence shown in SEQ ID NO: 150.

[0232]

[54] The molecule according to any one of

[41] to

[53] , wherein the first polypeptide consists of an amino acid sequence selected from the group consisting of the amino acid sequence of positions 20 to 745 of the amino acid sequence of SEQ ID NO: 85, the amino acid sequence of positions 20 to 745 of the amino acid sequence of SEQ ID NO: 87, the amino acid sequence of positions 20 to 745 of the amino acid sequence of SEQ ID NO: 88, the amino acid sequence of positions 20 to 745 of the amino acid sequence of SEQ ID NO: 89, the amino acid sequence of positions 20 to 745 of the amino acid sequence of SEQ ID NO: 90, the amino acid sequence of positions 20 to 745 of the amino acid sequence of SEQ ID NO: 91, the amino acid sequence of positions 20 to 745 of the amino acid sequence of SEQ ID NO: 92, the amino acid sequence of positions 20 to 745 of the amino acid sequence of SEQ ID NO: 93, the amino acid sequence of positions 20 to 745 of the amino acid sequence of SEQ ID NO: 94, the amino acid sequence of positions 20 to 745 of the amino acid sequence of SEQ ID NO: The amino acid sequence from positions 20 to 745 of the amino acid sequence shown in SEQ ID NO: 95, the amino acid sequence from positions 20 to 745 of the amino acid sequence shown in SEQ ID NO: 96, and the amino acid sequence from positions 20 to 745 of the amino acid sequence shown in SEQ ID NO: 86, the amino acid sequence from positions 20 to 745 of the amino acid sequence shown in SEQ ID NO: 149, and the amino acid sequence from positions 20 to 745 of the amino acid sequence shown in SEQ ID NO: 150.

[0233]

[55] The molecule according to any one of

[18] to

[23] , the molecule contains a first polypeptide, a second polypeptide and a third polypeptide, wherein the first polypeptide contains, from the N-terminus to the C-terminus, an scFv that specifically binds to human HLA / NY-ESO, a variable region and a constant region CH1 of an antibody heavy chain that specifically binds to CD3, and an immunoglobulin Fc region (i); the second polypeptide contains a hinge region and an Fc region (ii) of an immunoglobulin; and the third polypeptide contains an antibody light chain consisting of a variable region and a constant region. Preferably, 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 the constant region CH1 of the antibody heavy chain.

[0234]

[56] The molecule according to

[55] , wherein the scFv that specifically binds to human HLA / NY-ESO is the antibody or binding fragment thereof that specifically binds to human HLA / NY-ESO as described in [1] as an scFv.

[0235]

[57] The molecule according to

[55] , wherein the scFv that specifically binds to human HLA / NY-ESO is the antibody or binding fragment thereof that specifically binds to human HLA / NY-ESO as described in [2] as an scFv.

[0236]

[58] The molecule according to

[55] , wherein the scFv that specifically binds to human HLA / NY-ESO is the antibody or binding fragment thereof that specifically binds to human HLA / NY-ESO as described in [3] as an scFv.

[0237]

[59] The molecule according to

[55] , wherein the scFv that specifically binds to human HLA / NY-ESO is the antibody or binding fragment thereof that specifically binds to human HLA / NY-ESO as described in [4] as an scFv.

[0238]

[60] The molecule according to

[55] , wherein the scFv that specifically binds to human HLA / NY-ESO is the antibody or binding fragment thereof that specifically binds to human HLA / NY-ESO as described in [5] as an scFv.

[0239]

[61] The molecule according to any one of

[18] to

[21] , wherein the antibody or binding fragment thereof that specifically binds to CD3 is Fab.

[0240]

[62] The molecule according to any one of

[56] to

[61] , wherein the Fab that specifically binds to CD3 is the antibody or binding fragment thereof that specifically binds to CD3 as described in

[18] as Fab.

[0241]

[63] The molecule according to any one of

[56] to

[61] , wherein the Fab that specifically binds to CD3 is the antibody or binding fragment thereof that specifically binds to CD3 as described in

[19] as Fab.

[0242]

[64] The molecule according to any one of

[56] to

[61] , wherein the Fab that specifically binds to CD3 is the antibody or binding fragment thereof that specifically binds to CD3 as described in

[20] or

[21] as Fab.

[0243]

[65] The molecule according to any one of

[55] to

[64] , wherein the first polypeptide comprises the amino acid sequence from positions 21 to 394 of the amino acid sequence shown in SEQ ID NO: 160.

[0244]

[66] The molecule according to any one of

[55] to

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

[0245] (i) the amino acid sequence from positions 20 to 724 of the amino acid sequence shown in SEQ ID NO: 160;

[0246] (ii) the amino acid sequence from positions 20 to 719 of the amino acid sequence shown in SEQ ID NO: 197;

[0247] (iii) the amino acid sequence from position 20 to position 719 of the amino acid sequence shown in SEQ ID NO: 198.

[0248]

[67] The molecule according to any one of

[55] to

[66] , wherein the second polypeptide comprises the amino acid sequence from positions 20 to 246 of the amino acid sequence shown in SEQ ID NO: 84.

[0249]

[68] The molecule according to any one of

[56] to

[67] , wherein the third polypeptide comprises the amino acid sequence from positions 21 to 127 of the amino acid sequence shown in SEQ ID NO: 161.

[0250]

[69] The molecule according to any one of

[56] to

[68] , wherein the third polypeptide comprises the amino acid sequence from positions 21 to 233 of the amino acid sequence shown in SEQ ID NO: 161.

[0251]

[70] The molecule according to any one of

[15] to

[69] , wherein in the amino acid sequence of one or more polypeptides contained in the molecule, one or two amino acids are missing from the carboxyl terminus of the sequence.

[0252]

[71] A polynucleotide comprising a nucleotide sequence encoding the amino acid sequence contained in the molecule described in any one of

[15] to

[70] .

[0253]

[72] A vector comprising the polynucleotide described in

[71] .

[0254]

[73] A host cell containing the polynucleotide described in

[71] or the vector described in

[72] .

[0255]

[74] A method for producing a molecule that specifically binds to human HLA / NY-ESO and human CD3, the method comprising: (i) culturing the host cell described in

[73] ; and (ii) purifying the antibody or its binding fragment from the culture obtained in step (i).

[0256]

[75] A molecule that specifically binds to human HLA / NY-ESO and human CD3, the molecule being obtained by the method described in

[74] .

[0257]

[76] A pharmaceutical composition comprising as an active ingredient: the molecule described in any one of

[15] to

[70] and

[75] ; the polynucleotide described in

[71] ; the vector described in

[72] ; or the host cell described in

[73] .

[0258]

[77] The pharmaceutical composition according to

[14] or

[76] , which is an anticancer agent.

[0259]

[78] The pharmaceutical composition according to

[77] , wherein the cancer is one or more cancers selected from the group consisting of the following cancers: 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, gastric cancer, duodenal cancer, small intestinal cancer, colorectal cancer, rectal cancer, appendix cancer, anal cancer, liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, adrenal cancer, bladder cancer, Bladder cancer, prostate cancer, uterine cancer, vaginal cancer, liposarcoma, angiosarcoma, chondrosarcoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, undifferentiated pleomorphic sarcoma, myxofibrosarcoma, malignant peripheral nerve sheath tumor, retroperitoneal sarcoma, synovial sarcoma, uterine sarcoma, gastrointestinal stromal tumor, leiomyosarcoma, epithelioid sarcoma, B-cell lymphoma, NK / T-cell lymphoma, Hodgkin lymphoma, myeloid leukemia, lymphocytic leukemia, myeloproliferative disorders, myelodysplastic syndrome, multiple myeloma, testicular cancer, and ovarian cancer.

[0260]

[79] The pharmaceutical composition according to any one of

[76] to

[78] , which is used in combination with other pharmaceutical agents.

[0261] This specification incorporates the disclosure of Japanese Patent Application No. 2020-061476, which is the basis of priority for this application.

[0262] Effects of the Invention

[0263] According to the present invention, antibodies that bind to HLA-A2 / NY-ESO, as well as novel bispecific antibodies (bispecific molecules) that bind to both HLA-A2 / NY-ESO and CD3, can be obtained. Furthermore, novel pharmaceutical compositions containing such antibodies (molecules) as active ingredients can be obtained. These antibodies or molecules exhibit cytotoxic activity and can be used as therapeutic or preventive agents for cancer and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0264] Figure 1This table shows the normalized gMFI of the anti-HLA / NY-ESO scFvs NYA-0001, 1143, 1154, 1163, 2023, 2027, 2035, 2044, 2045, 2047, 2048, 2060, 2061, 2143, NYC-0003, and 0004 on T2 cells supplemented with various point mutation peptides. * / Underlined characters indicate that the normalized gMFI of each scFv on T2 cells supplemented with the NY-ESO peptide was less than half.

[0265] Figure 2A The table shows the information of selected homologous peptides. The binding activity to HLA-A0201 was predicted using NetMHCPan2.8, and the 50% inhibitory concentration (IC 50 )express.

[0266] Figure 2B This table shows the normalized gMFI of anti-HLA / NY-ESO scFvs NYA-0001, 1143, 1154, 1163, 2023, 2027, 2035, 2044, 2045, 2047, 2048, 2060, 2061, 2143, NYC-0003, and 0004 on T2 cells supplemented with various homologous peptides. * / Underlined values ​​indicate values ​​greater than the normalized gMFI of each scFv on T2 cells supplemented with DMSO.

[0267] Figure 3It is a diagram showing the configuration of the antibody shown in this example. (a) scFv: It is a configuration in which the antibody H chain variable region (VH, described later) and the antibody L chain variable region (VL, described later) (both in white) are connected by a linker. In this example, scFv against HLA-A2 / NY-ESO and CD3 were evaluated. (b) Fab: It is a configuration consisting of VH (white) and the antibody H chain constant region (CH1: checkered pattern) and VL (white) and the antibody L chain constant region (horizontal line). In this example, anti-HLA-A2 / NY-ESO Fab and the like were evaluated. (c) taFv: It is a configuration in which two scFvs (white and upper right slanted) are connected by a linker. In this example, taFv containing anti-HLA-A2 / NY-ESO scFv and anti-CD3 scFv was evaluated. (d) taFv-heterodimer Fc type: This is a configuration in which an Fc (upper left slash) (also referred to as the first polypeptide) with a heterodimer-forming mutation is added to the C-terminus of the taFv and is heterozygous with another Fc (blackened: also referred to as the second polypeptide). In this example, taFv-heterodimer Fc containing anti-HLA-A2 / NY-ESO scFv and anti-CD3 scFv were evaluated. (e) taFv-Fab-heterodimer Fc type: This is a configuration in which Fab is added to the above-mentioned taFv-heterodimer Fc type. In this example, taFv containing anti-HLA-A2 / NY-ESO scFv and anti-CD3 scFv, as well as taFv-Fab-heterodimer Fc using HLA-A2 / NY-ESO Fab, were evaluated. (f) represents a first polypeptide common to both the taFv-heterodimer Fc type and the taFv-Fab-heterodimer Fc type. The first polypeptide comprises, from the N-terminus to the C-terminus, an scFv that specifically binds to human HLA / NY-ESO, an scFv that specifically binds to CD3, and an Fc region (i). (g) 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 including a hinge region and the Fc region (ii). (i) represents a third polypeptide of the taFv-Fab-heterodimer Fc type. The third polypeptide comprises an immunoglobulin light chain.

[0268] Figure 4AThis graph shows that the Fc-added 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 cytotoxic activity against U266B1 cells expressing endogenous human NY-ESO in the presence of human PBMCs. Error bars in the graph represent standard deviations (n=3).

[0269] Figure 4B This graph shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules NYF-0016 and NYF-0058 have cytotoxic activity against U266B1 cells expressing endogenous human NY-ESO in the presence of human PBMCs. Error bars in the graph represent standard deviations (n=3).

[0270] Figure 4C This graph shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules NYF-0023, NYF-0045, NYF-0048, NYF-0060, and NYF-0061 have cytotoxic activity against U266B1 cells expressing endogenous human NY-ESO in the presence of human PBMCs. Error bars in the graph represent standard deviations (n=3).

[0271] Figure 4D This graph shows that the Fc-added 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 cytotoxic activity against NCI-H1703 cells expressing endogenous human NY-ESO in the presence of human PBMCs. Error bars in the graph represent standard deviations (n=3).

[0272] Figure 4E This graph shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules NYF-0016 and NYF-0058 have cytotoxic activity against NCI-H1703 cells expressing endogenous human NY-ESO in the presence of human PBMCs. Error bars in the graph represent standard deviations (n=3).

[0273] Figure 4FThis graph shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules NYF-0023, NYF-0045, NYF-0048, NYF-0060, and NYF-0061 have cytotoxic activity against NCI-H1703 cells expressing endogenous human NY-ESO in the presence of human PBMCs. Error bars in the graph represent standard deviations (n=3).

[0274] Figure 4G This graph shows that the Fc-added 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 exhibited no cytotoxic activity against AGS cells that do not express endogenous human NY-ESO in the presence of human PBMCs. Error bars in the graph represent standard deviations (n ​​= 3).

[0275] Figure 4H This graph shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules NYF-0016 and NYF-0058 exhibit no cytotoxic activity against AGS cells that do not express endogenous human NY-ESO in the presence of human PBMCs. Error bars in the graph represent standard deviations (n=3).

[0276] Figure 4I This graph shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules NYF-0023, NYF-0045, NYF-0048, NYF-0060, and NYF-0061 exhibit no cytotoxic activity against AGS cells that do not express endogenous human NY-ESO in the presence of human PBMCs. Error bars in the graph represent standard deviations (n=3).

[0277] Figure 4J This graph shows that the Fc-added 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 exhibited no cytotoxic activity against CFPAC-1 cells that do not express endogenous human NY-ESO in the presence of human PBMCs. Error bars in the graph represent standard deviations (n ​​= 3).

[0278] Figure 4KThis graph shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules NYF-0016 and NYF-0058 exhibit no cytotoxic activity against CFPAC-1 cells that do not express endogenous human NY-ESO in the presence of human PBMCs. Error bars in the graph represent standard deviations (n=3).

[0279] Figure 4L This graph shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules NYF-0023, NYF-0045, NYF-0048, NYF-0060, and NYF-0061 exhibit no cytotoxic activity against CFPAC-1 cells that do not express endogenous human NY-ESO in the presence of human PBMCs. Error bars in the graph represent standard deviations (n=3).

[0280] Figure 5A This graph shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules NYF-0016, NYF-0019, NYF-0044, and NYF-0047 have anti-tumor activity in a human PBMC metastasis model. Error bars in the graph represent standard error (n=4 on day 32 for NYF-0044 only; n=5 for the others).

[0281] Figure 5B This graph shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules NYF-0016, NYF-0022, NYF-0023, NYF-0027, NYF-0035, and NYF-0058 have anti-tumor activity in a human PBMC metastasis model. Error bars in the graph represent standard error (n=5, n=6 for the blank control group only).

[0282] Figure 5C This graph shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules NYF-0016, NYF-0045, NYF-0048, NYF-0060, and NYF-0061 have anti-tumor activity in a human PBMC metastasis model. Error bars in the graph represent standard error (n=5).

[0283] Figure 6AThe diagrams represent the configurations of the antibodies described in this example. (a) Hybrid: Two Fcs with heterodimer-forming mutations (hatched and black) are added to the C-terminus of the Fab and the C-terminus of the scFv, respectively, and the configuration is formed by the association of these two Fcs. In this example, a hybrid containing an anti-HLA-A2 / NY-ESO Fab and an anti-CD3 scFv was evaluated. (b) Dual: Two Fcs with heterodimer-forming mutations (hatched and black) are added to the C-terminus of two different scFvs, respectively, and the configuration is formed by the association of these two Fc hybrids. In this example, a dual containing an anti-HLA-A2 / NY-ESO scFv and an anti-CD3 scFv was evaluated. (c) scFv-Fab-heterodimer Fc: This configuration consists of an Fc (upper left slash) with a heterodimer-forming mutation added to the C-terminus of the scFv and Fab linked via a linker, and then associated with another Fc (black). In this example, scFv-Fab-heterodimer Fcs containing anti-CD3 scFv (upper right slash) and anti-HLA-A2 / NY-ESO Fab were evaluated. Separately, scFv-Fab-heterodimer Fcs containing anti-HLA-A2 / NY-ESO scFv (upper right slash) and anti-CD3 Fab were also evaluated.

[0284] Figure 6B The diagrams show the configurations of the antibodies described in this example. (a) taFv-heterodimer Fc type: Figure 3 (d). This configuration is a configuration in which an Fc (upper left slant line) into which a mutation that forms a heterodimer is introduced is added to the C-terminal side of taFv, and is associated with another Fc (painted in black) and a heterozygote. In this example, a taFv-heterodimer Fc type consisting of anti-HLA-A2 / NY-ESO scFv and anti-CD3 scFv was evaluated. (b) taFv (reverse)-heterodimer Fc type: a configuration in which the order of the two scFvs in the above-mentioned taFv-heterodimer Fc type is reversed. In this example, a taFv (reverse)-heterodimer Fc type consisting of anti-CD3 scFv and anti-HLA-A2 / NY-ESO scFv was evaluated. (c) represents the first polypeptide of the taFv (reverse)-heterodimer Fc type. The first polypeptide comprises, from the N-terminus to 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) represents a second polypeptide in the form of a taFv (reverse) heterodimer Fc. The second polypeptide comprises a hinge region and an Fc region (ii).

[0285] Figure 7AThis graph shows the cytotoxic activity of various anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules (hybrid (NYG-3143), diabody (NYG-2143), and taFv-heterodimeric Fcbody (NYF-0011)) against U266B1 cells expressing endogenous human NY-ESO in the presence of human PBMCs. Error bars in the graph represent standard deviation (n = 3).

[0286] Figure 7B This graph shows the cytotoxic activity of various anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules (scFv-Fab-heterodimeric Fc antibody (NYF-0003), taFv-heterodimeric Fc antibody (NYF-0010), and taFv (reverse)-heterodimeric Fc antibody (NYF-0004)) against U266B1 cells expressing endogenous human NY-ESO in the presence of human PBMCs. Error bars in the graph represent standard deviation (n = 3).

[0287] Figure 8 is the amino acid sequence of the peptide in NY-ESO (SEQ ID NO: 1).

[0288] Figure 9 is the amino acid sequence of the peptide in MAGEC-1 (SEQ ID NO: 2).

[0289] Figure 10 This is primer 1 (SEQ ID NO: 3) used for scFv sequence analysis.

[0290] Figure 11 This is primer 2 (SEQ ID NO: 4) used for scFv sequence analysis.

[0291] Figure 12 The nucleotide sequence of the heavy chain variable region of NYA-0001 (SEQ ID NO: 5).

[0292] Figure 13 is the amino acid sequence of the heavy chain variable region of NYA-0001 (SEQ ID NO: 6).

[0293] Figure 14 The nucleotide sequence of the light chain variable region of NYA-0001 (SEQ ID NO: 7).

[0294] Figure 15 is the amino acid sequence of the light chain variable region of NYA-0001 (SEQ ID NO: 8).

[0295] Figure 16 is the nucleotide sequence of the heavy chain variable region of NYA-0060 (SEQ ID NO: 9).

[0296] Figure 17 is the amino acid sequence of the heavy chain variable region of NYA-0060 (SEQ ID NO: 10).

[0297] Figure 18 The nucleotide sequence of the light chain variable region of NYA-0060 (SEQ ID NO: 11).

[0298] Figure 19 is the amino acid sequence of the light chain variable region of NYA-0060 (SEQ ID NO: 12).

[0299] Figure 20 The nucleotide sequence of the heavy chain variable region of NYA-0068 (SEQ ID NO: 13).

[0300] Figure 21 is the amino acid sequence of the heavy chain variable region of NYA-0068 (SEQ ID NO: 14).

[0301] Figure 22 The nucleotide sequence of the light chain variable region of NYA-0068 (SEQ ID NO: 15).

[0302] Figure 23 is the amino acid sequence of the light chain variable region of NYA-0068 (SEQ ID NO: 16).

[0303] Figure 24 is the nucleotide sequence of the heavy chain variable region of NYA-0082 (SEQ ID NO: 17).

[0304] Figure 25 is the amino acid sequence of the heavy chain variable region of NYA-0082 (SEQ ID NO: 18).

[0305] Figure 26 The nucleotide sequence of the light chain variable region of NYA-0082 (SEQ ID NO: 19).

[0306] Figure 27 is the amino acid sequence of the light chain variable region of NYA-0082 (SEQ ID NO: 20).

[0307] Figure 28 is the nucleotide sequence of the NYA-1163 tag additive (SEQ ID NO: 21).

[0308] Figure 29 is the nucleotide sequence of the NYA-2023 tag additive (SEQ ID NO: 22).

[0309] Figure 30 is the nucleotide sequence of the NYA-2027 tag additive (SEQ ID NO: 23).

[0310] Figure 31 is the nucleotide sequence of the NYA-1143 tag additive (SEQ ID NO: 24).

[0311] Figure 32 is the nucleotide sequence of the NYA-2143 tag additive (SEQ ID NO: 25).

[0312] Figure 33 The amino acid sequence of the NYA-1163 tag additive (SEQ ID NO: 26), where NYA-1163 is amino acid numbered 21 to 266.

[0313] Figure 34 The amino acid sequence of the NYA-2023 tag additive (SEQ ID NO: 27), where NYA-2023 is amino acid numbered 21 to 266.

[0314] Figure 35 The amino acid sequence of the NYA-2027 tag additive (SEQ ID NO: 28), where NYA-2027 is amino acid numbered 21 to 266.

[0315] Figure 36 The amino acid sequence of the NYA-1143 tag additive (SEQ ID NO: 29), wherein NYA-1143 is amino acid numbered 21 to 266.

[0316] Figure 37 The amino acid sequence of the NYA-2143 tag additive (SEQ ID NO: 30), where NYA-2143 is amino acid numbered 21 to 266.

[0317] Figure 38 is the nucleotide sequence of the NYA-1154 tag additive (SEQ ID NO: 31).

[0318] Figure 39 The amino acid sequence of the NYA-1154 tag additive (SEQ ID NO: 32), where NYA-1154 is amino acid numbered 21 to 266.

[0319] Figure 40 This is the amino acid sequence (SEQ ID NO: 33) of a truncation of HLA-A*0201 (GenBank: ASA47534.1).

[0320] Figure 41 is the amino acid sequence of β2-microglobulin (SEQ ID NO: 34).

[0321] Figure 42 is the nucleotide sequence of the NYA-2035 tag additive (SEQ ID NO: 35).

[0322] Figure 43 The amino acid sequence of the NYA-2035 tag additive (SEQ ID NO: 36), where NYA-2035 is amino acid numbered 21 to 266.

[0323] Figure 44 is the amino acid sequence of NYA-1143-VH01 (SEQ ID NO: 37).

[0324] Figure 45 is the amino acid sequence of NYA-1143-VH02 (SEQ ID NO: 38).

[0325] Figure 46 is the amino acid sequence of NYA-1143-VH03 (SEQ ID NO: 39).

[0326] Figure 47 is the amino acid sequence of NYA-1143-VL01 (SEQ ID NO: 40).

[0327] Figure 48 is the nucleotide sequence of the NYA-2044 tag additive (SEQ ID NO: 41).

[0328] Figure 49 is the nucleotide sequence of the NYA-2045 tag additive (SEQ ID NO: 42).

[0329] Figure 50 is the nucleotide sequence of the NYA-2047 tag addition (SEQ ID NO: 43).

[0330] Figure 51 is the nucleotide sequence of the NYA-2048 tag additive (SEQ ID NO: 44).

[0331] Figure 52 is the nucleotide sequence of the NYA-2060 tag additive (SEQ ID NO: 45).

[0332] Figure 53 is the nucleotide sequence of the NYA-2061 tag additive (SEQ ID NO: 46).

[0333] Figure 54The amino acid sequence of the NYA-2044 tag additive (SEQ ID NO: 47), where NYA-2044 is amino acid numbered 21 to 266.

[0334] Figure 55 The amino acid sequence of the NYA-2045 tag additive (SEQ ID NO: 48), where NYA-2045 is amino acid numbered 21 to 266.

[0335] Figure 56 is the amino acid sequence of NYA-0082 (SEQ ID NO: 49).

[0336] Figure 57 The amino acid sequence of the NYA-2047 tag additive (SEQ ID NO: 50), where NYA-2047 is amino acid numbered 21 to 266.

[0337] Figure 58 The amino acid sequence of the NYA-2048 tag additive (SEQ ID NO: 51), where NYA-2048 is amino acid numbered 21 to 266.

[0338] Figure 59 The amino acid sequence of the NYA-2060 tag additive (SEQ ID NO: 52), where NYA-2060 is amino acid numbered 21 to 266.

[0339] Figure 60 The amino acid sequence of the NYA-2061 tag additive (SEQ ID NO: 53), where NYA-2061 is amino acid numbered 21 to 266.

[0340] Figure 61 The amino acid sequences of the heavy chains CDRH1 to CDRH3 and the light chains CDRL1 to L3 of NYA-0001 are shown (SEQ ID NO: 54 to SEQ ID NO: 59).

[0341] Figure 62 is the amino acid sequence of CDRL1 of NYA-2023 (SEQ ID NO: 60).

[0342] Figure 63 is the amino acid sequence of CDRL3 of NYA-2027 (SEQ ID NO: 61).

[0343] Figure 64 The amino acid sequences of CDRH3 and CDRL3 of NYA-1154 (SEQ ID NO: 62 and SEQ ID NO: 63) are shown.

[0344] Figure 65The amino acid sequence of CDRL1 of NYA-0035 (SEQ ID NO: 64) is shown.

[0345] Figure 66 is the nucleotide sequence of the NYC-0003 tag additive (SEQ ID NO: 65).

[0346] Figure 67 is the nucleotide sequence of the NYC-0004 tag additive (SEQ ID NO: 66).

[0347] Figure 68 The amino acid sequence of the NYC-0003 tag additive (SEQ ID NO: 67), where NYC-0003 is amino acid numbered 21 to 263.

[0348] Figure 69 The amino acid sequence of the NYC-0004 tag additive (SEQ ID NO: 68), where NYC-0004 is amino acid numbered 21 to 263.

[0349] Figure 70 is the nucleotide sequence of the NYA-0001 tag additive (SEQ ID NO: 69).

[0350] Figure 71 The amino acid sequence of the NYA-0001 tag additive (SEQ ID NO: 70), where NYA-0001 is amino acid numbered 21 to 266.

[0351] Figure 72 is the nucleotide sequence of HCl (SEQ ID NO: 71).

[0352] Figure 73 is the nucleotide sequence of NYF-0016-HC2 (SEQ ID NO: 72).

[0353] Figure 74 is the nucleotide sequence of NYF-0019-HC2 (SEQ ID NO: 73).

[0354] Figure 75 is the nucleotide sequence of NYF-0022-HC2 (SEQ ID NO: 74).

[0355] Figure 76 is the nucleotide sequence of NYF-0023-HC2 (SEQ ID NO: 75).

[0356] Figure 77 is the nucleotide sequence of NYF-0027-HC2 (SEQ ID NO: 76).

[0357] Figure 78 is the nucleotide sequence of NYF-0035-HC2 (SEQ ID NO: 77).

[0358] Figure 79 is the nucleotide sequence of NYF-0044-HC2 (SEQ ID NO: 78).

[0359] Figure 80 is the nucleotide sequence of NYF-0045-HC2 (SEQ ID NO: 79).

[0360] Figure 81 is the nucleotide sequence of NYF-0047-HC2 (SEQ ID NO: 80).

[0361] Figure 82 is the nucleotide sequence of NYF-0048-HC2 (SEQ ID NO: 81).

[0362] Figure 83 is the nucleotide sequence of NYF-0060-HC2 (SEQ ID NO: 82).

[0363] Figure 84 is the nucleotide sequence of NYF-0061-HC2 (SEQ ID NO: 83).

[0364] Figure 85 is the amino acid sequence of HCl (SEQ ID NO: 84).

[0365] Figure 86 The amino acid sequence of NYF-0016-HC2 (SEQ ID NO: 85) is shown, with NYA-1143 representing amino acid numbers 21 to 266 and C3E-7085 representing amino acid numbers 272 to 511.

[0366] Figure 87 The amino acid sequence of NYF-0019-HC2 (SEQ ID NO: 86) is shown, with NYA-2143 representing amino acid numbers 21 to 266 and C3E-7085 representing amino acid numbers 272 to 511.

[0367] Figure 88 The amino acid sequence of NYF-0022-HC2 (SEQ ID NO: 87) is shown, with NYA-1163 representing amino acid numbers 21 to 266 and C3E-7085 representing amino acid numbers 272 to 511.

[0368] Figure 89The amino acid sequence of NYF-0023-HC2 (SEQ ID NO: 88) is shown, with NYA-2023 representing amino acid numbers 21 to 266 and C3E-7085 representing amino acid numbers 272 to 511.

[0369] Figure 90 The amino acid sequence of NYF-0027-HC2 (SEQ ID NO: 89) is shown, with NYA-2027 representing amino acid numbers 21 to 266 and C3E-7085 representing amino acid numbers 272 to 511.

[0370] Figure 91 The amino acid sequence of NYF-0035-HC2 (SEQ ID NO: 90) is shown, with amino acid numbers 21 to 266 for NYA-2035 and 272 to 511 for C3E-7085.

[0371] Figure 92 The amino acid sequence of NYF-0044-HC2 (SEQ ID NO: 91) is shown, wherein NYA-2044 represents amino acid numbers 21 to 266, and C3E-7085 represents amino acid numbers 272 to 511.

[0372] Figure 93 The amino acid sequence of NYF-0045-HC2 (SEQ ID NO: 92) is shown, with NYA-2045 representing amino acid numbers 21 to 266 and C3E-7085 representing amino acid numbers 272 to 511.

[0373] Figure 94 The amino acid sequence of NYF-0047-HC2 (SEQ ID NO: 93) is shown, wherein NYA-2047 represents amino acid numbers 21 to 266, and C3E-7085 represents amino acid numbers 272 to 511.

[0374] Figure 95 The amino acid sequence of NYF-0048-HC2 (SEQ ID NO: 94) is shown, wherein NYA-2048 represents amino acid numbers 21 to 266, and C3E-7085 represents amino acid numbers 272 to 511.

[0375] Figure 96 The amino acid sequence of NYF-0060-HC2 (SEQ ID NO: 95) is shown, wherein NYA-2060 represents amino acid numbers 21 to 266, and C3E-7085 represents amino acid numbers 272 to 511.

[0376] Figure 97The amino acid sequence of NYF-0061-HC2 (SEQ ID NO: 96) is shown, wherein NYA-2061 represents amino acid numbers 21 to 266, and C3E-7085 represents amino acid numbers 272 to 511.

[0377] Figure 98 is the nucleotide sequence of NYA-0001-Fab-HCl-k delete (SEQ ID NO: 97).

[0378] Figure 99 is the nucleotide sequence of NYA-0001-LC (SEQ ID NO: 98).

[0379] Figure 100 The amino acid sequence of NYA-0001-Fab-HCl-k delete (SEQ ID NO: 99) is shown, and the heavy chain variable region of NYA-0001 is amino acid numbers 20 to 139.

[0380] Figure 101 The amino acid sequence of NYA-0001-LC (SEQ ID NO: 100) is shown, and the light chain variable region of NYA-0001 is amino acid numbers 21 to 131.

[0381] Figure 102 is the nucleotide sequence of NYA-1143-Fab-HCl-k delete (SEQ ID NO: 101).

[0382] Figure 103 is the nucleotide sequence of NYA-1143-LC (SEQ ID NO: 102).

[0383] Figure 104 The nucleotide sequence of C3E-7085-HC2-k deleteC (SEQ ID NO: 103).

[0384] Figure 105 The amino acid sequence of NYA-1143-Fab-HCl-k delete (SEQ ID NO: 104) is amino acid numbered 20 to 139 of the heavy chain variable region of NYA-1143.

[0385] Figure 106 The amino acid sequence of NYA-1143-LC (SEQ ID NO: 105) is shown, and the light chain variable region of NYA-1143 is amino acid numbers 21 to 131.

[0386] Figure 107The amino acid sequence of C3E-7085-HC2-k delete (SEQ ID NO: 106), where C3E-7085 represents amino acids 21 to 260.

[0387] Figure 108 is the nucleotide sequence of NYA-1143-HCl-k delete (SEQ ID NO: 107).

[0388] Figure 109 The amino acid sequence of NYA-1143-HCl-k delete (SEQ ID NO: 108), wherein NYA-1143 is amino acid numbered 21 to 266.

[0389] Figure 110 The nucleotide sequence of C3E-7085-NYA-1154-Fab-HC2-k delete (SEQ ID NO: 109).

[0390] Figure 111 is the nucleotide sequence of NYA-1154-LC (SEQ ID NO: 110).

[0391] Figure 112 is the nucleotide sequence of OAA-HCl-k delete (SEQ ID NO: 111).

[0392] Figure 113 The amino acid sequence of C3E-7085-NYA-1154-Fab-HC2-k delete (SEQ ID NO: 112) is shown, where amino acids 21 to 260 of C3E-7085 and amino acids 266 to 285 of the heavy chain variable region of NYA-1154 are shown.

[0393] Figure 114 The amino acid sequence of NYA-1154-LC (SEQ ID NO: 113) is amino acid numbers 21 to 131 of the light chain variable region of NYA-1154.

[0394] Figure 115 is the amino acid sequence of OAA-HCl-k delete (SEQ ID NO: 114).

[0395] Figure 116 The nucleotide sequence of NYF-0010-HC2-k delete (SEQ ID NO: 115).

[0396] Figure 117 The nucleotide sequence of NYF-0004-HC2-k delete (SEQ ID NO: 116).

[0397] Figure 118 The nucleotide sequence of NYF-0011-HC2-k delete (SEQ ID NO: 117).

[0398] Figure 119 The amino acid sequence of NYF-0010-HC2-k delete (SEQ ID NO: 18) is shown, wherein NYA-1154 represents amino acids 21 to 266, and C3E-7085 represents amino acids 272 to 511.

[0399] Figure 120 The amino acid sequence of NYF-0004-HC2-k delete (SEQ ID NO: 119) is shown, wherein C3E-7085 represents amino acids 21 to 260, and NYA-1154 represents amino acids 272 to 511.

[0400] Figure 121 The amino acid sequence of NYF-0011-HC2-k delete (SEQ ID NO: 120) is shown, with NYA-1143 representing amino acid numbers 21 to 266 and C3E-7085 representing amino acid numbers 272 to 511.

[0401] Figure 122 is the amino acid sequence of the point mutation NY-ESO peptide 1F (SEQ ID NO: 121).

[0402] Figure 123 is the amino acid sequence of the point mutation NY-ESO peptide 2M (SEQ ID NO: 122).

[0403] Figure 124 is the amino acid sequence of the point mutation NY-ESO peptide 3A (SEQ ID NO: 123).

[0404] Figure 125 is the amino acid sequence of the point mutation NY-ESO peptide 4A (SEQ ID NO: 124).

[0405] Figure 126 is the amino acid sequence of the point mutation NY-ESO peptide 5A (SEQ ID NO: 125).

[0406] Figure 127 is the amino acid sequence of the point mutation NY-ESO peptide 6L (SEQ ID NO: 126).

[0407] Figure 128 is the amino acid sequence of the point mutation NY-ESO peptide 7F (SEQ ID NO: 127).

[0408] Figure 129is the amino acid sequence of the point mutation NY-ESO peptide 8A (SEQ ID NO: 128).

[0409] Figure 130 is the amino acid sequence of the point mutation NY-ESO peptide 9A (SEQ ID NO: 129).

[0410] Figure 131 is the amino acid sequence of the gp100 peptide (SEQ ID NO: 130).

[0411] Figure 132 is the amino acid sequence of the homologous peptide DOLPP1 (SEQ ID NO: 131).

[0412] Figure 133 The amino acid sequence of the homologous peptide IL20RB (SEQ ID NO: 132).

[0413] Figure 134 is the amino acid sequence of the homologous peptide PRKD2 (SEQ ID NO: 133).

[0414] Figure 135 is the amino acid sequence of the homologous peptide CD163 (SEQ ID NO: 134).

[0415] Figure 136 is the amino acid sequence of the homologous peptide P2RY8 (SEQ ID NO: 135).

[0416] Figure 137 is the amino acid sequence of C3E-7034 (SEQ ID NO: 136).

[0417] Figure 138 is the amino acid sequence of C3E-7036 (SEQ ID NO: 137).

[0418] Figure 139 is the amino acid sequence of C3E-7085 (SEQ ID NO: 138).

[0419] Figure 140 is the amino acid sequence of C3E-7088 (SEQ ID NO: 139).

[0420] Figure 141 is the amino acid sequence of C3E-7093 (SEQ ID NO: 140).

[0421] Figure 142 The amino acid sequences of heavy chain CDRH1 to CDRH3 and light chain CDRL1 to CDRL3 of C3E-7085 (SEQ ID NO: 141 to SEQ ID NO: 146).

[0422] Figure 143 is the amino acid sequence of C3E-7078 (SEQ ID NO: 147).

[0423] Figure 144 is the nucleotide sequence of NYF-0014-HC2 (SEQ ID NO: 148).

[0424] Figure 145 The amino acid sequence of NYF-0014-HC2 (SEQ ID NO: 149) is shown, with amino acid numbers 21 to 266 for NYA-0001 and 272 to 511 for C3E-7085.

[0425] Figure 146 The amino acid sequence of NYF-0082-HC2 (SEQ ID NO: 150) is shown, with amino acid numbers 21 to 266 for NYA-0082 and 272 to 511 for C3E-7085.

[0426] Figure 147 is the amino acid sequence of human CD3ε (SEQ ID NO: 151).

[0427] Figure 148 The full-length nucleotide sequence of NYZ-0038-HC2 (SEQ ID NO: 152) is shown.

[0428] Figure 149 The full-length nucleotide sequence of NYZ-0082-HC2 (SEQ ID NO: 153) is shown.

[0429] Figure 150 The full-length nucleotide sequence of NYZ-0083-HC2 (SEQ ID NO: 154) is shown.

[0430] Figure 151 The full-length amino acid sequence of NYZ-0038-HC2 (SEQ ID NO: 155) is shown, with NYA-2061 representing amino acid numbers 21 to 266 and C3E-7096 representing amino acid numbers 272 to 511.

[0431] Figure 152 The full-length amino acid sequence of NYZ-0082-HC2 (SEQ ID NO: 156) is shown, with NYA-3061 representing amino acid numbers 21 to 271 and C3E-7096 representing amino acid numbers 277 to 516.

[0432] Figure 153The full-length amino acid sequence of NYZ-0083-HC2 (SEQ ID NO: 157) is shown, with NYA-3061 representing amino acid numbers 21 to 271 and C3E-7097 representing amino acid numbers 277 to 516.

[0433] Figure 154 The full-length nucleotide sequence of NYZ-1010-HC2 is shown (SEQ ID NO: 158).

[0434] Figure 155 The full-length nucleotide sequence of C3E-7085-LC (SEQ ID NO: 159) is shown.

[0435] Figure 156 The amino acid sequence of the full length of NYZ-1010-HC2 (SEQ ID NO: 160) is shown, with amino acid numbers 21 to 271 of NYA-3061 and amino acid numbers 277 to 394 of the heavy chain variable region of C3E-7085.

[0436] Figure 157 The full-length amino acid sequence of C3E-7085-LC (SEQ ID NO: 161) is shown.

[0437] Figure 158A This table shows the normalized gMFI of the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules NYZ-0038, 0082, 0083, and 1010 on CD3e knockout T2 cells supplemented with various point mutation peptides. * / Underlined characters indicate a normalized gMFI of less than half that of the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules on CD3e knockout T2 cells supplemented with NY-ESO peptides. *Bold characters indicate a normalized gMFI of less than one-quarter that of the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules.

[0438] Figure 158B This table shows the normalized gMFI of the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules NYZ-0038, 0082, 0083, and 1010 on CD3e knockout T2 cells supplemented with various homologous peptides. * / Underlined values ​​indicate a higher value than the normalized gMFI of each Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule on CD3e knockout T2 cells supplemented with DMSO.

[0439] Figure 159AThis graph shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules NYZ-0038, 0082, and 0083 have cytotoxic activity against U266B1 cells expressing endogenous human NY-ESO in the presence of human PBMCs. Error bars in the graph represent standard deviations (n=3).

[0440] Figure 159B This graph shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule NYZ-1010 has cytotoxic activity against U266B1 cells expressing endogenous human NY-ESO in the presence of human PBMCs. Error bars in the graph represent standard deviations (n=3).

[0441] Figure 159C This graph shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules NYZ-0082 and 1010 have cytotoxic activity against NCI-H1703 cells expressing endogenous human NY-ESO in the presence of human PBMCs. Error bars in the graph represent standard deviations (n=3).

[0442] Figure 159D This graph shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules NYZ-0038 and 0083 have cytotoxic activity against NCI-H1703 cells expressing endogenous human NY-ESO in the presence of human PBMCs. Error bars in the graph represent standard deviations (n=3).

[0443] Figure 159E This graph shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules NYZ-0038, 0082, and 0083 exhibit no cytotoxic activity against AGS cells that do not express endogenous human NY-ESO in the presence of human PBMCs. Error bars in the graph represent standard deviations (n=3).

[0444] Figure 159F This graph shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule NYZ-1010 exhibits no cytotoxic activity against AGS cells that do not express endogenous human NY-ESO in the presence of human PBMCs. Error bars in the graph represent standard deviations (n=3).

[0445] Figure 159G This graph shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules NYZ-0082 and 1010 exhibit no cytotoxic activity against CFPAC-1 cells that do not express endogenous human NY-ESO in the presence of human PBMCs. Error bars in the graph represent standard deviations (n=3).

[0446] Figure 159H This graph shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules NYZ-0038 and 0083 exhibit no cytotoxic activity against CFPAC-1 cells that do not express endogenous human NY-ESO in the presence of human PBMCs. Error bars in the graph represent standard deviations (n=3).

[0447] Figure 160A This graph shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule NYZ-0038 has anti-tumor activity in a human PBMC metastasis model. Error bars in the graph represent standard error (n=5).

[0448] Figure 160B This graph shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule NYZ-0082 has anti-tumor activity in a human PBMC metastasis model. Error bars in the graph represent standard error (n=5).

[0449] Figure 160C This graph shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule NYZ-0083 has anti-tumor activity in a human PBMC metastasis model. Error bars in the graph represent standard error (n=5).

[0450] Figure 160D This graph shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule NYZ-1010 has anti-tumor activity in a human PBMC metastasis model. Error bars in the graph represent standard error (n=5).

[0451] Figure 161 is the amino acid sequence of the peptide linker (SEQ ID NO: 162).

[0452] Figure 162 This is a graph showing the multimer content (%) when various Fc-added anti-HLA / NY-ESO-anti-CD3 bispecific molecules were acid-treated.

[0453] Figure 163 This is a graph showing the results of the solution stability evaluation of anti-HLA / NY-ESO scFv-heterodimer Fc.

[0454] Figure 164 The full-length nucleotide sequence of NYA-3061 is shown (SEQ ID NO: 163).

[0455] Figure 165The full-length amino acid sequence of NYA-3061 is shown (SEQ ID NO: 164).

[0456] Figure 166 The full-length nucleotide sequence of NYC-0005 is shown (SEQ ID NO: 165).

[0457] Figure 167 The full-length amino acid sequence of NYC-0005 is shown (SEQ ID NO: 166).

[0458] Figure 168 The full-length nucleotide sequence of NYC-0006 is shown (SEQ ID NO: 167).

[0459] Figure 169 The full-length amino acid sequence of NYC-0006 is shown (SEQ ID NO: 168).

[0460] Figure 170 The full-length nucleotide sequence of NYC-0007 is shown (SEQ ID NO: 169).

[0461] Figure 171 The full-length amino acid sequence of NYC-0007 is shown (SEQ ID NO: 170).

[0462] Figure 172 The full-length nucleotide sequence of NYC-0008 (SEQ ID NO: 171) is shown.

[0463] Figure 173 The full-length amino acid sequence of NYC-0008 is shown (SEQ ID NO: 172).

[0464] Figure 174 The full-length nucleotide sequence of NYC-0009 (SEQ ID NO: 173) is shown.

[0465] Figure 175 The full-length amino acid sequence of NYC-0009 (SEQ ID NO: 174) is shown.

[0466] Figure 176 The full-length nucleotide sequence of NYC-0010 (SEQ ID NO: 175) is shown.

[0467] Figure 177 The full-length amino acid sequence of NYC-0010 (SEQ ID NO: 176) is shown.

[0468] Figure 178 The full-length nucleotide sequence of HC-h (SEQ ID NO: 177) is shown.

[0469] Figure 179 is the full-length amino acid sequence of HC-h (SEQ ID NO: 178).

[0470] Figure 180 The full-length nucleotide sequence of NYD-2047-HC-k is shown (SEQ ID NO: 179).

[0471] Figure 181 The full-length amino acid sequence of NYD-2047-HC-k is shown (SEQ ID NO: 180).

[0472] Figure 182 The full-length nucleotide sequence of NYD-2061-HC-k is shown (SEQ ID NO: 181).

[0473] Figure 183 The full-length amino acid sequence of NYD-2061-HC-k is shown (SEQ ID NO: 182).

[0474] Figure 184 The full-length nucleotide sequence of NYD-3061-HC-k is shown (SEQ ID NO: 183).

[0475] Figure 185 The full-length amino acid sequence of NYD-3061-HC-k is shown (SEQ ID NO: 184).

[0476] Figure 186 The full-length nucleotide sequence of NYC-0011-HC-k is shown (SEQ ID NO: 185).

[0477] Figure 187 The full-length amino acid sequence of NYC-0011-HC-k is shown (SEQ ID NO: 186).

[0478] Figure 188 The full-length nucleotide sequence of NYC-0012-HC-k is shown (SEQ ID NO: 187).

[0479] Figure 189 The full-length amino acid sequence of NYC-0012-HC-k is shown (SEQ ID NO: 188).

[0480] Figure 190 The full-length nucleotide sequence of NYC-0013-HC-k is shown (SEQ ID NO: 189).

[0481] Figure 191 The full-length amino acid sequence of NYC-0013-HC-k is shown (SEQ ID NO: 190).

[0482] Figure 192The full-length nucleotide sequence of NYC-0014-HC-k is shown (SEQ ID NO: 191).

[0483] Figure 193 The full-length amino acid sequence of NYC-0014-HC-k is shown (SEQ ID NO: 192).

[0484] Figure 194 The full-length nucleotide sequence of NYC-0015-HC-k is shown (SEQ ID NO: 193).

[0485] Figure 195 The full-length amino acid sequence of NYC-0015-HC-k is shown (SEQ ID NO: 194).

[0486] Figure 196 The full-length nucleotide sequence of NYC-0016-HC-k is shown (SEQ ID NO: 195).

[0487] Figure 197 The full-length amino acid sequence of NYC-0016-HC-k is shown (SEQ ID NO: 196).

[0488] Figure 198 The amino acid sequence of the full length of NYZ-1007-HC2 (SEQ ID NO: 197) is shown, with amino acids 21 to 266 of NYA-2061 and 272 to 389 of the heavy chain variable region of C3E-7085.

[0489] Figure 199 The amino acid sequence of the full length of NYZ-1017-HC2 (SEQ ID NO: 198), amino acid numbers 21 to 266 of NYA-2047, and amino acid numbers 277 to 389 of the heavy chain variable region of C3E-7085. DETAILED DESCRIPTION

[0490] The present invention is described in detail below.

[0491] 1. Definition

[0492] In the present invention, "gene" refers to a nucleotide chain or its complementary chain containing a base sequence of amino acids encoding a protein. For example, polynucleotides, oligonucleotides, DNA, mRNA, cDNA, cRNA, etc., which are nucleotide chains or their complementary chains containing a base sequence of amino acids encoding a protein, are all included in the meaning of "gene". A gene is a single-stranded, double-stranded or triple-stranded nucleotide, an associate of a DNA chain and an RNA chain, a nucleotide chain with a mixture of ribonucleotides (RNA) and deoxyribonucleotides (DNA), and a double-stranded or triple-stranded nucleotide chain containing such a nucleotide chain are also included in the meaning of "gene". In the present invention, base sequence and nucleotide sequence have the same meaning.

[0493] In the present invention, "polynucleotide," "nucleotide chain," "nucleic acid," and "nucleic acid molecule" are synonymous. For example, DNA, RNA, probes, oligonucleotides, primers, and the like are also included in the meaning of "polynucleotide." A polynucleotide is a polynucleotide composed of a single strand, a double strand, or three or more strands. Also included in the meaning of "polynucleotide" are complexes of DNA and RNA chains, polynucleotides in which ribonucleotides (RNA) and deoxyribonucleotides (DNA) are mixed in a single polynucleotide chain, and complexes containing double strands or three or more strands of such polynucleotide chains.

[0494] In the present invention, "polypeptide", "peptide" and "protein" have the same meaning.

[0495] In the present invention, "antigen" may be used to mean "immunogen".

[0496] In the present invention, "cell" also includes various cells derived from individual animals, secondary cultured cells, primary cultured cells, cell lines, recombinant cells, and microorganisms.

[0497] In the present invention, "antibody" and "immunoglobulin" are synonymous. However, in the context of the anti-HLA / NY-ESO antibodies of the present invention, "antibody" refers to an immunoglobulin having a constant region and a variable region. Antibodies may be natural antibodies or immunoglobulins produced by partial or complete synthesis, and are not particularly limited in the present invention. The anti-HLA / NY-ESO antibodies of the present invention are encompassed by the term "molecule," described below.

[0498] In the present invention, the "NY-ESO peptide" refers to a peptide consisting of 9 amino acids from position 157 to position 165 of NY-ESO-1 and LAGE-1 (SLLMWITQC, SEQ ID NO: 1).

[0499] In the present invention, "HLA-A2 / NY-ESO" refers to a complex of NY-ESO peptide and histocompatibility leukocyte antigen-A2 (HLA-A2), and is also referred to as "HLA / NY-ESO".

[0500] In the present invention, an "anti-HLA-A2 / NY-ESO antibody" refers to an antibody that binds to HLA-A2 / NY-ESO, in other words, an antibody that recognizes HLA-A2 / NY-ESO. Similarly, an "anti-HLA-A2 / NY-ESO scFv" refers to an scFv that binds to HLA / NY-ESO, in other words, an scFv that recognizes HLA-A2 / NY-ESO. "Anti-HLA-A2 / NY-ESO antibody" and "anti-HLA-A2 / NY-ESO scFv" are also referred to as "anti-HLA / NY-ESO antibody" and "anti-HLA / NY-ESO scFv," respectively.

[0501] The basic structure of a four-chain antibody consists of two identical light chains (L chains) and two identical heavy chains (H chains). The light chain is bound to the heavy chain by a 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 chain and heavy chain has regularly spaced intrachain disulfide bonds. The heavy and light chains have constant regions with very high amino acid sequence similarity, and variable regions with low amino acid sequence similarity. The light chain has a variable region (VL) connected to a constant region (CL) at its amino terminus. The heavy chain has a variable region (VH) connected to three constant regions (CH1 / CH2 / CH3) at its amino terminus. The VL and VH pair, with the CL juxtaposed with the first constant region (CH1) of the heavy chain. The VL and VH pair to form a single antigen-binding site.

[0502] The constant regions of the antibodies of the present invention are not particularly limited, but for the antibodies of the present invention used to treat or prevent human diseases, it is preferred to use constant regions of human antibodies. Examples of human antibody heavy chain constant regions include Cγβ, Cβ, Cβ, Cβ, Cβ, Cβ, Cβ, Cβ, and Cε. Examples of human antibody light chain constant regions include Cβ, Cβ, and Cβ.

[0503] Fab is composed of the VH of the heavy chain followed by CH1, and the VL of the light chain followed by CL. VH and VL contain complementarity determining regions (CDRs).

[0504] Fc (also known as the Fc region) is the carboxyl-terminal region of the heavy chain constant region, contains CH2 and CH3, and is a dimer. The Fc of the present invention can be either a native sequence Fc or a mutant Fc (referred to as a "mutant Fc") in which a mutation has occurred in the native sequence. In the multispecific and bispecific molecules of the present invention, the preferred Fc region is a mutant Fc, and more preferably a set of Fc regions capable of forming heterodimers. An example of a set of Fc regions is a combination of Fc (i) contained in the first polypeptide and Fc (ii) contained in the second polypeptide, described later. As long as a set of Fc regions can associate (form a heterodimer), there are no restrictions.

[0505] As mutant Fc, examples include but are not limited to: a modified Fc region (including a heterodimeric Fc region) contained in a heteromultimer with improved stability disclosed in WO2013 / 063702; an Fc contained in a heteromultimer comprising a CH3 region of an immunoglobulin derived from an IgG antibody having "protrusions" and "gaps" disclosed in WO1996 / 27011; an Fc contained in a heterodimer comprising a CH3 region in which one or more amino acid residues are substituted with charged amino acids, thereby being electrostatically favorable, disclosed in WO2009 / 089004; a heterodimeric Fc region contained in a heterodimer using conformational mutations and / or pI (isoelectric point) mutations disclosed in WO2014 / 110601; an Fc contained in a heterodimer comprising a modified CH3 region that eliminates or reduces binding to protein A disclosed in WO2010 / 151792, etc.

[0506] The variable region is composed of two regions: regions of extreme variability called hypervariable regions (HVRs) and relatively invariant regions separated from them called framework regions (FRs). The variable regions of native heavy and light chains contain four FRs connected by three hypervariable regions. The hypervariable regions of each chain are held in close proximity to those of other chains by the FRs, contributing to the formation of the antibody's antigen-binding site.

[0507] It is known that the heavy and light chains of antibody molecules each have three complementarity determining regions (CDRs). Complementarity determining regions, also called hypervariable regions, are located within the variable regions of the heavy and light chains of antibodies and are sites of particularly high mutability in the primary structure. They are usually separated into three locations in the primary structure of the heavy and light chain polypeptide chains. In the present invention, the complementarity determining regions of antibodies are designated as CDRH1, CDRH2, and CDRH3, starting from the amino-terminal side of the heavy chain amino acid sequence; and CDRL1, CDRL2, and CDRL3, starting from the amino-terminal side of the light chain amino acid sequence, for the complementarity determining regions of the light chain. These sites are conformationally close to each other and determine the specificity for the bound antigen.

[0508] In the present invention, the position and length of CDR are determined according to the definition of IMGT (Developmental and Comparative Immunology 27 (2003) 55-77).

[0509] FR is the variable region excluding CDR residues. The variable region generally has four FRs: FR1, FR2, FR3, and FR4.

[0510] The CDRs and FRs contained in the heavy and light chains are arranged in the order FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4 and FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4, respectively, from the amino terminus to the carboxyl terminus.

[0511] The positions of CDRs and FRs can also be determined according to various definitions known in the art, such as those of Kabat, Chothia, AbM, contact, etc., in addition to IMGT.

[0512] In the present invention, "antigen-binding fragment of an antibody" refers to a partial fragment of an antibody composed of a heavy chain variable region and a light chain variable region, which has binding activity with an antigen. Examples of "antigen-binding fragment of an antibody" include, but are not limited to, antigen-binding fragments such as Fab, F(ab')2, scFv, Fab', Fv, and single-domain antibodies (sdAb). The antigen-binding fragment of the antibody is obtained by treating the full-length molecule of the antibody protein with enzymes such as papain and pepsin. In addition, it can also be a recombinant protein produced in an appropriate host cell using a recombinant gene. In the present invention, "antibody binding fragment" and "antigen-binding fragment of an antibody" have the same meaning.

[0513] In the present invention, the "site" to which an antibody binds, that is, the "site" recognized by an antibody, refers to a portion of a peptide or a portion of a higher-order structure on an antigen to which the antibody binds or recognizes.

[0514] In the present invention, this site is also referred to as an epitope or an antibody binding site. In the present invention, a "mutant antibody" refers to a polypeptide that has an amino acid sequence formed by substitution, deletion, or addition (including insertion) of amino acids in the amino acid sequence of the original antibody (hereinafter collectively referred to as "mutation"), and binds to HLA / NY-ESO of the present invention. The number of mutated amino acids in the mutant antibody is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, or 50. Such mutant antibodies are also included in the "antibody" of the present invention.

[0515] In the present invention, the "several" in "one or several" means 2 to 10.

[0516] In this specification, "molecule" refers to molecules including the above-mentioned antibodies and antigen-binding fragments of antibodies, and also includes multispecific molecules composed of antibodies or multiple antigen-binding fragments derived from these antibodies.

[0517] In this specification, "a multispecific molecule", "a multispecific molecule" and "a multispecific molecule" have the same meaning, and there is no particular limitation as long as it is a molecule that can bind to multiple different epitopes on a molecule and / or different epitopes on two or more molecules. As multispecific molecules, antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL) are also included. Such multispecific molecules include, but are not limited to: full-length antibody molecules having two or more different heavy and light chains, i.e., IgG-type multispecific molecules; and molecules composed of two or more antigen-binding fragments having VL and VH, i.e., molecules derived from a combination of Fab, Fab', Fv, scFv, sdAb, etc., i.e., tandem scFv, diabodies, single-chain diabodies, tribodies, etc. In addition, multispecific molecules also include: molecules produced by genetically or chemically linking proteins having antigen binding properties without an immunoglobulin backbone in the antigen-binding fragment. In the present invention, such multispecific molecules may also be referred to as "multispecific antibodies", "multispecific antibodies", etc., except when antibodies or antigen-binding fragments thereof are not included.

[0518] Examples of the activities and properties exhibited by the anti-HLA / NY-ESO antibodies or antigen-binding fragments thereof, or molecules of the present invention include biological activity and physicochemical properties (also known as physicochemical properties). Specifically, examples include various biological activities such as cytotoxic activity, ADCC activity, and anti-tumor activity (all described below); antigen- and epitope-binding activity; stability during production and storage; and physicochemical properties such as thermal stability.

[0519] As used herein, "hybridization under stringent conditions" refers to hybridization in a solution containing 5×SSC at 65°C, followed by washing under the following conditions, or equivalent conditions: washing in a solution containing 2×SSC-0.1% SDS at 65°C for 20 minutes, washing in an aqueous solution containing 0.5×SSC-0.1% SDS at 65°C for 20 minutes, and washing in an aqueous solution containing 0.2×SSC-0.1% SDS at 65°C for 20 minutes. SSC refers to an aqueous solution of 150 mM NaCl-15 mM sodium citrate, and n×SSC means n-fold concentration of SSC.

[0520] As used herein, "cytotoxicity" refers to pathological changes in cells in some form. This is not limited to direct trauma but also encompasses any structural or functional damage to cells, including DNA cleavage and base dimer formation, chromosome cleavage, damage to the cell division apparatus, and decreased activity of various enzymes. As used herein, "cytotoxic activity" refers to the induction of such cytotoxicity.

[0521] In the present invention, "antibody-dependent cellular cytotoxicity activity" refers to "antibody dependent cellular cytotoxicity (ADCC) activity", which means the activity of NK cells to damage target cells such as tumor cells through antibodies.

[0522] In the present invention, "T cell-redirected cytotoxic activity" refers to the aforementioned cytotoxicity induced by a multispecific molecule comprising an anti-tumor antigen antibody and an anti-HLA / NY-ESO antibody. Specifically, the anti-tumor antigen antibody binds to target tumor cells, while the anti-HLA / NY-ESO antibody binds to T cells, thereby bringing the target tumor cells and T cells into close proximity and inducing cytotoxicity through T cell activation. This molecule can also be included in a pharmaceutical composition.

[0523] 2. Antigen

[0524] 2-1.HLA / NY-ESO antigens

[0525] The term "HLA / NY-ESO" used in the present invention has the same meaning as HLA / NY-ESO protein.

[0526] HLA / NY-ESO is a triple complex of HLA-A2, β2-microglobulin, and NY-ESO peptide. HLA-A2 is an HLA allele and is known to be the most common allele in Caucasians. HLA forms a triple complex with β2-microglobulin and a peptide fragment of its own protein within the endoplasmic reticulum of the cell, presenting it to the extracellular space for recognition by the TCR (T Cell Receptor) of T cells. NY-ESO peptide (SLLMWITQC, SEQ ID NO: 1, Figure 8 ) is a peptide consisting of 9 amino acids from positions 157 to 165 of NY-ESO-1 and LAGE-1. There are reports that the NY-ESO peptide is presented by HLA-A2.

[0527] 2-2.CD3 antigen

[0528] The term "CD3" used in the present invention has the same meaning as CD3 protein.

[0529] CD3 is expressed on T cells as part of a multimolecular T cell receptor complex, which is a complex of five polypeptides: γ chain, δ chain, ε chain, ζ chain, and η chain (molecular weights are 25,000-28,000, 21,000, 20,000, 16,000, and 22,000, respectively).

[0530] Examples of the CD3 complex include the γ, δ, ε, ζ, and η chains. These chains are also referred to as subunits. Anti-CD3 antibodies bind to T cells, thereby inducing cytotoxicity through T cell activation. Many anti-CD3 antibodies bind to CD3ε.

[0531] The nucleotide sequence of the cDNA encoding human CD3ε is registered with NCBI / GenBank under the accession number NM_000733 (NM_000733.3); the amino acid sequence of human CD3ε is registered with NCBI / GenPept under the accession number NP_000724 (NM_000724.1). The nucleotide sequence of the cDNA encoding cynomolgus monkey CD3 is registered with GenBank under the accession number NM_001283615.1. The amino acid sequence of human CD3ε is recorded in the sequence listing as SEQ ID NO: 151 ( Figure 147 ).

[0532] 2-3. Preparation of Antigen

[0533] The antigen proteins HLA / NY-ESO and CD3 used in the present invention (hereinafter referred to as HLA / NY-ESO and CD3) can be prepared by the following methods: purification and isolation from animal tissues (including body fluids), cells derived from the animal tissues, or cultures of the cells; genetic recombination; in vitro translation; chemical synthesis, etc.

[0534] The cDNA of the antigen protein can be obtained, for example, by the so-called PCR method, which is a method of using a cDNA library of an organ expressing the mRNA of the antigen protein as a template and primers that specifically amplify the cDNA of the antigen protein to perform a polymerase chain reaction (hereinafter referred to as "PCR") (Saiki, RK, et al., Science (1988) 239, 487-49).

[0535] It hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to a nucleotide sequence encoding the antigen protein expressed in humans or rats, and a polynucleotide encoding a protein having the same biological activity as the antigen protein is also contained in the cDNA of the antigen protein.

[0536] Then, the cDNA of the antigen protein also includes a polynucleotide that hybridizes with a splicing mutant transcribed from a gene locus encoding the antigen protein expressed in humans or rats or with the splicing mutant under stringent conditions and encodes a protein having the same biological activity as the antigen protein.

[0537] The nucleotide sequence of the antigen protein gene is also included in the nucleotide sequence of the antigen protein gene, which is composed of the amino acid sequence of the human or rat antigen protein, or the amino acid sequence after removing the signal sequence from these sequences in which one or several amino acids are substituted, deleted or added, and which has the same biological activity as the antigen protein.

[0538] Proteins composed of an amino acid sequence encoded by a splicing mutant transcribed from the gene locus of the antigen protein of human or rat, or an amino acid sequence in which one or several amino acids are substituted, deleted or added, and having biological activity equivalent to that of the antigen protein, are also included in the antigen protein.

[0539] 2-4. Binding specificity to antigenic proteins

[0540] The anti-HLA / NY-ESO antibodies and antigen-binding fragments thereof of the present invention recognize HLA / NY-ESO. That is, they bind to HLA / NY-ESO antigens. Currently, HLA / NY-ESO is not known to exist in non-human animals such as mice, rats, and cynomolgus monkeys.

[0541] The anti-CD3 antibodies and binding fragments thereof included in the multispecific molecules of the present invention recognize the CD3 antigen, i.e., bind to the CD3 antigen. These anti-CD3 antibodies preferably bind to human CD3, monkey CD3, and more preferably to human CD3 and cynomolgus monkey CD3. Furthermore, these preferred anti-CD3 antibodies do not bind to rat and / or mouse CD3.

[0542] The anti-tumor activity of the multispecific molecules of the present invention can be evaluated, for example, by (i) transplanting human cancer cells, human cancer tissues, etc., into non-human animals, preferably rats or mice, more preferably rats or mice with deficient endogenous effector function (e.g., immunodeficient rats or mice), transplanted with human peripheral blood lymphocytes, or (ii) transplanting mouse cancer cells, etc., into non-human animals, preferably rats or mice, into which the human CD3 gene has been knocked in, for evaluation. Using these immunodeficient or knock-in animals for evaluation enables various assays, immunohistochemistry, etc., to be performed in vivo using mice and / or rats, and is therefore preferred in the development of pharmaceuticals and non-clinical studies containing the multispecific molecules of the present invention.

[0543] In the present invention, "recognition" or "binding" refers to binding that is not nonspecific adsorption. As a criterion for determining whether recognition or binding occurs, for example, the dissociation constant (hereinafter referred to as "KD") can be cited. Preferably, the KD value of the antibody of the present invention for HLA / NY-ESO or CD3 is 1×10 -5 M or less, 5×10 -6 M or less, 2×10 -6 M or less, 1×10 -6 M or less; the preferred KD value for HLA / NY-ESO is 5×10 -7 M or less, 2×10 -7 M or less, 1×10 -7 M or less, 5×10 -8 M or less, 2×10 -8 M or less, 1×10 -8 M or less, 5×10 -9 M or less or 2×10 -9 M or less, more preferably 1×10 -9M or less. Examples of the anti-HLA / NY-ESO scFv of the present invention having excellent antigen-binding activity include NYA-1143, NYA-2023, NYA-2143, NYA-2044, NYA-2045, NYA-2060, NYA-2061, and NYA-3061. NYA-1143, NYA-2044, NYA-2045, and NYA-2143 have a KD value of 1×10 for HLA / NY-ESO. -9 M or less (Example 4, etc.).

[0544] The binding between the antigen and the antibody in the present invention can be measured or determined by biomolecular interaction analysis systems such as SPR and BLI, or ELISA and RIA. The binding between the antigen and the antibody expressed on the cell surface can be measured by flow cytometry analysis.

[0545] The SPR method (surface plasmon resonance analysis) is used as an analytical method for measuring the association rate constant (Ka value) and dissociation rate constant (Kd value) by chemical kinetic (kinetic) analysis to determine the dissociation constant (KD value) as an affinity index. 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 Co., Ltd.), and Autolab SPR (trademark) (manufactured by Metrohm).

[0546] BLI (BioLayer Interferometry) is a method for measuring interactions between biomolecules using biolayer interferometry. Examples of equipment used to analyze interactions using BLI include the Octet system (manufactured by Pall ForteBio).

[0547] The ELISA (Enzyme-linked ImmunoSorbent Assay) method uses specific antibodies or antigens to capture the target antigen or antibody in a sample solution, and then uses an enzymatic reaction to detect and quantify the amount. Enzyme activity is detected by integrating an enzyme-labeled antigen or antibody into the reaction system. Enzyme activity is measured using a substrate whose absorption spectrum changes due to the reaction, and the absorbance is converted to a numerical value by measuring the absorbance.

[0548] Cell-ELISA is a method that captures the target substance located on the cell surface of each cell and detects and quantifies it using an enzymatic reaction.

[0549] The RIA method (Radio Immunoassay) labels an antibody with a radioactive substance and measures the radioactivity derived from the antibody, thereby enabling quantification of the antibody.

[0550] Flow cytometry involves optically analyzing individual cells by dispersing them into a fluid and finely flowing the fluid. Fluorescently labeled antibodies bind to cell surface antigens through an antigen-antibody reaction. The fluorescence intensity of the cell-bound labeled antibodies is measured to quantify the antigen-binding capacity of the antibodies.

[0551] Examples of antibodies having excellent antigen-binding specificity among the anti-HLA / NY-ESO antibodies of the present invention include anti-HLA / NY-ESO scFvs NYA-0001, NYA-1143, NYA-1163, NYA-2023, NYA-2027, NYA-2035, NYA-2044, NYA-2045, NYA-2047, NYA-2048, NYA-2060, NYA-2061, NYA-2143, and NYA-3061 (Example 6, etc.).

[0552] 3. Antibodies or binding fragments thereof that specifically bind to HLA / NY-ESO

[0553] 3-1. Anti-HLA / NY-ESO or its binding fragment

[0554] The present invention provides antibodies or binding fragments thereof that recognize and bind to HLA / NY-ESO.

[0555] As previously described, HLA / NY-ESO is a complex consisting of HLA-A2 and the 9mer NY-ESO peptide (SLLMWITQC, SEQ ID NO: 1). The NY-ESO peptide is an intracellular protein derived from the cancer-testis antigen NY-ESO-1 or LAGE-1. HLA / NY-ESO is presented on the surface of cancer cells.

[0556] The anti-HLA / NY-ESO antibodies and antigen-binding fragments of the antibodies of the present invention (hereinafter also referred to as antibodies of the present invention, etc.) can be either monoclonal antibodies or polyclonal antibodies. The isotype of the monoclonal antibodies of the present invention is not particularly limited, and examples thereof include IgG such as IgG1, IgG2, IgG3, and IgG4, IgM, IgA such as IgA1 and IgA2, IgD, and Ig. The isotype and subclass of the monoclonal antibodies can be determined, for example, by agar two-way immunodiffusion, ELISA, RIA, and the like. Examples of the monoclonal antibodies of the present invention include antibodies from non-human animals (non-human animal antibodies), human antibodies, chimeric antibodies (also referred to as "chimeric antibodies"), and humanized antibodies, and preferably human antibodies can be used. The scope of the antibodies of the present invention also includes mutants of the antibodies (hereinafter referred to as "mutant antibodies"). For example, the scope of human antibodies also includes human mutant antibodies.

[0557] Examples of non-human animal antibodies include antibodies from vertebrates such as mammals and birds. Examples of antibodies from mammals include antibodies from rodents such as mouse antibodies and rat antibodies. Examples of antibodies from birds include chicken antibodies.

[0558] Examples of chimeric antibodies include, but are not limited to, antibodies in which the variable region of a non-human animal antibody is combined with the constant region of a human antibody (human immunoglobulin).

[0559] Examples of humanized antibodies include, but are not limited to: antibodies formed by transplanting the CDRs in the variable region of a non-human animal antibody into a human antibody (variable region of a human immunoglobulin); antibodies formed by transplanting a portion of the framework region of a non-human animal antibody in addition to the CDRs into a human antibody; antibodies formed by replacing one or more amino acids from any of the above non-human animal antibodies with human amino acids, etc.

[0560] Antibodies can be produced by various known methods. As a known method, they can be produced by methods using hybridomas and cellular immunization methods, or by genetic recombination methods. In addition, methods for obtaining human antibodies displayed on phages screened from human antibody libraries are also known. For example, a phage display method can be used in which the variable regions of human antibodies are expressed as scFv on the surface of phages to select phages that bind to the antigen. By analyzing the genes of phages selected for binding to the antigen, the DNA sequence encoding the variable regions of human antibodies that bind to the antigen can be determined. As long as the DNA sequence of the scFv that binds to the antigen is clear, an expression vector containing the sequence can be prepared, introduced into an appropriate host and expressed, thereby obtaining human antibodies (WO92 / 01047, WO92 / 20791, WO93 / 06213, WO93 / 11236, WO93 / 19172, WO95 / 01438, WO95 / 15388, Annu. Rev. Immunol (1994) 12, 433-455).

[0561] The highly active antibody thus obtained can be used as a lead antibody, and the gene encoding the lead antibody can be mutated to produce a mutant with higher activity (hereinafter referred to as a "mutant antibody").

[0562] Preferred examples of CDRH1 to CDRH3 contained in the heavy chain of the anti-HLA / NY-ESO antibody or antigen-binding fragment thereof of the present invention include: Figure 61 ), CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 55 ( Figure 61 ), and CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 56 ( Figure 61 ) or the amino acid sequence shown in SEQ ID NO: 56 ( Figure 61 ) wherein the amino acid at position 6 is N (Asn). More preferably, the following heavy chain variable region CDRH1 to CDRH3 combinations are mentioned: Figure 13 ) shown in NYA-0001 heavy chain variable region; SEQ ID NO: 18 ( Figure 25 ) of the NYA-0082 heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 27 ( Figure 34 ) of the amino acid sequence shown in SEQ ID NO: 21 to 140; the NYA-2023 heavy chain variable region consisting of amino acid numbers 21 to 140 of SEQ ID NO: 28 ( Figure 35) of the amino acid sequence shown in SEQ ID NO: 21 to 140; the NYA-2027 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence shown in SEQ ID NO: 29 ( Figure 36 ) of the amino acid sequence shown in SEQ ID NO: 21 to 140; the NYA-1143 heavy chain variable region consisting of amino acid numbers 21 to 140 of SEQ ID NO: 26 ( Figure 33 ) of the amino acid sequence shown in SEQ ID NO: 21 to 140; the NYA-1163 heavy chain variable region consisting of amino acid numbers 21 to 140 of SEQ ID NO: 27 ( Figure 34 ) of the amino acid sequence shown in SEQ ID NO: 21 to 140; the NYA-2023 heavy chain variable region consisting of amino acid numbers 21 to 140 of SEQ ID NO: 28 ( Figure 35 ) of the amino acid sequence shown in SEQ ID NO: 21 to 140; the NYA-2027 heavy chain variable region consisting of SEQ ID NO: 36 ( Figure 43 ) of the amino acid sequence shown in SEQ ID NO: 21 to 140; the NYA-2035 heavy chain variable region consisting of SEQ ID NO: 47 ( Figure 54 ) of the amino acid sequence shown in SEQ ID NO: 21 to 140; the NYA-2044 heavy chain variable region consisting of amino acid numbers 21 to 140 of SEQ ID NO: 48 ( Figure 55 ) of the amino acid sequence shown in SEQ ID NO: 50 ( Figure 57 ) of the amino acid sequence shown in SEQ ID NO: 51 ( Figure 58 ) of the amino acid sequence shown in SEQ ID NO: 52 ( Figure 59 ) of the amino acid sequence shown in SEQ ID NO: 53 ( Figure 60 ) of the amino acid sequence shown in SEQ ID NO: 21 to 140; or, a NYA-2061 heavy chain variable region consisting of amino acid numbers 21 to 140 of SEQ ID NO: 30 ( Figure 37 ) The NYA-2143 heavy chain variable region consists of amino acid numbers 21 to 140 of the amino acid sequence shown in ).

[0563] In addition, SEQ ID NO: 156 ( Figure 152 ) and a combination of CDRH1 to CDRH3 contained in the NYA-3061 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence shown in FIG.

[0564] Preferred examples of CDRL1 to CDRL3 contained in the light chain of the anti-HLA / NY-ESO antibody or antigen-binding fragment thereof of the present invention include: Figure 61 ) or the amino acid sequence shown in SEQ ID NO: 57 ( Figure 61 ) wherein the 7th amino acid is W (Trp) or the 8th amino acid is K (Lys), and CDRL1 consisting of SEQ ID NO: 58 ( Figure 61 ), and CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 59 ( Figure 61 ) or the amino acid sequence shown in SEQ ID NO: 59 ( Figure 61 ) A combination of CDRL3 consisting of an amino acid sequence in which the second amino acid in the amino acid sequence shown is A (Ala) or S (Ser).

[0565] More preferably, the following combination of CDRL1 to CDRL3 contained in the light chain variable region can be mentioned: Figure 15 ) shown in NYA-0001 light chain variable region; SEQ ID NO: 20 ( Figure 27 ) of the NYA-0082 light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 29 ( Figure 36 ) of the amino acid sequence shown in SEQ ID NO: 156 to 266; the NYA-1143 light chain variable region consisting of amino acid numbers 156 to 266 of SEQ ID NO: 26 ( Figure 33 ) of the amino acid sequence shown in SEQ ID NO: 27 ( Figure 34 ) of the amino acid sequence shown in SEQ ID NO: 28 ( Figure 35 ) of the amino acid sequence shown in SEQ ID NO: 36 ( Figure 43 ) of the amino acid sequence shown in SEQ ID NO: 156 to 266; the NYA-2035 light chain variable region consisting of amino acid numbers 156 to 266 of SEQ ID NO: 47 ( Figure 54 ) of the amino acid sequence shown in SEQ ID NO: 156 to 266; the NYA-2044 light chain variable region consisting of amino acid numbers 156 to 266 of SEQ ID NO: 48 ( Figure 55 ) of the amino acid sequence shown in SEQ ID NO: 50 ( Figure 57) of the amino acid sequence shown in SEQ ID NO: 51 ( Figure 58 ) of the amino acid sequence shown in SEQ ID NO: 52 ( Figure 59 ) of the amino acid sequence shown in SEQ ID NO: 53 ( Figure 60 ) of the amino acid sequence shown in SEQ ID NO: 156 to 266; or, a NYA-2061 light chain variable region consisting of amino acid numbers 156 to 266 of the amino acid sequence shown in SEQ ID NO: 30 ( Figure 37 ) The NYA-2143 light chain variable region consists of amino acid numbers 156 to 266 of the amino acid sequence shown in ).

[0566] In addition, SEQ ID NO: 156 ( Figure 152 ) and a combination of CDRL1 to CDRL3 contained in the NYA-3061 light chain variable region consisting of amino acid numbers 161 to 271 of the amino acid sequence shown in FIG.

[0567] Preferred examples of CDRH1 to CDRH3 contained in the heavy chain and CDRL1 to CDRL3 contained in the light chain of the anti-HLA / NY-ESO antibody or antigen-binding fragment thereof of the present invention include: Figure 61 ) shown in the amino acid sequence, CDRH1 consisting of SEQ ID NO: 55 ( Figure 61 ) of the amino acid sequence shown in SEQ ID NO: 56 ( Figure 61 ) or the amino acid sequence shown in SEQ ID NO: 56 ( Figure 61 ) wherein the sixth amino acid in the amino acid sequence is N (Asn), a heavy chain CDRH3 consisting of SEQ ID NO: 57 ( Figure 61 ) or the amino acid sequence shown in SEQ ID NO: 57 ( Figure 61 ), wherein the amino acid at position 7 is N (Asn) and / or the amino acid at position 8 is K (Lys), CDRL1 consisting of an amino acid sequence represented by SEQ ID NO: 58 ( Figure 61 ), and CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 59 ( Figure 61) or a combination of CDRL3 consisting of an amino acid sequence in which the second amino acid in the amino acid sequence of SEQ ID NO: 59 is A (Ala) or S (Ser). More preferably, the following combinations of CDRH1 to CDRH3 and CDRL1 to CDRL3 are included in the heavy chain variable region and light chain variable region, respectively: the NYA-0001 heavy chain variable region and light chain variable region consisting of the amino acid sequences shown in SEQ ID NO: 6 and SEQ ID NO: 8, respectively; the NYA-0001 heavy chain variable region and light chain variable region consisting of the amino acid sequences shown in SEQ ID NO: 29 ( Figure 36 ) of the amino acid sequence shown in SEQ ID NO: 26 ( Figure 33 ) of the amino acid sequence shown in SEQ ID NO: 27 ( Figure 34 ) of the amino acid sequence shown in SEQ ID NO: 28 ( Figure 35 ) of the amino acid sequence shown in SEQ ID NO: 36 ( Figure 43 ) of the amino acid sequence shown in SEQ ID NO: 47 ( Figure 54 ) of the amino acid sequence shown in SEQ ID NO: 48 ( Figure 55 ) of the amino acid sequence shown in SEQ ID NO: 50 ( Figure 57 ) of the amino acid sequence shown in SEQ ID NO: 51 ( Figure 58 ) of the amino acid sequence shown in SEQ ID NO: 52 ( Figure 59 ) of the amino acid sequence shown in SEQ ID NO: 53 ( Figure 60) of the amino acid sequence shown in SEQ ID NO: 21 to 140 and 156 to 266 of NYA-2061 heavy chain variable region and light chain variable region; or, a NYA-2061 heavy chain variable region and light chain variable region consisting of amino acid numbers 21 to 140 and 156 to 266 of SEQ ID NO: 30 ( Figure 37 ) The NYA-2143 heavy chain variable region and light chain variable region are composed of amino acid numbers 21 to 140 and 156 to 266 of the amino acid sequence shown in ).

[0568] In addition, SEQ ID NO: 156 ( Figure 152 ) of the amino acid sequence shown in , and the NYA-3061 heavy chain variable region and light chain variable region comprise a combination of CDRH1 to CDRH3 and CDRL1 to CDRL3, respectively.

[0569] Preferred examples of the heavy chain variable regions of the anti-HLA / NY-ESO antibodies or antigen-binding fragments thereof of the present invention include the aforementioned heavy chain CDRs or heavy chain variable regions comprising combinations thereof. More preferred examples include the NYA-0001 heavy chain variable region, NYA-0082 heavy chain variable region, NYA-1143 heavy chain variable region, NYA-1163 heavy chain variable region, NYA-2023 heavy chain variable region, 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 NYA-3061 heavy chain variable region. The amino acid sequences of each heavy chain variable region are described above.

[0570] Preferred examples of the light chain variable regions of the anti-HLA / NY-ESO antibodies or antigen-binding fragments thereof of the present invention include the above-mentioned light chain CDRs or light chain variable regions comprising combinations thereof. More preferred examples include the NYA-0001 light chain variable region, NYA-0082 light chain variable region, NYA-1143 light chain variable region, NYA-1163 light chain variable region, NYA-2023 light chain variable region, 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. The amino acid sequences of each light chain variable region are as described above.

[0571] Preferred examples of the heavy chain variable region and light chain variable region of the anti-HLA / NY-ESO antibody or antigen-binding fragment thereof of the present invention include the heavy chain and light chain CDRs described above, or heavy chain variable regions and light chain variable regions comprising a combination thereof. More preferred examples include: 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. The heavy chain variable region and light chain variable region of NYA-2060, the heavy chain variable region and light chain variable region of NYA-2061, and the heavy chain variable region and light chain variable region of NYA-2143, and the heavy chain variable region and light chain variable region of NYA-3061 are also included.

[0572] Preferred examples of the heavy chains of the anti-HLA / NY-ESO antibodies or antigen-binding fragments thereof of the present invention include heavy chains comprising the preferred or more preferred heavy chain variable regions described above. More preferred examples include the 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.

[0573] Preferred examples of the light chains of the anti-HLA / NY-ESO antibodies or antigen-binding fragments thereof of the present invention include light chains comprising the preferred or more preferred light chain variable regions described above. More preferred examples include NYA-0001 light chain, NYA-0082 light chain, NYA-1143 light chain, NYA-1163 light chain, NYA-2023 light chain, NYA-2027 light chain, NYA-2035 light chain, NYA-2044 light chain, NYA-2045 light chain, NYA-2047 light chain, NYA-2048 light chain, NYA-2060 light chain, NYA-2061 light chain, NYA-2143 light chain, and NYA-3061 light chain.

[0574] Preferred examples of the heavy and light chains of the anti-HLA / NY-ESO antibodies or antigen-binding fragments thereof of the present invention include those comprising the preferred or more preferred heavy chain variable regions and light chain variable regions described above, respectively. More preferred examples include combinations of heavy and light chains 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.

[0575] An antigen-binding fragment of an antibody refers to a fragment or modified form thereof that retains at least antigen-binding activity, among the functions of the antibody. Typical functions of the antibody include antigen-binding activity, activity that modulates antigen activity, antibody-dependent cellular cytotoxicity, and complement-dependent cytotoxicity. Examples of functions of the antibodies and multispecific molecules of the present invention, including those containing the antibodies and multispecific molecules of the present invention, include T cell redirection, T cell activation, and cytotoxic activity against cancer cells resulting from T cell activation.

[0576] Antigen-binding fragments of antibodies are not particularly limited as long as they are fragments of the antibody that retain at least antigen-binding activity. Examples include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv (scFv) in which heavy and light chain Fvs are linked via an appropriate linker, and single-domain antibodies (sdAb). Molecules containing portions other than the antigen-binding fragment of the antibody of the present invention, such as scFv with a linker, are also encompassed within the meaning of the antigen-binding fragment of the antibody of the present invention.

[0577] Molecules formed by deleting one, several, or more amino acids from the amino-terminus and / or carboxyl-terminus of an antibody protein, which retain at least a portion of the functions of the antibody, are also included within the meaning of an antigen-binding fragment of an antibody. Modified antigen-binding fragments of such antibodies are also included within the antibodies, antigen-binding fragments thereof, or modified products thereof (described below) of the present invention.

[0578] One form of the antibody or antigen-binding fragment thereof of the present invention is scFv. ScFvs are obtained by linking the heavy chain variable region and light chain variable region of an antibody with a polypeptide linker (Pluckthun A. The Pharmacology of Monoclonal Antibodies 113, Rosenburg and Moore eds., Springer-Verlag, New York, 269-315 (1994); Nature Biotechnology (2005), 23, 1126-1136). Furthermore, tandem scFvs, produced by linking two scFvs with a polypeptide linker, can also be used as bispecific molecules. Furthermore, triads composed of three or more scFvs can also be used as bispecific molecules.

[0579] As scFvs specific for HLA / NY-ESO (also referred to as "anti-HLA / NY-ESO scFvs"), preferably, scFvs comprising the aforementioned CDRH1 to CDRH3 and CDRL1 to CDRL3 can be exemplified; more preferably, scFvs comprising the aforementioned heavy chain variable region and light chain variable region can be exemplified; further preferably, NYA-0001 (SEQ ID NO: 70 ( Figure 71 ) of the amino acid sequence shown in SEQ ID NO: 21 to 266); NYA-0082 (comprising SEQ ID NO: 18 ( Figure 25 ) and the amino acid sequence shown in SEQ ID NO: 20 ( Figure 27 ) shown in the amino acid sequence); NYA-1143 (SEQ ID NO: 29 ( Figure 36 ) of the amino acid sequence shown in (amino acid numbers 21 to 266); NYA-1163 (SEQ ID NO: 26 ( Figure 33 ) of the amino acid sequence shown in (amino acid numbers 21 to 266); NYA-2023 (SEQ ID NO: 27 ( Figure 34 ) of the amino acid sequence shown in (amino acid numbers 21 to 266); NYA-2027 (SEQ ID NO: 28 ( Figure 35 ) of the amino acid sequence shown in amino acid numbers 21 to 266); NYA-2035 (SEQ ID NO: 36 ( Figure 43 ) of the amino acid sequence shown in (amino acid numbers 21 to 266); NYA-2044 (SEQ ID NO: 47 ( Figure 54 ) of the amino acid sequence shown in (amino acid numbers 21 to 266); NYA-2045 (SEQ ID NO: 48 ( Figure 55 ) of the amino acid sequence shown in (amino acid numbers 21 to 266); NYA-2047 (SEQ ID NO: 50 ( Figure 57 ) of the amino acid sequence shown in (amino acid numbers 21 to 266); NYA-2048 (SEQ ID NO: 51 ( Figure 58 ) of the amino acid sequence shown in (amino acid numbers 21 to 266); NYA-2060 (SEQ ID NO: 52 ( Figure 59 ) of the amino acid sequence shown in (amino acid numbers 21 to 266); NYA-2061 (SEQ ID NO: 53 ( Figure 60 ) of the amino acid sequence shown in (amino acid numbers 21 to 266); and NYA-2143 (SEQ ID NO: 30 ( Figure 37 ) of the amino acid sequence shown in ). In addition, NYA-3061 (SEQ ID NO: 156 ( Figure 152 ) of the amino acid sequence shown in (amino acid numbers 21 to 271).

[0580] Preferred forms of anti-HLA / NY-ESO scFv include forms in which a FLAG-His tag is fused to the carboxyl terminus (also simply referred to as a "tag additive"). Preferred tag additives include: NYA-0001 tag additive (SEQ ID NO: 70 ( Figure 71 ) amino acid numbers 20 to 292); NYA-1143 tag addition (SEQ ID NO: 29 ( Figure 36 ) amino acid numbers 20 to 292); NYA-1163 tag addition (SEQ ID NO: 26 ( Figure 33 ) amino acid numbers 20 to 292); NYA-2023 tag addition (SEQ ID NO: 27 ( Figure 34 ) amino acid numbers 20 to 292); NYA-2027 tag addition (SEQ ID NO: 28 ( Figure 35 ) amino acid numbers 20 to 292); NYA-2035 tag addition (SEQ ID NO: 36 ( Figure 43 ) amino acid numbers 20 to 292); NYA-2044 tag addition (SEQ ID NO: 47 ( Figure 54 ) amino acid numbers 20 to 292); NYA-2045 tag addition (SEQ ID NO: 48 ( Figure 55 ) amino acid numbers 20 to 292); NYA-2047 tag addition (SEQ ID NO: 50 ( Figure 57 ) amino acid numbers 20 to 292); NYA-2048 tag addition (SEQ ID NO: 51 ( Figure 58) amino acid numbers 20 to 292); NYA-2060 tag addition (SEQ ID NO: 52 ( Figure 59 ) amino acid numbers 20 to 292); NYA-2061 tag addition (SEQ ID NO: 53 ( Figure 60 ) amino acid numbers 20 to 292); and NYA-2143 tag addition (SEQ ID NO: 30 ( Figure 37 ) amino acid numbers 20 to 292).

[0581] Among these Fc-added bispecific molecules, NYA-2023 and its tag additions, NYA-2047 and its tag additions, NYA-2048 and its tag additions, NYA-2060 and its tag additions, and NYA-2061 and its tag additions have excellent biological activity and physicochemical properties and are therefore more preferred.

[0582] In addition, when the anti-HLA / NY-ESO scFv and its tag addition are expressed in host cells, a signal peptide can be added to its amino terminus. Examples of amino acid sequences of anti-HLA / NY-ESO scFv tag additions to which a signal peptide is added include SEQ ID NO: 70, SEQ ID NO: 29, SEQ ID NO: 26 to SEQ ID NO: 28, SEQ ID NO: 36, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 50 to SEQ ID NO: 53, and SEQ ID NO: 30 (SEQ ID NO: 51). Figure 71 、 36 , 33-35, 43, 54, 55, 57-60 and 37).

[0583] scFv can be obtained using a phage display method in which the variable region of an antibody is expressed as a single-chain antibody (scFv) on the surface of phage, and phage that binds to the antigen is selected (Nature Biotechnology (2005), 23, (9), pp. 1105-1116). By analyzing the genes of phage selected for antigen binding, the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined. Once the DNA sequence of the scFv that binds to the antigen is determined, an expression vector containing the sequence can be prepared, introduced into an appropriate host, and expressed, thereby obtaining human antibodies (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).

[0584] The antibodies of the present invention may also be antibodies having a single heavy chain variable region without a light chain sequence. Such antibodies are called single domain antibodies (sdAbs) or nanobodies, and there are reports that they have antigen binding ability (Muyldemans S. et al., Protein Eng., (1994) 7 (9), 1129-35, Hamers-Casterman C. et al., Nature (1993) 363 (6428), 446-448). These antibodies are also included in the meaning of the antigen-binding fragment of the antibody in the present invention.

[0585] In addition, the present invention includes single-chain immunoglobulins (single-chain immunoglobulins) formed by linking the full-length sequences of the heavy and light chains of an antibody using an appropriate linker (Lee, H.S., et al., Molecular Immunology (1999) 36, 61-71; Shirrmann, T. et al., mAbs (2010), 2(1), 1-4). By dimerizing such single-chain immunoglobulins, a structure and activity similar to those of the original tetrameric antibody can be maintained. The anti-HLA / NY-ESO antibody of the present invention may also be a single-chain immunoglobulin.

[0586] In the scFv of the present invention, the heavy chain variable region and the light chain variable region may be linked via a disulfide bond.

[0587] The anti-HLA / NY-ESO antibodies of the present invention, if they bind to HLA / NY-ESO, may be composed of portions from multiple different antibodies. Examples include antibodies formed by swapping heavy and / or light chains between multiple different antibodies, antibodies formed by swapping the full length of heavy and / or light chains, antibodies formed by swapping only the variable regions or only the constant regions, and antibodies formed by swapping all or part of the CDRs. The heavy and light chain variable regions of a chimeric antibody may also be derived from different anti-HLA / NY-ESO antibodies of the present invention. The heavy chain CDR1 to heavy chain CDR3 and light chain CDR1 to light chain CDR3 in the heavy and light chain variable regions of a humanized antibody may be derived from two or more anti-HLA / NY-ESO antibodies of the present invention. The heavy chain CDR1 to heavy chain CDR3 and light chain CDR1 to light chain CDR3 in the heavy and light chain variable regions of a humanized antibody may also be a combination of CDRs possessed by two or more anti-HLA / NY-ESO antibodies of the present invention.

[0588] The anti-HLA / NY-ESO antibodies of the present invention also include antibodies that contain an amino acid sequence encoded by a nucleotide sequence contained in a polynucleotide that hybridizes under stringent conditions to a complementary strand of a polynucleotide containing a nucleotide sequence encoding the amino acid sequence contained in the anti-HLA / NY-ESO antibodies of the present invention, and that bind to HLA / NY-ESO.

[0589] Alternatively, the antibody or antigen-binding fragment thereof may comprise an amino acid sequence comprised in the heavy chain variable region of the anti-HLA / NY-ESO antibody or antigen-binding fragment thereof of the present invention (preferably the amino acid sequence of amino acids 21 to 140 of the amino acid sequence set forth in SEQ ID NO: 27, the amino acid sequence set forth in SEQ ID NO: 38, the amino acid sequence set forth in SEQ ID NO: 39, or the amino acid sequence of amino acids 21 to 140 of the amino acid sequence set forth in SEQ ID NO: 160, SEQ ID NO: 197, or SEQ ID NO: 198), and / or an amino acid sequence comprised in the light chain variable region (preferably the amino acid sequence of amino acids 156 to 266 of the amino acid sequence set forth in SEQ ID NO: 27, the amino acid sequence of amino acids 156 to 266 of the amino acid sequence set forth in SEQ ID NO: 52, the amino acid sequence set forth in SEQ ID NO: 40, the amino acid sequence of amino acids 161 to 271 of the amino acid sequence set forth in SEQ ID NO: 160, or the amino acid sequence of amino acids 197 to 198). An antibody or antigen-binding fragment thereof that binds to HLA / NY-ESO and 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 of amino acids 156 to 266 of the amino acid sequence shown in NO: 198.

[0590] Regarding the position and length of the light chain variable region, when determined using a definition different from the IMGT definition (e.g., Kabat, Chothia, AbM, contact, etc.), the amino acid sequence of the light chain variable region determined according to the IMGT definition may contain one or more amino acids, such as arginine and glycine, at the carboxyl terminus. Antibodies or binding fragments thereof having such light chain variable regions are also encompassed by the antibodies or binding fragments thereof of the present invention.

[0591] The antibodies of the present invention may also be antibodies wherein mutations are introduced into the binding fragments of the anti-HLA / NY-ESO antibodies of the present invention to optimize binding ability to HLA / NY-ESO, particularly human and / or cynomolgus monkey HLA / NY-ESO. Specific methods for introducing mutations include random mutagenesis using mismatch PCR, site-specific amino acid mutation introduction using the NNK library, site-specific mutation introduction using structural information, and combinations thereof.

[0592] 3-2. Mutants of anti-HLA / NY-ESO antibodies (mutant antibodies)

[0593] Mutants of the anti-HLA / NY-ESO antibodies of the present invention preferably exhibit the following functions: reduced susceptibility to protein degradation or oxidation; maintenance or improvement of biological activity and function, or inhibition of decline or change in biological activity and function; improvement or modulation of antigen binding ability; or imparting physicochemical or functional properties. It is known that changes in specific amino acid side chains on the surface of proteins can alter the function and activity of the protein. Examples include deamidation of asparagine side chains and isomerization of aspartic acid side chains. Mutants that substitute other amino acids to prevent such amino acid side chain changes are also encompassed by the scope of the present invention.

[0594] Examples of mutant antibodies of the present invention include antibodies having an amino acid sequence formed by conservative amino acid substitutions in the amino acid sequence of the antibody. Conservative amino acid substitutions refer to substitutions made within a group of amino acids associated with the amino acid side chains.

[0595] Preferred amino acid groups are as follows: acidic group = aspartic acid, glutamic acid; basic group = lysine, arginine, histidine; nonpolar group = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and uncharged polar group = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Other preferred amino acid groups are as follows: aliphatic hydroxyl group = serine and threonine; amide group = asparagine and glutamine; aliphatic group = alanine, valine, leucine, and isoleucine; and aromatic group = phenylalanine, tryptophan, and tyrosine. Amino acid substitutions in the mutant antibody are preferably made within a range that does not diminish the antigen-binding activity of the original antibody.

[0596] The following are also included in the anti-HLA / NY-ESO antibodies of the present invention, antigen-binding fragments thereof, mutants thereof (mutated antibodies or antigen-binding fragments thereof), or molecules of the present invention: mutant antibodies, antigen-binding fragments thereof, and molecules comprising the same, which have an amino acid sequence in which conservative amino acid substitutions and / or other mutations are made in the amino acid sequence of an antibody of the present invention, such as NYA-2023, and bind to HLA / NY-ESO; chimeric antibodies, humanized antibodies, human antibodies, antigen-binding fragments thereof, and molecules comprising the same, which have an amino acid sequence in which conservative amino acid substitutions and / or other mutations are made in any of the amino acid sequences of CDRH1 to CDRH3 and CDRL1 to CDRL3 derived from an antibody of the present invention, such as NYA-2023, and bind to HLA / NY-ESO, and which contain the CDRs thereof.

[0597] 3-3. Anti-HLA / NY-ESO Antibody Binding Fragments

[0598] As one aspect of the present invention, provided are antigen-binding fragments of the anti-HLA / NY-ESO antibodies of the present invention (hereinafter simply referred to as "binding fragments"). The binding fragments of the anti-HLA / NY-ESO antibodies of the present invention include binding fragments of chimeric, humanized, or human antibodies. An antibody binding fragment refers to a fragment or modified form thereof that retains at least antigen binding among the functions of the antibody. Typical functions of the antibody include antigen-binding activity, activity that modulates antigen activity (e.g., agonist activity), activity that internalizes the antigen into cells, and activity that inhibits or promotes interaction with substances that interact with the antigen.

[0599] There are no particular limitations on antibody binding fragments, as long as they retain at least antigen binding activity among the activities possessed by the antibody. Examples of such antibody binding fragments include, but are not limited to, Fab, Fab', F(ab')2, single-chain Fab (scFab) formed by linking the carboxyl terminus of a Fab light chain to the amino terminus of a Fab heavy chain via an appropriate linker, Fv, single-chain Fv (scFv) formed by linking Fvs of heavy and light chains via an appropriate linker, and single-domain antibodies (sdAbs) having a single heavy chain variable region without a light chain sequence, also known as nanobodies (Muyldemans S. et al., Protein Eng., (1994) 7(9), 1129-35, Hamers-Casterman C. et al., Nature (1993) 363(6428), 446-448). Molecules containing portions 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.

[0600] 3-4. Modified Anti-HLA / NY-ESO Antibodies or Their Binding Fragments, and Complexes

[0601] The present invention provides modified substances of antibodies or their binding fragments. The modified substances of the antibodies or their binding fragments of the present invention refer to modified substances formed by chemical or biological modifications to the antibodies or their binding fragments of the present invention. Chemical modifications include chemical bonds to the chemical part of the amino acid backbone, chemical modifications of N- or O-linked carbohydrate chains, etc. Biological modifications include post-translational modifications (e.g., addition of sugar chains to N- or O-links, processing of the amino terminal region or the carboxyl terminal region, deamidation, isomerization of aspartic acid, oxidation of methionine), modifications formed by expression using prokaryotic host cells to add methionine residues to the amino terminal, etc. In addition, substances labeled to enable detection or separation of the antibodies or antigens of the present invention, such as enzyme labels, fluorescent labels, affinity labels, are also included in the meaning of the modified substances. Such modified substances of the antibodies or their binding fragments of the present invention can be used to improve the stability and retention in the blood of the original antibodies or their binding fragments of the present invention, reduce antigenicity, detect or separate the antibodies or antigens, etc.

[0602] Examples of the chemical moiety contained in the chemical modification include water-soluble polymers such as polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, and polyvinyl alcohol.

[0603] Examples of biomodified products include those modified by enzyme treatment, cell treatment, etc., fusions to which other peptides such as tags are added by genetic recombination, and those produced using cells expressing endogenous or exogenous sugar chain modifying enzymes as hosts.

[0604] The modification can be made at any position in the antibody or its binding fragment, or at a desired position, and the same modification or two or more different modifications can be made at one or two or more positions.

[0605] However, deletion of these heavy chain sequences, or modification of the heavy chain or light chain sequences, does not affect the antigen-binding ability and effector functions (such as complement activation and antibody-dependent cellular cytotoxicity) of the antibody.

[0606] Therefore, the present invention also includes antibodies that have undergone such deletions or modifications. For example, examples include: deletions in which one or two amino acids are deleted at the carboxyl terminus of the heavy chain (Journal of Chromatography A; 705; 129-134 (1995)); deletions in which the two amino acid residues glycine and lysine at the carboxyl terminus of the heavy chain are deleted, and the new proline residue at the carboxyl terminus is amidated (Analytical Biochemistry, 360: 75-83 (2007)); antibodies in which the amino-terminal glutamine or glutamic acid residue of the heavy chain or light chain of the antibody is pyroglutamylated (International Patent Publication No. WO2013 / 147153), etc. (these are collectively referred to as "deletions"). However, as long as the antigen binding ability and effector function are maintained, the deletions at the carboxyl termini of the heavy and light chains of the antibodies involved in the present invention are not limited to the above types. When the antibody of the present invention contains two or more chains (e.g., heavy chains), the two or more chains (e.g., heavy chains) may be either full-length heavy chains or heavy chains selected from the group consisting of the aforementioned deletions, or a combination of any two. The quantitative ratio or molecular ratio of each deletion will be affected by the type of cultured mammalian cells producing the antibody of the present invention and the culture conditions. However, as the main component of the antibody of the present invention, one example is a case where both heavy chains have a single amino acid residue deleted at the carboxyl terminus.

[0607] Furthermore, even if one or more amino acids derived from an expression vector and / or a signal sequence, etc., are added to the amino and / or carboxyl termini of an antibody or antigen-binding fragment thereof of the present invention (an antibody or antigen-binding fragment thereof included in a molecule, multispecific molecule, bispecific molecule, etc. of the present invention) (and part or all of these are modified as described above), as long as the desired antigen-binding activity is maintained, such modified antibodies or antigen-binding fragments are included in the scope of the modified antibodies or antigen-binding fragments of the present invention, and molecules including such modified antibodies or antigen-binding fragments are also included in the scope of the molecules of the present invention.

[0608] In the present invention, the term "antibody or antigen-binding fragment thereof" includes the meaning of "modified forms of antibodies or antigen-binding fragments thereof." Furthermore, the term "antibody or antigen-binding fragment thereof" encompassed in the molecules, multispecific molecules, bispecific molecules, and the like of the present invention also includes the meaning of "modified forms of antibodies or antigen-binding fragments thereof."

[0609] Furthermore, antibody-dependent cellular cytotoxicity can be enhanced by regulating the sugar chain modifications (glycosylation, defucosylation, etc.) associated with the antibodies of the present invention. Techniques for regulating antibody sugar chain modifications include, but are not limited to, International Patent Publications Nos. WO99 / 54342, WO00 / 61739, and WO02 / 31140.

[0610] The present invention also encompasses complexes (immunoconjugates) formed by linking the above-mentioned antibodies with other molecules. Examples of antibody-drug complexes formed by conjugating an antibody to a radioactive substance or a pharmacologically active compound (drug) include ADCs (Methods Mol Biol. (2013) 1045: 1-27; Nature Biotechnology (2005) 23, pp. 1137-1146).

[0611] Furthermore, the present invention also encompasses complexes formed by linking these antibodies to other functional polypeptides. Examples of such antibody-peptide complexes include complexes of antibodies and albumin-binding polypeptides (Protein Eng Des Sel. (2012) (2): 81-8).

[0612] The modified products of the above-mentioned antibodies, antibodies with regulated sugar chain modification, and complexes are included in the antibodies of the present invention; and binding fragments of the modified products of the above-mentioned antibodies, antibodies with regulated sugar chain modification, and complexes are included in the binding fragments of the antibodies of the present invention.

[0613] 4. Antibody Production Methods

[0614] The antibodies of the present invention can be prepared, for example, by inserting DNA encoding the heavy chain variable region or DNA encoding the light chain variable region into an expression vector, transforming host cells for expression with the vector, and culturing the host cells to allow the cells to produce the antibodies of the present invention as recombinant antibodies.

[0615] Regarding antibody-encoding DNA, DNA encoding the heavy chain variable region can be ligated with DNA encoding the heavy chain constant region to obtain DNA encoding the heavy chain; conversely, DNA encoding the light chain variable region can be ligated with DNA encoding the light chain constant region to obtain DNA encoding the light chain.

[0616] Regarding the anti-HLA / NY-ESO antibodies of the present invention, the DNA encoding the heavy chain and the DNA encoding the light chain can be inserted into an expression vector, used to transform host cells, and cultured to produce the anti-HLA / NY-ESO antibodies of the present invention. In this case, the DNA encoding the heavy chain and the DNA encoding the light chain can be introduced into the same expression vector and used to transform the host cells, or the DNA encoding the heavy chain and the DNA encoding the light chain can be introduced into separate expression vectors and used to transform the host cells. Alternatively, the DNA encoding the heavy and light chain variable regions can be introduced into a vector that already contains DNA encoding the heavy and light chain constant regions. Furthermore, the vector can contain DNA encoding a signal peptide that promotes secretion of the antibody from the host cells. In this case, the DNA encoding the signal peptide is ligated in-frame to the DNA encoding the antibody. After antibody production, the signal peptide can be removed to obtain the antibody as a mature protein.

[0617] In this case, DNA encoding the heavy chain variable region, DNA encoding the light chain variable region, DNA obtained by linking the heavy chain variable region-encoding DNA and the heavy chain constant region-encoding DNA, and DNA obtained by linking the light chain variable region-encoding DNA and the light chain constant region-encoding DNA can also be functionally linked to elements such as a promoter, an enhancer, and a polyadenylation signal. Here, "functionally linked" means that the elements are linked so that they can achieve their functions.

[0618] As long as the expression vector can be replicated in hosts such as animal cells, bacteria, yeast, there is no particular limitation, and examples thereof include known plasmids, phages, etc. As the vector for constructing the expression vector, examples thereof include: pcDNA (trademark) (Thermo Fisher Scientific), Flexi (registered trademark) vector (Promega), pUC19, pUEX2 (Amersham), pGEX-4T, pKK233-2 (Pharmacia), pMAM-neo (Clontech), etc. As host cells, prokaryotic cells such as Escherichia coli and Bacillus subtilis can be used, and eukaryotic cells such as yeast and animal cells can also be used, preferably eukaryotic cells. For example, as animal cells, human embryonic kidney cell line HEK293 cells, Chinese hamster ovary (CHO) cells, etc. can be used. The expression vector can be introduced into the host cell by a known method to transform the host cell. For example, electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. can be mentioned. The produced antibodies can be purified using common protein separation and purification methods. For example, affinity chromatography, other chromatography, filtration membranes, ultrafiltration membranes, salting out, dialysis, etc. can be appropriately selected and combined.

[0619] 5. Molecules that bind to HLA / NY-ESO

[0620] The molecules of the present invention comprise the anti-HLA / NY-ESO antibodies of the present invention or antigen-binding fragments thereof. The molecules of the present invention are preferably multispecific molecules having two or more antigen-binding sites. That is, the molecules of the present invention are molecules capable of binding to two or more distinct epitopes on a single molecule or to distinct epitopes on two or more molecules, and comprise multiple distinct antigen-binding fragments. Such multispecific molecules include, but are not limited to, IgG-type multispecific molecules; multispecific molecules having two or more variable regions; antibody fragments such as tandem scFv (taFv), single-chain diabodies, diabodies, and tribodies, and antibody fragments linked by covalent or non-covalent bonds. The multispecific molecules may also comprise an Fc.

[0621] The multispecific molecules of the present invention may comprise, in addition to the anti-HLA / NY-ESO antibodies or antigen-binding fragments thereof of the present invention, one or more other antibodies or antigen-binding fragments thereof. Examples of antigen-binding fragments of other antibodies include Fab, F(ab)', Fv, scFv, and sdAb.

[0622] Preferred multispecific molecules of the present invention further comprise an anti-CD3 antibody or antigen-binding fragment thereof, which also specifically binds to CD3.

[0623] The anti-CD3 antibodies or antigen-binding fragments thereof included in the multispecific molecule of the present invention are not particularly limited as long as they bind to human CD3, but preferably also bind to CD3 of non-human primates such as cynomolgus monkeys. More preferred anti-CD3 antibodies or antigen-binding fragments thereof include antibodies or antigen-binding fragments thereof comprising the following heavy chain variable regions and light chain variable regions: a heavy chain variable region CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 141; a heavy chain variable region CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 142; a heavy chain variable region CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 143; a light chain variable region CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 144; a light chain variable region CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 145; and a light chain variable region CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 146 (refer to the above). Figure 142 ).

[0624] More preferred antibodies or antigen-binding fragments thereof containing CDRH1 to CDRH3 and CDRL1 to CDRL3 include antibodies or antigen-binding fragments thereof containing the following heavy chain variable region: Figure 137) of the amino acid sequence shown in SEQ ID NO: 2 to 119; C3E-7034 heavy chain variable region consisting of the amino acid sequence numbered 2 to 119 of SEQ ID NO: 137 ( Figure 138 ) of the amino acid sequence shown in SEQ ID NO: 2 to 119; the C3E-7036 heavy chain variable region consisting of the amino acid sequence numbered 2 to 119 of SEQ ID NO: 138 ( Figure 139 ) of the amino acid sequence shown in SEQ ID NO: 2 to 119; a C3E-7085 heavy chain variable region consisting of an amino acid sequence numbered 2 to 119 of SEQ ID NO: 139 ( Figure 140 ) of the amino acid sequence shown in SEQ ID NO: 2 to 119; a C3E-7088 heavy chain variable region consisting of an amino acid sequence numbered 2 to 119 of SEQ ID NO: 140 ( Figure 141 ) of the amino acid sequence shown in SEQ ID NO: 2 to 119; a C3E-7093 heavy chain variable region consisting of an amino acid sequence numbered 2 to 119 of SEQ ID NO: 155 ( Figure 151 ) of the amino acid sequence shown in SEQ ID NO: 272 to 389; C3E-7096 heavy chain variable region consisting of the amino acid sequence of amino acid numbers 272 to 389 of SEQ ID NO: 156 ( Figure 152 ) of the amino acid sequence shown in SEQ ID NO: 277 to 394; the C3E-7096 heavy chain variable region consisting of the amino acid sequence numbered 277 to 394 of SEQ ID NO: 157 ( Figure 153 ) The C3E-7097 heavy chain variable region consists of the amino acid sequence of amino acids 277 to 394 of the amino acid sequence shown in ).

[0625] In addition, more preferred antibodies or antigen-binding fragments thereof containing CDRH1 to CDRH3 and CDRL1 to CDRL3 include antibodies or antigen-binding fragments thereof containing the following light chain variable region: Figure 137 ) of the amino acid sequence shown in SEQ ID NO: 135 to 243; C3E-7034 light chain variable region consisting of the amino acid sequence of amino acid numbers 135 to 243 of SEQ ID NO: 137 ( Figure 138 ) of the amino acid sequence shown in SEQ ID NO: 138 ( Figure 139 ) of the amino acid sequence shown in SEQ ID NO: 139 ( Figure 140 ) of the amino acid sequence shown in SEQ ID NO: 135 to 243; a C3E-7088 light chain variable region consisting of an amino acid sequence numbered 135 to 243 of SEQ ID NO: 140 ( Figure 141) of the amino acid sequence shown in SEQ ID NO: 155 ( Figure 151 ) of the amino acid sequence shown in SEQ ID NO: 156 ( Figure 152 ) of the amino acid sequence shown in SEQ ID NO: 157 ( Figure 153 ) The C3E-7097 light chain variable region consists of the amino acid sequence of amino acid numbers 410 to 516 of the amino acid sequence shown in ).

[0626] More preferred antibodies or antigen-binding fragments thereof containing CDRH1 to CDRH3 and CDRL1 to CDRL3 include antibodies or antigen-binding fragments thereof containing the following combination of heavy chain variable regions and light chain variable regions: Figure 137 ) shown in SEQ ID NO: 137 ( Figure 138 ) shown in SEQ ID NO: 147 ( Figure 143 ) shown in SEQ ID NO: 138 ( Figure 139 ) shown in SEQ ID NO: 139 ( Figure 140 ) shown in SEQ ID NO: 140 ( Figure 141 ) shown in SEQ ID NO: 155 ( Figure 151 ) shown in SEQ ID NO: 156 ( Figure 152 ) shown in SEQ ID NO: 157 ( Figure 153 ) is a combination of the C3E-7097 heavy chain variable region and light chain variable region consisting of amino acid numbers 277 to 394 and 410 to 516 shown in ).

[0627] In addition, more preferred antibodies or antigen-binding fragments thereof containing CDRH1 to CDRH3 and CDRL1 to CDRL3 include antibodies or antibody-binding fragments containing the following scFv and any of these scFvs: Figure 137 ) of the amino acid sequence shown in SEQ ID NO: 2 to 243; C3E-7034scFv consisting of the amino acid sequence of SEQ ID NO: 137 ( Figure 138 ) of the amino acid sequence shown in SEQ ID NO: 2 to 241; C3E-7036scFv consisting of the amino acid sequence of SEQ ID NO: 147 ( Figure 143 ) of the amino acid sequence shown in SEQ ID NO: 2 to 243 of the amino acid sequence; C3E-7078scFv consisting of SEQ ID NO: 138 ( Figure 139 ) of the amino acid sequence shown in SEQ ID NO: 2 to 241; C3E-7085scFv consisting of the amino acid sequence of SEQ ID NO: 139 ( Figure 140 ) of the amino acid sequence shown in SEQ ID NO: 2 to 243; C3E-7088scFv consisting of the amino acid sequence of SEQ ID NO: 140 ( Figure 141 ) is a C3E-7093 scFv consisting of the amino acid sequence of amino acids 2 to 243 of the amino acid sequence shown in ). Preferred forms of scFv specific for CD3 (also called "anti-CD3 scFv") include forms in which a FLAG-His tag is attached to the carboxyl terminus (also simply referred to as "tag addition"). Preferred tag additions include C3E-7034 (SEQ ID NO: 136, Figure 137 ), C3E-7036 (SEQ ID NO: 137, Figure 138 ), C3E-7085 (SEQ ID NO: 138, Figure 139 )、C3E-7088 (SEQ ID NO: 139, Figure 140 ) and C3E-7093 (SEQ ID NO: 140, Figure 141 ), more preferably C3E-7085.

[0628] Preferred examples of multispecific molecules of the present invention include bispecific molecules. "Bispecific" refers to the ability to bind to two distinct epitopes on the same molecule, or to distinct epitopes on two molecules, and encompasses antibodies or antigen-binding fragments exhibiting such bispecificity. The bispecific molecules of the present invention bind to both HLA / NY-ESO and CD3.

[0629] Examples of the bispecific molecules of the present invention include molecules having the following structures (configurations).

[0630] In a bispecific molecule of the double scFv type, two scFvs that bind to different epitopes are connected to one Fc of the dimer through a linker, or are directly bound without a linker. Alternatively, two scFvs that bind to different epitopes are connected to CH and CL through a linker, and then to one Fc of the dimer through a linker. The bispecific molecule is a configuration in which a mutant Fc is associated with a heterozygote, wherein the introduced mutation forms a heterodimer downstream of each of the two different scFvs. A bispecific molecule of the double scFv type is called a bitype bispecific molecule, or simply a bitype ( Figure 6A (b)).

[0631] In the present invention, for example, a bispecific molecule composed of an anti-HLA-A2 / NY-ESO scFv and an anti-CD3 scFv can be used.

[0632] Alternatively, the bispecific molecule of the present invention may be a molecule comprising a Fab and a scFv that bind to different epitopes, wherein the first antibody Fab is bound to one Fc of the dimer via a linker, and the second antibody scFv is bound to the other Fc via a linker. This bispecific molecule is a configuration in which a mutant Fc is associated with a hybrid, wherein the introduced mutation forms a heterodimer downstream of each of the Fab and scFv. This bispecific molecule is called a hybrid bispecific molecule or a hybrid ( Figure 6A (a)) In the present invention, for example, a hybrid composed of anti-HLA-A2 / NY-ESO Fab and anti-CD3 scFv can be used.

[0633] Alternatively, it may be a bispecific molecule in which the first antibody Fab and the second antibody scFv are bound to one Fc of the dimer via a linker. In this case, either Fab can be bound to Fc and scFv to the Fab, or scFv can be bound to Fc and Fab to the scFv. Preferably, Fab is bound and scFv is bound to the Fab. The binding of Fab to scFv can bind the variable region of scFv to Fab via a linker. The bispecific molecule is a configuration in which a mutation is introduced into the Fc, wherein the introduced mutation forms a heterodimer downstream of the connection of scFv to Fab via a linker. This bispecific molecule is called a scFv-Fab-heterodimer Fc type bispecific molecule, or a scFv-Fab-heterodimer Fc type ( Figure 6A (c)).

[0634] In the present invention, for example, a scFv-Fab-heterodimer Fc type composed of anti-CD3 scFv and anti-HLA-A2 / NY-ESO Fab can be used.

[0635] In addition, a taFv ( Figure 3 (c)) can be bound to one Fc of the dimer through a linker, or can be bound directly without a linker. This bispecific molecule is called a taFv-heterodimer Fc type bispecific molecule, or a taFv-heterodimer Fc type ( Figure 3 (d)). This bispecific molecule is a configuration in which a mutant Fc is introduced and a heterozygote is associated, wherein the introduced mutation forms a heterodimer downstream of the taFv. There is no restriction on the order in which the first antibody and the second antibody are linked in the taFv. When the order of linking the first antibody and the second antibody in the taFv is reversed, the original bispecific molecule is called the taFv-heterodimer Fc type, and is called the taFv (reverse)-heterodimer Fc type (also called the taFv (reverse)-Fc type).

[0636] Figure 6A (a) shows the structure of the hybrid bispecific molecule, Figure 6A (b) shows the structure of a bispecific molecule. Figure 6A (c) shows the structure of scFv-Fab-heterodimer Fc type bispecific molecule. 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, Figure 3(e) shows the structure of the taFv-Fab-heterodimer Fc type bispecific molecule. Figure 6B (a) shows the structure of the taFv-heterodimer Fc-type bispecific molecule (with Figure 3 (d) the same), Figure 6B (b) shows the structure of taFv (reverse)-heterodimer Fc-type bispecific molecule, Figure 6B (c) shows the structure of the first polypeptide contained in the taFv (reverse)-heterodimer Fc-type bispecific molecule, Figure 6B (d) shows the structure of the second polypeptide contained in the taFv (reverse)-heterodimer Fc-type bispecific molecule.

[0637] The bispecific molecule of the present invention has a structure in which multiple polypeptides are associated.

[0638] In the present invention, for example, as taFv, taFv of anti-HLA-A2 / NY-ESO scFv and anti-CD3 scFv can be used. Regarding the taFv-heterodimer Fc-type bispecific molecule, preferably, (a) contains a first polypeptide and a second polypeptide, the first polypeptide containing, from the N-terminus to the C-terminus, an scFv that specifically binds to HLA / NY-ESO, an scFv that specifically binds to CD3, and an immunoglobulin Fc region (i); the second polypeptide contains the hinge region and Fc region (ii) of the immunoglobulin; more preferably, (b) the first polypeptide and the second polypeptide associate in the Fc region (i) and the Fc region (ii). The Fc regions of the first polypeptide and the second polypeptide may also contain mutations for forming heterodimers. An example of a taFv-heterodimer Fc-type bispecific molecule is shown in Figure 3 (d). Figure 3 As shown in (d), the Fc region (i) of the first polypeptide binds to the Fc region (ii) of the second polypeptide, which is indicated in black, and the first and second polypeptides are associated. Figure 3 (f) represents a first polypeptide, Figure 3 (g) represents the second polypeptide. For example, Figure 3 In (d), the scFv indicated by white is the anti-HLA-A2 / NY-ESO scFv, and the scFv indicated by the upper right slash is the anti-CD3 scFv.

[0639] A more preferred taFv-heterodimer Fc-type bispecific molecule of the present invention comprises the following amino acid sequence: the amino acid sequence from positions 21 to 511 of the amino acid sequence of SEQ ID NO: 85, the amino acid sequence from positions 21 to 511 of the amino acid sequence of SEQ ID NO: 87, the amino acid sequence from positions 21 to 511 of the amino acid sequence of SEQ ID NO: 88, the amino acid sequence from positions 21 to 511 of the amino acid sequence of SEQ ID NO: 89, the amino acid sequence from positions 21 to 511 of the amino acid sequence of SEQ ID NO: 90, the amino acid sequence from positions 21 to 511 of the amino acid sequence of SEQ ID NO: 91, the amino acid sequence from positions 21 to 511 of the amino acid sequence of SEQ ID NO: 92, the amino acid sequence from positions 21 to 511 of the amino acid sequence of SEQ ID NO: 93, the amino acid sequence from positions 21 to 511 of the amino acid sequence of SEQ ID NO: 94, the amino acid sequence from positions 21 to 511 of the amino acid sequence set forth in SEQ ID NO: 95, the amino acid sequence from positions 21 to 511 of the amino acid sequence set forth in SEQ ID NO: 96, the amino acid sequence from positions 21 to 511 of the amino acid sequence set forth in SEQ ID NO: 86, the amino acid sequence from positions 21 to 511 of the amino acid sequence set forth in SEQ ID NO: 149, the amino acid sequence from positions 21 to 511 of the amino acid sequence set forth in SEQ ID NO: 150, the amino acid sequence from positions 20 to 511 of the amino acid sequence set forth in SEQ ID NO: 155, the amino acid sequence from positions 20 to 516 of the amino acid sequence set forth in SEQ ID NO: 156, or the amino acid sequence from positions 20 to 516 of the amino acid sequence set forth in SEQ ID NO: 157.A further preferred taFv-heterodimer Fc-type bispecific molecule comprises a first polypeptide comprising the following amino acid sequence: the amino acid sequence from positions 529 to 745 of the amino acid sequence set forth in SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 86, SEQ ID NO: 149, SEQ ID NO: 150, or SEQ ID NO: 155; or the amino acid sequence from positions 534 to 750 of the amino acid sequence set forth in SEQ ID NO: 156 or SEQ ID NO: 157. A further preferred taFv-heterodimer Fc-type bispecific molecule comprises a first polypeptide consisting of the following amino acid sequence: the amino acid sequence from positions 21 to 745 of the amino acid sequence set forth in SEQ ID NO: 85; the amino acid sequence from positions 21 to 745 of the amino acid sequence set forth in SEQ ID NO: 87; the amino acid sequence from positions 21 to 745 of the amino acid sequence set forth in SEQ ID NO: 88; the amino acid sequence from positions 21 to 745 of the amino acid sequence set forth in SEQ ID NO: 89; the amino acid sequence from positions 21 to 745 of the amino acid sequence set forth in SEQ ID NO: 90; the amino acid sequence from positions 21 to 745 of the amino acid sequence set forth in SEQ ID NO: 91; the amino acid sequence from positions 21 to 745 of the amino acid sequence set forth in SEQ ID NO: 92; the amino acid sequence from positions 21 to 745 of the amino acid sequence set forth in SEQ ID NO: 93; the amino acid sequence from positions 21 to 745 of the amino acid sequence set forth in SEQ ID NO: 94; the amino acid sequence from positions 21 to 745 of the amino acid sequence set forth in SEQ ID NO:95; the amino acid sequence from positions 21 to 745 of the amino acid sequence set forth in SEQ ID NO:96; the amino acid sequence from positions 21 to 745 of the amino acid sequence set forth in SEQ ID NO:86; the amino acid sequence from positions 21 to 745 of the amino acid sequence set forth in SEQ ID NO:149; the amino acid sequence from positions 21 to 745 of the amino acid sequence set forth in SEQ ID NO:150; or the amino acid sequence from positions 20 to 745 of the amino acid sequence set forth in SEQ ID NO:155.Alternatively, it consists of the amino acid sequence from positions 20 to 750 of the amino acid sequence shown in SEQ ID NO: 156 or the amino acid sequence from positions 20 to 750 of the amino acid sequence shown in SEQ ID NO: 157.

[0640] The second polypeptide contained in the preferred taFv-heterodimer Fc bispecific molecule of the present invention comprises the hinge region and mutant Fc from a human antibody, and the second polypeptide contained in the more preferred taFv-heterodimer Fc bispecific molecule comprises the amino acid sequence from positions 20 to 246 of the amino acid sequence shown in SEQ ID NO:84.

[0641] Among them, the following antibodies can be exemplified as preferred taFv-heterodimer Fc-type bispecific molecules of the present invention: NYF-0016, which is a combination of a first polypeptide consisting of amino acids 21 to 745 of the amino acid sequence set forth in SEQ ID NO: 85 and a second polypeptide consisting of amino acids 21 to 246 of the amino acid sequence set forth in SEQ ID NO: 84; NYF-0022, which is a combination of a first polypeptide consisting of amino acids 21 to 745 of the amino acid sequence set forth in SEQ ID NO: 87 and a second polypeptide consisting of amino acids 21 to 246 of the amino acid sequence set forth in SEQ ID NO: 84; NYF-0023, which is a combination of a first polypeptide consisting of amino acids 21 to 745 of the amino acid sequence set forth in SEQ ID NO: 88 and a second polypeptide consisting of amino acids 21 to 246 of the amino acid sequence set forth in SEQ ID NO: 84; and NYF-0044, which is a combination of a first polypeptide consisting of amino acids 21 to 745 of the amino acid sequence set forth in SEQ ID NO: 89 and a second polypeptide consisting of amino acids 21 to 246 of the amino acid sequence set forth in SEQ ID NO: 81. NYF-0027, which is formed by the association of a first polypeptide consisting of amino acids from positions 21 to 745 of the amino acid sequence set forth in SEQ ID NO: 90 and a second polypeptide consisting of amino acids from positions 21 to 246 of the amino acid sequence set forth in SEQ ID NO: 84; NYF-0035, which is formed by the association of a first polypeptide consisting of amino acids from positions 21 to 745 of the amino acid sequence set forth in SEQ ID NO: 91 and a second polypeptide consisting of amino acids from positions 21 to 246 of the amino acid sequence set forth in SEQ ID NO: 84; NYF-0044, which is formed by the association of a first polypeptide consisting of amino acids from positions 21 to 745 of the amino acid sequence set forth in SEQ ID NO: 92 and a second polypeptide consisting of amino acids from positions 21 to 246 of the amino acid sequence set forth in SEQ ID NO: 84; NYF-0047, formed by the association of a first polypeptide consisting of amino acids 21 to 745 of the amino acid sequence set forth in SEQ ID NO:93 and a second polypeptide consisting of amino acids 21 to 246 of the amino acid sequence set forth in SEQ ID NO:84; NYF-0048, formed by the association of a first polypeptide consisting of amino acids 21 to 745 of the amino acid sequence set forth in SEQ ID NO:94 and a second polypeptide consisting of amino acids 21 to 246 of the amino acid sequence set forth in SEQ ID NO:84;NYF-0060, which is formed by the association of a first polypeptide consisting of amino acids from positions 21 to 745 of the amino acid sequence set forth in SEQ ID NO: 95 and a second polypeptide consisting of amino acids from positions 21 to 246 of the amino acid sequence set forth in SEQ ID NO: 84; NYF-0061, which is formed by the association of a first polypeptide consisting of amino acids from positions 21 to 745 of the amino acid sequence set forth in SEQ ID NO: 96 and a second polypeptide consisting of amino acids from positions 21 to 246 of the amino acid sequence set forth in SEQ ID NO: 84; NYF-0019, which is formed by the association of a first polypeptide consisting of amino acids from positions 21 to 745 of the amino acid sequence set forth in SEQ ID NO: 86 and a second polypeptide consisting of amino acids from positions 21 to 246 of the amino acid sequence set forth in SEQ ID NO: 84; a first polypeptide consisting of amino acids from positions 21 to 745 of the amino acid sequence set forth in SEQ ID NO: 149 and a second polypeptide consisting of amino acids from positions 21 to 246 of the amino acid sequence set forth in SEQ ID NO: NYF-0014, which is formed by the association of a first polypeptide consisting of amino acids 21 to 745 of the amino acid sequence set forth in SEQ ID NO: 150 and a second polypeptide consisting of amino acids 21 to 246 of the amino acid sequence set forth in SEQ ID NO: 84; and NYF-0082, which is formed by the association of a first polypeptide consisting of amino acids 21 to 745 of the amino acid sequence set forth in SEQ ID NO: 150 and a second polypeptide consisting of amino acids 21 to 246 of the amino acid sequence set forth in SEQ ID NO: 84.

[0642] In addition, the following are examples of preferred taFv-heterodimer Fc-type bispecific molecules of the present invention: NYZ-0038, which is formed by the association of a first polypeptide consisting of amino acids 20 to 745 of the amino acid sequence set forth in SEQ ID NO: 155 and a second polypeptide consisting of amino acids 20 to 246 of the amino acid sequence set forth in SEQ ID NO: 84; NYZ-0082, which is formed by the association of a first polypeptide consisting of amino acids 20 to 750 of the amino acid sequence set forth in SEQ ID NO: 156 and a second polypeptide consisting of amino acids 20 to 246 of the amino acid sequence set forth in SEQ ID NO: 84; and NYZ-0083, which is formed by the association of a first polypeptide consisting of amino acids 20 to 750 of the amino acid sequence set forth in SEQ ID NO: 157 and a second polypeptide consisting of amino acids 20 to 246 of the amino acid sequence set forth in SEQ ID NO: 84.

[0643] Among them, NYF-0023, NYF-0047, NYF-0048, NYF-0060, NYF-0061, NYZ-0038, NYZ-0082, and NYZ-0083 are preferred because they have particularly excellent biological activities and physicochemical properties.

[0644] In addition, the first antibody taFv can be directly bound to one Fc of the dimer with or without a linker, and the first antibody or second antibody Fab can be directly bound to the other Fc with or without a linker. This bispecific molecule is a configuration in which Fab is added upstream of the Fc region (ii) (blackened) side of the second polypeptide of the taFv-heterodimer Fc type. This bispecific molecule is called a taFv-Fab-heterodimer Fc type bispecific molecule, or a taFv-Fab-heterodimer Fc type ( Figure 3 ).

[0645] In the present invention, as taFv included in the taFv-Fab-heterodimer Fc-type bispecific molecule, for example, taFv comprising anti-HLA-A2 / NY-ESO scFv and anti-CD3 scFv can be used; as Fab, for example, HLA / NY-ESO Fab can be used.

[0646] Regarding the taFv-Fab-heterodimer Fc-type bispecific molecule, preferably, (a) contains a first polypeptide, a second polypeptide and a third polypeptide, wherein the first polypeptide contains, from the N-terminus to the C-terminus, an scFv that specifically binds to human HLA / NY-ESO, an scFv that specifically binds to CD3, and an immunoglobulin Fc region (i); the second polypeptide is composed of an immunoglobulin heavy chain containing an Fc region (ii); and the third polypeptide is composed of 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 Figure 3 (e), the first polypeptide is shown in Figure 3 (f), the second polypeptide is shown in Figure 3 (h), the third polypeptide is shown in Figure 3 (i). like Figure 3As shown in (e), the Fc region (i) of the first polypeptide is bound to the second polypeptide (shown in black) consisting of an immunoglobulin heavy chain containing the Fc region (ii). The Fc region (i) of the first polypeptide and the Fc region (ii) of the second polypeptide are bound together, and an immunoglobulin light chain is bound to the second polypeptide. This preferred taFv-Fab-heterodimer Fc-type bispecific molecule can also be formed by binding a taFv-heterodimer Fc-type bispecific molecule comprising a taFv and an immunoglobulin Fc region to a Fab. Preferred amino acid sequences comprised by the first polypeptide contained in the taFv-Fab heterodimeric Fc-type bispecific molecule include: the amino acid sequence from positions 21 to 511 of the amino acid sequence set forth in SEQ ID NO: 85, the amino acid sequence from positions 21 to 511 of the amino acid sequence set forth in SEQ ID NO: 87, the amino acid sequence from positions 21 to 511 of the amino acid sequence set forth in SEQ ID NO: 88, the amino acid sequence from positions 21 to 511 of the amino acid sequence set forth in SEQ ID NO: 89, the amino acid sequence from positions 21 to 511 of the amino acid sequence set forth in SEQ ID NO: 90, the amino acid sequence from positions 21 to 511 of the amino acid sequence set forth in SEQ ID NO: 91, the amino acid sequence from positions 21 to 511 of the amino acid sequence set forth in SEQ ID NO: 92, the amino acid sequence from positions 21 to 511 of the amino acid sequence set forth in SEQ ID NO: 93, the amino acid sequence from positions 21 to 511 of the amino acid sequence set forth in SEQ ID NO: 94, the amino acid sequence from positions 21 to 511 of the amino acid sequence set forth in SEQ ID NO: 95, The amino acid sequence from positions 21 to 511 of the amino acid sequence shown in SEQ ID NO: 95, the amino acid sequence from positions 21 to 511 of the amino acid sequence shown in SEQ ID NO: 96, the amino acid sequence from positions 21 to 511 of the amino acid sequence shown in SEQ ID NO: 86, the amino acid sequence from positions 21 to 511 of the amino acid sequence shown in SEQ ID NO: 149; and the amino acid sequence from positions 21 to 511 of the amino acid sequence shown in SEQ ID NO: 150.More preferably, the first polypeptide contained in the taFv-Fab heterodimeric Fc-type bispecific molecule consists of the following amino acid sequence: the amino acid sequence of positions 21 to 745 of the amino acid sequence set forth in SEQ ID NO: 85; the amino acid sequence of positions 21 to 745 of the amino acid sequence set forth in SEQ ID NO: 87; the amino acid sequence of positions 21 to 745 of the amino acid sequence set forth in SEQ ID NO: 88; the amino acid sequence of positions 21 to 745 of the amino acid sequence set forth in SEQ ID NO: 89; the amino acid sequence of positions 21 to 745 of the amino acid sequence set forth in SEQ ID NO: 90; the amino acid sequence of positions 21 to 745 of the amino acid sequence set forth in SEQ ID NO: 91; the amino acid sequence of positions 21 to 745 of the amino acid sequence set forth in SEQ ID NO: 92; the amino acid sequence of positions 21 to 745 of the amino acid sequence set forth in SEQ ID NO: 93; the amino acid sequence of positions 21 to 745 of the amino acid sequence set forth in SEQ ID NO: 94; The amino acid sequence from positions 21 to 745 of the amino acid sequence shown in SEQ ID NO: 95; the amino acid sequence from positions 21 to 745 of the amino acid sequence shown in SEQ ID NO: 96; and the amino acid sequence from positions 21 to 745 of the amino acid sequence shown in SEQ ID NO: 86; the amino acid sequence from positions 21 to 745 of the amino acid sequence shown in SEQ ID NO: 149; or the amino acid sequence from positions 21 to 745 of the amino acid sequence shown in SEQ ID NO: 150.

[0647] The second polypeptide contained in the preferred taFv-Fab-heterodimer Fc type bispecific molecule of the present invention comprises the variable region, CH1 region and hinge region of the heavy chain of a human antibody or a humanized antibody, and a mutant Fc; the second polypeptide contained in the more preferred taFv-Fab-heterodimer Fc type bispecific molecule comprises the amino acid sequence from positions 20 to 242 of the amino acid sequence shown in SEQ ID NO:99.

[0648] The third polypeptide contained in the preferred taFv-Fab-heterodimer Fc-type bispecific molecule of the present invention comprises the variable region and constant region of the light chain of a human antibody or a humanized antibody; the third polypeptide contained in the more preferred taFv-Fab-heterodimer Fc-type bispecific molecule comprises positions 21 to 131 of the amino acid sequence shown in SEQ ID NO:100.

[0649] The variable region and CH1 region of the second polypeptide and the third polypeptide contained in the preferred taFv-Fab-heterodimer Fc-type bispecific molecule constitute Fab, and the preferred Fab is Fab of an anti-HLA / NY-ESO antibody, for example, Fab of NYA-0001.

[0650] In the present invention, as the scFv included in the scFv-Fab-heterodimer Fc-type bispecific molecule, for example, anti-HLA-A2 / NY-ESO scFv or anti-CD3 scFv can be used; as the Fab, for example, HLA / NY-ESO Fab or anti-CD3 Fab can be used.

[0651] Regarding the scFv-Fab-heterodimer Fc type bispecific molecule, preferably, (a) contains a first polypeptide, a second polypeptide and a third polypeptide, the first polypeptide containing, from N-terminus to C-terminus, an scFv that specifically binds to human HLA / NY-ESO, a variable region and a constant region CH1 of an antibody heavy chain that specifically binds to CD3, and an immunoglobulin Fc region (i); the second polypeptide contains the hinge region and the Fc region (ii) of an immunoglobulin; the third polypeptide contains an antibody light chain composed of a variable region and a constant region; 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 associates with the third polypeptide (antibody light chain) in the variable region and constant region CH1 of the antibody heavy chain. The Fc regions of the first polypeptide and the second polypeptide can be either wild type or contain mutations that form heterodimers. An example of a scFv-Fab-heterodimer Fc type bispecific molecule is shown in Figure 6A (c). Figure 6A The right half of (c) is the first polypeptide and the third polypeptide, and the left half is the second polypeptide. Figure 6A As shown in (c), the Fc region (i) of the first polypeptide associates with the Fc region (ii) of the second polypeptide indicated in black, and the first polypeptide and the third polypeptide associate. Figure 6A In (c), the scFv indicated by the upper right slash is the anti-HLA-A2 / NY-ESO scFv, and the Fab indicated by the white, checkered pattern, and horizontal lines is the anti-CD3 Fab.

[0652] Preferred amino acid sequences comprised by the first polypeptide of the scFv-Fab-heterodimer Fc-type bispecific molecule include amino acid sequences from positions 21 to 394 of the amino acid sequence shown in SEQ ID NO: 160, and more preferably amino acid sequences from positions 20 to 724.

[0653] In addition, as preferred other amino acid sequences contained in the first polypeptide contained in the scFv-Fab-heterodimer Fc-type bispecific molecule, the amino acid sequence of positions 21 to 389 of the amino acid sequence shown in SEQ ID NO: 197 can be mentioned, and more preferably the amino acid sequence of positions 20 to 719 can be mentioned.

[0654] In addition, as preferred other amino acid sequences contained in the first polypeptide contained in the scFv-Fab-heterodimer Fc-type bispecific molecule, the amino acid sequence of positions 21 to 389 of the amino acid sequence shown in SEQ ID NO: 198 can be mentioned, and more preferably the amino acid sequence of positions 20 to 719 can be mentioned.

[0655] The second polypeptide contained in the preferred scFv-Fab-heterodimer Fc bispecific molecule of the present invention comprises the hinge region and mutant Fc from a human antibody. More preferably, the second polypeptide contained in the scFv-Fab-heterodimer Fc bispecific molecule comprises the amino acid sequence from positions 20 to 246 of the amino acid sequence shown in SEQ ID NO:84.

[0656] The third polypeptide contained in the preferred scFv-Fab-heterodimer Fc-type bispecific molecule of the present invention comprises a light chain derived from a human antibody. A more preferred third polypeptide contained in the scFv-Fab-heterodimer Fc-type bispecific molecule includes amino acids 21 to 127 of the amino acid sequence set forth in SEQ ID NO: 161, and more preferably, amino acids 21 to 233.

[0657] Among them, NYZ-1010, which is composed of the following first polypeptide, second polypeptide, and third polypeptide, can be exemplified as a preferred scFv-Fab-heterodimer Fc-type bispecific molecule of the present invention: a first polypeptide consisting of the amino acid sequence from positions 20 to 724 of the amino acid sequence set forth in SEQ ID NO: 160; a second polypeptide consisting of positions 20 to 246 of the amino acid sequence set forth in SEQ ID NO: 84; and a third polypeptide consisting of positions 21 to 233 of the amino acid sequence set forth in SEQ ID NO: 161.

[0658] In addition, NYZ-1007, which is composed of the following first, second, and third polypeptides, can also be exemplified as a preferred scFv-Fab-heterodimer Fc-type bispecific molecule of the present invention: a first polypeptide consisting of the amino acid sequence from positions 20 to 719 of the amino acid sequence set forth in SEQ ID NO: 197; a second polypeptide consisting of the amino acid sequence from positions 20 to 246 of the amino acid sequence set forth in SEQ ID NO: 84; and a third polypeptide consisting of the amino acid sequence from positions 21 to 233 of the amino acid sequence set forth in SEQ ID NO: 161.

[0659] In addition, NYZ-1017, which is composed of the following first, second, and third polypeptides, can also be exemplified as a preferred scFv-Fab-heterodimer Fc-type bispecific molecule of the present invention: a first polypeptide consisting of the amino acid sequence from positions 20 to 719 of the amino acid sequence set forth in SEQ ID NO: 198; a second polypeptide consisting of amino acid sequences from positions 20 to 246 of the amino acid sequence set forth in SEQ ID NO: 84; and a third polypeptide consisting of amino acid sequences from positions 21 to 233 of the amino acid sequence set forth in SEQ ID NO: 161.

[0660] The one, two, or three or more peptides contained in the bispecific molecule of the present invention may be the aforementioned "deletion variants," i.e., one or two (or more) amino acids at their carboxyl termini, particularly at the carboxyl termini of the antibody heavy chain, are mutated (including deleted). For example, the carboxyl terminus of the first polypeptide contained in NYZ-1010, one of the preferred scFv-Fab-heterodimer Fc-type bispecific molecules of the present invention, may be any one of Lys at position 724 of SEQ ID NO: 160, Gly at position 723 with a deletion of one amino acid, or a mixture thereof. Similarly, the carboxyl terminus of the second polypeptide contained in the preferred scFv-Fab-heterodimer Fc-type bispecific molecule of the present invention may be any one of Lys at position 246 of SEQ ID NO: 84, Gly at position 245 with a deletion of one amino acid, or a mixture thereof.

[0661] The scFv and Fab contained in the bispecific molecule of the present invention are preferably scFv and Fab of a humanized antibody or a human antibody, and the Fc is preferably the Fc of a human antibody.

[0662] The variable regions contained in the bispecific molecules of the present invention can be combined in the order of heavy chain variable region and light chain variable region from the amino terminal side of the antibody, or can be combined in the order of light chain variable region and heavy chain variable region. A linker (optional) may also be present between the two variable regions. In addition, a glycine residue (optional) may also be present at the amino terminus of the variable region on the amino terminal side. The bispecific molecules of the tandem scFv type can also be combined with a linker, a FLAG tag, and / or a His tag (optional) at the carboxyl terminus of the variable region on the carboxyl terminal side. As one of the preferred forms, it can be exemplified that, starting from the amino terminus, the heavy chain variable region, the first linker, the light chain variable region, the second linker, the FLAG tag, and the His tag are combined in sequence.

[0663] The linker includes a single-chain polypeptide or a single-chain oligopeptide, or a synthetic substance such as PEG, nucleotide, sugar chain, compound, etc. In addition, there is no particular limitation as long as it can bind two polypeptides, and a known linker can be used.

[0664] As for the length of the linker, for example, the length of the peptide linker is 5 to 30 amino acids. When the bispecific molecule contains multiple linkers, peptide linkers of the same length or different lengths may be used.

[0665] Examples of peptide linkers include the cycle (Gly·Gly·Gly·Gly·Ser) (SEQ ID NO: 161), but one or more amino acid residues other than Gly and Ser may be added thereto.

[0666] In summary, among the structures (configurations) that the multispecific antibodies of the present invention, especially bispecific antibodies, can adopt, preferably taFv-heterodimer Fc type, taFv-Fab-heterodimer Fc type, and scFv-Fab-heterodimer Fc type; more preferably taFv-heterodimer Fc type. From N-terminus to C-terminus, the configuration arranged in the order of anti-HLA / NY-ESO scFv and anti-CD3 scFv (taFv-heterodimer Fc type) is further preferred compared with the configuration arranged in reverse (taFv (reverse)-heterodimer Fc type) (Example 11, etc.). In addition, other more preferred configurations are scFv-Fab-heterodimer Fc type.

[0667] The present invention also includes the following molecules: molecules comprising an amino acid sequence encoded by a nucleotide sequence contained in a polynucleotide that hybridizes under stringent conditions to a complementary strand of a polynucleotide containing a nucleotide sequence encoding the amino acid sequence contained in the molecule of the present invention, and that binds to HLA / NY-ESO, preferably further to CD3.

[0668] The present invention also includes molecules comprising an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identical to an amino acid sequence comprised by a molecule of the present invention, and that binds to HLA / NY-ESO, preferably also to CD3.

[0669] The antibodies of the present invention, their binding fragments and multispecific antibodies comprising them have excellent biological activity, physicochemical properties (hereinafter referred to as "physicochemical properties"), safety, in vivo kinetics and other properties. (a) As biological activity or its index, examples include: antigen binding activity, in vitro cytotoxic activity, in vivo anti-tumor activity, etc. For example, the dissociation constant (KD value) for HLA / NY-ESO is 100nM or less or 50nM or less, preferably 20nM or less or 10nM or less, and more preferably 5nM or less. In addition, for example, for the cell lines U266B1 and / or NCI-H1703 expressing endogenous human NY-ESO, human peripheral blood mononuclear cells are used as ECs of the cytotoxic activity exerted by effector cells. 50 The value is 20 nM or less, preferably 10 nM or less, and more preferably 5 nM or less (as for the determination and calculation of in vitro cytotoxic activity, the method described in Example 8 can be cited but is not limited thereto). In addition, for example, 0.1 mL of 6×10 7 cells / mL suspension was transplanted subcutaneously into NOG mice, and 4 days later, 3.75×10 7The cell / mL suspension is transplanted into the tail vein, and starting 14 days later, the volume of the tumor after the antibody is administered three times is measured once a week. At this time, the tumor proliferation inhibitory activity is 50% or more, preferably 75% or more, and more preferably 90% or more relative to the control group administered with the solvent (as the determination and calculation of in vivo anti-tumor activity, the method described in Example 9 can be cited but not limited to). (b) The impurities contained in biological drugs are related to the safety of the drugs, so it is necessary to set appropriate regulations and manage whether there is an increase during manufacturing and storage. Among them, HMWS (aggregates) is one of the main impurities, which can lead to immunogenicity risks and reduced efficacy, so this item should be managed with extra strictness. Regarding the management of impurities, evaluation must be carried out not only during manufacturing, but also during the manufacturing steps and after manufacturing, including the stability over time (whether there is an increase). Since the shelf life of the drug is set based on the results of long-term stability tests, antibodies that are stable over time can have a longer shelf life. Therefore, as indicators for screening suitable antibodies for use in biopharmaceuticals, that is, the physicochemical properties of the present invention, acid resistance (inhibiting the generation of HMWS, etc.) and solution stability (inhibiting the generation of HMWS, etc.) can be cited. As other indicators, host cells suitable for the production of the antibodies of the present invention or their binding fragments or molecules containing them can be cited. For example, recombinant cells obtained by introducing genes encoding the amino acid sequences contained therein into Expi293F cells have high production and yield in culture. The preferred antibodies of the present invention, their antigen-binding fragments, and multispecific antibodies containing them that have these physicochemical properties can do the following: their solutions can be exposed to acidic conditions, and their manufacture can be implemented or easily implemented, such as chromatography methods such as protein A and ion exchange, virus inactivation and other steps; their solutions can suppress the generation of HMWS at a low level, so their manufacture, formulation, circulation and storage of drugs containing them can be implemented or easily implemented; they can be produced efficiently. Regarding acid resistance, for example, when the mixture is kept at pH 3.5 and room temperature for 1 hour, and then HMWS is measured and calculated by size exclusion chromatography, the content of HMWS produced is 5% or less, preferably 2% or less, and more preferably 1% or less (as for the determination and calculation of HMWS content for acid resistance evaluation, the method described in Example 19-1 can be cited as an example but is not limited to it). Regarding solution stability, for example, when the mixture is dissolved in a solution consisting of 25 mM histidine and 5% sorbitol, the pH value is adjusted to 6.0 and the concentration is adjusted to 25 mg / ml, and the mixture is stored at 25°C for 6 days, and then HMWS is measured and calculated by size exclusion chromatography, the content of HMWS is 20% or less, preferably 10% or less (as for the determination and calculation of HMWS content for solution stability evaluation, the method described in Example 19-2 can be cited as an example but is not limited to it). As for the determination and calculation of production efficiency or yield, the methods described in Examples 20 and 21 can be cited as an example but are not limited to it.(c) Safety or safety indicators include antigen recognition properties and observations upon administration. For example, the antibody recognizes multiple amino acids on the wild-type NY-ESO peptide and does not bind to homologous peptides with amino acid sequences similar but not identical to the wild-type NY-ESO peptide, resulting in a low risk of side effects due to off-target effects. Furthermore, immunogenicity screening based on the ISPRI network (EpiVax, Inc.) has been predicted to be low, with a low risk of side effects such as antibody-induced cytokine production. Furthermore, for example, when NYF-0023, NYF-0045, NYF-0047, NYF-0048, NYF-0060, NYF-0061, NYZ-0082, or NYZ-1010, which are included in the bispecific antibodies of the present invention, were administered to Balb / c mice, no adverse effects on blood half-life were observed, nor was weight loss or other significant toxicity observed. In addition, a single administration of NYZ-0082 or NYZ-1010 to cynomolgus monkeys did not reveal adverse effects on blood half-life. No changes in general state, body weight, food intake, temperature, or plasma cytokine levels were observed after administration. (d) Examples of kinetics or indicators thereof include blood half-life. For example, administration of several bispecific antibodies obtained in the present invention to Balb / c mice or cynomolgus monkeys did not reveal adverse effects on blood half-life. Antibodies, binding fragments, and molecules of the present invention, which possess excellent biological activity, physicochemical properties, safety, and in vivo kinetics, can be preferably included in pharmaceutical compositions. Preferred antibodies or antigen-binding fragments thereof of the present invention having the antigen-binding activity described in (a) and the various properties described in (b) include, but are not limited to, NYA-1143, NYA-1163, NYA-2023, NYA-2027, NYA-2035, NYA-2044, NYA-2045, NYA-2047, NYA-2048, NYA-2060, NYA-2031, NYA-2047, NYA-2061, NYA-2143, and NYA-3061; more preferably, include, but are not limited to, NYA-2047, NYA-2061, NYA-2143, and NYA-3061.In addition, preferred multispecific antibodies of the present invention having the properties described in (a) to (d) include, but are not limited to, 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 preferred examples include, but are not limited to, NYF-0061, NYZ-0038, NYZ-0082, NYZ-0088, NYZ-1007, NYZ-1010, and NYZ-1017.

[0670] In the present invention, the "site" to which an antibody binds, i.e., the "site" recognized by an antibody, refers to a portion of a peptide or a portion of a higher-order structure on an antigen to which the antibody binds or recognizes. In the present invention, this site is also referred to as an epitope or an antibody binding site. Examples of sites on HLA / NY-ESO that the anti-HLA / NY-ESO antibodies of the present invention bind to or recognize include multiple amino acids in an HLA / NY-ESO peptide and portions of a higher-order structure.

[0671] "Antibodies or their binding fragments that bind to the same site" as the antibodies or binding fragments of the present invention are also included in the present invention. "Antibodies that bind to the same site" as a certain antibody refer to other antibodies that bind to the site on the antigen molecule recognized by the antibody. If a partial peptide or partial conformation on the antigen molecule to which the first antibody binds binds to the second antibody, it can be determined that the first antibody and the second antibody bind to the same site. When the first antibody of the present invention has the antigen binding activity described in (a) above, the second antibody that binds to the same site on HLA / NY-ESO is very likely to have the same activity, and the second antibody is also included in the present invention. In addition, if the antibody that competes with the first antibody of the present invention in binding to HLA / NY-ESO also has the antigen binding activity described in (a), it is also included in the present invention. Such antibodies that bind to the site on HLA / NY-ESO recognized by the monoclonal antibodies of the present invention, antibodies that compete with the monoclonal antibodies of the present invention in binding to HLA / NY-ESO, and binding fragments thereof preferably have the in vitro cytotoxic activity and in vivo anti-tumor activity described in (a) and one or more of the properties described in (b) to (d), more preferably have three or more properties, and most preferably have all of the properties.

[0672] The binding site of an antibody can be determined by methods well known to those skilled in the art, such as immunoassays. For example, the binding site can be determined by appropriately removing the amino acid sequence of the antigen from the C-terminus or N-terminus, preparing a series of peptides, studying the reactivity of the antibody to these peptides, and, after determining the approximate recognition site, synthesizing short peptides and studying the reactivity of the antibody to these short peptides. Alternatively, for example, the binding site can be determined by deleting or replacing specific sites or regions in the amino acid sequence of the antigen or antigen fragment peptide with other amino acid sequences, or by introducing mutations into the amino acid sequence, and studying the reactivity of the antibody to these peptides. Antigen fragment peptides can be prepared using techniques such as genetic recombination and peptide synthesis.

[0673] When an antibody binds to or recognizes part of the higher-order structure of an antigen, X-ray structural analysis can be used to identify the amino acid residues on the antigen adjacent to the antibody, thereby determining the binding site of the antibody. For example, by combining and crystallizing an antibody or its fragment and an antigen or its fragment, structural analysis can be performed to identify the amino acid residues on the antigen that have an interaction distance with the antibody. The interaction distance is The following are preferably The following are more preferably One or more amino acid residues that have an interaction distance with the antibody may constitute the antigen binding site (epitope) of the antibody. When there are two or more amino acid residues, the amino acids may not be adjacent to each other in the primary sequence.

[0674] The anti-HLA / NY-ESO antibodies or binding fragments thereof of the present invention can specifically recognize multiple amino acids present in the amino acid sequence of HLA / NY-ESO. Antibodies or binding fragments thereof that recognize these multiple amino acids, antibodies or binding fragments thereof that compete with the antibodies or binding fragments of the present invention for binding to HLA / NY-ESO, or antibodies or binding fragments thereof that have an interaction distance with these multiple amino acids are also encompassed by the present invention. Furthermore, multispecific antibodies comprising such antibodies or binding fragments are also encompassed by the present invention.

[0675] 6. Pharmaceutical Compositions

[0676] The present invention includes an anticancer agent comprising the anti-HLA / NY-ESO antibody or the multispecific molecule binding to HLA / NY-ESO of the present invention as an active ingredient.

[0677] The anticancer agent of the present invention can be used for one or more selected from the following cancers or tumors: carcinoma, sarcoma, lymphoma, leukemia, myeloma, germ cell tumor, brain tumor, carcinoid tumor, neuroblastoma, retinoblastoma, Wilms' tumor. Specifically, with respect to cancer, examples 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, gastric cancer, duodenal cancer, small intestine cancer, colorectal cancer, rectal cancer, appendix cancer, anal cancer, liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, adrenal cancer, bladder cancer, prostate cancer, uterine cancer, vaginal cancer, etc.; with respect to sarcoma, examples include: liposarcoma, angiosarcoma, chondrosarcoma, rhabdomyosarcoma, Ewing's sarcoma, osteosarcoma, unclassified Examples of the cancerous tumors include myeloid leukemia, myxofibrosarcoma, malignant peripheral nerve sheath tumor, retroperitoneal sarcoma, synovial sarcoma, uterine sarcoma, gastrointestinal stromal tumor, leiomyosarcoma, epithelioid sarcoma, etc.; examples of lymphomas include B-cell lymphoma, NK / T-cell lymphoma, Hodgkin's lymphoma, etc.; examples of leukemias include myeloid leukemia, lymphocytic leukemia, myeloproliferative diseases, myelodysplastic syndrome, etc.; examples of myeloma include multiple myeloma, etc.; examples of germ cell tumors include testicular cancer, ovarian cancer, etc.; examples of brain tumors include glioma, meningioma, etc.

[0678] The anticancer agent of the present invention may contain: a therapeutically effective amount of an anti-HLA / NY-ESO antibody or a multispecific molecule that binds to HLA / NY-ESO, as well as pharmaceutically acceptable carriers, diluents, solubilizers, emulsifiers, preservatives, adjuvants, etc. "Pharmaceutically acceptable carriers" and the like can be appropriately selected from a wide range depending on the type of target disease and the form of administration of the agent. The method of administration of the anticancer agent of the present invention can be appropriately selected, for example, injection, local infusion, intraperitoneal administration, selective intravenous infusion, intravenous injection, subcutaneous injection, organ perfusion fluid infusion, etc. can also be used. In addition, solutions for injection can be formulated using carriers composed of saline solution, glucose solution, or a mixture of saline and glucose solution, various buffer solutions, etc. Alternatively, the agent can be formulated in a powdered state and mixed with the above-mentioned liquid carrier to prepare an injection solution at the time of use.

[0679] Other methods of administration can also be appropriately selected along with formulation development. For example, for oral administration, oral liquids, powders, pills, capsules, and tablets can be used. For oral liquids, suspensions and syrups can be formulated using the following: water; sugars such as sucrose, sorbitol, and fructose; glycols such as PEG; oils such as sesame oil and soybean oil; preservatives such as alkyl parabens; and flavorings such as strawberry and mint. Powders, pills, capsules, and tablets can be formulated using the following: 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 the preferred unit administration forms of the compositions of the present invention due to their ease of administration. Tablets and capsules are manufactured using a solid carrier.

[0680] The effective amount of an anti-HLA / NY-ESO antibody or multispecific molecule that binds to HLA / NY-ESO for treatment varies depending on the nature of the condition being treated, the age, and condition of the patient, and is ultimately determined by the physician. For example, a single dose ranges from 0.0001 mg to 100 mg per kg of body weight. The prescribed dosage may be administered once every 1 to 180 days, or divided into two, three, four, or more daily doses administered at appropriate intervals.

[0681] The present invention also encompasses polynucleotides encoding the amino acid sequences contained in the anti-HLA / NY-ESO antibodies or multispecific molecules that bind to HLA / NY-ESO of the present invention, vectors containing the polynucleotides, cells containing the polynucleotides or vectors, and pharmaceutical compositions containing any of the polynucleotides, vectors, and cells as active ingredients. The pharmaceutical compositions are preferably anticancer agents.

[0682] In addition, the pharmaceutical composition of the present invention can be used in combination with other pharmaceutical agents. Other pharmaceutical agents include chemotherapeutic agents, radiotherapy agents, biopharmaceuticals, etc., without particular limitation, and can be administered or applied simultaneously with or at different times as the pharmaceutical composition of the present invention, and can be administered or applied as a single formulation comprising the pharmaceutical composition of the present invention or as two or more different formulations or therapies.

[0683] [Example]

[0684] The present invention will be described in more detail below in the examples, but the present invention is not limited to these examples.

[0685] Unless otherwise specified, the genetic manipulation procedures in the following examples were performed according to the methods described in "Molecular Cloning" (Sambrook, J., Fritsch, EF, and Maniatis, T., Cold Spring Harbor Laboratory Press, 1989) and other experimental manuals used by those skilled in the art. Alternatively, when commercially available reagents and kits were used, the procedures were performed according to the instructions provided. Primer synthesis required for gene synthesis and vector construction was contracted to FASMAC Co., Ltd., Thermo Fisher Scientific, and Eurofins Genomics, as needed.

[0686] (Example 1) Obtaining anti-HLA / NY-ESO antibodies from a human antibody phage library

[0687] 1) Preparation of HLA / NY-ESO antigen protein

[0688] Inclusion bodies prepared from Escherichia coli (BL21(DE3), Agilent Technologies) expressing a truncated form of HLA-A*0201 (GenBank: ASA47534.1) (with a biotin ligase recognition sequence added, SEQ ID NO: 33) and β2-microglobulin (UniProtKB-P61769, SEQ ID NO: 34), respectively, and the NY-ESO peptide: SLLMWITQC (SEQ ID NO: 1 in the sequence listing) were refolded by significant dilution. The resulting mixture was then passed through a gel filtration column (Superdex 200 10 / 300, GE Healthcare) to prepare an HLA-A*0201 / β2-microglobulin / NY-ESO peptide complex (hereinafter referred to as HLA / NY-ESO).

[0689] As a negative control antigen, the MAGEC-1 peptide ILFGISLREV (SEQ ID NO: 2 in the sequence listing) was refolded in the same manner to prepare an HLA-A*0201 / β2-microglobulin / MAGEC-1 peptide complex (hereinafter referred to as HLA / MC1). Subsequently, the prepared HLA / NY-ESO and HLA / MC1 were biotinylated using Escherichia coli biotin ligase and separated by gel filtration column (Superdex200 10 / 300, GE Healthcare) to prepare the biotinylated proteins.

[0690] 1)-2 Isolation of scFv with HLA / NY-ESO Binding Ability

[0691] scFvs binding to HLA / NY-ESO were isolated from a human antibody phage library. First, phage were added to Dynabeads Streptavidin M-280 (Thermo Fisher Scientific) immobilized with biotinylated HLA / MC1, and unbound phage were recovered. Next, Dynabeads Streptavidin M-280Ag immobilized with biotinylated HLA / NY-ESO were added, and unbound phage were removed by washing using a magnetic stand (DynaMag-2, Thermo Fisher Scientific).

[0692] Phage that bind to HLA / NY-ESO were then infected with Escherichia coli (XL-1 Blue, Agilent Technologies), and HLA / NY-ESO-binding phage were recovered and amplified. After three rounds of screening, the polyclonal phagemid was transferred to an E. coli expression vector containing a FLAG tag and a His tag at the carboxyl terminus of the scFv. The scFv was then transformed into E. coli and expressed in the presence of IPTG (Isopropyl-β-D-thiogalactopyranoside) (Sigma-Aldrich) for ELISA screening.

[0693] 1)-3 Screening of HLA / NY-ESO binding scFv by ELISA

[0694] To each well of a 384-well Maxi-Sorp plate (Black, Nunc), 50 μL of NeutrAvidin (Life Technologies) diluted to 1 μg / mL in PBS (0.01M phosphate-buffered saline (pH 7.4) containing 0.138M sodium chloride and 0.0027M potassium chloride, Sigma-Aldrich) was added and immobilized overnight at 4°C. After washing three times with PBS (ELISA buffer) containing 0.05% Tween-20 (Bio-Rad), the plate was diluted to 1 μg / mL in PBS, and the biotinylated HLA / NY-ESO used in Example 1)-2 was added. The plate was shaken at room temperature for 1 hour. After washing three times with ELISA buffer, the plate was blocked with Blocker Casein (Thermo Fisher Scientific) and washed three times with ELISA buffer. A culture of E. coli expressing scFv was then added and reacted at room temperature for 2 hours. After washing three times with ELISA buffer, 50 μL of horseradish peroxidase (HRP)-labeled anti-FLAG antibody (Sigma-Aldrich) diluted 5000-fold with ELISA buffer was added and 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 was measured after 10 minutes using a plate reader (Envision 2104 Multilabel Reader, PerkinElmer) to isolate ELISA-positive clones binding to HLA / NY-ESO.

[0695] 1) Determination of the nucleotide and amino acid sequences of the 4-ELISA-positive clone NY-R119

[0696] From the ELISA-positive clones obtained in 1)-3, NY-R119 was selected as an scFv with strong HLA / NY-ESO binding and excellent recognition specificity. The nucleotide sequences of the heavy and light chain variable regions of NY-R119 were analyzed using the DyeTerminator method (BigDye (registered trademark) Terminator v3.1, Thermo Fisher Scientific). The primer sequences used for sequence analysis are shown below.

[0697] Primer A: 5'-CTCTTCGCTATTACGCCAGCTGGCGA-3' (SEQ ID NO: 3 in the sequence listing) Figure 10 ))

[0698] Primer B: 5'-ATAACAATTTCACACAGGAAACAGCTATGA-3' (SEQ ID NO: 4 in the sequence listing) Figure 11 ))

[0699] The nucleotide sequence of the cDNA encoding the heavy chain variable region of NY-R119 is shown in SEQ ID NO: 5 ( Figure 12 ), and the amino acid sequence is shown in SEQ ID NO: 6 ( Figure 13 ).

[0700] The nucleotide sequence of the cDNA encoding the light chain variable region of NY-R119 is shown in SEQ ID NO: 7 ( Figure 14 ), and the amino acid sequence is shown in SEQ ID NO: 8 ( Figure 15 ).

[0701] The CDR sequences of NY-R119 in the CDR definition of IMGT are as follows: CDRH1 is shown in SEQ ID NO: 54 ( Figure 61 ), CDRH2 is shown in SEQ ID NO: 55 ( Figure 61 ), CDRH3 is shown in SEQ ID NO: 56 ( Figure 61 ), CDRL1 is shown in SEQ ID NO: 57 ( Figure 61 ), CDRL2 is shown in SEQ ID NO: 58 ( Figure 61 ), CDRL3 is shown in SEQ ID NO: 59 ( Figure 61 ).

[0702] 1)-5NYA-0001 production

[0703] 1) Construction of 5-1NYA-0001 expression vector

[0704] To prepare various evaluation samples, an expression vector for NY-R119 was constructed for mammalian cell culture. NY-R119 expressed in mammalian cells was designated NYA-0001. The scFv portion, heavy and light chain variable regions, and CDRH1-3 and CDRL1-3 of NY-R119 and NYA-0001 share the same amino acid sequence. Using the In-Fusion HD Cloning Kit (CLONTECH), a DNA fragment encoding NYA-0001 was inserted into a mammalian cell expression vector based on the pcDNA3.3 (Thermo Fisher Scientific) backbone to construct the NYA-0001 expression vector.

[0705] The nucleotide sequence of the constructed NYA-0001 expression vector was reanalyzed, and the full-length nucleotide sequence of NYA-0001 was confirmed to be SEQ ID NO: 69 ( Figure 70 ) shown in the nucleotide sequence. In addition, in the above nucleotide sequence, the NYA-0001 full-length amino acid sequence encoded by the sequence is SEQ ID NO: 70 ( Figure 71 ). In this sequence, the amino acid sequence from positions 1 to 19 is a signal sequence, the amino acid sequence from positions 21 to 266 is NYA-0001, and the amino acid sequence from positions 267 to 292 is a Flag-His tag. In addition, the amino acid sequence from positions 46 to 53 is CDRH1, the amino acid sequence from positions 71 to 78 is CDRH2, and the amino acid sequence from positions 117 to 129 is CDRH3. In addition, the amino acid sequence from positions 181 to 188 is CDRL1, the amino acid sequence from positions 206 to 208 is CDRL2, and the amino acid sequence from positions 245 to 256 is CDRL3.

[0706] 1) Expression and purification of 5-2NYA-0001

[0707] Expi293F cells (Thermo Fisher Scientific) were subcultured according to the manual. The culture medium of Expi293F cells in the logarithmic growth phase was diluted to 2.5×10 6 cells / mL for the production of NYA-0001. To 20 mL of Opti-Pro SFM medium (Thermo Fisher Scientific), 0.3 mg of the NYA-0001 expression vector and 0.9 mg of polyethyleneimine (Polyscience #24765) were added and stirred gently. The mixture was allowed to stand for 5 minutes before being added to Expi293F cells. The cells were cultured in a 37°C, 8% CO2 incubator at 135 rpm for 6 days. The resulting culture supernatant 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 the supernatant, and purifying the supernatant by passing it through a gel filtration column (Superdex 200 Increase, GE Healthcare) equilibrated with 25 mM histidine, 300 mM NaCl, 5% sorbitol, and pH 6.0. Purified protein samples were analyzed by size exclusion chromatography (SEC) and used for various evaluations after the purity and concentration were determined.

[0708] (Example 2) Preparation of NYA-0001 mutant

[0709] 2) Obtaining 1NYA-0001 mutants

[0710] Using the NYA-0001 gene as a template, a phage library was constructed using a method for introducing mutations by PCR (error-prone-based library), or a method for constructing a library by synthesizing oligomers with 20 random amino acid mutations for each residue in all CDRs (oligo-based library). Screening for clones with high binding ability was performed, and NYA-0060, NYA-0068, and NYA-0082 were obtained as high-binding mutants, and the nucleotide sequences of each were determined.

[0711] The nucleotide sequence of the obtained cDNA encoding the heavy chain variable region of NYA-0060 is shown in SEQ ID NO: 9 ( Figure 16 ), and the amino acid sequence is shown in SEQ ID NO: 10 ( Figure 17 ).

[0712] The nucleotide sequence of the obtained cDNA encoding the light chain variable region of NYA-0060 is shown in SEQ ID NO: 11 ( Figure 18 ), and the amino acid sequence is shown in SEQ ID NO: 12 ( Figure 19 ).

[0713] The nucleotide sequence of the obtained cDNA encoding the heavy chain variable region of NYA-0068 is shown in SEQ ID NO: 13 ( Figure 20 ), and the amino acid sequence is shown in SEQ ID NO: 14 ( Figure 21 ).

[0714] The nucleotide sequence of the obtained cDNA encoding the light chain variable region of NYA-0068 is shown in SEQ ID NO: 15 ( Figure 22 ), and the amino acid sequence is shown in SEQ ID NO: 16 ( Figure 23 ).

[0715] The nucleotide sequence of the obtained cDNA encoding the heavy chain variable region of NYA-0082 is shown in SEQ ID NO: 17 ( Figure 24 ), and the amino acid sequence is shown in SEQ ID NO: 18 ( Figure 25 ).

[0716] The nucleotide sequence of the obtained cDNA encoding the light chain variable region of NYA-0082 is shown in SEQ ID NO: 19 ( Figure 26 ), and the amino acid sequence is shown in SEQ ID NO: 20 ( Figure 27 ).

[0717] 2)-2 Using the mutation sites and combinations identified from NYA-0060, NYA-0068, and NYA-0082, we created mutants with enhanced binding capacity

[0718] Using the In-Fusion HD cloning kit (CLONTECH), DNA fragments encoding NYA-1163 and NYA-2023 with the respective mutation sites of NYA-0060 and NYA-0068 were inserted into a mammalian cell expression vector with pcDNA3.3 (Thermo Fisher Scientific) as the backbone to construct a mammalian cell scFv expression vector.

[0719] In addition, NYA-2027, NYA-1143, and NYA-2143 were designed based on the mutation sites identified in NYA-0060, NYA-0068, and NYA-0082, and their combinations. Each mammalian cell-based scFv expression vector was constructed by introducing site-specific mutations into the NYA-0001 expression vector constructed in 1)-5, or by inserting a DNA fragment encoding the desired scFv into a mammalian cell-based expression vector using the pcDNA3.3 (Thermo Fisher Scientific) backbone using the In-Fusion HD Cloning Kit (CLONTECH).

[0720] The nucleotide sequence of the constructed scFv expression vector was reanalyzed, and the full-length nucleotide sequence of NYA-1163 was confirmed to be SEQ ID NO: 21 ( Figure 28 ) shown in the nucleotide sequence.

[0721] The full-length nucleotide sequence of NYA-2023 was confirmed to be SEQ ID NO: 22 ( Figure 29 ) shown in the nucleotide sequence.

[0722] The full-length nucleotide sequence of NYA-2027 was confirmed to be SEQ ID NO: 23 ( Figure 30 ) shown in the nucleotide sequence.

[0723] The full-length nucleotide sequence of NYA-1143 was confirmed to be SEQ ID NO: 24 ( Figure 31 ) shown in the nucleotide sequence.

[0724] The full-length nucleotide sequence of NYA-2143 was confirmed to be SEQ ID NO: 25 ( Figure 32 ) shown in the nucleotide sequence.

[0725] In addition, the full-length amino acid sequences of NYA-1163, NYA-2023, NYA-2027, NYA-1143, and NYA-2143 encoded by the nucleotide sequences were confirmed.

[0726] The full-length amino acid sequence of NYA-1163 is SEQ ID NO: 26 ( Figure 33 ). In this sequence, the amino acid sequence from positions 1 to 19 is a signal sequence, the amino acid sequence from positions 21 to 266 is NYA-1163, and the amino acid sequence from positions 267 to 292 is a Flag-His tag. In addition, the amino acid sequence from positions 46 to 53 is CDRH1, the amino acid sequence from positions 71 to 78 is CDRH2, and the amino acid sequence from positions 117 to 129 is CDRH3. In addition, 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.

[0727] The full-length amino acid sequence of NYA-2023 is SEQ ID NO: 27 ( Figure 34 ) is an amino acid sequence shown in FIG. In this sequence, the amino acid sequence from positions 1 to 19 is a signal sequence, the amino acid sequence from positions 21 to 266 is NYA-2023, and the amino acid sequence from positions 267 to 292 is a Flag-His tag. In addition, the amino acid sequence from positions 46 to 53 is CDRH1, the amino acid sequence from positions 71 to 78 is CDRH2, and the amino acid sequence from positions 117 to 129 is CDRH3. In addition, 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.

[0728] The full-length amino acid sequence of NYA-2027 is SEQ ID NO: 28 ( Figure 35 ). In this sequence, the amino acid sequence from positions 1 to 19 is a signal sequence, the amino acid sequence from positions 21 to 266 is NYA-2027, and the amino acid sequence from positions 267 to 292 is a Flag-His tag. In addition, the amino acid sequence from positions 46 to 53 is CDRH1, the amino acid sequence from positions 71 to 78 is CDRH2, and the amino acid sequence from positions 117 to 129 is CDRH3. In addition, 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.

[0729] The full-length amino acid sequence of NYA-1143 is SEQ ID NO: 29 ( Figure 36 ). In this sequence, the amino acid sequence from positions 1 to 19 is a signal sequence, the amino acid sequence from positions 21 to 266 is NYA-1143, and the amino acid sequence from positions 267 to 292 is a Flag-His tag. In addition, the amino acid sequence from positions 46 to 53 is CDRH1, the amino acid sequence from positions 71 to 78 is CDRH2, and the amino acid sequence from positions 117 to 129 is CDRH3. In addition, 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.

[0730] The full-length amino acid sequence of NYA-2143 is SEQ ID NO: 30 ( Figure 37 ). In this sequence, the amino acid sequence from positions 1 to 19 is a signal sequence, the amino acid sequence from positions 21 to 266 is NYA-2143, and the amino acid sequence from positions 267 to 292 is a Flag-His tag. In addition, the amino acid sequence from positions 46 to 53 is CDRH1, the amino acid sequence from positions 71 to 78 is CDRH2, and the amino acid sequence from positions 117 to 129 is CDRH3. In addition, 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.

[0731] It should be noted that the CDR sequences of NYA-1163 are identical to those of NYA-0001. In addition, the CDR sequences of NYA-1143, NYA-2143, and NYA-2023 are identical, and CDRH1 is shown in SEQ ID NO: 54 ( Figure 61 ), CDRH2 is shown in SEQ ID NO: 55 ( Figure 61 ), CDRH3 is shown in SEQ ID NO: 56 ( Figure 61 ), CDRL1 is shown in SEQ ID NO: 60 ( Figure 62 ), CDRL2 is shown in SEQ ID NO: 58 ( Figure 61 ), CDRL3 is shown in SEQ ID NO: 59 ( Figure 61 ). In addition, regarding the CDR sequence of NYA-2027, CDRH1 is shown in SEQ ID NO: 54 ( Figure 61 ), CDRH2 is shown in SEQ ID NO: 55 ( Figure 61 ), CDRH3 is shown in SEQ ID NO: 56 ( Figure 61), CDRL1 is shown in SEQ ID NO: 57 ( Figure 61 ), CDRL2 is shown in SEQ ID NO: 58 ( Figure 61 ), CDRL3 is shown in SEQ ID NO: 61 ( Figure 63 ).

[0732] In addition, NYA-1154 was designed and combined with the binding mutation sites found during the screening of high-binding clones. Site-specific mutations were introduced into the NYA-0001 expression vector to construct the NYA-1154 expression vector using pcDNA3.3 (Thermo Fisher Scientific) as the backbone. The nucleotide sequence of the constructed scFv expression vector was reanalyzed and confirmed to be SEQ ID NO: 31 ( Figure 38 ) is shown in the nucleotide sequence. In the above nucleotide sequence, the full-length amino acid sequence of NYA-1154 encoded by the sequence was confirmed (SEQ ID NO: 32 ( Figure 39 )). In this sequence, the amino acid sequence from positions 1 to 19 is a signal sequence, the amino acid sequence from positions 21 to 266 is NYA-1154, and the amino acid sequence from positions 267 to 292 is a Flag-His tag. In addition, the amino acid sequence from positions 46 to 53 is CDRH1, the amino acid sequence from positions 71 to 78 is CDRH2, and the amino acid sequence from positions 117 to 129 is CDRH3. In addition, 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.

[0733] The CDR sequences of NYA-1154 are as follows: CDRH1 is shown in SEQ ID NO: 54 ( Figure 61 ), CDRH2 is shown in SEQ ID NO: 55 ( Figure 61 ), CDRH3 is shown in SEQ ID NO: 62 ( Figure 64 ), CDRL1 is shown in SEQ ID NO: 57 ( Figure 61 ), CDRL2 is shown in SEQ ID NO: 58 ( Figure 61 ), CDRL3 is shown in SEQ ID NO: 63 ( Figure 64 ).

[0734] NYA-1163, NYA-2023, NYA-2027, NYA-1143, NYA-2143, and NYA-1154 were expressed and purified using the same method as in 1)-5-2. Purified protein samples were analyzed by SEC, and after purity and concentration were determined, they were used in various assays.

[0735] 2)-3 Production of NYA-1143 mutants

[0736] 2)-3-1 Production of NYA-2035

[0737] As a mutant of NYA-1143, NYA-2035 was designed, and a site-specific mutation was introduced into the NYA-1143 expression vector in mammalian cells to construct the NYA-2035 expression vector. The nucleotide sequence of the constructed scFv expression vector was reanalyzed and confirmed to be SEQ ID NO: 35 ( Figure 42 ) shown in the nucleotide sequence. In the above nucleotide sequence, the full-length amino acid sequence of NYA-2035 encoded by the sequence was determined (SEQ ID NO: 36 ( Figure 43 )). In this sequence, the amino acid sequence at positions 1 to 19 is a signal sequence, the amino acid sequence at positions 21 to 266 is NYA-2035, and the amino acid sequence at positions 267 to 292 is a Flag-His tag. In addition, the amino acid sequence at positions 46 to 53 is CDRH1, the amino acid sequence at positions 71 to 78 is CDRH2, and the amino acid sequence at positions 117 to 129 is CDRH3. In addition, 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 CDR definition of IMGT are: CDRH1 is shown in SEQ ID NO: 54 ( Figure 61 ), CDRH2 is shown in SEQ ID NO: 55 ( Figure 61 ), CDRH3 is shown in SEQ ID NO: 56 ( Figure 61 ), CDRL1 is shown in SEQ ID NO: 64 ( Figure 65 ), CDRL2 is shown in SEQ ID NO: 58 ( Figure 61 ), CDRL3 is shown in SEQ ID NO: 59 ( Figure 61 ).

[0738] 2) Preparation of 3-2NYA-1143 CDR grafted mutants

[0739] To improve physicochemical properties, NYA-1143 CDR grafted mutants were designed. The sequences of the framework regions of NYA-1143's VH and VL were compared with the human subgroup consensus sequences and framework regions of germline sequences given 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, for the VH, the human germline sequence IGHV3_30*15 and the consensus sequence of human gamma chain subgroup 3 were selected as receptors with high sequence identity in their framework regions; for the VL, the human germline sequence IGLV1-44*01 was selected as a receptor with high sequence identity in its framework regions. Next, the amino acid sequences of the framework regions of each receptor were aligned with the amino acid sequence of NYA-1143, and residues with different amino acids were identified.

[0740] Next, using the three-dimensional model of NYA-1143 and referring to the criteria given by Queen et al. (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)), framework residues to be transferred to the receptor were selected. Using the above method, NYA-1143-VH01, NYA-1143-VH02, and NYA-1143-VH03 were designed as CDR grafted mutant amino acid sequences of the VH portion of NYA-1143. The relevant amino acid sequences are SEQ ID NO: 37 ( Figure 44 )、SEQ ID NO:38( Figure 45 )、SEQ ID NO:39( Figure 46 ). NYA-1143-VL01 was designed as the CDR grafted mutant amino acid sequence of the VL portion of NYA-1143. Each of the amino acid sequences is SEQ ID NO: 40 ( Figure 47 ).

[0741] The various scFvs listed below were designed by combining the designed VH and VL sequences. The scFv in which the VH portion of NYA-1143 was replaced with the amino acid sequence of NYA-1143-VH01 was named NYA-2044. Furthermore, the scFv in which the VL portion of NYA-2044 was replaced with the amino acid sequence of NYA-1143-VL01 was named NYA-2045.

[0742] The scFv in which the VH portion of NYA-1143 was replaced with the amino acid sequence of NYA-1143-VH02 was named NYA-2047, and the scFv in which the VL portion of NYA-2047 was replaced with the amino acid sequence of NYA-1143-VL01 was named NYA-2048.

[0743] The scFv in which the VH portion of NYA-1143 was replaced with the amino acid sequence of NYA-1143-VH03 was named NYA-2060, and the scFv in which the VL portion of NYA-2060 was replaced with the amino acid sequence of NYA-1143-VL01 was named NYA-2061.

[0744] Regarding the various NYA-1143 CDR grafted mutants designed, DNA fragments were fully synthesized (FASMAC Co., Ltd.) and combined using the In-Fusion HD Cloning Kit (CLONTECH), thereby constructing a mammalian cell scFv expression vector with pcDNA3.3 (Thermo Fisher Scientific) as the backbone.

[0745] The nucleotide sequence of the constructed scFv expression vector was reanalyzed, and the full-length nucleotide sequence of NYA-2044 was confirmed to be SEQ ID NO: 41 ( Figure 48 ) shown in the nucleotide sequence.

[0746] The full-length nucleotide sequence of NYA-2045 was confirmed to be SEQ ID NO: 42 ( Figure 49 ) shown in the nucleotide sequence.

[0747] The full-length nucleotide sequence of NYA-2047 was confirmed to be SEQ ID NO: 43 ( Figure 50 ) shown in the nucleotide sequence.

[0748] The full-length nucleotide sequence of NYA-2048 was confirmed to be SEQ ID NO: 44 ( Figure 51 ) shown in the nucleotide sequence.

[0749] The full-length nucleotide sequence of NYA-2060 was confirmed to be SEQ ID NO: 45 ( Figure 52 ) shown in the nucleotide sequence.

[0750] The full-length nucleotide sequence of NYA-2061 was confirmed to be SEQ ID NO: 46 ( Figure 53 ) shown in the nucleotide sequence.

[0751] Furthermore, the full-length amino acid sequences of NYA-2044, NYA-2045, NYA-2047, NYA-2048, NYA-2060, and NYA-2061 encoded by the aforementioned nucleotide sequences were determined. It should be noted that the CDR sequences of NYA-2044, NYA-2045, NYA-2047, NYA-2048, NYA-2060, and NYA-2061 are identical to those of NYA-1143.

[0752] The full-length amino acid sequence of NYA-2044 is SEQ ID NO: 47 ( Figure 54 ). In this sequence, the amino acid sequence from positions 1 to 19 is a signal sequence, the amino acid sequence from positions 21 to 266 is NYA-2044, and the amino acid sequence from positions 267 to 292 is a Flag-His tag. In addition, the amino acid sequence from positions 46 to 53 is CDRH1, the amino acid sequence from positions 71 to 78 is CDRH2, and the amino acid sequence from positions 117 to 129 is CDRH3. In addition, 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.

[0753] The full-length amino acid sequence of NYA-2045 is SEQ ID NO: 48 ( Figure 55 ). In this sequence, the amino acid sequence from positions 1 to 19 is a signal sequence, the amino acid sequence from positions 21 to 266 is NYA-2045, and the amino acid sequence from positions 267 to 292 is a Flag-His tag. In addition, the amino acid sequence from positions 46 to 53 is CDRH1, the amino acid sequence from positions 71 to 78 is CDRH2, and the amino acid sequence from positions 117 to 129 is CDRH3. In addition, 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.

[0754] The full-length amino acid sequence of NYA-2047 is SEQ ID NO: 50 ( Figure 57). In this sequence, the amino acid sequence from positions 1 to 19 is a signal sequence, the amino acid sequence from positions 21 to 266 is NYA-2047, and the amino acid sequence from positions 267 to 292 is a Flag-His tag. In addition, the amino acid sequence from positions 46 to 53 is CDRH1, the amino acid sequence from positions 71 to 78 is CDRH2, and the amino acid sequence from positions 117 to 129 is CDRH3. In addition, 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.

[0755] The full-length amino acid sequence of NYA-2048 is SEQ ID NO: 51 ( Figure 58 ). In this sequence, the amino acid sequence from positions 1 to 19 is a signal sequence, the amino acid sequence from positions 21 to 266 is NYA-2048, and the amino acid sequence from positions 267 to 292 is a Flag-His tag. In addition, the amino acid sequence from positions 46 to 53 is CDRH1, the amino acid sequence from positions 71 to 78 is CDRH2, and the amino acid sequence from positions 117 to 129 is CDRH3. In addition, 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.

[0756] The full-length amino acid sequence of NYA-2060 is SEQ ID NO: 52 ( Figure 59 ). In this sequence, the amino acid sequence from positions 1 to 19 is a signal sequence, the amino acid sequence from positions 21 to 266 is NYA-2060, and the amino acid sequence from positions 267 to 292 is a Flag-His tag. In addition, the amino acid sequence from positions 46 to 53 is CDRH1, the amino acid sequence from positions 71 to 78 is CDRH2, and the amino acid sequence from positions 117 to 129 is CDRH3. In addition, 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.

[0757] The full-length amino acid sequence of NYA-2061 is SEQ ID NO: 53 ( Figure 60). In this sequence, the amino acid sequence from positions 1 to 19 is a signal sequence, the amino acid sequence from positions 21 to 266 is NYA-2061, and the amino acid sequence from positions 267 to 292 is a Flag-His tag. In addition, the amino acid sequence from positions 46 to 53 is CDRH1, the amino acid sequence from positions 71 to 78 is CDRH2, and the amino acid sequence from positions 117 to 129 is CDRH3. In addition, 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.

[0758] 2) Expression and purification of 3-3NYA-1143 mutants

[0759] 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 and purified using the same method as in 1)-5-2. Purified protein samples were used for analytical SEC, and after purity and concentration were determined, they were used in various assays.

[0760] (Example 3) Preparation of scFv of anti-HLA / NY-ESO reference antibody

[0761] scFvs of antibodies 3M4E5 and T1 (WO2010 / 106431) with high binding ability to HLA / NY-ESO were designed. The scFv of 3M4E5 was named NYC-0003, and the scFv of T1 was named NYC-0004.

[0762] The DNA fragments of NYC-0003 and NYC-0004 were fully synthesized (Thermo Fisher Scientific), and a scFv expression vector for mammalian cells using pcDNA3.3 (Thermo Fisher Scientific) as the backbone was constructed using the In-Fusion HD Cloning Kit (CLONTECH).

[0763] The nucleotide sequence of the constructed scFv expression vector was reanalyzed, and it was confirmed that the full-length nucleotide sequence of NYC-0003 and NYC-0004 was SEQ ID NO: 65 ( Figure 66 ) and SEQ ID NO: 66 ( Figure 67 ) shown in the nucleotide sequence. In addition, in the above nucleotide sequence, the full-length amino acid sequence of NYC-0003 encoded by the sequence is SEQ ID NO: 67 ( Figure 68) and the full-length amino acid sequence of NYC-0004 is SEQ ID NO: 68 ( Figure 69 ) shown in the amino acid sequence.

[0764] NYC-0003 and NYC-0004 were expressed and purified using the same method as in 1)-5-2. Each purified protein sample was used for analytical SEC, and after determining purity and concentration, it was used in various assays.

[0765] (Example 4) Evaluation of HLA / NY-ESO Binding Ability Using Biacore

[0766] Using Biacore T200, anti-HLA / NY-ESO scFv was captured (captured) as a ligand to an immobilized anti-His antibody, and the antigen was measured as the analyte. The antigen used was HLA / NY-ESO prepared in 1)-1. According to the kit instructions, the anti-His antibody (His capture kit, GE Healthcare) was immobilized on the sensor chip CM5 (GE Healthcare). Each anti-HLA / NY-ESO scFv evaluated, diluted to 0.5 μg / mL with HBS-EP+ (GE Healthcare), was contacted at 10 μL / min for 60 seconds and immobilized. Then, HLA / NY-ESO of various concentrations diluted with HBS-EP+ was used as the analyte, and each sample was added at a flow rate of 30 μL / min for 120 seconds, and K was calculated by single-cycle kinetic analysis measured at intervals of 600 seconds. D The results are recorded in Table 1. The results show that compared with the parent antibody NYA-0001, all its mutants bind strongly to HLA / NY-ESO. In addition, compared with NYC-0004, NYA-1143, NYA-2023, NYA-2143, NYA-2044, NYA-2045, NYA-2060 and NYA-2061 bind more strongly, among which NYA-1143, NYA-2044, NYA-2045, NYA-2143 have a K D The concentration was shown to be below 1 nM.

[0767] [Table 1]

[0768] Clone Name <![CDATA[K D (nM)]]> NYA-0001 33.8 NYA-1143 1.0 NYA-1163 13.0 NYA-2023 2.4 NYA-2027 11.2 NYA-2035 3.2 NYA-2044 0.8 NYA-2045 0.8 NYA-2047 5.3 NYA-2048 6.3 NYA-2060 1.1 NYA-2061 1.5 NYA-2143 0.9 NYC-0003 8.1 NYC-0004 2.8

[0769] (Example 5) Analysis of Recognition Amino Acids in the NY-ESO Peptide of Anti-HLA / NY-ESO scFv

[0770] Human lymphoblastoid fusion cell line T2 (ATCC) cells were adjusted to an appropriate concentration using AIM-V medium (Thermo Fisher Scientific) containing 20% ​​FBS, and NY-ESO peptide (SEQ ID NO: 1), point mutant NY-ESO peptides 1F, 2M, 3A, 4A, 5A, 6L, 7F, 8A, 9A (SEQ ID NO: 121 ( Figure 122 )、SEQ ID NO:122( Figure 123 )、SEQ ID NO:123( Figure 124 )、SEQ ID NO:124( Figure 125 )、SEQ ID NO:125( Figure 126 )、SEQ ID NO:126( Figure 127 )、SEQ ID NO:127( Figure 128 )、SEQ ID NO:128( Figure 129 )、SEQ ID NO:129( Figure 130 ))、gp100 peptide (SEQ ID NO:130( Figure 131 ) (all from Sigma Genosys) were dissolved in DMSO to a 5mM solution to a final concentration of 50μM; or 1 / 100 of DMSO was added and incubated at 37°C for 4 hours. After washing twice with AIM-V medium containing 20% ​​FBS, the solution was prepared to an appropriate concentration with PBS containing 5% FBS, and the LIVE / DEAD fixable dead cell staining kit (Thermo Fisher Scientific) was added and allowed to stand at 4°C for 30 minutes. After washing twice with PBS containing 5% FBS, the cells were divided into two groups and fixed with PBS containing 5% FBS at 10 5Cells / well were seeded into a 96-well U-bottom microplate, and the supernatant was removed after centrifugation. For one group of cells, anti-HLA / NY-ESO scFv diluted to 100 nM in PBS containing 5% FBS was added at 25 μL / well and allowed to stand at 4°C for 30 minutes. After washing twice with PBS containing 5% FBS, Penta-His Alexa Fluor 488 (QIAGEN) diluted in PBS containing 5% FBS was added at 25 μL / well and allowed to stand at 4°C for 30 minutes. After washing twice with PBS containing 5% FBS, anti-mouse IgG Alexa Fluor 488 (Thermo Fisher Scientific) diluted in PBS containing 5% FBS was added at 25 μL / well and allowed to stand at 4°C for 30 minutes. After washing twice with PBS containing 5% FBS, the cells were fixed overnight with Mildform 10N (Fujifilm Wako Pure Chemical Industries, Ltd.) and resuspended in PBS containing 5% FBS. To normalize the amount of HLA / peptide complexes, 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 were added to another set of cells and incubated at 4°C for 30 minutes. After washing twice with PBS containing 5% FBS, the cells were fixed overnight with Mildform 10N (Fujifilm Wako Pure Chemical Industries, Ltd.) and resuspended in PBS containing 5% FBS. These cell suspensions were analyzed using a flow cytometer (Canto II, Becton Dickinson). Data analysis was performed using FlowJo (Treestar), and the geometric mean fluorescence intensity (gMFI) of Alexa Fluor 488 in the dead cell-depleted fraction of T2 cells was measured. The normalized gMFI, representing the binding activity of each scFv, was calculated using the following formula, normalized to the amount of HLA / peptide complex on T2 cells.

[0771] Normalized gMFI = (A / B) / ((C / D) / (E / F))

[0772] A / B = relative gMFI

[0773] A: gMFI of T2 cells supplemented with DMSO or each peptide

[0774] B: gMFI of T2 cells in DMSO without antibody or with each peptide

[0775] (C / D) / (E / F) = Corrected value of the amount of HLA / peptide complex in T2 cells supplemented with DMSO or each peptide

[0776] C: gMFI of T2 cells supplemented with DMSO containing HLA-A2 antibody or with each peptide

[0777] D: gMFI of T2 cells supplemented with DMSO containing mouse IgG2b antibody or with each peptide

[0778] E: gMFI of T2 cells supplemented with DMSO containing HLA-A2 antibody

[0779] F: gMFI of T2 cells supplemented with DMSO containing mouse IgG2b antibody

[0780] like Figure 1 As shown, the binding of anti-HLA / NY-ESO scFvs NYA-0001, 1143, 2044, 2045, 2047, 2048, 2060, and 2061 to T2, a peptide with point mutations introduced at amino acids 1, 4, 5, and 7, was reduced to less than half compared to the wild-type NY-ESO peptide, indicating that they recognized amino acids 1, 4, 5, and 7 of the NY-ESO peptide. Similarly, NYA-1154 recognized amino acids 1 and 5, NYA-1163 recognized amino acids 1, 3, 4, 5, 6, and 7, NYA-2023, 2027, and 2035 recognized amino acids 1, 4, and 5, and NYA-2143 recognized amino acids 1, 5, and 7. Furthermore, NYC-0003 and 0004 also recognized amino acids 4 and 5.

[0781] (Example 6) Evaluation of Antigen Binding Specificity of Anti-HLA / NY-ESO scFv

[0782] In order to search for human peptides (hereinafter referred to as "homologous peptides") that are similar but not identical to the NY-ESO peptide: SLLMWITQC (SEQ ID NO: 1) in the human proteome (Swiss-Prot), we searched for 9-mer peptides that were identical at positions 1, 4, and 5, which are recognized by many antibodies of the present invention. The binding properties of the retrieved 9-mer peptides to HLA-A0201 were predicted using NetMHCPan2.8, and those with a predicted IC50 of 500 nM or less were selected. Figure 2A The 9-mer peptide shown was used as a homologous peptide for evaluating the binding specificity of anti-HLA / NY-ESO scFv. T2 cells were adjusted to an appropriate concentration using AIM-V medium (Thermo Fisher Scientific) containing 20% ​​FBS, and the NY-ESO peptide (SEQ ID NO: 1) and homologous peptides DOLPP1, IL20RB, PRKD2, CD163, and P2RY8 (SEQ ID NO: 131 ( Figure 132 )、SEQ ID NO:132( Figure 133 )、SEQ ID NO:133( Figure 134 )、SEQ ID NO:134( Figure 135 )、SEQ ID NO:135( Figure 136 ))、gp100 peptide (SEQ ID NO: 130 ( Figure 131 ) (all from Sigma Genosys) were dissolved in DMSO to 5 mM, and the final concentration of the solution was adjusted to 50 μM. Alternatively, 1 / 100 of the amount of DMSO was added to the solution, and the binding of each antibody was evaluated by the same method as in Example 5. The normalized gMFI, representing the binding of each scFv, normalized to the amount of HLA / peptide complex on T2 cells, was calculated using the following formula.

[0783] Normalized gMFI = (A / B) / ((C / D) / (E / F))

[0784] A / B = relative gMFI

[0785] A: gMFI of T2 cells supplemented with DMSO or each peptide

[0786] B: gMFI of T2 cells in DMSO without antibody or with each peptide

[0787] (C / D) / (E / F) = DMSO or HLA / peptide complex correction value of T2 cells to which each peptide was added

[0788] C: gMFI of T2 cells supplemented with DMSO containing HLA-A2 antibody or with each peptide

[0789] D: gMFI of T2 cells supplemented with DMSO containing mouse IgG2b antibody or with each peptide

[0790] E: gMFI of T2 cells supplemented with DMSO containing HLA-A2 antibody

[0791] F: gMFI of T2 cells supplemented with DMSO containing mouse IgG2b antibody

[0792] like Figure 2B As shown, the anti-HLA / NY-ESO scFvs NYA-0001, 1143, 1163, 2023, 2027, 2035, 2044, 2045, 2047, 2048, 2060, 2061, and 2143 did not bind to cells supplemented with the homologous peptides, indicating high specificity. On the other hand, NYA-1154, NYC-0003, and 0004 were observed to partially bind to T2 cells supplemented with the homologous peptides.

[0793] (Example 7) Preparation of Fc-added anti-HLA / NY-ESO-anti-CD3 bispecific molecules

[0794] 7) Preparation of 1Fc-added anti-HLA / NY-ESO-anti-CD3 bispecific molecule expression vector

[0795] 7)-1-1taFv-heterodimer Fc-type bispecific molecule expression vector

[0796] To evaluate taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecules, expression vectors for each molecule were designed. The anti-HLA / NY-ESO antibodies used were 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. The anti-CD3 antibody used was the humanized anti-CD3 scFv C3E-7085 (WO2018 / 117237). The heterodimeric Fc sequence used an Fc sequence introduced with mutations that reduce effector function and allow heteromultimer formation (WO2014 / 190441).

[0797] A DNA fragment encoding an Fc (HC1 or HC2) with a mutation that reduces effector function and forms heteromultimers was synthesized (FASMAC Co., Ltd.), and an expression vector for mammalian cells with pcDNA3.3 (Thermo Fisher Scientific) as the backbone was prepared using the In-Fusion HD Cloning Kit (CLONTECH), and named "p_HC1".

[0798] An expression vector for mammalian cells was constructed by introducing a DNA fragment encoding HC2 into the carboxyl terminus of taFv linked to NYA-1143 and C3E-7085 via a GGGGS linker and named "p_NYF-0016-HC2".

[0799] By introducing site-specific mutations into p_NYF-0016-HC2, an expression vector for mammalian cells was constructed, in which a DNA fragment encoding HC2 was introduced into the carboxyl terminus of taFv linked to NYA-2143 and C3E-7085 via a GGGGS linker. The vector was named "p_NYF-0019-HC2."

[0800] Using the In-Fusion HD Cloning Kit (CLONTECH), the base sequence of NYA-1143 encoding p_NYF-0016-HC2 was partially replaced with a DNA fragment encoding NYA-1163. This created an expression vector for mammalian cells named "p_NYF-0022-HC2" in which the DNA fragment encoding HC2 was introduced into the carboxyl terminus of taFv linked to NYA-1163 and C3E-7085 using a GGGGS linker.

[0801] By introducing site-specific mutations into p_NYF-0016-HC2, an expression vector for mammalian cells was constructed in which a DNA fragment encoding HC2 was introduced into the carboxyl terminus of taFv linked to NYA-2023 and C3E-7085 via a GGGGS linker. The vector was named "p_NYF-0023-HC2."

[0802] By introducing site-specific mutations into a mammalian cell expression vector containing a DNA fragment encoding HC2 at the carboxyl terminus of a taFv linked to NYA-0001 and C3E-7085 using a GGGGS linker, an expression vector for mammalian cells containing a DNA fragment encoding HC2 at the carboxyl terminus of a taFv linked to NYA-2027 and C3E-7085 using a GGGGS linker was created and named "p_NYF-0027-HC2."

[0803] By introducing site-specific mutations into p_NYF-0016-HC2, an expression vector for mammalian cells was constructed in which a DNA fragment encoding HC2 was introduced into the carboxyl terminus of taFv linked to NYA-2035 and C3E-7085 via a GGGGS linker. The vector was named "p_NYF-0035-HC2."

[0804] Using the In-Fusion HD Cloning Kit (CLONTECH), the base sequence of NYA-1143 encoding p_NYF-0016-HC2 was partially replaced with a DNA fragment encoding NYA-2044. This created an expression vector for mammalian cells named "p_NYF-0044-HC2" in which the DNA fragment encoding HC2 was introduced into the carboxyl terminus of taFv linked to NYA-2044 and C3E-7085 using a GGGGS linker.

[0805] Using the In-Fusion HD Cloning Kit (CLONTECH), the base sequence of NYA-1143 encoding p_NYF-0016-HC2 was partially replaced with a DNA fragment encoding NYA-2045. This created an expression vector for mammalian cells named "p_NYF-0045-HC2" in which the DNA fragment encoding HC2 was introduced into the carboxyl terminus of taFv linked to NYA-2045 and C3E-7085 using a GGGGS linker.

[0806] Using the In-Fusion HD Cloning Kit (CLONTECH), the base sequence of NYA-1143 encoding p_NYF-0016-HC2 was partially replaced with a DNA fragment encoding NYA-2047. This created an expression vector for mammalian cells named "p_NYF-0047-HC2" in which the DNA fragment encoding HC2 was introduced into the carboxyl terminus of taFv linked to NYA-2047 and C3E-7085 using a GGGGS linker.

[0807] Using the In-Fusion HD Cloning Kit (CLONTECH), the base sequence of NYA-1143 encoding p_NYF-0016-HC2 was partially replaced with a DNA fragment encoding NYA-2048. This created an expression vector for mammalian cells named "p_NYF-0048-HC2" in which the DNA fragment encoding HC2 was introduced into the carboxyl terminus of taFv linked to NYA-2048 and C3E-7085 using a GGGGS linker.

[0808] By introducing site-specific mutations into p_NYF-0044-HC2, an expression vector for mammalian cells was constructed in which a DNA fragment encoding HC2 was introduced into the carboxyl terminus of taFv linked to NYA-2060 and C3E-7085 via a GGGGS linker. This vector was named "p_NYF-0060-HC2."

[0809] By introducing site-specific mutations into p_NYF-0045-HC2, an expression vector for mammalian cells was constructed in which a DNA fragment encoding HC2 was introduced into the carboxyl terminus of taFv linked to NYA-2061 and C3E-7085 via a GGGGS linker. This vector was named "p_NYF-0061-HC2."

[0810] The nucleotide sequence of p_HC1 was reanalyzed and the full-length nucleotide sequence of HC1 was confirmed to be SEQ ID NO: 71 ( Figure 72 ) shown in the nucleotide sequence.

[0811] The nucleotide sequence of p_NYF-0016-HC2 was reanalyzed and the full-length nucleotide sequence of NYF-0016-HC2 was confirmed to be SEQ ID NO: 72 ( Figure 73 ) shown in the nucleotide sequence.

[0812] The nucleotide sequence of p_NYF-0019-HC2 was reanalyzed and the full-length nucleotide sequence of NYF-0019-HC2 was confirmed to be SEQ ID NO: 73 ( Figure 74 ) shown in the nucleotide sequence.

[0813] The nucleotide sequence of p_NYF-0022-HC2 was reanalyzed and the full-length nucleotide sequence of NYF-0022-HC2 was confirmed to be SEQ ID NO: 74 ( Figure 75 ) shown in the nucleotide sequence.

[0814] The nucleotide sequence of p_NYF-0023-HC2 was reanalyzed and the full-length nucleotide sequence of NYF-0023-HC2 was confirmed to be SEQ ID NO: 75 ( Figure 76 ) shown in the nucleotide sequence.

[0815] The nucleotide sequence of p_NYF-0027-HC2 was reanalyzed and the full-length nucleotide sequence of NYF-0027-HC2 was confirmed to be SEQ ID NO: 76 ( Figure 77 ) shown in the nucleotide sequence.

[0816] The nucleotide sequence of p_NYF-0035-HC2 was reanalyzed and the full-length nucleotide sequence of NYF-0035-HC2 was confirmed to be SEQ ID NO: 77 ( Figure 78 ) shown in the nucleotide sequence.

[0817] The nucleotide sequence of p_NYF-0044-HC2 was reanalyzed and the full-length nucleotide sequence of NYF-0044-HC2 was confirmed to be SEQ ID NO: 78 ( Figure 79 ) shown in the nucleotide sequence.

[0818] The nucleotide sequence of p_NYF-0045-HC2 was reanalyzed and the full-length nucleotide sequence of NYF-0045-HC2 was confirmed to be SEQ ID NO: 79 ( Figure 80 ) shown in the nucleotide sequence.

[0819] The nucleotide sequence of p_NYF-0047-HC2 was reanalyzed and the full-length nucleotide sequence of NYF-0047-HC2 was confirmed to be SEQ ID NO: 80 ( Figure 81 ) shown in the nucleotide sequence.

[0820] The nucleotide sequence of p_NYF-0048-HC2 was reanalyzed and the full-length nucleotide sequence of NYF-0048-HC2 was confirmed to be SEQ ID NO: 81 ( Figure 82 ) shown in the nucleotide sequence.

[0821] The nucleotide sequence of p_NYF-0060-HC2 was reanalyzed and the full-length nucleotide sequence of NYF-0060-HC2 was confirmed to be SEQ ID NO: 82 ( Figure 83 ) shown in the nucleotide sequence.

[0822] The nucleotide sequence of p_NYF-0061-HC2 was reanalyzed and the full-length nucleotide sequence of NYF-0061-HC2 was confirmed to be SEQ ID NO: 83 ( Figure 84 ) shown in the nucleotide sequence.

[0823] In addition, in the above-mentioned nucleotide sequence, the full-length amino acid sequences 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 sequence were confirmed.

[0824] The full-length amino acid sequence of HCl is SEQ ID NO: 84 ( Figure 85 ). In this sequence, the amino acid sequence from position 1 to position 19 is a signal sequence, and the amino acid sequence from position 20 to position 246 is HC.

[0825] The full-length amino acid sequence of NYF-0016-HC2 is SEQ ID NO: 85 ( Figure 86 ). In this sequence, amino acids 1 to 19 are a signal sequence, amino acids 21 to 511 are NYA-1143-C3E-7085taFv, amino acids 512 to 513 are a linker, and amino acids 514 to 745 are HC2.

[0826] The full-length amino acid sequence of NYF-0019-HC2 is SEQ ID NO: 86 ( Figure 87 ). In this sequence, amino acids 1 to 19 are a signal sequence, amino acids 21 to 511 are NYA-2143-C3E-7085taFv, amino acids 512 to 513 are a linker, and amino acids 514 to 745 are HC2.

[0827] The full-length amino acid sequence of NYF-0022-HC2 is SEQ ID NO: 87 ( Figure 88 ). In this sequence, amino acids 1 to 19 are a signal sequence, amino acids 21 to 511 are NYA-1163-C3E-7085taFv, amino acids 512 to 513 are a linker, and amino acids 514 to 745 are HC2.

[0828] The full-length amino acid sequence of NYF-0023-HC2 is SEQ ID NO: 88 ( Figure 89 ). In this sequence, amino acids 1 to 19 are a signal sequence, amino acids 21 to 511 are NYA-2023-C3E-7085taFv, amino acids 512 to 513 are a linker, and amino acids 514 to 745 are HC2.

[0829] The full-length amino acid sequence of NYF-0027-HC2 is SEQ ID NO: 89 ( Figure 90 ). In this sequence, amino acids 1 to 19 are a signal sequence, amino acids 21 to 511 are NYA-2027-C3E-7085taFv, amino acids 512 to 513 are a linker, and amino acids 514 to 745 are HC2.

[0830] The full-length amino acid sequence of NYF-0035-HC2 is SEQ ID NO: 90 ( Figure 91 ). In this sequence, amino acids 1 to 19 are a signal sequence, amino acids 21 to 511 are NYA-2035-C3E-7085taFv, amino acids 512 to 513 are a linker, and amino acids 514 to 745 are HC2.

[0831] The full-length amino acid sequence of NYF-0044-HC2 is SEQ ID NO: 91 ( Figure 92 ). In this sequence, amino acids 1 to 19 are a signal sequence, amino acids 21 to 511 are NYA-2044-C3E-7085taFv, amino acids 512 to 513 are a linker, and amino acids 514 to 745 are HC2.

[0832] The full-length amino acid sequence of NYF-0045-HC2 is SEQ ID NO: 92 ( Figure 93 ). In this sequence, amino acids 1 to 19 are a signal sequence, amino acids 21 to 511 are NYA-2045-C3E-7085taFv, amino acids 512 to 513 are a linker, and amino acids 514 to 745 are HC2.

[0833] The full-length amino acid sequence of NYF-0047-HC2 is SEQ ID NO: 93 ( Figure 94 ). In this sequence, amino acids 1 to 19 are a signal sequence, amino acids 21 to 511 are NYA-2047-C3E-7085taFv, amino acids 512 to 513 are a linker, and amino acids 514 to 745 are HC2.

[0834] The full-length amino acid sequence of NYF-0048-HC2 is SEQ ID NO: 94 ( Figure 95 ). In this sequence, amino acids 1 to 19 are a signal sequence, amino acids 21 to 511 are NYA-2048-C3E-7085taFv, amino acids 512 to 513 are a linker, and amino acids 514 to 745 are HC2.

[0835] The full-length amino acid sequence of NYF-0060-HC2 is SEQ ID NO: 95 ( Figure 96 ). In this sequence, amino acids 1 to 19 are a signal sequence, amino acids 21 to 511 are NYA-2060-C3E-7085taFv, amino acids 512 to 513 are a linker, and amino acids 514 to 745 are HC2.

[0836] The full-length amino acid sequence of NYF-0061-HC2 is SEQ ID NO: 96 ( Figure 97 ). In this sequence, amino acids 1 to 19 are a signal sequence, amino acids 21 to 511 are NYA-2061-C3E-7085taFv, amino acids 512 to 513 are a linker, and amino acids 514 to 745 are HC2.

[0837] 7) Preparation of 1-2taFv-Fab-heterodimer Fc-type bispecific molecule expression vector

[0838] A vector for expressing a taFv-Fab-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule was designed. The anti-HLA / NY-ESO antibody used was NYA-0001. The anti-CD3 antibody used was C3E-7085 (WO2018 / 117237), a humanized anti-CD3 scFv. The heterodimeric Fc sequence used an Fc sequence introduced with mutations that reduce effector function and allow for heteromultimer formation (WO2014 / 190441).

[0839] A mammalian cell expression vector was constructed, containing the NYA-0001 heavy chain variable region, the CH1 region from human IgG, and a DNA fragment encoding the HCl-k delete that reduces effector function. The vector was named "p_NYA-0001-Fab-HCl-k delete." Separately, a mammalian cell expression vector was constructed, containing the NYA-0001 light chain variable region and a DNA fragment encoding the CL region from human IgG. The vector was named "p_NYA-0001-LC."

[0840] The nucleotide sequence of p_NYA-0001-Fab-HCl-k delete was reanalyzed, and the full-length nucleotide sequence of NYA-0001-Fab-HCl-k delete was confirmed to be SEQ ID NO: 97 ( Figure 98 ) shown in the nucleotide sequence.

[0841] The nucleotide sequence of p_NYA-0001-LC was reanalyzed and the full-length nucleotide sequence of NYA-0001-LC was confirmed to be SEQ ID NO: 98 ( Figure 99 ) shown in the nucleotide sequence.

[0842] In addition, the NYA-0001-Fab-HCl-k delete and the full-length amino acid sequence of NYA-0001-LC encoded by the sequence were confirmed in the above-mentioned nucleotide sequence.

[0843] The full-length amino acid sequence of NYA-0001-Fab-HCl-k delete is SEQ ID NO: 99 ( Figure 100 ) is an amino acid sequence shown in SEQ ID NO: 54 (). In this sequence, the amino acid sequence from positions 1 to 19 is a signal sequence, the amino acid sequence from positions 20 to 139 is a variable region, and the amino acid sequence from positions 140 to 468 is a constant region. In addition, the amino acid sequence from positions 45 to 52 is CDRH1 (SEQ ID NO: 54 ( Figure 61 )), the amino acid sequence from position 70 to position 77 is CDRH2 (SEQ ID NO: 55 ( Figure 61 )), the amino acid sequence from position 116 to position 128 is CDRH3 (SEQ ID NO: 56 ( Figure 61 )).

[0844] The full-length amino acid sequence of NYA-0001-LC is SEQ ID NO: 100 ( Figure 101 ) is the amino acid sequence shown. The amino acid sequence from position 1 to position 20 is the signal sequence, the amino acid sequence from position 21 to position 131 is the variable region, and the amino acid sequence from position 132 to position 237 is the constant region. In addition, amino acid numbers 46 to 53 are CDRL1 (SEQ ID NO: 57 ( Figure 61 )), amino acid numbers 71 to 73 are CDRL2 (SEQ ID NO: 58 ( Figure 61 )), amino acid numbers 110 to 121 are CDRL3 (SEQ ID NO: 59 ( Figure 61 )).

[0845] 7) Expression of 2Fc-added anti-HLA / NY-ESO-anti-CD3 bispecific molecules

[0846] 7) Expression of 2-1taFv-heterodimer Fc-type bispecific molecules

[0847] Expi293F cells (Thermo Fisher Scientific) were subcultured according to the manual. The culture medium of Expi293F cells in the logarithmic growth phase was diluted to 2.5×10 6cells / mL for the production of various bispecific molecules. 0.3 mg of a vector mixture of p_NYF-0016-HC2 and p_HC1 in a ratio of 1:1.5 and 0.9 mg of polyethyleneimine (Polyscience #24765) were added to 20 mL of Opti-Pro SFM medium (Thermo Fisher Scientific) and stirred steadily. After standing for 5 minutes, the mixture was added to Expi293F cells. The cells were cultured in a 37°C, 8% CO2 incubator at 135 rpm for 6 days. The culture supernatant was filtered with a 0.2 μm filter membrane (Millopore) to obtain the culture supernatant of the taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0016). Each vector constituting NYF-0016 was expressed, and the resulting amino acid sequence is shown in SEQ ID NO: 85 ( Figure 86 ) and SEQ ID NO:84( Figure 85 ).

[0848] By the same method, p_NYF-0019-HC2 and p_HC1 were used to express and prepare the culture supernatant of the taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0019). The amino acid sequence obtained by expressing each vector constituting NYF-0019 is shown in SEQ ID NO: 86 in the sequence listing. Figure 87 ) and SEQ ID NO:84( Figure 85 ).

[0849] Using p_NYF-0022-HC2 and p_HC1, culture supernatant of taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0022) was expressed and prepared. The amino acid sequence obtained by expressing each vector constituting NYF-0022 is shown in SEQ ID NO: 87 ( Figure 88 ) and SEQ ID NO:84( Figure 85 ).

[0850] Using p_NYF-0023-HC2 and p_HC1, culture supernatant of taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0023) was expressed and prepared. The amino acid sequence obtained by expressing each vector constituting NYF-0023 is shown in SEQ ID NO: 88 ( Figure 89 ) and SEQ ID NO:84( Figure 85 ).

[0851] Using p_NYF-0027-HC2 and p_HC1, culture supernatant of taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0027) was expressed and prepared. The amino acid sequence obtained by expressing each vector constituting NYF-0027 is shown in SEQ ID NO: 89 in the sequence listing. Figure 90 ) and SEQ ID NO:84( Figure 85 ).

[0852] Using p_NYF-0035-HC2 and p_HC1, culture supernatant of taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0035) was expressed and prepared. The amino acid sequence obtained by expressing each vector constituting NYF-0035 is shown in SEQ ID NO: 90 ( Figure 91 ) and SEQ ID NO:84( Figure 85 ).

[0853] Using p_NYF-0044-HC2 and p_HC1, culture supernatant of taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0044) was expressed and prepared. The amino acid sequence obtained by expressing each vector constituting NYF-0044 is shown in SEQ ID NO: 91 in the sequence listing ( Figure 92 ) and SEQ ID NO:84( Figure 85 ).

[0854] Using p_NYF-0045-HC2 and p_HC1, culture supernatant of taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0045) was expressed and prepared. The amino acid sequence obtained by expressing each vector constituting NYF-0045 is shown in SEQ ID NO: 92 ( Figure 93 ) and SEQ ID NO:84( Figure 85 ).

[0855] Using p_NYF-0047-HC2 and p_HC1, culture supernatant of taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0047) was expressed and prepared. The amino acid sequence obtained by expressing each vector constituting NYF-0047 is shown in SEQ ID NO: 93 ( Figure 94 ) and SEQ ID NO:84( Figure 85 ).

[0856] Using p_NYF-0048-HC2 and p_HC1, culture supernatant of taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0048) was expressed and prepared. The amino acid sequence obtained by expressing each vector constituting NYF-0048 is shown in SEQ ID NO: 94 ( Figure 95 ) and SEQ ID NO:84( Figure 85 ).

[0857] Using p_NYF-0060-HC2 and p_HC1, culture supernatant of taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0060) was expressed and prepared. The amino acid sequence obtained by expressing each vector constituting NYF-0060 is shown in SEQ ID NO: 95 ( Figure 96 ) and SEQ ID NO:84( Figure 85 ).

[0858] Using p_NYF-0061-HC2 and p_HC1, culture supernatant of taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0061) was expressed and prepared. The amino acid sequence obtained by expressing each vector constituting NYF-0061 is shown in SEQ ID NO: 96 ( Figure 97 ) and SEQ ID NO:84( Figure 85 ).

[0859] 7) Expression of 2-2taFv-Fab-heterodimer Fc-type bispecific molecules

[0860] By the same method as 7)-2-1, a mixture of p_NYF-0023-HC2, p_NYA-0001-Fab-HCl-k delete, and p_NYA-0001-LC in a ratio of 1:1:1.5 was used to express and prepare a culture supernatant of a taFv-Fab-heterodimer Fc-type bispecific molecule (NYF-0058). The amino acid sequence obtained by expressing each vector constituting NYF-0058 is shown in SEQ ID NO: 88 (Figure 1). 89) Amino acid numbers 20 to 745, SEQ ID NO: 99 ( Figure 100 ) of amino acid numbers 20 to 468, and SEQ ID NO: 100 ( Figure 101 ) are numbered 21 to 237.

[0861] 7) Purification of 3Fc-added anti-HLA / NY-ESO-anti-CD3 bispecific molecules

[0862] Various bispecific molecules were purified from the culture supernatant obtained in 7)-2 by a two-stage step of protein A affinity chromatography and gel filtration chromatography.

[0863] The culture supernatant was loaded onto a MabSelectSuRe column (GE Healthcare Bioscience, also referred to simply as "GE Healthcare") equilibrated with PBS at pH 7.4 to adsorb the target bispecific molecule. Non-adsorbed components were removed with PBS, and the adsorbed components were eluted with acetate buffer at pH 3.5. The eluted fraction was adjusted to a neutral pH with Tris buffer at pH 9.5, concentrated, and applied to a Superdex 200 10 / 300 gel filtration column (GE Healthcare Bioscience) pre-equilibrated with 25 mM histidine, 300 mM NaCl, and 5% sorbitol at pH 5.5. From the peak fractions obtained by gel filtration chromatography, the fraction corresponding to the target heterodimer was recovered and confirmed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to confirm the formation of the target anti-HLA / NY-ESO-anti-CD3 bispecific molecule. The purified protein sample was subjected to analytical SEC to determine purity and concentration, and then used for various evaluations.

[0864] (Example 8) Evaluation of Cytotoxic Activity of Fc-Added Anti-HLA-A2 / NY-ESO-Anti-CD3 Bispecific Molecule

[0865] 8)-1 Preparation of target cells

[0866] Endogenous human NY-ESO-expressing cell lines (U266B1 and NCI-H1703) and non-endogenous human NY-ESO-expressing cell lines (AGS and CFPAC-1) were cultured in RPMI1640 medium (Fujifilm Wako Pure Chemical Industries, Ltd.) containing 10% FBS to a concentration of 1 × 10 6 100 μL of chromium-51 radionuclide (PerkinElmer) was added per mL of each cell suspension and incubated at 37°C, 5% CO2 for 2 hours. The cells were washed twice with RPMI1640 medium containing 10% FBS and resuspended in RPMI1640 medium containing 10% FBS to a concentration of 1×10 cells / mL. 5 cells / mL, and the product was used as the target cell.

[0867] 8)-2 Preparation of effector cells

[0868] Commercially available frozen human PBMCs (Cellular Technology Limited) were thawed at 37°C, transferred to RPMI1640 medium containing 10% FBS and supplemented with Anti-aggregate Wash reagent (Cellular Technology Limited), washed twice, and then prepared into 1×10 6 cells / mL, as effector cells.

[0869] 8)-3 Cytotoxicity assay

[0870] Each target cell obtained in step 8)-1 was added to a 96-well U-bottom microplate at 50 μL / well. Various Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules prepared in Example 7 at various concentrations were added at 50 μL / well. The effector cells prepared in Example 8)-2 were added at 100 μL / well. After centrifugation at 1000 rpm for 1 minute at room temperature, the cells were incubated at 37°C and 5% CO₂ for 20 to 24 hours. 50 μL of the supernatant was collected on a LumaPlate (PerkinElmer), dried at 50°C for approximately 2 hours, and then assayed using a plate reader (TopCount, PerkinElmer). The experiment was repeated three times, and the cell lysis rate was calculated using the following formula.

[0871] Cell lysis rate (%) = (AB) / (CB) × 100

[0872] A: Count of sample wells.

[0873] B: Average value of background (wells without antibody addition) counts (n=3). 50 μL of detection medium was added when the antibody was added. The same procedures as for the sample wells were performed except for this. C: Average value of maximum release (wells in which target cells were dissolved with a surfactant) counts (n=3). 50 μL of detection medium was added when the antibody was added. 100 μL of surfactant was added, and 50 μL was transferred to the LumaPlate in the same manner as for the sample wells, and the measurement was performed.

[0874] like Figure 4A As shown in Figure 5-F, various anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules showed cytotoxic activity against cell lines expressing endogenous human NY-ESO (U266B1 and NCI-H1703). EC values ​​were calculated using the Four Parameter Logistics Curve formula of the Sigmaplot analysis software (version 12.0). 50The results for the U266B1 cell line are recorded in Table 2, and the results for the NCI-H1703 cell line are recorded in Table 3. Not Applicable (NA) means that the correlation coefficient R value is not calculated and the curve fitting cannot be performed. Figure 4G-L As shown, no cytotoxic activity was observed against cell lines (AGS and CFPAC-1) that do not express endogenous human NY-ESO.

[0875] [Table 2]

[0876] name <![CDATA[EC 50 (nM)]]> 1 NYF-0016 0.30 2 NYF-0019 0.20 3 NYF-0022 1.30 4 NYF-0023 0.90 5 NYF-0027 2.68 6 NYF-0035 0.97 7 NYF-0044 0.10 8 NYF-0045 0.18 9 NYF-0047 0.93 10 NYF-0048 1.10 11 NYF-0058 0.95 12 NYF-0060 0.39 13 NYF-0061 0.40

[0877] [Table 3]

[0878]

[0879]

[0880] *NA = Not Applicable

[0881] (Example 9) In vivo activity evaluation of Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules in a human PBMC metastasis model

[0882] Human squamous cell lung cancer cell line NCI-H1703 (ATCC) was prepared into 6×10 7 cells / mL, 0.1 mL was transplanted subcutaneously into NOG mice (female, 6-7 weeks old) (day 0). On day 4, human PBMCs were prepared into 3.75×10 7 cells / mL, and 0.2 mL was transplanted into the tail vein. Starting approximately one week later (day 6-7), the long and short diameters (mm) of the tumors were measured with a digital caliper over time, and the estimated tumor volume was calculated using the formula shown below.

[0883] Estimated tumor volume (mm 3 ) = the mean of the estimated tumor volumes of each individual

[0884] Estimated tumor volume of each individual = long diameter × short diameter 2 / 2

[0885] On day 14, the patients were divided into groups according to tumor volume, with n=5-6 per group, and various anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules were administered intravenously (1 mg / kg, except for NYF-0058, which was administered at 1.5 mg / kg for comparison at the same molar amount). Administration was carried out on days 14, 21, and 28. Antitumor effects were confirmed in the groups administered with various anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules ( Figure 5A-C The tumor growth inhibition (%) on days 31 to 32 was calculated using the following formula and is shown in Table 4.

[0886] Tumor growth inhibition (%) = 100 - (estimated tumor volume of each group / estimated tumor volume of blank control × 100)

[0887] [Table 4]

[0888]

[0889]

[0890] (Example 10) Preparation of various Fc-added anti-HLA / NY-ESO-anti-CD3 bispecific molecules

[0891] 10)-1 Preparation of expression vectors for various Fc-added anti-HLA / NY-ESO-anti-CD3 bispecific molecules

[0892] 10)-1-1 Preparation of hybrid bispecific molecule expression vector

[0893] 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-CD3 scFv, was used as the anti-CD3 antibody. The heterodimeric Fc sequence used an Fc sequence introduced with mutations that reduce effector function and allow for heteromultimer formation (WO2014 / 190441).

[0894] A mammalian cell expression vector was constructed, containing DNA fragments encoding the heavy chain variable region of NYA-1143, the CH1 region from human IgG, and the Fc region with mutations that reduce effector function and allow heteromultimer formation. The vector was named "p_NYA-1143-Fab-HC1-k delete." Separately, a mammalian cell expression vector was constructed, containing DNA fragments encoding the light chain variable region of NYA-1143 and the CL region from human IgG. The vector was named "p_NYA-1143-LC." Furthermore, a mammalian cell expression vector, "p_C3E-7085-HC2-k delete," was constructed, containing a DNA fragment encoding the Fc region with mutations introduced into the carboxyl terminus of a humanized anti-CD3 scFv (C3E-7085) that reduce effector function and allow heteromultimer formation.

[0895] The nucleotide sequence of p_NYA-1143-Fab-HCl-k delete was reanalyzed, and the full-length nucleotide sequence of NYA-1143-Fab-HCl-k delete was confirmed to be SEQ ID NO: 101 ( Figure 102 ) shown in the nucleotide sequence.

[0896] The nucleotide sequence of p_NYA-1143-LC was reanalyzed and the full-length nucleotide sequence of NYA-1143-LC was confirmed to be SEQ ID NO: 102 ( Figure 103 ) shown in the nucleotide sequence.

[0897] The nucleotide sequence of p_C3E-7085-HC2-k delete was reanalyzed and the full-length nucleotide sequence of C3E-7085-HC2-k delete was confirmed to be SEQ ID NO: 103 ( Figure 104 ) shown in the nucleotide sequence.

[0898] In addition, the full-length amino acid sequences of NYA-1143-Fab-HCl-k delete, NYA-1143-LC, and C3E-7085-HC2-k delete encoded by the nucleotide sequence were confirmed.

[0899] The full-length amino acid sequence of NYA-1143-Fab-HCl-k delete is SEQ ID NO: 104 ( Figure 105) is an amino acid sequence shown in SEQ ID NO: 54 (). In this sequence, the amino acid sequence from positions 1 to 19 is a signal sequence, the amino acid sequence from positions 21 to 139 is a variable region, and the amino acid sequence from positions 140 to 468 is a constant region. In addition, the amino acid sequence from positions 45 to 52 is CDRH1 (SEQ ID NO: 54 ( Figure 61 )), the amino acid sequence from position 70 to position 77 is CDRH2 (SEQ ID NO: 55 ( Figure 61 )), the amino acid sequence from position 116 to position 128 is CDRH3 (SEQ ID NO: 56 ( Figure 61 )).

[0900] The full-length amino acid sequence of NYA-1143-LC is SEQ ID NO: 105 ( Figure 106 ) is the amino acid sequence shown. The amino acid sequence from position 1 to position 20 is the signal sequence, the amino acid sequence from position 21 to position 131 is the variable region, and the amino acid sequence from position 132 to position 237 is the constant region. In addition, amino acid numbers 46 to 53 are CDRL1 (SEQ ID NO: 60 ( Figure 62 )), amino acid numbers 71 to 73 are CDRL2 (SEQ ID NO: 58 ( Figure 61 )), amino acid numbers 110 to 121 are CDRL3 (SEQ ID NO: 59 ( Figure 61 )).

[0901] The full-length amino acid sequence of C3E-7085-HC2-k delete is SEQ ID NO: 106 ( Figure 107 ). The amino acid sequence from positions 1 to 19 is a signal sequence, the amino acid sequence from positions 21 to 260 is C3E-7085, the amino acid sequence from positions 261 to 262 is a linker, and the amino acid sequence from positions 263 to 493 is an Fc sequence into which a mutation that reduces effector function and forms a heterodimer has been introduced.

[0902] 10)-1-2 Preparation of bispecific molecule expression vector

[0903] To evaluate the dual-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule, the following expression vector was designed. The mammalian cell expression vector "p_NYA-1143-HCl-k delete" was constructed. This expression vector contained a DNA fragment encoding the Fc region with a mutation that reduces effector function and allows for heteromultimer formation, introduced into the carboxyl terminus of NYA-1143.

[0904] The nucleotide sequence of p_NYA-1143-HCl-k delete was reanalyzed and the full-length nucleotide sequence of NYA-1143-HCl-k delete was confirmed to be SEQ ID NO: 107 ( Figure 108 ) shown in the nucleotide sequence. In addition, the NYA-1143-HCl-k delete full-length amino acid sequence encoded by the sequence was confirmed. The NYA-1143-HCl-k delete full-length amino acid sequence is SEQ ID NO: 108 ( Figure 109 ). In this sequence, amino acids 1 to 19 represent a signal sequence, amino acids 21 to 266 represent NYA-1143, amino acids 267 to 268 represent a linker, and amino acids 269 to 499 represent an Fc sequence into which a mutation that reduces effector function and allows heterodimer formation has been introduced.

[0905] 10) Preparation of 1-3scFv-Fab-heterodimer Fc-type bispecific molecule expression vector

[0906] To evaluate scFv-Fab-heterodimer Fc-based anti-HLA / NY-ESO-anti-CD3 bispecific molecules, expression vectors for each molecule were designed. NYA-1154 was used for the anti-HLA / NY-ESO antibody, and C3E-7085 was used for the anti-CD3 antibody. The heterodimeric Fc sequence employed an Fc sequence incorporating mutations that reduced effector function and facilitated heteromultimer formation.

[0907] An expression vector for mammalian cells was created, containing DNA fragments encoding the heavy chain variable region of NYA-1154 at the carboxyl terminus of C3E-7085, the CH1 region from human IgG, and the Fc region with mutations that reduce effector function and allow heteromultimer formation. The vector was named "p_C3E-7085-NYA-1154-Fab-HC2-k delete." Separately, an expression vector for mammalian cells was created, containing the light chain variable region of NYA-1154 and a DNA fragment encoding the CL region from human IgG. The vector was named "p_NYA-1154-LC." Furthermore, an expression vector for mammalian cells was created, containing a DNA fragment encoding the HC1-k delete. The vector was named "p_OAA-HC1-k delete."

[0908] The nucleotide sequence of p_C3E-7085-NYA-1154-Fab-HC2-k delete was reanalyzed, and the full-length nucleotide sequence of C3E-7085-NYA-1154-Fab-HC2-k delete was confirmed to be SEQ ID NO: 109 ( Figure 110 ) shown in the nucleotide sequence.

[0909] The nucleotide sequence of p_NYA-1154-LC was reanalyzed and the full-length nucleotide sequence of NYA-1154-LC was confirmed to be SEQ ID NO: 110 ( Figure 111 ) shown in the nucleotide sequence.

[0910] The nucleotide sequence of p_OAA-HCl-k delete was reanalyzed and the full-length nucleotide sequence of OAA-HCl-k delete was confirmed to be SEQ ID NO: 111 ( Figure 112 ) shown in the nucleotide sequence.

[0911] In addition, 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 nucleotide sequence were confirmed.

[0912] The full-length amino acid sequence of C3E-7085-NYA-1154-Fab-HC2-k delete is SEQ ID NO: 112 ( Figure 113 ). The amino acid sequence from positions 1 to 19 is a signal sequence, the amino acid sequence from positions 21 to 260 is C3E-7085, the amino acid sequence from positions 261 to 385 is a linker, and the amino acid sequence from positions 266 to 385 is NYA-1154 - heavy chain variable region. In addition, the amino acid sequence from amino acid numbers 386 to 714 is HC2-kdelete.

[0913] The full-length amino acid sequence of NYA-1154-LC is SEQ ID NO: 113 ( Figure 114 ) is the amino acid sequence shown. The amino acid sequence from position 1 to position 20 is the signal sequence, the amino acid sequence from position 21 to position 131 is the variable region, and the amino acid sequence from position 132 to position 237 is the constant region. In addition, amino acid numbers 46 to 53 are CDRL1 (SEQ ID NO: 57 ( Figure 61 )), amino acid numbers 71 to 73 are CDRL2 (SEQ ID NO: 58 ( Figure 61)), amino acid numbers 110 to 121 are CDRL3 (SEQ ID NO: 63 ( Figure 64 )).

[0914] The full-length amino acid sequence of OAA-HCl-k delete is SEQ ID NO: 114 ( Figure 115 ) In this sequence, the amino acid sequence from positions 1 to 19 is a signal sequence, and the amino acid sequence from positions 20 to 245 is an OAA-HC1-k delete.

[0915] 10)-1-4 Preparation of various taFv-heterodimer Fc-type bispecific molecule expression vectors for configuration evaluation

[0916] To evaluate various taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecules for configuration evaluation, 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-CD3 scFv, was used as an anti-CD3 antibody. The heterodimeric Fc sequence used an Fc sequence in which mutations that reduced effector function and promoted heteromultimer formation were introduced.

[0917] An expression vector for mammalian cells was constructed by introducing a DNA fragment encoding HC2-k delete into the carboxyl terminus of taFv in which NYA-1154 and C3E-7085 were linked via a GGGGS linker. The vector was named "p_NYF-0010-HC2-k delete."

[0918] An expression vector for mammalian cells was constructed by introducing a DNA fragment encoding HC2-k delete into the carboxyl terminus of taFv in which C3E-7085 and NYA-1154 were linked via a GGGGS linker. The vector was named "p_NYF-0004-HC2-k delete".

[0919] An expression vector for mammalian cells was constructed by introducing a DNA fragment encoding HC2-k delete into the carboxyl terminus of taFv in which NYA-1143 and C3E-7085 were linked via a GGGGS linker. The vector was named "p_NYF-0011-HC2-k delete."

[0920] The nucleotide sequence of p_NYF-0010-HC2-k delete was reanalyzed and the full-length nucleotide sequence of NYF-0010-HC2-k delete was confirmed to be SEQ ID NO: 115 ( Figure 116 ) shown in the nucleotide sequence.

[0921] The nucleotide sequence of p_NYF-0004-HC2-k delete was reanalyzed and the full-length nucleotide sequence of NYF-0004-HC2-k delete was confirmed to be SEQ ID NO: 116 ( Figure 117 ) shown in the nucleotide sequence.

[0922] The nucleotide sequence of p_NYF-0011-HC2-k delete was reanalyzed and the full-length nucleotide sequence of NYF-0011-HC2-k delete was confirmed to be SEQ ID NO: 117 ( Figure 118 ) shown in the nucleotide sequence.

[0923] In addition, the full-length amino acid sequences of NYF-0010-HC2-k delete, NYF-0004-HC2-k delete, and NYF-0011-HC2-k delete encoded by the nucleotide sequence were confirmed.

[0924] The full-length amino acid sequence of NYF-0010-HC2-k delete is SEQ ID NO: 118 ( Figure 119 ). In this sequence, the amino acid sequence from positions 1 to 19 is a signal sequence, the amino acid sequence from positions 21 to 511 is NYA-1154-C3E-7085taFv, the amino acid sequence from positions 512 to 513 is a linker, and the amino acid sequence from positions 514 to 744 is HC2-kdelete.

[0925] The full-length amino acid sequence of NYF-0004-HC2-k delete is SEQ ID NO: 119 ( Figure 120 ). In this sequence, amino acids 1 to 19 are a signal sequence, amino acids 21 to 511 are C3E-7085-NYA-1154taFv, amino acids 512 to 513 are a linker, and amino acids 514 to 744 are an HC2-k delete.

[0926] The full-length amino acid sequence of NYF-0011-HC2-k delete is SEQ ID NO: 120 ( Figure 121). In this sequence, amino acids 1 to 19 are a signal sequence, amino acids 21 to 511 are NYA-1143-C3E-7085taFv, amino acids 512 to 513 are a linker, and amino acids 514 to 745 are an HC2-k delete.

[0927] 10)-2 Expression of various Fc-added anti-HLA / NY-ESO-anti-CD3 bispecific molecules

[0928] 10)-2-1 Expression of Hybrid Bispecific Molecules

[0929] By the same method as 7)-2-1, a mixture of vectors containing p_NYA-1143-Fab-HCl-k delete, p_C3E-7085-HC2-k delete, and p_NYA-1143-LC at a ratio of 1:1:1.5 was used to express and prepare a culture supernatant of a hybrid bispecific molecule (NYG-3143). The amino acid sequence obtained by expressing each vector constituting NYG-3143 is shown in SEQ ID NO: 104 ( Figure 105 )、SEQ ID NO:105( Figure 106 ) and SEQ ID NO: 106 ( Figure 107 ).

[0930] 10) Expression of bispecific molecules

[0931] The culture supernatant of the bispecific molecule (NYG-2143) was expressed and prepared using a mixture of vectors containing p_NYA-1143-HCl-kdelete and p_C3E-7085-HC2-kdelete at a ratio of 2:1 using the same method as 7)-2-1. The amino acid sequence obtained by expressing each vector constituting NYG-2143 is shown in SEQ ID NO: 108 ( Figure 109 ) and SEQ ID NO: 106 ( Figure 107 ).

[0932] 10) Expression of 2-3scFv-Fab-heterodimer Fc-type bispecific molecules

[0933] By the same method as 7)-2-1, a mixture of p_C3E-7085-NYA-1154-Fab-HC2-k delete, p_OAA-HCl-k delete, and p_NYA-1154-LC in a ratio of 1:1:1.5 was used to express and prepare a culture supernatant of a scFv-Fab-heterodimer Fc type (scFv-Fab-Fc type) bispecific molecule (NYF-0003). The amino acid sequence obtained by expressing each vector constituting NYF-0003 is shown in SEQ ID NO: 112 ( Figure 113 )、SEQ ID NO:113( Figure 114 ) and SEQ ID NO: 114 ( Figure 115 ).

[0934] 10)-2-4 Expression of various taFv-heterodimer Fc-type bispecific molecules for configuration evaluation

[0935] By the same method as 7)-2-1, p_NYF-0010-HC2-k delete and p_OAA-HC1-k delete were used to express and prepare the culture supernatant of taFv-heterodimer Fc type (taFv-Fc type) anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0010). The amino acid sequence obtained by expressing each vector constituting NYF-0010 is shown in SEQ ID NO: 118 ( Figure 119 ) and SEQ ID NO...

Claims

1. An antibody or a binding fragment thereof that specifically binds to human HLA / NY-ESO, the antibody or the binding fragment thereof comprising: A heavy chain CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 54; A heavy chain CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 55; A heavy chain CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 56; A light chain CDRL1 consisting of an amino acid sequence in which the 7th amino acid in the amino acid sequence shown in SEQ ID NO: 57 is W; A light chain CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 58; and Light chain CDRL3 consisting of the amino acid sequence shown in SEQ ID NO:

59.

2. The antibody or binding fragment thereof according to claim 1, wherein the antibody or binding fragment thereof comprises the following heavy chain variable region and light chain variable region: A heavy chain variable region consisting of the amino acid sequence from positions 21 to 140 of the amino acid sequence shown in SEQ ID NO: 53, and a light chain variable region consisting of the amino acid sequence from positions 156 to 266 of the amino acid sequence shown in SEQ ID NO: 53; or, A heavy chain variable region consisting of the amino acid sequence from positions 21 to 140 of the amino acid sequence shown in SEQ ID NO: 156, and a light chain variable region consisting of the amino acid sequence from positions 161 to 271 of the amino acid sequence shown in SEQ ID NO:

156.

3. The antibody or binding fragment thereof according to claim 1, wherein the antibody or binding fragment thereof is scFv.

4. The antibody or binding fragment thereof according to claim 3, wherein the antibody or binding fragment thereof is the following scFv: An scFv consisting of the amino acid sequence from positions 21 to 266 of the amino acid sequence shown in SEQ ID NO: 53; or, A scFv consisting of the amino acid sequence from positions 21 to 271 of the amino acid sequence shown in SEQ ID NO:

156.

5. A polynucleotide encoding the antibody or binding fragment thereof according to claim 1. A vector comprising the polynucleotide according to claim 5.

7. A host cell, comprising the polynucleotide according to claim 5 or the vector according to claim 6.

8. A method for producing an antibody or a binding fragment thereof that specifically binds to human HLA / NY-ESO, the method comprising: (i) a step of culturing the host cell according to claim 7; and (ii) a step of purifying the antibody or binding fragment thereof from the culture obtained in step (i).

9. An antibody or a binding fragment thereof that specifically binds to human HLA / NY-ESO, wherein the antibody or the binding fragment thereof is obtained by the method according to claim 8. 10 . A pharmaceutical composition comprising the following as an active ingredient: the antibody or binding fragment thereof according to any one of claims 1 to 4 and 9 .

11. A pharmaceutical composition comprising the polynucleotide according to claim 5 as an active ingredient.

12. A pharmaceutical composition comprising the carrier according to claim 6 as an active ingredient.

13. A pharmaceutical composition comprising the cell according to claim 7 as an active ingredient.

14. A molecule that specifically binds to human HLA / NY-ESO, comprising the antibody or binding fragment thereof according to any one of claims 1 to 4 and 9.

15. The molecule of claim 14, which is a multispecific antibody.

16. The molecule of claim 14, which is a bispecific antibody.

17. The molecule according to any one of claims 14 to 16, comprising an antibody or a binding fragment thereof that specifically binds to CD3.

18. The molecule according to claim 17, wherein The antibody or its binding fragment that specifically binds to CD3 is an antibody or its binding fragment that specifically binds to CD3 that contains the following chains: (CCH1) heavy chain CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 141; (CCH2) a heavy chain CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 142, or a heavy chain CDRH2 consisting of an amino acid sequence shown in SEQ ID NO: 142 wherein the third amino acid is N or S; (CCH3) a heavy chain CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 143; (CCL1) light chain CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 144; (CCL2) a light chain CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 145, or a light chain CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 145 wherein the second amino acid is N; and, (CCL3) Light chain CDRL3 consisting of the amino acid sequence shown in SEQ ID NO:

146.

19. The molecule according to claim 17, wherein The antibody or its binding fragment that specifically binds to CD3 contains: (CH1CL1) a heavy chain variable region consisting of the amino acid sequence from positions 2 to 119 of the amino acid sequence shown in SEQ ID NO: 136, and a light chain variable region consisting of the amino acid sequence from positions 135 to 243 of the amino acid sequence shown in SEQ ID NO: 136; (CH2CL2) a heavy chain variable region consisting of the amino acid sequence from positions 2 to 119 of the amino acid sequence shown in SEQ ID NO: 137, and a light chain variable region consisting of the amino acid sequence from positions 135 to 241 of the amino acid sequence shown in SEQ ID NO: 137; (CH3CL3) a heavy chain variable region consisting of the amino acid sequence from positions 2 to 119 of the amino acid sequence shown in SEQ ID NO: 147, and a light chain variable region consisting of the amino acid sequence from positions 135 to 243 of the amino acid sequence shown in SEQ ID NO: 147; (CH4CL4) a heavy chain variable region consisting of the amino acid sequence from positions 2 to 119 of the amino acid sequence shown in SEQ ID NO: 138, and a light chain variable region consisting of the amino acid sequence from positions 135 to 241 of the amino acid sequence shown in SEQ ID NO: 138; (CH5CL5) a heavy chain variable region consisting of the amino acid sequence from positions 2 to 119 of the amino acid sequence shown in SEQ ID NO: 139, and a light chain variable region consisting of the amino acid sequence from positions 135 to 243 of the amino acid sequence shown in SEQ ID NO: 139; (CH6CL6) a heavy chain variable region consisting of the amino acid sequence from positions 2 to 119 of the amino acid sequence shown in SEQ ID NO: 140, and a light chain variable region consisting of the amino acid sequence from positions 135 to 243 of the amino acid sequence shown in SEQ ID NO: 140; (CH7CL7) a heavy chain variable region consisting of the amino acid sequence from positions 272 to 389 of the amino acid sequence shown in SEQ ID NO: 155, and a light chain variable region consisting of the amino acid sequence from positions 405 to 511 of the amino acid sequence shown in SEQ ID NO: 155; (CH8CL8) a heavy chain variable region consisting of the amino acid sequence from positions 277 to 394 of the amino acid sequence shown in SEQ ID NO: 156, and a light chain variable region consisting of the amino acid sequence from positions 410 to 516 of the amino acid sequence shown in SEQ ID NO: 156; or, (CH9CL9) a heavy chain variable region consisting of the amino acid sequence at positions 277 to 394 of the amino acid sequence shown in SEQ ID NO: 157, and a light chain variable region consisting of the amino acid sequence at positions 410 to 516 of the amino acid sequence shown in SEQ ID NO:

157.

20. The molecule according to claim 17, wherein The antibody or its binding fragment that specifically binds to CD3 is scFv.

21. The molecule according to claim 20, wherein scFvs include: (CS1) a scFv consisting of the amino acid sequence from positions 2 to 243 of the amino acid sequence shown in SEQ ID NO: 136; (CS2) a scFv consisting of the amino acid sequence from position 2 to position 241 of the amino acid sequence shown in SEQ ID NO: 137; (CS3) a scFv consisting of the amino acid sequence from positions 2 to 243 of the amino acid sequence shown in SEQ ID NO: 147; (CS4) a scFv consisting of the amino acid sequence from position 2 to position 241 of the amino acid sequence shown in SEQ ID NO: 138; (CS5) a scFv consisting of the amino acid sequence from positions 2 to 243 of the amino acid sequence shown in SEQ ID NO: 139; (CS6) a scFv consisting of the amino acid sequence from positions 2 to 243 of the amino acid sequence shown in SEQ ID NO: 140; (CS7) a scFv consisting of the amino acid sequence from positions 272 to 511 of the amino acid sequence shown in SEQ ID NO: 155; (CS8) a scFv consisting of the amino acid sequence from positions 277 to 516 of the amino acid sequence shown in SEQ ID NO: 156; or, (CS9) An scFv consisting of the amino acid sequence from positions 277 to 516 of the amino acid sequence shown in SEQ ID NO:

157.

22. The molecule according to claim 17, wherein the molecule comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises, from N-terminus to 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 the second polypeptide comprises an Fc region (ii).

23. The molecule of claim 22, further associated in Fc region (i) and Fc region (ii).

24. The molecule according to claim 22, wherein The scFv that specifically binds to human HLA / NY-ESO is the antibody or binding fragment thereof that specifically binds to human HLA / NY-ESO according to claim 1 as scFv.

25. The molecule according to claim 22, wherein The scFv that specifically binds to human HLA / NY-ESO is the antibody or binding fragment thereof that specifically binds to human HLA / NY-ESO according to claim 2 as scFv.

26. The molecule according to claim 22, wherein The scFv that specifically binds to human HLA / NY-ESO is the antibody or binding fragment thereof that specifically binds to human HLA / NY-ESO according to claim 4 as scFv.

27. The molecule according to claim 22, wherein The scFv that specifically binds to CD3 is the antibody or binding fragment thereof that specifically binds to CD3 according to claim 18 as scFv.

28. The molecule according to claim 22, wherein The scFv that specifically binds to CD3 is the antibody or binding fragment thereof that specifically binds to CD3 according to claim 19 as scFv.

29. The molecule according to claim 22, wherein The scFv that specifically binds to CD3 is the antibody or binding fragment thereof that specifically binds to CD3 according to claim 21 as scFv.

30. The molecule according to claim 22, comprising the amino acid sequence from positions 21 to 511 of the amino acid sequence shown in SEQ ID NO: 96, the amino acid sequence from positions 21 to 516 of the amino acid sequence shown in SEQ ID NO: 156, or the amino acid sequence from positions 21 to 516 of the amino acid sequence shown in SEQ ID NO:

157.

31. The molecule according to claim 22, wherein The first polypeptide contains the amino acid sequence from positions 529 to 745 of the amino acid sequence shown in SEQ ID NO: 96, the amino acid sequence from positions 534 to 750 of the amino acid sequence shown in SEQ ID NO: 156, or the amino acid sequence from positions 534 to 750 of the amino acid sequence shown in SEQ ID NO:

157.

32. The molecule according to claim 22, wherein The first polypeptide consists of the following amino acid sequence: the amino acid sequence from positions 20 to 745 of the amino acid sequence shown in SEQ ID NO: 96, the amino acid sequence from positions 20 to 750 of the amino acid sequence shown in SEQ ID NO: 156, or the amino acid sequence from positions 20 to 750 of the amino acid sequence shown in SEQ ID NO:

157.

33. The molecule according to claim 22, wherein The second polypeptide contains the amino acid sequence from positions 20 to 246 of the amino acid sequence shown in SEQ ID NO:

84.

34. The molecule according to claim 22, wherein One or two amino acids are deleted from the carboxyl terminus of the amino acid sequence of at least one polypeptide contained in the molecule.

35. The molecule according to claim 17, comprising a first polypeptide, a second polypeptide and a third polypeptide, wherein the first polypeptide comprises, from N-terminus to C-terminus, a scFv that specifically binds to human HLA / NY-ESO, a variable region and a constant region CH1 of an antibody heavy chain that specifically binds to CD3, and an immunoglobulin Fc region (i); the second polypeptide comprises a hinge region and an Fc region of an immunoglobulin (ii); and the third polypeptide comprises an antibody light chain consisting of a variable region and a constant region.

36. The molecule according to claim 35, 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 associated with the third polypeptide in the variable region and the constant region CH1 of the antibody heavy chain.

37. The molecule according to claim 35, wherein The third polypeptide contains the antibody light chain that specifically binds to CD3, which is composed of a variable region and a constant region.

38. The molecule according to claim 35, wherein The scFv that specifically binds to human HLA / NY-ESO is the antibody or binding fragment thereof that specifically binds to human HLA / NY-ESO according to claim 1 as scFv.

39. The molecule according to claim 35, wherein The scFv that specifically binds to human HLA / NY-ESO is the antibody or binding fragment thereof that specifically binds to human HLA / NY-ESO according to claim 2 as scFv.

40. The molecule according to claim 35, wherein The scFv that specifically binds to human HLA / NY-ESO is the antibody or binding fragment thereof that specifically binds to human HLA / NY-ESO according to claim 3 as scFv.

41. The molecule according to claim 35, wherein The scFv that specifically binds to human HLA / NY-ESO is the antibody or binding fragment thereof that specifically binds to human HLA / NY-ESO according to claim 4 as scFv.

42. The molecule according to claim 35, wherein The antibody or its binding fragment that specifically binds to CD3 is Fab.

43. The molecule according to claim 35, wherein The Fab that specifically binds to CD3 is the antibody that specifically binds to CD3 according to claim 18 or a binding fragment thereof as Fab.

44. The molecule according to claim 35, wherein The Fab that specifically binds to CD3 is the antibody that specifically binds to CD3 according to claim 19 or a binding fragment thereof as Fab.

45. The molecule according to claim 35, wherein The Fab that specifically binds to CD3 is the antibody that specifically binds to CD3 according to claim 21 or a binding fragment thereof as Fab.

46. ​​The molecule according to claim 35, wherein The first polypeptide contains the amino acid sequence from positions 21 to 394 of the amino acid sequence shown in SEQ ID NO:

160.

47. The molecule according to claim 35, wherein The first polypeptide comprises the amino acid sequence described in (i) or (ii) below: (i) the amino acid sequence from position 20 to position 724 of the amino acid sequence shown in SEQ ID NO: 160; (ii) the amino acid sequence from position 20 to position 719 of the amino acid sequence shown in SEQ ID NO:

197.

48. The molecule according to claim 35, wherein The second polypeptide contains the amino acid sequence from positions 20 to 246 of the amino acid sequence shown in SEQ ID NO:

84.

49. The molecule according to claim 35, wherein The third polypeptide contains the amino acid sequence from positions 21 to 127 of the amino acid sequence shown in SEQ ID NO:

161.

50. The molecule according to claim 35, wherein The third polypeptide contains the amino acid sequence from positions 21 to 233 of the amino acid sequence shown in SEQ ID NO:

161.

51. The molecule according to claim 35, wherein One or two amino acids are deleted from the carboxyl terminus of the amino acid sequence of one or more polypeptides contained in the molecule.

52. A polynucleotide comprising a nucleotide sequence encoding the molecule of claim 17.

53. A vector comprising the polynucleotide of claim 52.

54. A host cell comprising the polynucleotide of claim 52 or the vector of claim 53.

55. A method for producing a molecule that specifically binds to human HLA / NY-ESO and human CD3, the method comprising: (i) a step of culturing the host cell according to claim 54; and (ii) a step of purifying the antibody or binding fragment thereof from the culture obtained in step (i).

56. A molecule that specifically binds to human HLA / NY-ESO and human CD3, wherein the molecule is obtained by the method of claim 55.

57. A polynucleotide comprising a nucleotide sequence encoding the molecule of claim 22.

58. A vector comprising the polynucleotide of claim 57.

59. A host cell comprising the polynucleotide of claim 57 or the vector of claim 58.

60. A method for producing a molecule that specifically binds to human HLA / NY-ESO and human CD3, the method comprising: (i) a step of culturing the host cell according to claim 59; and (ii) a step of purifying the antibody or binding fragment thereof from the culture obtained in step (i).

61. A molecule that specifically binds to human HLA / NY-ESO and human CD3, wherein the molecule is obtained by the method of claim 60.

62. A polynucleotide comprising a nucleotide sequence encoding the molecule of claim 35.

63. A vector comprising the polynucleotide of claim 62.

64. A host cell comprising the polynucleotide of claim 62 or the vector of claim 63.

65. A method for producing a molecule that specifically binds to human HLA / NY-ESO and human CD3, the method comprising: (i) a step of culturing the host cell according to claim 64; and (ii) a step of purifying the antibody or binding fragment thereof from the culture obtained in step (i).

66. A molecule that specifically binds to human HLA / NY-ESO and human CD3, wherein the molecule is obtained by the method of claim 65.

67. A pharmaceutical composition comprising the molecule of claim 17 as an active ingredient.

68. A pharmaceutical composition comprising the polynucleotide of claim 52 as an active ingredient.

69. A pharmaceutical composition comprising the carrier according to claim 53 as an active ingredient.

70. A pharmaceutical composition comprising the host cell according to claim 54 as an active ingredient.

71. A pharmaceutical composition comprising the molecule of claim 22 as an active ingredient.

72. A pharmaceutical composition comprising the polynucleotide of claim 57 as an active ingredient.

73. A pharmaceutical composition comprising the carrier according to claim 58 as an active ingredient.

74. A pharmaceutical composition comprising the host cell according to claim 59 as an active ingredient.

75. A pharmaceutical composition comprising the molecule of claim 35 as an active ingredient.

76. A pharmaceutical composition comprising the polynucleotide of claim 62 as an active ingredient.

77. A pharmaceutical composition comprising the carrier according to claim 63 as an active ingredient.

78. A pharmaceutical composition comprising the host cell according to claim 64 as an active ingredient.

79. The pharmaceutical composition according to claim 67, which is an anticancer agent, wherein The cancer is one or more cancers selected from the group consisting of renal cancer, melanoma, thyroid cancer, lung cancer, breast cancer, esophageal cancer, gastric cancer, colorectal cancer, rectal cancer, liver cancer, bile duct cancer, bladder cancer, prostate cancer, uterine cancer, liposarcoma, chondrosarcoma, rhabdomyosarcoma, osteosarcoma, undifferentiated pleomorphic sarcoma, myxofibrosarcoma, malignant peripheral nerve sheath tumor, synovial sarcoma, uterine sarcoma, leiomyosarcoma, B-cell lymphoma, myeloid leukemia, multiple myeloma, testicular cancer and ovarian cancer.

80. The pharmaceutical composition of claim 79, wherein the lung cancer is non-small cell lung cancer or small cell lung cancer.

81. The pharmaceutical composition of claim 80, wherein the non-small cell lung cancer is adenocarcinoma, squamous cell carcinoma, or large cell carcinoma.

82. The pharmaceutical composition according to claim 71, which is an anticancer agent, wherein The cancer is one or more cancers selected from the group consisting of renal cancer, melanoma, thyroid cancer, lung cancer, breast cancer, esophageal cancer, gastric cancer, colorectal cancer, rectal cancer, liver cancer, bile duct cancer, bladder cancer, prostate cancer, uterine cancer, liposarcoma, chondrosarcoma, rhabdomyosarcoma, osteosarcoma, undifferentiated pleomorphic sarcoma, myxofibrosarcoma, malignant peripheral nerve sheath tumor, synovial sarcoma, uterine sarcoma, leiomyosarcoma, B-cell lymphoma, myeloid leukemia, multiple myeloma, testicular cancer and ovarian cancer.

83. The pharmaceutical composition of claim 82, wherein the lung cancer is non-small cell lung cancer or small cell lung cancer.

84. The pharmaceutical composition of claim 83, wherein the non-small cell lung cancer is adenocarcinoma, squamous cell carcinoma, or large cell carcinoma.

85. The pharmaceutical composition according to claim 75, which is an anticancer agent, wherein The cancer is one or more cancers selected from the group consisting of renal cancer, melanoma, thyroid cancer, lung cancer, breast cancer, esophageal cancer, gastric cancer, colorectal cancer, rectal cancer, liver cancer, bile duct cancer, bladder cancer, prostate cancer, uterine cancer, liposarcoma, chondrosarcoma, rhabdomyosarcoma, osteosarcoma, undifferentiated pleomorphic sarcoma, myxofibrosarcoma, malignant peripheral nerve sheath tumor, synovial sarcoma, uterine sarcoma, leiomyosarcoma, B-cell lymphoma, myeloid leukemia, multiple myeloma, testicular cancer and ovarian cancer.

86. The pharmaceutical composition of claim 85, wherein the lung cancer is non-small cell lung cancer or small cell lung cancer.

87. The pharmaceutical composition of claim 86, wherein the non-small cell lung cancer is adenocarcinoma, squamous cell carcinoma, or large cell carcinoma.

88. The pharmaceutical composition of claim 67, which is used in combination with other pharmaceutical agents.

89. The pharmaceutical composition of claim 71, which is used in combination with other pharmaceutical agents.

90. The pharmaceutical composition of claim 75, which is used in combination with other pharmaceutical agents.

91. A bispecific molecule that specifically binds to human HLA / NY-ESO and human CD3, the bispecific molecule comprising a first polypeptide and a second polypeptide, the first polypeptide comprising, from N-terminus to C-terminus, a scFv that specifically binds to human HLA / NY-ESO, a scFv that specifically binds to CD3, and an Fc region (i); the second polypeptide comprises a hinge region and an Fc region of an immunoglobulin (ii), The bispecific molecule associates in the Fc region (i) and in the Fc region (ii), The first polypeptide comprises: the amino acid sequence of positions 21 to 745 of the amino acid sequence shown in SEQ ID NO: 96, the amino acid sequence of positions 20 to 745 of the amino acid sequence shown in SEQ ID NO: 155, the amino acid sequence of positions 20 to 750 of the amino acid sequence shown in SEQ ID NO: 156, or the amino acid sequence of positions 20 to 750 of the amino acid sequence shown in SEQ ID NO: 157, or an amino acid sequence in which one or two amino acids are deleted from the carboxyl terminus of the amino acid sequence, The second polypeptide contains the amino acid sequence from positions 20 to 246 of the amino acid sequence shown in SEQ ID NO: 84, or an amino acid sequence in which one or two amino acids are deleted from the carboxyl terminus of the amino acid sequence.

92. A bispecific molecule that specifically binds to human HLA / NY-ESO and human CD3, the bispecific molecule comprising a first polypeptide, a second polypeptide and a third polypeptide, the first polypeptide comprising, from N-terminus to C-terminus, a scFv that specifically binds to human HLA / NY-ESO, a variable region and a constant region CH1 of an antibody heavy chain that specifically binds to CD3, and an immunoglobulin Fc region (i); the second polypeptide comprises an immunoglobulin hinge region and an Fc region (ii); the third polypeptide comprises an antibody light chain consisting of a variable region and a constant region, The first polypeptide and the second polypeptide are associated in the Fc region (i) and the Fc region (ii), the first polypeptide is associated with the third polypeptide in the variable region and the constant region CH1 of the antibody heavy chain, The first polypeptide comprises the amino acid sequence of positions 20 to 724 of the amino acid sequence shown in SEQ ID NO: 160 or the amino acid sequence of positions 20 to 719 of the amino acid sequence shown in SEQ ID NO: 197, or an amino acid sequence in which one or two amino acids are deleted from the carboxyl terminus of the amino acid sequence, The second polypeptide comprises the amino acid sequence of positions 20 to 246 of the amino acid sequence shown in SEQ ID NO: 84, or an amino acid sequence in which one or two amino acids are deleted from the carboxyl terminus of the amino acid sequence, The third polypeptide contains the amino acid sequence from positions 21 to 127 of the amino acid sequence shown in SEQ ID NO: 161, or the amino acid sequence from positions 21 to 233 of the amino acid sequence shown in SEQ ID NO:

161.

93. A pharmaceutical composition comprising the molecule of claim 91 as an active ingredient.

94. A pharmaceutical composition comprising the molecule of claim 92 as an active ingredient.

95. The pharmaceutical composition according to claim 93, which is an anticancer agent, wherein The cancer is one or more cancers selected from the group consisting of renal cancer, melanoma, thyroid cancer, lung cancer, breast cancer, esophageal cancer, gastric cancer, colorectal cancer, rectal cancer, liver cancer, bile duct cancer, bladder cancer, prostate cancer, uterine cancer, liposarcoma, chondrosarcoma, rhabdomyosarcoma, osteosarcoma, undifferentiated pleomorphic sarcoma, myxofibrosarcoma, malignant peripheral nerve sheath tumor, synovial sarcoma, uterine sarcoma, leiomyosarcoma, B-cell lymphoma, myeloid leukemia, multiple myeloma, testicular cancer and ovarian cancer.

96. The pharmaceutical composition of claim 95, wherein the lung cancer is non-small cell lung cancer or small cell lung cancer.

97. The pharmaceutical composition of claim 96, wherein the non-small cell lung cancer is adenocarcinoma, squamous cell carcinoma, or large cell carcinoma.

98. The pharmaceutical composition according to claim 94, which is an anticancer agent, wherein The cancer is one or more cancers selected from the group consisting of renal cancer, melanoma, thyroid cancer, lung cancer, breast cancer, esophageal cancer, gastric cancer, colorectal cancer, rectal cancer, liver cancer, bile duct cancer, bladder cancer, prostate cancer, uterine cancer, liposarcoma, chondrosarcoma, rhabdomyosarcoma, osteosarcoma, undifferentiated pleomorphic sarcoma, myxofibrosarcoma, malignant peripheral nerve sheath tumor, synovial sarcoma, uterine sarcoma, leiomyosarcoma, B-cell lymphoma, myeloid leukemia, multiple myeloma, testicular cancer and ovarian cancer.

99. The pharmaceutical composition of claim 98, wherein the lung cancer is non-small cell lung cancer or small cell lung cancer.

100. The pharmaceutical composition of claim 99, wherein the non-small cell lung cancer is adenocarcinoma, squamous cell carcinoma, or large cell carcinoma.

101. The pharmaceutical composition of claim 93, which is used in combination with other pharmaceutical agents.

102. The pharmaceutical composition of claim 93, comprising other pharmaceutical agents.

103. The pharmaceutical composition of claim 94, which is used in combination with other pharmaceutical agents.

104. The pharmaceutical composition of claim 94, comprising other pharmaceutical agents.

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