Bispecific antibodies

Abstract

The invention relates to bispecific antibodies. The purpose of the present invention is to provide: a novel antibody which can be used as an anti-tumor agent; and an anti-tumor agent which contains, as an active ingredient, a molecule including the antibody or the like. The antibody or the binding fragment thereof contains the following heavy chains and light chains: a heavy chain CDRH1 consisting of an amino acid sequence as shown in SEQ ID NO: 54; a heavy chain CDRH2 composed of an amino acid sequence represented by SEQ ID NO: 55; a heavy chain CDRH3 composed of an amino acid sequence represented by SEQ ID NO: 56; the light chain CDRL1 is composed of an amino acid sequence as shown in SEQ ID NO: 57, or the light chain CDRL1 is composed of an amino acid sequence with the seventh amino acid being W and / or the eighth amino acid being K in the amino acid sequence as shown in SEQ ID NO: 57; a light chain CDRL2 composed of an amino acid sequence as shown in SEQ ID NO: 58; and a light chain CDRL3 composed of an amino acid sequence as shown in SEQ ID NO: 59, or a light chain CDRL3 composed of an amino acid sequence of which the second amino acid is A or S in the amino acid sequence as shown in SEQ ID NO: 57.

Description

[0001] This application is a divisional application of the application with the filing date of March 29, 2021, application number 202180009615.9, and invention title "Bispecific Antibody". Technical Field

[0002] The present invention relates to antibodies, bispecific antibodies, etc. that can be used for treating cancer. Background Art

[0003] NY-ESO-1 is a molecule identified from esophageal cancer by the method of serological analysis of recombinant cDNA expression libraries (SEREX) (Non-Patent Document 1). The alias of LAGE-1 is also called NY-ESO-2, which is a molecule identified by representational difference analysis of tumor cDNA libraries (Non-Patent Document 2). Although it is known that these molecules are only expressed in the testis in normal tissues, their functions are not yet clear. There are reports that NY-ESO-1 and LAGE-1 are expressed in a wide variety 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 the expression of NY-ESO-1 in metastatic lesions of melanoma is higher than that in primary lesions (Non-Patent Document 4); in urothelial carcinoma, the expression of NY-ESO-1 and LAGE-1 is higher in intermediate-stage cancer than in early-stage cancer (Non-Patent Document 5); the expression of NY-ESO-1 in high-risk myeloma with chromosomal abnormalities is higher than that in myeloma with normal chromosomes (Non-Patent Document 6). From these information, it can be seen that NY-ESO-1 and LAGE-1 are attracting attention as molecules with high cancer specificity, and a large amount of research and development is being carried out as drug discovery targets for cancer vaccine therapy, but so far there is no approved drug.

[0004] It is known that the NY-ESO peptide SLLMWITQC of 9-mer at positions 157 to 165 of NY-ESO-1 and LAGE-1 forms a complex (HLA / NY-ESO peptide complex) with HLA (Histocompatibility Leukocyte Antigen)-A2 and is presented extracellularly (Non-Patent Document 7). As described above, since the expression of NY-ESO-1 and LAGE-1 is cancer-specific, it is suggested that the HLA / NY-ESO peptide complex is a cancer-specific therapeutic target present only on cancer cells that are HLA-A2 positive and NY-ESO-1 or LAGE-1 positive (Non-Patent Document 8). Moreover, as molecules that bind to the HLA / NY-ESO peptide complex, TCR (Patent Documents 1 and 2) and antibodies (Patent Documents 3 and 8) have been reported.

[0005] As one of the uses of binding molecules against cancer target molecules, there are CD3 bispecific antibodies (Non-Patent Documents 9 and 10), whose mechanism is to induce T cells to cancer cells, thereby redirecting T cells to show antitumor effects caused by cytotoxicity. As a currently marketed CD3 bispecific antibody drug, Blinatumomab, which is a BiTE (bispecfic T-cell engager) against CD19, can be cited. It has been approved for 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 is very short when the tandem scFv (taFv) without the Fc region, which is a bispecific antibody configuration, is administered to patients (Non-Patent Document 11).

[0006] As bispecific antibodies with a heterodimeric Fc region having a half-life in the blood similar to that of IgG-type antibodies, research and clinical trials of CD3 bispecific antibodies using various antibody configurations such as knobs-into-holes, CrossMAb, and DuoBody (registered trademark) (Patent Documents 4, 5, 6, and 7) are underway, and CD3 bispecific antibodies using antibodies against the HLA / NY-ESO peptide complex have been reported (Non-Patent Document 12).

[0007] Prior Art Documents

[0008] Patent Documents

[0009] Patent Document 1: WO2005 / 113595

[0010] Patent Document 2: WO2017 / 109496

[0011] Patent Document 3: WO2010 / 106431

[0012] Patent Document 4: WO1998 / 050431

[0013] Patent Document 5: WO2006 / 106905

[0014] Patent Document 6: WO2011 / 028952

[0015] Patent Document 7: WO2011 / 131746

[0016] Patent Document 8: WO2021 / 003357

[0017] Non - Patent Literature

[0018] Non - Patent Literature 1: Proc Natl Acad Sci U S A.;94(5): 1914 - 8(1997).

[0019] Non - Patent Literature 2: Int J Cancer.;76(6): 903 - 8(1998).

[0020] Non - Patent Literature 3: Immunol Cell Biol.;84(3): 303 - 17(2006).

[0021] Non - Patent Literature 4: J Surg Res.;98(2): 76 - 80(2001).

[0022] Non - Patent Literature 5: Cancer Res.;61(12): 4671 - 4(2001).

[0023] Non - Patent Literature 6: Blood.;105(10): 3939 - 44(2005).

[0024] Non - Patent Literature 7: J Exp Med.;187(2): 265 - 70(1998).

[0025] Non - Patent Literature 8: J Immunol.;176(12): 7308 - 16(2006).

[0026] Non - Patent Literature 9: Nature;314(6012); 628 - 31(1985).

[0027] Non - Patent Literature 10: Int Rev Immunol.;4(2); 159 - 73(1989).

[0028] Non-Patent Document 11: Drug Des Devel Ther.; 10; 757-765 (2016).

[0029] Non-Patent Document 12: Proceedings of the 21st Cancer Immunology Society; O13-4 Summary of the Invention

[0030]

Problems to be Solved by the Invention

[0031] An object of the present invention is to provide a novel anti-HLA-A2 / NY-ESO antibody that can be used as an anti-tumor agent, and an anti-tumor agent containing a molecule that binds to HLA-A2 / NY-ESO including the antibody as an active ingredient.

[0032]

Technical Solutions for Solving the Problems

[0033] The inventors of the present invention conducted intensive research to solve the above problems, created a novel anti-HLA-A2 / NY-ESO antibody, and a molecule that binds to HLA-A2 / NY-ESO including the antibody, thereby completing the present invention.

[0034] That is, the present invention includes the following.

[0035] [1] An antibody or a binding fragment thereof that specifically binds to human HLA / NY-ESO, and the antibody or the binding fragment thereof contains:

[0036] Heavy chain CDRH1 consisting of the amino acid sequence shown in SEQ ID NO:54;

[0037] Heavy chain CDRH2 consisting of the amino acid sequence shown in SEQ ID NO:55;

[0038] Heavy chain CDRH3 consisting of the amino acid sequence shown in SEQ ID NO:56;

[0039] Light chain CDRL1 consisting of the amino acid sequence shown in SEQ ID NO:57, or 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 and / or the 8th amino acid is K;

[0040] Light chain CDRL2 consisting of the amino acid sequence shown in SEQ ID NO:58; and,

[0041] Light chain CDRL3 consisting of the amino acid sequence shown in SEQ ID NO:59, or light chain CDRL3 consisting of an amino acid sequence in which the 2nd amino acid in the amino acid sequence shown in SEQ ID NO:59 is A or S.

[0042] [2] The antibody or binding fragment thereof according to [1], wherein the antibody or binding fragment thereof comprises one or more sets selected from the group consisting of the following (i) to (v) for CDRH1 to CDRH3 and CDRL1 to CDRL3:

[0043] (i) Heavy chain CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 54;

[0044] Heavy chain CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 55;

[0045] Heavy chain CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 56;

[0046] Light chain CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 57;

[0047] Light chain CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 58; and,

[0048] Light chain CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 59;

[0049] (ii) Heavy chain CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 54;

[0050] Heavy chain CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 55;

[0051] Heavy chain CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 56;

[0052] Light chain CDRL1 consisting of the amino acid sequence in SEQ ID NO: 57 where the 7th amino acid is W;

[0053] Light chain CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 58; and,

[0054] Light chain CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 59;

[0055] (iii) Heavy chain CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 54;

[0056] Heavy chain CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 55;

[0057] Heavy chain CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 56;

[0058] The light chain CDRL1 consisting of the amino acid sequence shown in SEQ ID NO:57;

[0059] The light chain CDRL2 consisting of the amino acid sequence shown in SEQ ID NO:58; and,

[0060] The light chain CDRL3 consisting of the amino acid sequence in which the 2nd amino acid in the amino acid sequence shown in SEQ ID NO:59 is A;

[0061] (iv) The heavy chain CDRH1 consisting of the amino acid sequence shown in SEQ ID NO:54;

[0062] The heavy chain CDRH2 consisting of the amino acid sequence shown in SEQ ID NO:55;

[0063] The heavy chain CDRH3 consisting of the amino acid sequence shown in SEQ ID NO:56;

[0064] The light chain CDRL1 consisting of the amino acid sequence shown in SEQ ID NO:57;

[0065] The light chain CDRL2 consisting of the amino acid sequence shown in SEQ ID NO:58; and,

[0066] The light chain CDRL3 consisting of the amino acid sequence in which the 2nd amino acid in the amino acid sequence shown in SEQ ID NO:59 is S; and,

[0067] (v) The heavy chain CDRH1 consisting of the amino acid sequence shown in SEQ ID NO:54;

[0068] The heavy chain CDRH2 consisting of the amino acid sequence shown in SEQ ID NO:55;

[0069] The heavy chain CDRH3 consisting of the amino acid sequence shown in SEQ ID NO:56;

[0070] The light chain CDRL1 consisting of the amino acid sequence in which the 7th amino acid is W and the 8th amino acid is K in the amino acid sequence shown in SEQ ID NO:57;

[0071] The light chain CDRL2 consisting of the amino acid sequence shown in SEQ ID NO:58; and,

[0072] The light chain CDRL3 consisting of the amino acid sequence shown in SEQ ID NO:59.

[0073] [3] The antibody or its binding fragment according to [1], wherein the antibody or its binding fragment comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region consists of the amino acid sequence from amino acid number 21 to 140 of the amino acid sequence shown in SEQ ID NO: 27, or an amino acid sequence having a sequence identity of more than 95% with the amino acid sequence shown in SEQ ID NO: 38 or SEQ ID NO: 39; the light chain variable region consists of the amino acid sequence from amino acid number 156 to 266 of the amino acid sequence shown in SEQ ID NO: 27 or SEQ ID NO: 52, or an amino acid sequence having a sequence identity of more than 95% with the amino acid sequence shown in SEQ ID NO: 40.

[0074] [4] The antibody or its binding fragment according to [1], wherein the antibody or its binding fragment comprises the following heavy chain variable region and light chain variable region:

[0075] (H1) A heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 6;

[0076] (H2) A heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 18;

[0077] (H3) A heavy chain variable region consisting of the amino acid sequence from position 21 to position 140 of the amino acid sequence shown in SEQ ID NO: 29;

[0078] (H4) A heavy chain variable region consisting of the amino acid sequence from position 21 to position 140 of the amino acid sequence shown in SEQ ID NO: 26;

[0079] (H5) A heavy chain variable region consisting of the amino acid sequence from position 21 to position 140 of the amino acid sequence shown in SEQ ID NO: 27;

[0080] (H6) A heavy chain variable region consisting of the amino acid sequence from position 21 to position 140 of the amino acid sequence shown in SEQ ID NO: 28;

[0081] (H7) A heavy chain variable region consisting of the amino acid sequence from position 21 to position 140 of the amino acid sequence shown in SEQ ID NO: 36;

[0082] (H8) A heavy chain variable region consisting of the amino acid sequence from position 21 to position 140 of the amino acid sequence shown in SEQ ID NO: 47;

[0083] (H9) A heavy chain variable region consisting of the amino acid sequence from position 21 to position 140 of the amino acid sequence shown in SEQ ID NO: 48;

[0084] (H10) The heavy chain variable region consisting of the amino acid sequence from position 21 to position 140 of the amino acid sequence shown in SEQ ID NO:50;

[0085] (H11) The heavy chain variable region consisting of the amino acid sequence from position 21 to position 140 of the amino acid sequence shown in SEQ ID NO:51;

[0086] (H12) The heavy chain variable region consisting of the amino acid sequence from position 21 to position 140 of the amino acid sequence shown in SEQ ID NO:52;

[0087] (H13) The heavy chain variable region consisting of the amino acid sequence from position 21 to position 140 of the amino acid sequence shown in SEQ ID NO:53;

[0088] (H14) The heavy chain variable region consisting of the amino acid sequence from position 21 to position 140 of the amino acid sequence shown in SEQ ID NO:30; or,

[0089] (H15) The heavy chain variable region consisting of the amino acid sequence from position 21 to position 140 of the amino acid sequence shown in SEQ ID NO:156; and,

[0090] (L1) The light chain variable region consisting of the amino acid sequence shown in SEQ ID NO:8;

[0091] (L2) The light chain variable region consisting of the amino acid sequence shown in SEQ ID NO:20;

[0092] (L3) The light chain variable region consisting of the amino acid sequence from position 156 to position 266 of the amino acid sequence shown in SEQ ID NO:29;

[0093] (L4) The light chain variable region consisting of the amino acid sequence from position 156 to position 266 of the amino acid sequence shown in SEQ ID NO:26;

[0094] (L5) The light chain variable region consisting of the amino acid sequence from position 156 to position 266 of the amino acid sequence shown in SEQ ID NO:27;

[0095] (L6) The light chain variable region consisting of the amino acid sequence from position 156 to position 266 of the amino acid sequence shown in SEQ ID NO:28;

[0096] (L7) The light chain variable region consisting of the amino acid sequence from position 156 to position 266 of the amino acid sequence shown in SEQ ID NO:36;

[0097] (L8) A light chain variable region consisting of the amino acid sequence from position 156 to position 266 of the amino acid sequence shown in SEQ ID NO:47;

[0098] (L9) A light chain variable region consisting of the amino acid sequence from position 156 to position 266 of the amino acid sequence shown in SEQ ID NO:48;

[0099] (L10) A light chain variable region consisting of the amino acid sequence from position 156 to position 266 of the amino acid sequence shown in SEQ ID NO:50;

[0100] (L11) A light chain variable region consisting of the amino acid sequence from position 156 to position 266 of the amino acid sequence shown in SEQ ID NO:51;

[0101] (L12) A light chain variable region consisting of the amino acid sequence from position 156 to position 266 of the amino acid sequence shown in SEQ ID NO:52;

[0102] (L13) A light chain variable region consisting of the amino acid sequence from position 156 to position 266 of the amino acid sequence shown in SEQ ID NO:53;

[0103] (L14) A light chain variable region consisting of the amino acid sequence from position 156 to position 266 of the amino acid sequence shown in SEQ ID NO:30; or,

[0104] (L15) A light chain variable region consisting of the amino acid sequence from position 161 to position 271 of the amino acid sequence shown in SEQ ID NO:156.

[0105] [5] The antibody or its binding fragment according to [4], wherein the antibody or its binding fragment comprises the following heavy chain variable region and light chain variable region:

[0106] (H1L1) A heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO:6, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO:8;

[0107] (H2L2) 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;

[0108] (H3L3) A heavy chain variable region consisting of the amino acid sequence from position 21 to position 140 of the amino acid sequence shown in SEQ ID NO:29, and a light chain variable region consisting of the amino acid sequence from position 156 to position 266 of the amino acid sequence shown in SEQ ID NO:29;

[0109] (H4L4) The heavy chain variable region consisting of the amino acid sequence from position 21 to position 140 of the amino acid sequence shown in SEQ ID NO: 26, and the light chain variable region consisting of the amino acid sequence from position 156 to position 266 of the amino acid sequence shown in SEQ ID NO: 26;

[0110] (H5L5) The heavy chain variable region consisting of the amino acid sequence from position 21 to position 140 of the amino acid sequence shown in SEQ ID NO: 27, and the light chain variable region consisting of the amino acid sequence from position 156 to position 266 of the amino acid sequence shown in SEQ ID NO: 27;

[0111] (H6L6) The heavy chain variable region consisting of the amino acid sequence from position 21 to position 140 of the amino acid sequence shown in SEQ ID NO: 28, and the light chain variable region consisting of the amino acid sequence from position 156 to position 266 of the amino acid sequence shown in SEQ ID NO: 28;

[0112] (H7L7) The heavy chain variable region consisting of the amino acid sequence from position 21 to position 140 of the amino acid sequence shown in SEQ ID NO: 36, and the light chain variable region consisting of the amino acid sequence from position 156 to position 266 of the amino acid sequence shown in SEQ ID NO: 36;

[0113] (H8L8) The heavy chain variable region consisting of the amino acid sequence from position 21 to position 140 of the amino acid sequence shown in SEQ ID NO: 47, and the light chain variable region consisting of the amino acid sequence from position 156 to position 266 of the amino acid sequence shown in SEQ ID NO: 47;

[0114] (H9L9) The heavy chain variable region consisting of the amino acid sequence from position 21 to position 140 of the amino acid sequence shown in SEQ ID NO: 48, and the light chain variable region consisting of the amino acid sequence from position 156 to position 266 of the amino acid sequence shown in SEQ ID NO: 48;

[0115] (H10L10) The heavy chain variable region consisting of the amino acid sequence from position 21 to position 140 of the amino acid sequence shown in SEQ ID NO: 50, and the light chain variable region consisting of the amino acid sequence from position 156 to position 266 of the amino acid sequence shown in SEQ ID NO: 50;

[0116] (H11L11) A heavy chain variable region consisting of the amino acid sequence from position 21 to position 140 of the amino acid sequence shown in SEQ ID NO:51, and a light chain variable region consisting of the amino acid sequence from position 156 to position 266 of the amino acid sequence shown in SEQ ID NO:51;

[0117] (H12L12) A heavy chain variable region consisting of the amino acid sequence from position 21 to position 140 of the amino acid sequence shown in SEQ ID NO:52, and a light chain variable region consisting of the amino acid sequence from position 156 to position 266 of the amino acid sequence shown in SEQ ID NO:52;

[0118] (H13L13) A heavy chain variable region consisting of the amino acid sequence from position 21 to position 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 position 156 to position 266 of the amino acid sequence shown in SEQ ID NO:53;

[0119] (H14L14) A heavy chain variable region consisting of the amino acid sequence from position 21 to position 140 of the amino acid sequence shown in SEQ ID NO:30, and a light chain variable region consisting of the amino acid sequence from position 156 to position 266 of the amino acid sequence shown in SEQ ID NO:30; or,

[0120] (H15L15) A heavy chain variable region consisting of the amino acid sequence from position 21 to position 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 position 161 to position 271 of the amino acid sequence shown in SEQ ID NO:156.

[0121] [6] The antibody or its binding fragment according to any one of [1] to [5], wherein the antibody or its binding fragment is a scFv.

[0122] [7] The antibody or its binding fragment according to [6], wherein the antibody or its binding fragment is the following scFv:

[0123] (s1) A scFv consisting of the amino acid sequence from position 21 to position 266 of the amino acid sequence shown in SEQ ID NO:70;

[0124] (s2) A scFv containing 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;

[0125] (s3) An scFv consisting of the amino acid sequence from position 21 to position 266 of the amino acid sequence shown in SEQ ID NO:29;

[0126] (s4) An scFv consisting of the amino acid sequence from position 21 to position 266 of the amino acid sequence shown in SEQ ID NO:26;

[0127] (s5) An scFv consisting of the amino acid sequence from position 21 to position 266 of the amino acid sequence shown in SEQ ID NO:27;

[0128] (s6) An scFv consisting of the amino acid sequence from position 21 to position 266 of the amino acid sequence shown in SEQ ID NO:28;

[0129] (s7) An scFv consisting of the amino acid sequence from position 21 to position 266 of the amino acid sequence shown in SEQ ID NO:36;

[0130] (s8) An scFv consisting of the amino acid sequence from position 21 to position 266 of the amino acid sequence shown in SEQ ID NO:47;

[0131] (s9) An scFv consisting of the amino acid sequence from position 21 to position 266 of the amino acid sequence shown in SEQ ID NO:48;

[0132] (s10) An scFv consisting of the amino acid sequence from position 21 to position 266 of the amino acid sequence shown in SEQ ID NO:50;

[0133] (s11) An scFv consisting of the amino acid sequence from position 21 to position 266 of the amino acid sequence shown in SEQ ID NO:51;

[0134] (s12) An scFv consisting of the amino acid sequence from position 21 to position 266 of the amino acid sequence shown in SEQ ID NO:52;

[0135] (s13) An scFv consisting of the amino acid sequence from position 21 to position 266 of the amino acid sequence shown in SEQ ID NO:53;

[0136] (s14) An scFv consisting of the amino acid sequence from position 21 to position 266 of the amino acid sequence shown in SEQ ID NO:30; or,

[0137] (s15) An scFv consisting of the amino acid sequence from position 21 to position 271 of the amino acid sequence shown in SEQ ID NO:156.

[0138] [8] A polynucleotide that encodes the antibody or its binding fragment as described in any one of [1] to [7].

[0139] [9] A vector that contains the polynucleotide as described in [8].

[0140]

[10] A host cell that contains the polynucleotide as described in [8] or the vector as described in [9].

[0141]

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

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

[0142]

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

[11] .

[0143]

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

[0144] (i) Binding to the site on HLA-A2 / NY-ESO recognized by the antibody or its binding fragment as described in [7];

[0145] (ii) Competing with the antibody or its binding fragment as described in [7] for binding to human HLA-A2 / NY-ESO.

[0146]

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

[12] , and

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

[10] .

[0147]

[15] A molecule that specifically binds to human HLA / NY-ESO, the molecule containing the antibody or its binding fragment as described in any one of [1] to [7],

[12] , and

[13] .

[0148]

[16] The molecule according to

[15] , wherein the molecule is a multispecific antibody.

[0149]

[17] The molecule according to

[15] , wherein the molecule is a bispecific antibody.

[0150]

[18] The molecule according to any one of

[15] to

[17] , wherein the molecule contains an antibody or its binding fragment that specifically binds to CD3.

[0151]

[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 and contains the following chains:

[0152] (CCH1) A heavy chain CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 141;

[0153] (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 in which the 3rd amino acid in the amino acid sequence shown in SEQ ID NO: 142 is N or S;

[0154] (CCH3) A heavy chain CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 143;

[0155] (CCL1) A light chain CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 144;

[0156] (CCL2) A light chain CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 145, or a light chain CDRL2 consisting of an amino acid sequence in which the 2nd amino acid in the amino acid sequence shown in SEQ ID NO: 145 is N; and,

[0157] (CCL3) A light chain CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 146.

[0158]

[20] The molecule according to

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

[0159] (CH1) A heavy chain variable region consisting of the amino acid sequence from the 2nd to the 119th position of the amino acid sequence shown in SEQ ID NO: 136;

[0160] (CH2) A heavy chain variable region consisting of the amino acid sequence from the 2nd to the 119th position of the amino acid sequence shown in SEQ ID NO: 137;

[0161] (CH3) A heavy chain variable region consisting of the amino acid sequence from the 2nd to the 119th position of the amino acid sequence shown in SEQ ID NO: 147;

[0162] (CH4) A heavy chain variable region consisting of the amino acid sequence from the 2nd to the 119th position of the amino acid sequence shown in SEQ ID NO: 138;

[0163] (CH5) The heavy chain variable region consisting of the amino acid sequence from position 2 to position 119 of the amino acid sequence shown in SEQ ID NO: 139;

[0164] (CH6) The heavy chain variable region consisting of the amino acid sequence from position 2 to position 119 of the amino acid sequence shown in SEQ ID NO: 140;

[0165] (CH7) The heavy chain variable region consisting of the amino acid sequence from position 272 to position 389 of the amino acid sequence shown in SEQ ID NO: 155;

[0166] (CH8) The heavy chain variable region consisting of the amino acid sequence from position 277 to position 394 of the amino acid sequence shown in SEQ ID NO: 156; or,

[0167] (CH9) The heavy chain variable region consisting of the amino acid sequence from position 277 to position 394 of the amino acid sequence shown in SEQ ID NO: 157; and,

[0168] (CL1) The light chain variable region consisting of the amino acid sequence from position 135 to position 243 of the amino acid sequence shown in SEQ ID NO: 136;

[0169] (CL2) The light chain variable region consisting of the amino acid sequence from position 135 to position 241 of the amino acid sequence shown in SEQ ID NO: 137;

[0170] (CL3) The light chain variable region consisting of the amino acid sequence from position 135 to position 243 of the amino acid sequence shown in SEQ ID NO: 147;

[0171] (CL4) The light chain variable region consisting of the amino acid sequence from position 135 to position 241 of the amino acid sequence shown in SEQ ID NO: 138;

[0172] (CL5) The light chain variable region consisting of the amino acid sequence from position 135 to position 243 of the amino acid sequence shown in SEQ ID NO: 139;

[0173] (CL6) The light chain variable region consisting of the amino acid sequence from position 135 to position 243 of the amino acid sequence shown in SEQ ID NO: 140;

[0174] (CL7) The light chain variable region consisting of the amino acid sequence from position 405 to position 511 of the amino acid sequence shown in SEQ ID NO: 155;

[0175] (CL8) A light chain variable region consisting of the amino acid sequence from position 410 to position 516 of the amino acid sequence shown in SEQ ID NO: 156; or,

[0176] (CL9) A light chain variable region consisting of the amino acid sequence from position 410 to position 516 of the amino acid sequence shown in SEQ ID NO: 157.

[0177]

[21] The molecule according to

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

[0178] (CH1CL1) A heavy chain variable region consisting of the amino acid sequence from position 2 to position 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 position 135 to position 243 of the amino acid sequence shown in SEQ ID NO: 136;

[0179] (CH2CL2) A heavy chain variable region consisting of the amino acid sequence from position 2 to position 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 position 135 to position 241 of the amino acid sequence shown in SEQ ID NO: 137;

[0180] (CH3CL3) A heavy chain variable region consisting of the amino acid sequence from position 2 to position 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 position 135 to position 243 of the amino acid sequence shown in SEQ ID NO: 147;

[0181] (CH4CL4) A heavy chain variable region consisting of the amino acid sequence from position 2 to position 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 position 135 to position 241 of the amino acid sequence shown in SEQ ID NO: 138;

[0182] (CH5CL5) A heavy chain variable region consisting of the amino acid sequence from position 2 to position 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 position 135 to position 243 of the amino acid sequence shown in SEQ ID NO: 139;

[0183] (CH6CL6) A heavy chain variable region consisting of the amino acid sequence from position 2 to position 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 position 135 to position 243 of the amino acid sequence shown in SEQ ID NO: 140;

[0184] (CH7CL7) The heavy chain variable region consisting of the amino acid sequence from position 272 to position 389 of the amino acid sequence shown in SEQ ID NO: 155, and the light chain variable region consisting of the amino acid sequence from position 405 to position 511 of the amino acid sequence shown in SEQ ID NO: 155;

[0185] (CH8CL8) The heavy chain variable region consisting of the amino acid sequence from position 277 to position 394 of the amino acid sequence shown in SEQ ID NO: 156, and the light chain variable region consisting of the amino acid sequence from position 410 to position 516 of the amino acid sequence shown in SEQ ID NO: 156; or,

[0186] (CH9CL9) The heavy chain variable region consisting of the amino acid sequence from position 277 to position 394 of the amino acid sequence shown in SEQ ID NO: 157, and the light chain variable region consisting of the amino acid sequence from position 410 to position 516 of the amino acid sequence shown in SEQ ID NO: 157.

[0187]

[22] The molecule according to any one of

[18] to

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

[0188]

[23] The molecule according to

[22] , wherein the scFv includes:

[0189] (CS1) The scFv consisting of the amino acid sequence from position 2 to position 243 of the amino acid sequence shown in SEQ ID NO: 136;

[0190] (CS2) The scFv consisting of the amino acid sequence from position 2 to position 241 of the amino acid sequence shown in SEQ ID NO: 137;

[0191] (CS3) The scFv consisting of the amino acid sequence from position 2 to position 243 of the amino acid sequence shown in SEQ ID NO: 147;

[0192] (CS4) The scFv consisting of the amino acid sequence from position 2 to position 241 of the amino acid sequence shown in SEQ ID NO: 138;

[0193] (CS5) The scFv consisting of the amino acid sequence from position 2 to position 243 of the amino acid sequence shown in SEQ ID NO: 139;

[0194] (CS6) The scFv consisting of the amino acid sequence from position 2 to position 243 of the amino acid sequence shown in SEQ ID NO: 140;

[0195] (CS7) An scFv consisting of the amino acid sequence from position 272 to position 511 of the amino acid sequence shown in SEQ ID NO: 155;

[0196] (CS8) An scFv consisting of the amino acid sequence from position 277 to position 516 of the amino acid sequence shown in SEQ ID NO: 156; or,

[0197] (CS9) An scFv consisting of the amino acid sequence from position 277 to position 516 of the amino acid sequence shown in SEQ ID NO: 157.

[0198]

[24] The molecule according to any one of

[18] to

[23] , the molecule comprising a first polypeptide and a second polypeptide, the first polypeptide sequentially containing 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 contains an Fc region (ii); preferably, the molecule associates in the Fc region (i) and the Fc region (ii).

[0199]

[25] The molecule according to

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

[0200]

[26] The molecule according to

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

[0201]

[27] The molecule according to

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

[0202]

[28] The molecule according to

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

[0203]

[29] The molecule according to

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

[0204]

[30] The molecule according to any one of

[19] to

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

[18] of the scFv.

[0205]

[31] The molecule according to any one of

[19] to

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

[19] of the scFv.

[0206]

[32] The molecule according to any one of

[19] to

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

[20] or

[21] of the scFv.

[0207]

[33] The molecule according to any one of

[19] to

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

[23] of the scFv.

[0208]

[34] The molecule according to any one of

[24] to

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

[0209]

[35] The molecule according to any one of

[24] to

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

[0210]

[36] The molecule according to any one of

[24] to

[34] , wherein the first polypeptide contains the amino acid sequence from position 529 to position 745 of the amino acid sequence shown 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 or SEQ ID NO: 150.

[0211]

[37] The molecule according to any one of

[24] to

[33] and

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

[0212]

[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 from position 20 to position 745 of the amino acid sequence shown in SEQ ID NO: 85, the amino acid sequence from position 20 to position 745 of the amino acid sequence shown in SEQ ID NO: 87, the amino acid sequence from position 20 to position 745 of the amino acid sequence shown in SEQ ID NO: 88, the amino acid sequence from position 20 to position 745 of the amino acid sequence shown in SEQ ID NO: 89, the amino acid sequence from position 20 to position 745 of the amino acid sequence shown in SEQ ID NO: 90, the amino acid sequence from position 20 to position 745 of the amino acid sequence shown in SEQ ID NO: 91, the amino acid sequence from position 20 to position 745 of the amino acid sequence shown in SEQ ID NO: 92, the amino acid sequence from position 20 to position 745 of the amino acid sequence shown in SEQ ID NO: 93, the amino acid sequence from position 20 to position 745 of the amino acid sequence shown in SEQ ID NO: 94, the amino acid sequence from position 20 to position 745 of the amino acid sequence shown in SEQ ID NO: 95, the amino acid sequence from position 20 to position 745 of the amino acid sequence shown in SEQ ID NO: 96, the amino acid sequence from position 20 to position 745 of the amino acid sequence shown in SEQ ID NO: 86, the amino acid sequence from position 20 to position 745 of the amino acid sequence shown in SEQ ID NO: 149, and the amino acid sequence from position 20 to position 745 of the amino acid sequence shown in SEQ ID NO: 150.

[0213]

[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 from position 20 to position 745 of the amino acid sequence shown in SEQ ID NO: 155, the amino acid sequence from position 20 to position 750 of the amino acid sequence shown in SEQ ID NO: 156; or the amino acid sequence from position 20 to position 750 of the amino acid sequence shown in SEQ ID NO: 157.

[0214]

[40] The molecule according to any one of

[24] to

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

[0215]

[41] The molecule according to any one of

[18] to

[23] , the molecule contains a first polypeptide, a second polypeptide and a third polypeptide,

[0216] The first polypeptide contains, in sequence 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);

[0217] The second polypeptide consists of an immunoglobulin heavy chain containing an Fc region (ii);

[0218] The third polypeptide consists of an immunoglobulin light chain,

[0219] Preferably, the second polypeptide and the third polypeptide associate,

[0220] The first polypeptide and the second polypeptide associate in their respective Fc regions.

[0221]

[42] The molecule according to

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

[0222]

[43] The molecule according to

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

[0223]

[44] The molecule according to

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

[0224]

[45] The molecule according to

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

[0225]

[46] The molecule according to

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

[0226]

[47] The molecule according to any one of

[42] to

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

[18] of the scFv.

[0227]

[48] The molecule according to any one of

[42] to

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

[19] of the scFv.

[0228]

[49] The molecule according to any one of

[42] to

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

[20] or

[21] of the scFv.

[0229]

[50] The molecule according to any one of

[42] to

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

[23] of the scFv.

[0230]

[51] The molecule according to any one of

[41] to

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

[0231]

[52] The molecule according to any one of

[41] to

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

[0232]

[53] The molecule according to any one of

[41] to

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

[0233]

[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 from position 20 to position 745 of the amino acid sequence shown in SEQ ID NO: 85, the amino acid sequence from position 20 to position 745 of the amino acid sequence shown in SEQ ID NO: 87, the amino acid sequence from position 20 to position 745 of the amino acid sequence shown in SEQ ID NO: 88, the amino acid sequence from position 20 to position 745 of the amino acid sequence shown in SEQ ID NO: 89, the amino acid sequence from position 20 to position 745 of the amino acid sequence shown in SEQ ID NO: 90, the amino acid sequence from position 20 to position 745 of the amino acid sequence shown in SEQ ID NO: 91, the amino acid sequence from position 20 to position 745 of the amino acid sequence shown in SEQ ID NO: 92, the amino acid sequence from position 20 to position 745 of the amino acid sequence shown in SEQ ID NO: 93, the amino acid sequence from position 20 to position 745 of the amino acid sequence shown in SEQ ID NO: 94, the amino acid sequence from position 20 to position 745 of the amino acid sequence shown in SEQ ID NO: 95, the amino acid sequence from position 20 to position 745 of the amino acid sequence shown in SEQ ID NO: 96, the amino acid sequence from position 20 to position 745 of the amino acid sequence shown in SEQ ID NO: 86, the amino acid sequence from position 20 to position 745 of the amino acid sequence shown in SEQ ID NO: 149, and the amino acid sequence from position 20 to position 745 of the amino acid sequence shown in SEQ ID NO: 150.

[0234]

[55] The molecule according to any one of

[18] to

[23] , the molecule contains a first polypeptide, a second polypeptide and a third polypeptide, the first polypeptide contains, from the N-terminus to the C-terminus, an scFv that specifically binds to human HLA / NY-ESO, the variable region and the constant region CH1 of the heavy chain of an antibody 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; 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 heavy chain of the antibody.

[0235]

[56] The molecule according to

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

[0236]

[57] The molecule according to

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

[0237]

[58] The molecule according to

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

[0238]

[59] The molecule according to

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

[0239]

[60] The molecule according to

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

[0240]

[61] The molecule according to any one of

[18] to

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

[0241]

[62] The molecule according to any one of

[56] to

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

[18] of the Fab.

[0242]

[63] The molecule according to any one of

[56] to

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

[19] of the Fab.

[0243]

[64] The molecule according to any one of

[56] to

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

[20] or

[21] of the Fab.

[0244]

[65] The molecule according to any one of

[55] to

[64] , wherein the first polypeptide contains the amino acid sequence from the 21st to the 394th amino acid sequence shown in SEQ ID NO:160.

[0245]

[66] The molecule according to any one of

[55] to

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

[0246] (i) The amino acid sequence from position 20 to position 724 of the amino acid sequence shown in SEQ ID NO: 160;

[0247] (ii) The amino acid sequence from position 20 to position 719 of the amino acid sequence shown in SEQ ID NO: 197;

[0248] (iii) The amino acid sequence from position 20 to position 719 of the amino acid sequence shown in SEQ ID NO: 198.

[0249]

[67] The molecule according to any one of

[55] to

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

[0250]

[68] The molecule according to any one of

[56] to

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

[0251]

[69] The molecule according to any one of

[56] to

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

[0252]

[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 deleted from the carboxyl terminus of the sequence.

[0253]

[71] A polynucleotide, which contains a nucleotide sequence encoding the amino acid sequence contained in the molecule according to any one of

[15] to

[70] .

[0254]

[72] A vector, which contains the polynucleotide described in

[71] .

[0255]

[73] A host cell, which contains the polynucleotide described in

[71] or the vector described in

[72] .

[0256]

[74] 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 described in

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

[0257]

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

[74] .

[0258]

[76] A pharmaceutical composition, the pharmaceutical composition containing the following as active ingredients: the molecule described in any one of

[15] to

[70] and

[75] ; the polynucleotide described in

[71] ; the carrier described in

[72] ; or the host cell described in

[73] .

[0259]

[77] The pharmaceutical composition according to

[14] or

[76] , the pharmaceutical composition being an anticancer agent.

[0260]

[78] The pharmaceutical composition according to

[77] , wherein the cancer is one or more cancers selected from the group consisting of: renal cancer, melanoma, squamous cell carcinoma, basal cell carcinoma, conjunctival carcinoma, 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, appendiceal cancer, anal cancer, liver cancer, gallbladder cancer, cholangiocarcinoma, pancreatic cancer, adrenal cancer, bladder cancer, prostate cancer, uterine cancer, vaginal cancer, liposarcoma, angiosarcoma, chondrosarcoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, undifferentiated pleomorphic sarcoma, 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 disease, myelodysplastic syndrome, multiple myeloma, testicular cancer and ovarian cancer.

[0261]

[79] The pharmaceutical composition according to any one of

[76] to

[78] , the pharmaceutical composition being used in combination with other medicaments.

[0262] This specification incorporates the disclosure of Japanese Patent Application No. 2020-061476, which is the basis of the priority of this application.

[0263] Advantages of the Invention

[0264] According to the present invention, an antibody that binds to HLA-A2 / NY-ESO, and a novel bispecific antibody (bispecific molecule) that binds to HLA-A2 / NY-ESO and binds to CD3 can be obtained. In addition, a novel pharmaceutical composition containing such an antibody (molecule) as an active ingredient can also be obtained. This antibody or this molecule, etc. has cytotoxic activity and can be used as a therapeutic or prophylactic agent for cancers and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0265] Figure 1It is a table showing the normalized gMFI of anti-HLA / NY-ESO scFv NYA-0001, 1143, 1154, 1163, 2023, 2027, 2035, 2044, 2045, 2047, 2048, 2060, 2061, 2143, NYC-0003, 0004 for T2 cells added with various point mutant peptides. * / Underline indicates that it is less than half of the normalized gMFI of each scFv for T2 cells added with NY-ESO peptide.

[0266] Figure 2A It is a table showing the information of selected homologous peptides. The binding to HLA-A0201 was predicted using NetMHCPan2.8 and is represented by the 50% inhibitory concentration (IC 50 )

[0267] Figure 2B It is a table showing the normalized gMFI of anti-HLA / NY-ESO scFv NYA-0001, 1143, 1154, 1163, 2023, 2027, 2035, 2044, 2045, 2047, 2048, 2060, 2061, 2143, NYC-0003, 0004 for T2 cells added with various homologous peptides. * / Underline indicates that it is a larger value compared to the normalized gMFI of each scFv for T2 cells added with DMSO.

[0268] Figure 3This is a diagram showing the configurations of the antibodies shown in this embodiment. (a) scFv: It is a configuration in which the variable region of the antibody heavy chain (VH, described later) and the variable region of the antibody light chain (VL, described later) (both are white) are connected by a linker. In this embodiment, scFvs against HLA-A2 / NY-ESO and CD3 were evaluated. (b) Fab: It is a configuration composed of VH (white), the constant region of the antibody heavy chain (CH1: checker pattern), VL (white), and the constant region of the antibody light chain (horizontal lines). In this embodiment, anti-HLA-A2 / NY-ESO Fab, etc. were evaluated. (c) taFv: It is a configuration in which two scFvs (white and upper right diagonal lines) are connected by a linker. In this embodiment, taFvs containing anti-HLA-A2 / NY-ESO scFv and anti-CD3 scFv were evaluated. (d) taFv-heterodimer Fc type: It is a configuration formed by adding an Fc (upper left diagonal line) (also called the first polypeptide) introducing a mutation for forming a heterodimer to the C-terminal side of taFv and associating it with another Fc (blackened: also called the second polypeptide). In this embodiment, taFv-heterodimer Fc containing anti-HLA-A2 / NY-ESO scFv and anti-CD3 scFv was evaluated. (e) taFv-Fab-heterodimer Fc type: It is a configuration obtained by adding Fab to the above taFv-heterodimer Fc type. In this embodiment, taFvs containing anti-HLA-A2 / NY-ESO scFv and anti-CD3 scFv, and taFv-Fab-heterodimer Fc using HLA-A2 / NY-ESO Fab were evaluated. (f) Represents the first polypeptide common to taFv-heterodimer Fc type and taFv-Fab-heterodimer Fc type. The first polypeptide sequentially contains an scFv specifically binding to human HLA / NY-ESO, an scFv specifically binding to CD3, and an Fc region (i) from the N-terminal to the C-terminal. (g) Represents the second polypeptide of taFv-heterodimer Fc type. The second polypeptide contains a hinge region and an Fc region (ii). (h) Represents the second polypeptide of taFv-Fab-heterodimer Fc type. The second polypeptide contains a hinge region and an immunoglobulin heavy chain including an Fc region (ii). (i) Represents the third polypeptide of taFv-Fab-heterodimer Fc type. The third polypeptide contains an immunoglobulin light chain.

[0269] Figure 4AIt is a figure showing that, in the co-presence of human PBMCs, 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. The error bars in the figure represent the standard deviation (n = 3).

[0270] Figure 4B It is a figure showing that, in the co-presence of human PBMCs, 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. The error bars in the figure represent the standard deviation (n = 3).

[0271] Figure 4C It is a figure showing that, in the co-presence of human PBMCs, 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. The error bars in the figure represent the standard deviation (n = 3).

[0272] Figure 4D It is a figure showing that, in the co-presence of human PBMCs, 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. The error bars in the figure represent the standard deviation (n = 3).

[0273] Figure 4E It is a figure showing that, in the co-presence of human PBMCs, 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. The error bars in the figure represent the standard deviation (n = 3).

[0274] Figure 4FThis figure shows the cytotoxic activity of the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules NYF-0023, NYF-0045, NYF-0048, NYF-0060, and NYF-0061 against NCI-H1703 cells expressing endogenous human NY-ESO in the presence of human PBMC. The error bars in the figure represent the standard deviation (n = 3).

[0275] Figure 4G This figure 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 did not exhibit cytotoxic activity against AGS cells that do not express endogenous human NY-ESO in the presence of human PBMC. The error bars in the figure represent the standard deviation (n = 3).

[0276] Figure 4H This figure shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules NYF-0016 and NYF-0058 did not exhibit cytotoxic activity against AGS cells that do not express endogenous human NY-ESO in the presence of human PBMC. The error bars in the figure represent the standard deviation (n = 3).

[0277] Fig. 4I This figure 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 did not exhibit cytotoxic activity against AGS cells that do not express endogenous human NY-ESO in the presence of human PBMC. The error bars in the figure represent the standard deviation (n = 3).

[0278] Figure 4J This figure 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 did not exhibit cytotoxic activity against CFPAC-1 cells that do not express endogenous human NY-ESO in the presence of human PBMC. The error bars in the figure represent the standard deviation (n = 3).

[0279] Figure 4KThis figure shows that in the co - existence of human PBMCs, the Fc - added anti - HLA - A2 / NY - ESO - anti - CD3 bispecific molecules NYF - 0016 and NYF - 0058 did not exhibit cytotoxic activity against CFPAC - 1 cells that do not express endogenous human NY - ESO. The error bars in the figure represent the standard deviation (n = 3).

[0280] Figure 4L This figure shows that in the co - existence of human PBMCs, the Fc - added anti - HLA - A2 / NY - ESO - anti - CD3 bispecific molecules NYF - 0023, NYF - 0045, NYF - 0048, NYF - 0060, and NYF - 0061 did not exhibit cytotoxic activity against CFPAC - 1 cells that do not express endogenous human NY - ESO. The error bars in the figure represent the standard deviation (n = 3).

[0281] Figure 5A This figure shows that the Fc - added anti - HLA - A2 / NY - ESO - anti - CD3 bispecific molecules NYF - 0016, NYF - 0019, NYF - 0044, and NYF - 0047 have antitumor activity in the human PBMC transfer model. The error bars in the figure represent the standard error (n = 4 for NYF - 0044 on day 32, and n = 5 for others).

[0282] Figure 5B This figure 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 antitumor activity in the human PBMC transfer model. The error bars in the figure represent the standard error (n = 5, and n = 6 for the blank control group only).

[0283] Figure 5C This figure 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 antitumor activity in the human PBMC transfer model. The error bars in the figure represent the standard error (n = 5).

[0284] Fig. 6AIt is a diagram showing the configurations of the antibodies shown in this example. (a) Heterodimeric type: It is a configuration formed by adding two Fcs (diagonal lines and blackened) with mutations introduced to form heterodimers to the C-terminal side of Fab and to the C-terminal side of scFv, respectively, and the association of these two Fcs. In this example, the heterodimeric type containing anti-HLA-A2 / NY-ESO Fab and anti-CD3 scFv was evaluated. (b) Bispecific type: It is a configuration formed by adding two Fcs (upper left diagonal lines and blackened) with mutations introduced to form heterodimers to the C-terminal side of each of two different scFvs, respectively, and the association of these two Fc heterozygotes. In this example, the bispecific type containing anti-HLA-A2 / NY-ESO scFv and anti-CD3 scFv was evaluated. (c) scFv-Fab-heterodimeric Fc type: It is a configuration formed by adding one Fc (upper left diagonal line) with a mutation introduced to form a heterodimer to the C-terminal side where scFv is connected to Fab via a linker, and the association with another Fc (blackened). In this example, the scFv-Fab-heterodimeric Fc type containing anti-CD3 scFv (upper right diagonal line) and anti-HLA-A2 / NY-ESO Fab was evaluated. Additionally, the scFv-Fab-heterodimeric Fc type containing anti-HLA-A2 / NY-ESO scFv (upper right diagonal line) and anti-CD3 Fab was also evaluated.

[0285] Figure 6B It is a diagram showing the configurations of the antibodies shown in this example. (a) taFv-heterodimeric Fc type: same as Figure 3 (d). This configuration is formed by adding one Fc (upper left diagonal line) with a mutation introduced to form a heterodimer to the C-terminal side of taFv, and the association with another Fc (blackened) and a heterozygote. In this example, the taFv-heterodimeric Fc type composed of anti-HLA-A2 / NY-ESO scFv and anti-CD3 scFv was evaluated. (b) taFv (reverse)-heterodimeric Fc type: It is a configuration formed by reversing the order of the two scFvs in the above taFv-heterodimeric Fc type. In this example, the taFv (reverse)-heterodimeric Fc type composed of anti-CD3 scFv and anti-HLA-A2 / NY-ESO scFv was evaluated. (c) The first polypeptide representing the taFv (reverse)-heterodimeric Fc type. The first polypeptide sequentially contains, from the N-terminal to the C-terminal, scFv that specifically binds to CD3, scFv that specifically binds to human HLA / NY-ESO, and the Fc region (i). (d) The second polypeptide representing the taFv (reverse)-heterodimeric Fc type. The second polypeptide contains a hinge region and the Fc region (ii).

[0286] Fig. 7AThis is a graph showing that in the presence of human PBMC, various anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules (hybrid (NYG-3143), diabody (NYG-2143), taFv-heterodimeric Fc body (NYF-0011)) exhibit cytotoxic activity against U266B1 cells expressing endogenous human NY-ESO. The error bars in the graph represent the standard deviation (n = 3).

[0287] Figure 7B This is a graph showing that in the presence of human PBMC, various anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules (scFv-Fab-heterodimeric Fc body (NYF-0003), taFv-heterodimeric Fc body (NYF-0010), taFv (reverse)-heterodimeric Fc body (NYF-0004)) exhibit cytotoxic activity against U266B1 cells expressing endogenous human NY-ESO. The error bars in the graph represent the standard deviation (n = 3).

[0288] Figure 8 This is the amino acid sequence of the peptide in NY-ESO (SEQ ID NO:1).

[0289] Fig. 9 This is the amino acid sequence of the peptide in MAGEC-1 (SEQ ID NO:2).

[0290] Fig.10 This is primer 1 (SEQ ID NO:3) for scFv sequence analysis.

[0291] Fig.11 This is primer 2 (SEQ ID NO:4) for scFv sequence analysis.

[0292] Fig.12 This is the nucleotide sequence of the heavy chain variable region of NYA-0001 (SEQ ID NO:5).

[0293] Fig.13 This is the amino acid sequence of the heavy chain variable region of NYA-0001 (SEQ ID NO:6).

[0294] Fig.14 This is the nucleotide sequence of the light chain variable region of NYA-0001 (SEQ ID NO:7).

[0295] Fig.15 This is the amino acid sequence of the light chain variable region of NYA-0001 (SEQ ID NO:8).

[0296] Fig.16 This is the nucleotide sequence of the heavy chain variable region of NYA-0060 (SEQ ID NO:9).

[0297] Fig.17 is the amino acid sequence of the heavy chain variable region of NYA-0060 (SEQ ID NO: 10).

[0298] Fig.18 is the nucleotide sequence of the light chain variable region of NYA-0060 (SEQ ID NO: 11).

[0299] Fig.19 is the amino acid sequence of the light chain variable region of NYA-0060 (SEQ ID NO: 12).

[0300] Fig. 20 is the nucleotide sequence of the heavy chain variable region of NYA-0068 (SEQ ID NO: 13).

[0301] Fig.21 is the amino acid sequence of the heavy chain variable region of NYA-0068 (SEQ ID NO: 14).

[0302] Fig. 22 is the nucleotide sequence of the light chain variable region of NYA-0068 (SEQ ID NO: 15).

[0303] Fig.23 is the amino acid sequence of the light chain variable region of NYA-0068 (SEQ ID NO: 16).

[0304] Fig.24 is the nucleotide sequence of the heavy chain variable region of NYA-0082 (SEQ ID NO: 17).

[0305] Fig.25 is the amino acid sequence of the heavy chain variable region of NYA-0082 (SEQ ID NO: 18).

[0306] Fig.26 is the nucleotide sequence of the light chain variable region of NYA-0082 (SEQ ID NO: 19).

[0307] Fig. 27 is the amino acid sequence of the light chain variable region of NYA-0082 (SEQ ID NO: 20).

[0308] Fig.28 is the nucleotide sequence of the NYA-1163 tag addition (SEQ ID NO: 21).

[0309] Fig.29 is the nucleotide sequence of the NYA-2023 tag addition (SEQ ID NO: 22).

[0310] Fig.30 It is the nucleotide sequence (SEQ ID NO:23) of the NYA-2027 tag additive.

[0311] Fig.31 It is the nucleotide sequence (SEQ ID NO:24) of the NYA-1143 tag additive.

[0312] Fig.32 It is the nucleotide sequence (SEQ ID NO:25) of the NYA-2143 tag additive.

[0313] Fig.33 It is the amino acid sequence (SEQ ID NO:26) of the NYA-1163 tag additive, and NYA-1163 is amino acid numbers 21 to 266.

[0314] Fig.34 It is the amino acid sequence (SEQ ID NO:27) of the NYA-2023 tag additive, and NYA-2023 is amino acid numbers 21 to 266.

[0315] Fig.35 It is the amino acid sequence (SEQ ID NO:28) of the NYA-2027 tag additive, and NYA-2027 is amino acid numbers 21 to 266.

[0316] Fig.36 It is the amino acid sequence (SEQ ID NO:29) of the NYA-1143 tag additive, and NYA-1143 is amino acid numbers 21 to 266.

[0317] Fig.37 It is the amino acid sequence (SEQ ID NO:30) of the NYA-2143 tag additive, and NYA-2143 is amino acid numbers 21 to 266.

[0318] Fig.38 It is the nucleotide sequence (SEQ ID NO:31) of the NYA-1154 tag additive.

[0319] Fig.39 It is the amino acid sequence (SEQ ID NO:32) of the NYA-1154 tag additive, and NYA-1154 is amino acid numbers 21 to 266.

[0320] Fig.40 It is the amino acid sequence (SEQ ID NO:33) of the truncation of HLA-A*0201 (GenBank: ASA47534.1).

[0321] Fig.41 It is the amino acid sequence (SEQ ID NO:34) of β2-microglobulin.

[0322] Fig.42 It is the nucleotide sequence (SEQ ID NO: 35) of the NYA-2035 tag additive.

[0323] Fig.43 It is the amino acid sequence (SEQ ID NO: 36) of the NYA-2035 tag additive, where NYA-2035 is amino acid numbers 21 to 266.

[0324] Fig.44 It is the amino acid sequence (SEQ ID NO: 37) of NYA-1143-VH01.

[0325] Fig.45 It is the amino acid sequence (SEQ ID NO: 38) of NYA-1143-VH02.

[0326] Fig.46 It is the amino acid sequence (SEQ ID NO: 39) of NYA-1143-VH03.

[0327] Fig.47 It is the amino acid sequence (SEQ ID NO: 40) of NYA-1143-VL01.

[0328] Fig.48 It is the nucleotide sequence (SEQ ID NO: 41) of the NYA-2044 tag additive.

[0329] Fig.49 It is the nucleotide sequence (SEQ ID NO: 42) of the NYA-2045 tag additive.

[0330] Fig.50 It is the nucleotide sequence (SEQ ID NO: 43) of the NYA-2047 tag additive.

[0331] Fig.51 It is the nucleotide sequence (SEQ ID NO: 44) of the NYA-2048 tag additive.

[0332] Fig.52 It is the nucleotide sequence (SEQ ID NO: 45) of the NYA-2060 tag additive.

[0333] Fig.53 It is the nucleotide sequence (SEQ ID NO: 46) of the NYA-2061 tag additive.

[0334] Fig.54is the amino acid sequence of the NYA-2044 tag addition (SEQ ID NO: 47), where NYA-2044 corresponds to amino acid numbers 21 to 266.

[0335] Fig.55 is the amino acid sequence of the NYA-2045 tag addition (SEQ ID NO: 48), where NYA-2045 corresponds to amino acid numbers 21 to 266.

[0336] Fig.56 is the amino acid sequence of NYA-0082 (SEQ ID NO: 49).

[0337] Fig.57 is the amino acid sequence of the NYA-2047 tag addition (SEQ ID NO: 50), where NYA-2047 corresponds to amino acid numbers 21 to 266.

[0338] Fig.58 is the amino acid sequence of the NYA-2048 tag addition (SEQ ID NO: 51), where NYA-2048 corresponds to amino acid numbers 21 to 266.

[0339] Fig.59 is the amino acid sequence of the NYA-2060 tag addition (SEQ ID NO: 52), where NYA-2060 corresponds to amino acid numbers 21 to 266.

[0340] Fig.60 is the amino acid sequence of the NYA-2061 tag addition (SEQ ID NO: 53), where NYA-2061 corresponds to amino acid numbers 21 to 266.

[0341] Fig.61 is the amino acid sequence of the heavy chain CDRH1 - CDRH3 and light chain CDRL1 - L3 of NYA-0001 (SEQ ID NO: 54 - SEQ ID NO: 59).

[0342] Fig.62 is the amino acid sequence of the CDRL1 of NYA-2023 (SEQ ID NO: 60).

[0343] Fig.63 is the amino acid sequence of the CDRL3 of NYA-2027 (SEQ ID NO: 61).

[0344] Fig.64 is the amino acid sequence of the CDRH3 and CDRL3 of NYA-1154 (SEQ ID NO: 62 and SEQ ID NO: 63).

[0345] Fig.65Is the amino acid sequence of CDRL1 of NYA-0035 (SEQ ID NO: 64).

[0346] Fig.66 Is the nucleotide sequence of the NYC-0003 tag addition (SEQ ID NO: 65).

[0347] Fig.67 Is the nucleotide sequence of the NYC-0004 tag addition (SEQ ID NO: 66).

[0348] Fig.68 Is the amino acid sequence of the NYC-0003 tag addition (SEQ ID NO: 67), where NYC-0003 corresponds to amino acid numbers 21 to 263.

[0349] Fig.69 Is the amino acid sequence of the NYC-0004 tag addition (SEQ ID NO: 68), where NYC-0004 corresponds to amino acid numbers 21 to 263.

[0350] Fig.70 Is the nucleotide sequence of the NYA-0001 tag addition (SEQ ID NO: 69).

[0351] Fig.71 Is the amino acid sequence of the NYA-0001 tag addition (SEQ ID NO: 70), where NYA-0001 corresponds to amino acid numbers 21 to 266.

[0352] Fig.72 Is the nucleotide sequence of HC1 (SEQ ID NO: 71).

[0353] Fig.73 Is the nucleotide sequence of NYF-0016-HC2 (SEQ ID NO: 72).

[0354] Fig.74 Is the nucleotide sequence of NYF-0019-HC2 (SEQ ID NO: 73).

[0355] Fig.75 Is the nucleotide sequence of NYF-0022-HC2 (SEQ ID NO: 74).

[0356] Fig.76 Is the nucleotide sequence of NYF-0023-HC2 (SEQ ID NO: 75).

[0357] Fig.77 Is the nucleotide sequence of NYF-0027-HC2 (SEQ ID NO: 76).

[0358] Fig.78 is the nucleotide sequence of NYF-0035-HC2 (SEQ ID NO:77).

[0359] Fig.79 is the nucleotide sequence of NYF-0044-HC2 (SEQ ID NO:78).

[0360] Fig.80 is the nucleotide sequence of NYF-0045-HC2 (SEQ ID NO:79).

[0361] Fig.81 is the nucleotide sequence of NYF-0047-HC2 (SEQ ID NO:80).

[0362] Fig.82 is the nucleotide sequence of NYF-0048-HC2 (SEQ ID NO:81).

[0363] Fig.83 is the nucleotide sequence of NYF-0060-HC2 (SEQ ID NO:82).

[0364] Fig.84 is the nucleotide sequence of NYF-0061-HC2 (SEQ ID NO:83).

[0365] Fig.85 is the amino acid sequence of HC1 (SEQ ID NO:84).

[0366] Fig.86 is the amino acid sequence of NYF-0016-HC2 (SEQ ID NO:85), NYA-1143 is amino acid numbers 21 to 266, and C3E-7085 is amino acid numbers 272 to 511.

[0367] Fig.87 is the amino acid sequence of NYF-0019-HC2 (SEQ ID NO:86), NYA-2143 is amino acid numbers 21 to 266, and C3E-7085 is amino acid numbers 272 to 511.

[0368] Fig.88 is the amino acid sequence of NYF-0022-HC2 (SEQ ID NO:87), NYA-1163 is amino acid numbers 21 to 266, and C3E-7085 is amino acid numbers 272 to 511.

[0369] Fig.89is the amino acid sequence of NYF-0023-HC2 (SEQ ID NO:88), where NYA-2023 corresponds to amino acid numbers 21 to 266, and C3E-7085 corresponds to amino acid numbers 272 to 511.

[0370] Fig.90 is the amino acid sequence of NYF-0027-HC2 (SEQ ID NO:89), where NYA-2027 corresponds to amino acid numbers 21 to 266, and C3E-7085 corresponds to amino acid numbers 272 to 511.

[0371] Fig.91 is the amino acid sequence of NYF-0035-HC2 (SEQ ID NO:90), where NYA-2035 corresponds to amino acid numbers 21 to 266, and C3E-7085 corresponds to amino acid numbers 272 to 511.

[0372] Fig.92 is the amino acid sequence of NYF-0044-HC2 (SEQ ID NO:91), where NYA-2044 corresponds to amino acid numbers 21 to 266, and C3E-7085 corresponds to amino acid numbers 272 to 511.

[0373] Fig.93 is the amino acid sequence of NYF-0045-HC2 (SEQ ID NO:92), where NYA-2045 corresponds to amino acid numbers 21 to 266, and C3E-7085 corresponds to amino acid numbers 272 to 511.

[0374] Fig.94 is the amino acid sequence of NYF-0047-HC2 (SEQ ID NO:93), where NYA-2047 corresponds to amino acid numbers 21 to 266, and C3E-7085 corresponds to amino acid numbers 272 to 511.

[0375] Fig.95 is the amino acid sequence of NYF-0048-HC2 (SEQ ID NO:94), where NYA-2048 corresponds to amino acid numbers 21 to 266, and C3E-7085 corresponds to amino acid numbers 272 to 511.

[0376] Fig.96 is the amino acid sequence of NYF-0060-HC2 (SEQ ID NO:95), where NYA-2060 corresponds to amino acid numbers 21 to 266, and C3E-7085 corresponds to amino acid numbers 272 to 511.

[0377] Fig.97is the amino acid sequence of NYF-0061-HC2 (SEQ ID NO:96), NYA-2061 is amino acid numbers 21 to 266, and C3E-7085 is amino acid numbers 272 to 511.

[0378] Fig.98 is the nucleotide sequence of NYA-0001-Fab-HC1-k delete (SEQ ID NO:97).

[0379] Fig.99 is the nucleotide sequence of NYA-0001-LC (SEQ ID NO:98).

[0380] Fig.100 is the amino acid sequence of NYA-0001-Fab-HC1-k delete (SEQ ID NO:99), and the heavy chain variable region of NYA-0001 is amino acid numbers 20 to 139.

[0381] Fig.101 is the amino acid sequence of NYA-0001-LC (SEQ ID NO:100), and the light chain variable region of NYA-0001 is amino acid numbers 21 to 131.

[0382] Fig.102 is the nucleotide sequence of NYA-1143-Fab-HC1-k delete (SEQ ID NO:101).

[0383] Fig.103 is the nucleotide sequence of NYA-1143-LC (SEQ ID NO:102).

[0384] Fig.104 is the nucleotide sequence of C3E-7085-HC2-k deleteC (SEQ ID NO:103).

[0385] Fig.105 is the amino acid sequence of NYA-1143-Fab-HC1-k delete (SEQ ID NO:104), and the heavy chain variable region of NYA-1143 is amino acid numbers 20 to 139.

[0386] Fig.106 is the amino acid sequence of NYA-1143-LC (SEQ ID NO:105), and the light chain variable region of NYA-1143 is amino acid numbers 21 to 131.

[0387] Fig.107is the amino acid sequence of C3E-7085-HC2-k delete (SEQ ID NO:106), where C3E-7085 corresponds to amino acid numbers 21 to 260.

[0388] Fig.108 is the nucleotide sequence of NYA-1143-HC1-k delete (SEQ ID NO:107).

[0389] Fig.109 is the amino acid sequence of NYA-1143-HC1-k delete (SEQ ID NO:108), where NYA-1143 corresponds to amino acid numbers 21 to 266.

[0390] Fig.110 is the nucleotide sequence of C3E-7085-NYA-1154-Fab-HC2-k delete (SEQ ID NO:109).

[0391] Fig.111 is the nucleotide sequence of NYA-1154-LC (SEQ ID NO:110).

[0392] Fig.112 is the nucleotide sequence of OAA-HC1-k delete (SEQ ID NO:111).

[0393] Fig.113 is the amino acid sequence of C3E-7085-NYA-1154-Fab-HC2-k delete (SEQ ID NO:112), where C3E-7085 corresponds to amino acid numbers 21 to 260, and the heavy chain variable region of NYA-1154 corresponds to amino acid numbers 266 to 285.

[0394] Fig.114 is the amino acid sequence of NYA-1154-LC (SEQ ID NO:113), where the light chain variable region of NYA-1154 corresponds to amino acid numbers 21 to 131.

[0395] Fig.115 is the amino acid sequence of OAA-HC1-k delete (SEQ ID NO:114).

[0396] Fig.116 is the nucleotide sequence of NYF-0010-HC2-k delete (SEQ ID NO:115).

[0397] Fig.117 is the nucleotide sequence of NYF-0004-HC2-k delete (SEQ ID NO:116).

[0398] Fig.118 It is the nucleotide sequence of NYF-0011-HC2-k delete (SEQ ID NO: 117).

[0399] Fig.119 It is the amino acid sequence of NYF-0010-HC2-k delete (SEQ ID NO: 18). For NYA-1154, the amino acid numbers are 21 to 266, and for C3E-7085, the amino acid numbers are 272 to 511.

[0400] Fig.120 It is the amino acid sequence of NYF-0004-HC2-k delete (SEQ ID NO: 119). For C3E-7085, the amino acid numbers are 21 to 260, and for NYA-1154, the amino acid numbers are 272 to 511.

[0401] Fig.121 It is the amino acid sequence of NYF-0011-HC2-k delete (SEQ ID NO: 120). For NYA-1143, the amino acid numbers are 21 to 266, and for C3E-7085, the amino acid numbers are 272 to 511.

[0402] Fig.122 It is the amino acid sequence of the point-mutated NY-ESO peptide 1F (SEQ ID NO: 121).

[0403] Fig.123 It is the amino acid sequence of the point-mutated NY-ESO peptide 2M (SEQ ID NO: 122).

[0404] Fig.124 It is the amino acid sequence of the point-mutated NY-ESO peptide 3A (SEQ ID NO: 123).

[0405] Fig.125 It is the amino acid sequence of the point-mutated NY-ESO peptide 4A (SEQ ID NO: 124).

[0406] Fig.126 It is the amino acid sequence of the point-mutated NY-ESO peptide 5A (SEQ ID NO: 125).

[0407] Fig.127 It is the amino acid sequence of the point-mutated NY-ESO peptide 6L (SEQ ID NO: 126).

[0408] Fig.128 It is the amino acid sequence of the point-mutated NY-ESO peptide 7F (SEQ ID NO: 127).

[0409] Fig.129It is the amino acid sequence of the point mutant NY-ESO peptide 8A (SEQ ID NO: 128).

[0410] Fig.130 It is the amino acid sequence of the point mutant NY-ESO peptide 9A (SEQ ID NO: 129).

[0411] Fig.131 It is the amino acid sequence of the gp100 peptide (SEQ ID NO: 130).

[0412] Fig.132 It is the amino acid sequence of the homologous peptide DOLPP1 (SEQ ID NO: 131).

[0413] Fig.133 It is the amino acid sequence of the homologous peptide IL20RB (SEQ ID NO: 132).

[0414] Fig.134 It is the amino acid sequence of the homologous peptide PRKD2 (SEQ ID NO: 133).

[0415] Fig.135 It is the amino acid sequence of the homologous peptide CD163 (SEQ ID NO: 134).

[0416] Fig.136 It is the amino acid sequence of the homologous peptide P2RY8 (SEQ ID NO: 135).

[0417] Fig.137 It is the amino acid sequence of C3E-7034 (SEQ ID NO: 136).

[0418] Fig.138 It is the amino acid sequence of C3E-7036 (SEQ ID NO: 137).

[0419] Fig.139 It is the amino acid sequence of C3E-7085 (SEQ ID NO: 138).

[0420] Fig.140 It is the amino acid sequence of C3E-7088 (SEQ ID NO: 139).

[0421] Fig.141 It is the amino acid sequence of C3E-7093 (SEQ ID NO: 140).

[0422] Fig.142 It is the amino acid sequence of the heavy chain CDRH1 - CDRH3 and the light chain CDRL1 - CDRL3 of C3E-7085 (SEQ ID NO: 141 - SEQ ID NO: 146).

[0423] Fig.143 is the amino acid sequence of C3E-7078 (SEQ ID NO: 147).

[0424] Fig.144 is the nucleotide sequence of NYF-0014-HC2 (SEQ ID NO: 148).

[0425] Fig.145 is the amino acid sequence of NYF-0014-HC2 (SEQ ID NO: 149), where NYA-0001 corresponds to amino acid numbers 21 to 266, and C3E-7085 corresponds to amino acid numbers 272 to 511.

[0426] Fig.146 is the amino acid sequence of NYF-0082-HC2 (SEQ ID NO: 150), where NYA-0082 corresponds to amino acid numbers 21 to 266, and C3E-7085 corresponds to amino acid numbers 272 to 511.

[0427] Fig.147 is the amino acid sequence of human CD3ε (SEQ ID NO: 151).

[0428] Fig.148 is the full-length nucleotide sequence of NYZ-0038-HC2 (SEQ ID NO: 152).

[0429] Fig.149 is the full-length nucleotide sequence of NYZ-0082-HC2 (SEQ ID NO: 153).

[0430] Fig.150 is the full-length nucleotide sequence of NYZ-0083-HC2 (SEQ ID NO: 154).

[0431] Fig.151 is the full-length amino acid sequence of NYZ-0038-HC2 (SEQ ID NO: 155), where NYA-2061 corresponds to amino acid numbers 21 to 266, and C3E-7096 corresponds to amino acid numbers 272 to 511.

[0432] Fig.152 is the full-length amino acid sequence of NYZ-0082-HC2 (SEQ ID NO: 156), where NYA-3061 corresponds to amino acid numbers 21 to 271, and C3E-7096 corresponds to amino acid numbers 277 to 516.

[0433] Fig.153is the amino acid sequence of the full length of NYZ-0083-HC2 (SEQ ID NO: 157), NYA-3061 is amino acid numbers 21 to 271, and C3E-7097 is amino acid numbers 277 to 516.

[0434] Fig.154 is the nucleotide sequence of the full length of NYZ-1010-HC2 (SEQ ID NO: 158).

[0435] Fig.155 is the nucleotide sequence of the full length of C3E-7085-LC (SEQ ID NO: 159).

[0436] Fig.156 is the amino acid sequence of the full length of NYZ-1010-HC2 (SEQ ID NO: 160), NYA-3061 is amino acid numbers 21 to 271, and the heavy chain variable region of C3E-7085 is amino acid numbers 277 to 394.

[0437] Fig.157 is the amino acid sequence of the full length of C3E-7085-LC (SEQ ID NO: 161).

[0438] Fig.158A is a table showing the normalized gMFI of the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules NYZ-0038, 0082, 0083, 1010 against CD3e knockout T2 cells added with various point mutant peptides. * / Underline indicates less than half compared to the normalized gMFI of each Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule against CD3e knockout T2 cells added with NY-ESO peptide. *Bold indicates less than one-fourth.

[0439] Fig.158B is a table showing the normalized gMFI of the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules NYZ-0038, 0082, 0083, 1010 against CD3e knockout T2 cells added with various homologous peptides. * / Underline indicates a larger value compared to the normalized gMFI of each Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule against CD3e knockout T2 cells added with DMSO.

[0440] Fig.159AThis figure shows the cytotoxic activity of the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules NYZ-0038, 0082, and 0083 against U266B1 cells expressing endogenous human NY-ESO in the presence of human PBMC. The error bars in the figure represent the standard deviation (n = 3).

[0441] Fig.159B This figure shows the cytotoxic activity of the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule NYZ-1010 against U266B1 cells expressing endogenous human NY-ESO in the presence of human PBMC. The error bars in the figure represent the standard deviation (n = 3).

[0442] Fig.159C This figure shows the cytotoxic activity of the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules NYZ-0082 and 1010 against NCI-H1703 cells expressing endogenous human NY-ESO in the presence of human PBMC. The error bars in the figure represent the standard deviation (n = 3).

[0443] Fig.159D This figure shows the cytotoxic activity of the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules NYZ-0038 and 0083 against NCI-H1703 cells expressing endogenous human NY-ESO in the presence of human PBMC. The error bars in the figure represent the standard deviation (n = 3).

[0444] Fig.159E This figure shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules NYZ-0038, 0082, and 0083 did not exhibit cytotoxic activity against AGS cells that do not express endogenous human NY-ESO in the presence of human PBMC. The error bars in the figure represent the standard deviation (n = 3).

[0445] Fig.159F This figure shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule NYZ-1010 did not exhibit cytotoxic activity against AGS cells that do not express endogenous human NY-ESO in the presence of human PBMC. The error bars in the figure represent the standard deviation (n = 3).

[0446] Figure 159G This figure shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules NYZ-0082 and 1010 did not exhibit cytotoxic activity against CFPAC-1 cells that do not express endogenous human NY-ESO in the presence of human PBMC. The error bars in the figure represent the standard deviation (n = 3).

[0447] Figure 159H This figure shows that in the co - existence of human PBMCs, the Fc - added anti - HLA - A2 / NY - ESO - anti - CD3 bispecific molecules NYZ - 0038 and 0083 did not show cytotoxic activity against CFPAC - 1 cells that do not express endogenous human NY - ESO. The error bars in the figure represent the standard deviation (n = 3).

[0448] Fig.160A This figure shows that the Fc - added anti - HLA - A2 / NY - ESO - anti - CD3 bispecific molecule NYZ - 0038 has anti - tumor activity in the human PBMC transfer model. The error bars in the figure represent the standard error (n = 5).

[0449] Fig.160B This figure shows that the Fc - added anti - HLA - A2 / NY - ESO - anti - CD3 bispecific molecule NYZ - 0082 has anti - tumor activity in the human PBMC transfer model. The error bars in the figure represent the standard error (n = 5).

[0450] Fig.160C This figure shows that the Fc - added anti - HLA - A2 / NY - ESO - anti - CD3 bispecific molecule NYZ - 0083 has anti - tumor activity in the human PBMC transfer model. The error bars in the figure represent the standard error (n = 5).

[0451] Fig.160D This figure shows that the Fc - added anti - HLA - A2 / NY - ESO - anti - CD3 bispecific molecule NYZ - 1010 has anti - tumor activity in the human PBMC transfer model. The error bars in the figure represent the standard error (n = 5).

[0452] Fig.161 This is the amino acid sequence of the peptide linker (SEQ ID NO:162).

[0453] Fig.162 This figure shows the content (%) of the multimer when various Fc - added anti - HLA / NY - ESO - anti - CD3 bispecific molecules are treated with acid.

[0454] Fig.163 This figure shows the results of the solution stability evaluation of the anti - HLA / NY - ESO scFv - heterodimer Fc.

[0455] Fig.164 This is the nucleotide sequence of the full - length NYA - 3061 (SEQ ID NO:163).

[0456] Fig.165is the amino acid sequence of the full length of NYA-3061 (SEQ ID NO: 164).

[0457] Fig.166 is the nucleotide sequence of the full length of NYC-0005 (SEQ ID NO: 165).

[0458] Fig.167 is the amino acid sequence of the full length of NYC-0005 (SEQ ID NO: 166).

[0459] Fig.168 is the nucleotide sequence of the full length of NYC-0006 (SEQ ID NO: 167).

[0460] Fig.169 is the amino acid sequence of the full length of NYC-0006 (SEQ ID NO: 168).

[0461] Fig.170 is the nucleotide sequence of the full length of NYC-0007 (SEQ ID NO: 169).

[0462] Fig.171 is the amino acid sequence of the full length of NYC-0007 (SEQ ID NO: 170).

[0463] Fig.172 is the nucleotide sequence of the full length of NYC-0008 (SEQ ID NO: 171).

[0464] Fig.173 is the amino acid sequence of the full length of NYC-0008 (SEQ ID NO: 172).

[0465] Fig.174 is the nucleotide sequence of the full length of NYC-0009 (SEQ ID NO: 173).

[0466] Fig.175 is the amino acid sequence of the full length of NYC-0009 (SEQ ID NO: 174).

[0467] Fig.176 is the nucleotide sequence of the full length of NYC-0010 (SEQ ID NO: 175).

[0468] Fig.177 is the amino acid sequence of the full length of NYC-0010 (SEQ ID NO: 176).

[0469] Fig.178 is the nucleotide sequence of the full length of HC-h (SEQ ID NO: 177).

[0470] Fig.179 is the amino acid sequence of the full-length HC-h (SEQ ID NO: 178).

[0471] Fig.180 is the nucleotide sequence of the full-length NYD-2047-HC-k (SEQ ID NO: 179).

[0472] Fig.181 is the amino acid sequence of the full-length NYD-2047-HC-k (SEQ ID NO: 180).

[0473] Fig.182 is the nucleotide sequence of the full-length NYD-2061-HC-k (SEQ ID NO: 181).

[0474] Fig.183 is the amino acid sequence of the full-length NYD-2061-HC-k (SEQ ID NO: 182).

[0475] Fig.184 is the nucleotide sequence of the full-length NYD-3061-HC-k (SEQ ID NO: 183).

[0476] Fig.185 is the amino acid sequence of the full-length NYD-3061-HC-k (SEQ ID NO: 184).

[0477] Fig.186 is the nucleotide sequence of the full-length NYC-0011-HC-k (SEQ ID NO: 185).

[0478] Fig.187 is the amino acid sequence of the full-length NYC-0011-HC-k (SEQ ID NO: 186).

[0479] Fig.188 is the nucleotide sequence of the full-length NYC-0012-HC-k (SEQ ID NO: 187).

[0480] Fig.189 is the amino acid sequence of the full-length NYC-0012-HC-k (SEQ ID NO: 188).

[0481] Fig.190 is the nucleotide sequence of the full-length NYC-0013-HC-k (SEQ ID NO: 189).

[0482] Fig.191 is the amino acid sequence of the full-length NYC-0013-HC-k (SEQ ID NO: 190).

[0483] Fig.192Is the full-length nucleotide sequence of NYC-0014-HC-k (SEQ ID NO: 191).

[0484] Fig.193 Is the full-length amino acid sequence of NYC-0014-HC-k (SEQ ID NO: 192).

[0485] Fig.194 Is the full-length nucleotide sequence of NYC-0015-HC-k (SEQ ID NO: 193).

[0486] Fig.195 Is the full-length amino acid sequence of NYC-0015-HC-k (SEQ ID NO: 194).

[0487] Fig.196 Is the full-length nucleotide sequence of NYC-0016-HC-k (SEQ ID NO: 195).

[0488] Fig.197 Is the full-length amino acid sequence of NYC-0016-HC-k (SEQ ID NO: 196).

[0489] Fig.198 Is the full-length amino acid sequence of NYZ-1007-HC2 (SEQ ID NO: 197), NYA-2061 is amino acid numbers 21 - 266, and the heavy chain variable region of C3E-7085 is amino acid numbers 272 - 389.

[0490] Fig.199 Is the full-length amino acid sequence of NYZ-1017-HC2 (SEQ ID NO: 198), NYA-2047 is amino acid numbers 21 - 266, and the heavy chain variable region of C3E-7085 is amino acid numbers 277 - 389. Detailed implementation manners

[0491] The present invention will be described in detail below.

[0492] 1. Definitions

[0493] In the present invention, "gene" refers to a nucleotide chain containing a base sequence encoding amino acids of a protein or its complementary chain. For example, polynucleotides, oligonucleotides, DNA, mRNA, cDNA, cRNA, etc., which are nucleotide chains containing a base sequence encoding amino acids of a protein or their complementary chains, are all included in the meaning of "gene". A gene is a single-stranded, double-stranded or triple-stranded or more nucleotide, an association of a DNA strand and an RNA strand, a nucleotide in which ribonucleotides (RNA) and deoxyribonucleotides (DNA) are mixed on one nucleotide chain, and a double-stranded or triple-stranded or more nucleotide containing such a nucleotide chain are also included in the meaning of "gene". In the present invention, the base sequence has the same meaning as the nucleotide sequence.

[0494] In the present invention, "polynucleotide", "nucleotide chain", "nucleic acid" and "nucleic acid molecule" have the same meaning. For example, DNA, RNA, probes, oligonucleotides, primers, etc. 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. An association of a DNA strand and an RNA strand, a polynucleotide in which ribonucleotides (RNA) and deoxyribonucleotides (DNA) are mixed on one polynucleotide chain, and an association of a double strand or three or more strands containing such a polynucleotide chain are also included in the meaning of "polynucleotide".

[0495] In the present invention, "polypeptide", "peptide" and "protein" have the same meaning.

[0496] In the present invention, "antigen" is sometimes used in the meaning of "immunogen".

[0497] In the present invention, "cell" also includes various cells derived from an animal individual, subcultured cells, primary cultured cells, cell lines, recombinant cells, and microorganisms, etc.

[0498] In the present invention, "antibody" has the same meaning as immunoglobulin. However, in the case of the anti-HLA / NY-ESO antibody of the present invention, "antibody" is used to mean an immunoglobulin having a constant region and a variable region. An antibody is a natural antibody or an immunoglobulin manufactured by partial synthesis or total synthesis, and there is no particular limitation in the present invention. The anti-HLA / NY-ESO antibody of the present invention is included in the "molecule" described below.

[0499] In the present invention, "NY-ESO peptide" refers to a peptide (SLLMWITQC, SEQ ID NO:1) composed of 9 amino acids at positions 157 to 165 of NY-ESO-1 and LAGE-1.

[0500] 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 denoted as "HLA / NY-ESO".

[0501] In the present invention, "anti-HLA-A2 / NY-ESO antibody" refers to an antibody that binds to HLA-A2 / NY-ESO, in other words, an antibody that recognizes HLA-A2 / NY-ESO. Similarly, "anti-HLA-A2 / NY-ESO scFv" refers to an scFv that binds to HLA / NY-ESO, in other words, an scFv that recognizes HLA-A2 / NY-ESO. "Anti-HLA-A2 / NY-ESO antibody" and "anti-HLA-A2 / NY-ESO scFv" are also denoted as "anti-HLA / NY-ESO antibody" and "anti-HLA / NY-ESO scFv", respectively.

[0502] The basic structure of a four-chain antibody is composed 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 according to the heavy chain isotype. Each light chain and heavy chain has regularly spaced intra-chain disulfide bonds. In the heavy chain and light chain, there are constant regions where the amino acid sequences show very high similarity, and variable regions where the amino acid sequence similarity is low. The light chain has a variable region (VL) contiguous with the constant region (CL) at the amino terminus. The heavy chain has a variable region (VH) contiguous with three constant regions (CH1 / CH2 / CH3) at the amino terminus. VL and VH pair, and CL is juxtaposed with the first constant region (CH1) of the heavy chain. VL and VH pair to form a single antigen-binding site.

[0503] The constant region of the antibody of the present invention is not particularly limited, but as the antibody of the present invention for treating or preventing human diseases, the constant region of a human antibody is preferably used. As the heavy chain constant region of a human antibody, for example, Cγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cμ, Cδ, Cε, etc. can be cited. As the light chain constant region of a human antibody, for example, Cκ, Cλ, etc. can be cited.

[0504] Fab is composed of VH and subsequent CH1 of the heavy chain, and VL and subsequent CL of the light chain. VH and VL contain Complementary Determining Regions (CDRs).

[0505] Fc (also referred to as the Fc region) is the carboxyl-terminal region of the constant region of the heavy chain, containing CH2 and CH3, and is a dimer. The Fc of the present invention can be either a natural-sequence Fc or a mutant Fc (referred to as "mutant Fc") that has a mutation in the natural sequence. In the multispecific molecules and bispecific molecules of the present invention, the preferred Fc region is mutant Fc, and more preferably a group of Fc that can form a heterodimer. As a group of Fc, a combination of Fc(i) contained in the first polypeptide and Fc(ii) contained in the second polypeptide described below can be cited. As long as a group of Fc can associate (form a heterodimer), there is no limitation.

[0506] Examples of the mutant Fc include, but are not limited to: a modified Fc region (including a heterodimer Fc region) contained in a heteropolymer having enhanced stability, as disclosed in WO2013 / 063702; an Fc contained in a heteropolymer and including the CH3 region of an immunoglobulin derived from an IgG antibody having a "protrusion" and a "void", as disclosed in WO1996 / 27011; an Fc contained in a heterodimer including a CH3 region in which one or more amino acid residues are substituted with charged amino acids to be electrostatically favorable, as disclosed in WO2009 / 089004; a heterodimer Fc region contained in a heterodimer using conformational mutation and / or pI (isoelectric point) mutation, as disclosed in WO2014 / 110601; an Fc of a heterodimer including a CH3 region containing a modification that abolishes or reduces the binding to protein A, as disclosed in WO2010 / 151792, etc.

[0507] The variable region is composed of the following two regions: a region with extremely high variability called the hypervariable region (HVR), and a relatively invariant region called the framework region (FR) separated from this region. The variable regions of natural heavy and light chains contain 4 FRs connected by 3 hypervariable regions, and the hypervariable regions of each chain are held very close together with the hypervariable regions of other chains through the FRs, contributing to the formation of the antigen-binding site of the antibody.

[0508] The heavy and light chains of a known antibody molecule each have three Complementarity Determining Regions (CDRs). The Complementarity Determining Regions are also called hypervariable regions and are located within the variable regions of the heavy and light chains of the antibody. They are sites with particularly high mutability in the primary structure. In the primary structure of the polypeptide chains of the heavy and light chains, they are usually separated into three regions each. In the present invention, for the Complementarity Determining Regions of an antibody, the Complementarity Determining Regions of the heavy chain are sequentially denoted as CDRH1, CDRH2, and CDRH3 starting from the amino-terminal side of the heavy chain amino acid sequence; the Complementarity Determining Regions of the light chain are sequentially denoted as CDRL1, CDRL2, and CDRL3 starting from the amino-terminal side of the light chain amino acid sequence. These sites are conformationally close to each other and determine the specificity for the antigen to which they bind.

[0509] In the present invention, the positions and lengths of the CDRs are determined by the definition of IMGT (Developmental and Comparative Immunology 27(2003)55-77).

[0510] FRs are variable regions other than CDR residues. The variable region generally has four FRs, namely FR1, FR2, FR3, and FR4.

[0511] The CDRs and FRs contained in the heavy and light chains are sequentially arranged as FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4, and FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4 from the amino-terminal to the carboxyl-terminal.

[0512] The positions of the CDRs and FRs can also be determined by various definitions known in the art, such as the definitions of Kabat, Chothia, AbM, contact, etc. other than IMGT.

[0513] In the present invention, an "antigen-binding fragment of an antibody" refers to a partial fragment of an antibody composed of the heavy chain variable region and the light chain variable region and having binding activity to an antigen. Examples of "antigen-binding fragments of an antibody" include, but are not limited to: Fab, F(ab’) 2 、scFv、Fab’、Fv, single-domain antibody (sdAb), and other antigen-binding fragments. The antigen-binding fragments of the antibody are obtained by treating the full-length molecule of the antibody protein with enzymes such as papain and pepsin. In addition, they can also be recombinant proteins produced using recombinant genes in appropriate host cells. In the present invention, "binding fragment of an antibody" has the same meaning as "antigen-binding fragment of an antibody".

[0514] In the present invention, the "site" to which the antibody binds, i.e., the "site" recognized by the antibody, refers to a partial peptide or partial higher-order structure on the antigen to which the antibody binds or recognizes.

[0515] In the present invention, this site is also called an epitope or the binding site of the antibody. In the present invention, a "mutant antibody" refers to a polypeptide having an amino acid sequence formed by substitution, deletion, addition (addition includes insertion) (hereinafter collectively referred to as "mutation") of amino acids in the amino acid sequence of the original antibody and binding to HLA / NY-ESO of the present invention. The number of mutant 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. The mutant antibody is also included in the "antibody" of the present invention.

[0516] In the present invention, "several" in "one or several" means 2 to 10.

[0517] In this specification, a "molecule" includes molecules such as the above-mentioned antibody and antigen-binding fragment of the antibody, and also includes a multi-specific molecule formed by an antibody or a plurality of antigen-binding fragments derived from these antibodies.

[0518] In this specification, "multi-specific molecule", "multi-specific molecule" and "multi-specific molecule" have the same meaning, and there is no particular limitation as long as it is a molecule capable of binding to a plurality of different epitopes on one molecule and / or different epitopes on two or more molecules. As a multi-specific molecule, it also includes an antibody containing a heavy chain variable region (VH) and a light chain variable region (VL). Such multi-specific molecules include, but are not limited to: full-length antibody molecules having two or more different heavy chains and light chains, i.e., IgG-type multi-specific molecules; and molecules composed of two or more antigen-binding fragments having VL and VH, i.e., molecules derived by combining Fab, Fab', Fv, scFv, sdAb, etc., i.e., tandem scFv, diabody, single-chain diabody, triabody, etc. In addition, multi-specific 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, except in the case of not containing an antibody or its antigen-binding fragment, such multi-specific molecules can also be called "multi-specific antibody", "multi-specific antibody", etc.

[0519] As the anti-HLA / NY-ESO antibody of the present invention or an antigen-binding fragment of the antibody, or the activities and properties exhibited by the molecule of the present invention, such as biological activities, physicochemical properties (also referred to as physical and chemical properties), etc. can be exemplified. Specifically, various biological activities such as cytotoxic activity, ADCC activity, anti-tumor activity (all described later); binding activity to antigens and epitopes; stability during production and storage; physicochemical properties such as thermal stability.

[0520] In the present invention, "hybridization under stringent conditions" means hybridization in a solution containing 5×SSC at 65°C, followed by washing under the following conditions respectively or hybridization under conditions equivalent thereto: 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 an SSC solution of n-fold concentration.

[0521] In the present invention, "cytotoxicity" means causing pathological changes in cells in some form, not limited to direct trauma, and also means all damages to cells in terms of structure and function such as DNA cleavage, formation of base dimers, chromosome cleavage, damage to the cell division apparatus, and decrease in various enzyme activities. In the present invention, "cytotoxic activity" means causing the above-mentioned cytotoxicity.

[0522] In the present invention, "antibody-dependent cellular cytotoxic activity" refers to "antibody dependent cellular cytotoxicity (ADCC) activity", which means the activity of NK cells causing damage to target cells such as tumor cells through antibodies.

[0523] In the present invention, "T cell redirected cytotoxic activity" means causing the above-mentioned cytotoxicity through a multispecific molecule including an anti-tumor antigen antibody and an anti-HLA / NY-ESO antibody. That is, it means that the anti-tumor antigen antibody binds to the target tumor cell, and the anti-HLA / NY-ESO antibody binds to the T cell, thereby bringing the target tumor cell closer to the T cell and inducing cytotoxicity through T cell activation. This molecule can also be included in a pharmaceutical composition.

[0524] 2. Antigen

[0525] 2-1. HLA / NY-ESO Antigen

[0526] The term "HLA / NY-ESO" used in the present invention has the same meaning as the HLA / NY-ESO protein.

[0527] HLA / NY-ESO is a ternary complex of HLA-A2, β2-microglobulin, and NY-ESO peptide. HLA-A2 is one of the alleles of HLA and is known to be the allele with the highest frequency in Caucasians. HLA forms a ternary complex with β2-microglobulin and peptide fragments of self-proteins in the endoplasmic reticulum of cells, presents it extracellularly, and is recognized by the TCR (T Cell Receptor) of T cells. The NY-ESO peptide (SLLMWITQC, SEQ ID NO:1, Figure 8 ) is a peptide composed of 9 amino acids at positions 157 to 165 of NY-ESO-1 and LAGE-1, and it has been reported that the NY-ESO peptide is presented by HLA-A2.

[0528] 2-2. CD3 Antigen

[0529] The term "CD3" used in the present invention has the same meaning as the CD3 protein.

[0530] CD3 is expressed on T cells as part of a multi-molecular T cell receptor complex and is a complex of 5 polypeptides (molecular weights are 25,000 - 28,000, 21,000, 20,000, 16,000, and 22,000 in sequence), namely the γ chain, δ chain, ε chain, ζ chain, and η chain.

[0531] As the CD3 complex, the γ, δ, ε, ζ, and η chains can be cited. These chains are also called subunits. Anti-CD3 antibodies bind to T cells, and thereby induce cytotoxicity through T cell activation. Many anti-CD3 antibodies bind to CD3ε.

[0532] The nucleotide sequence of the cDNA encoding human CD3ε is registered in NCBI / GenBank with the accession number NM_000733 (NM_000733.3); the amino acid sequence of human CD3ε is registered in NCBI / GenPept with the accession number NP_000724 (NP_000724.1). The nucleotide sequence of the cDNA encoding cynomolgus monkey CD3 is registered in GenBank with the accession number NM_001283615.1. The amino acid sequence of human CD3ε is described in SEQ ID NO:151 in the sequence listing ( Fig.147 )).

[0533] 2-3. Preparation of Antigen

[0534] The above-mentioned antigen proteins HLA / NY-ESO and CD3 used in the present invention (hereinafter, HLA / NY-ESO and CD3 are collectively referred to as the antigen protein) can be prepared in the following ways: purification and isolation from animal tissues (including body fluids), cells derived from the animal tissues, or cultures of the cells; gene recombination; in vitro translation; chemical synthesis, etc.

[0535] The cDNA of the antigen protein can be obtained, for example, by the so-called PCR method. The PCR method uses 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 polymerase chain reaction (hereinafter referred to as "PCR") (Saiki, R.K., et al., Science (1988) 239, 487-49).

[0536] Polynucleotides consisting of nucleotide sequences complementary to the nucleotide sequences encoding the antigen protein expressed in humans or rats and hybridizing under stringent conditions, and polynucleotides encoding proteins having biological activities equivalent to those of the antigen protein are also included in the cDNA of the antigen protein.

[0537] Furthermore, polynucleotides that hybridize with splicing mutants transcribed from the gene locus encoding the antigen protein expressed in humans or rats under stringent conditions and encode proteins having biological activities equivalent to those of the antigen protein are also included in the cDNA of the antigen protein.

[0538] Nucleotide sequences encoding proteins consisting of the amino acid sequences encoding the antigen protein of humans or rats, or amino acid sequences in which one or several amino acids are substituted, deleted, or added after removing the signal sequences from these sequences and having biological activities equivalent to those of the antigen protein are also included in the nucleotide sequences of the antigen protein gene.

[0539] Proteins consisting of the amino acid sequences encoded by splicing mutants transcribed from the gene locus of the antigen protein of humans or rats, or amino acid sequences in which one or several amino acids are substituted, deleted, or added in the amino acid sequences and having biological activities equivalent to those of the antigen protein are also included in the antigen protein.

[0540] 2-4. Binding Specificity of Antigen Protein

[0541] The anti-HLA / NY-ESO antibodies and antigen-binding fragments thereof of the present invention will recognize HLA / NY-ESO. That is, they will bind to the HLA / NY-ESO antigen. At present, it is not known whether HLA / NY-ESO exists in non-human animals such as mice, rats, and cynomolgus monkeys.

[0542] The anti-CD3 antibody and its binding fragments included in the multispecific molecule of the present invention recognize the CD3 antigen, i.e., they bind to the CD3 antigen. The anti-CD3 antibody etc. preferably bind to human CD3, cynomolgus monkey CD3 etc., and more preferably bind to human CD3 and cynomolgus monkey CD3. On the other hand, the preferred anti-CD3 antibody does not bind to rat and / or mouse CD3.

[0543] The anti-tumor activity of the multispecific molecule of the present invention can be evaluated, for example, by the following methods: (i) transplanting human cancer cells, human cancer tissues etc. into a non-human animal transplanted with human peripheral blood lymphocytes, preferably a rat or a mouse, more preferably a rat or a mouse with endogenous effector dysfunction (e.g., immunodeficient rat or mouse) for evaluation; or, (ii) transplanting mouse cancer cells etc. into which HLA and NY-ESO genes have been introduced into a non-human animal knocked in with the human CD3 gene, preferably a rat or a mouse for evaluation. Evaluating using these immunodeficient animals or knocked-in animals enables various detections, immunohistochemistry etc. to be carried out using the in vivo of the mouse and / or the rat, which is preferred in the drugs containing the multispecific molecule of the present invention, non-clinical development etc.

[0544] In the present invention, "recognition" i.e., "binding" means binding that is not non-specific adsorption. As the determination criterion for whether recognition i.e., whether binding occurs, for example, the dissociation constant (hereinafter referred to as "KD") can be cited. The present invention preferably has a KD value of 1×10 -5 M or less, 5×10 -6 M or less, 2×10 -6 M or less, 1×10 -6 M or less for the antibody etc. against HLA / NY-ESO or CD3; 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, and more preferably 1×10 -9M or less. As the anti-HLA / NY-ESO scFv of the present invention having excellent antigen-binding activity, NYA-1143, NYA-2023, NYA-2143, NYA-2044, NYA-2045, NYA-2060, NYA-2061, NYA-3061 can be exemplified. For HLA / NY-ESO, the KD values of NYA-1143, NYA-2044, NYA-2045, NYA-2143, etc. are 1×10 -9 M or less (Example 4, etc.).

[0545] The binding between the antigen and the antibody in the present invention can be measured or determined by a biomolecular interaction analysis system such as SPR method, BLI method, or ELISA method, RIA method, etc. The binding between the antigen expressed on the cell surface and the antibody can be measured by flow cytometry analysis method, etc.

[0546] The SPR method (surface plasmon resonance analysis method) is used as the following analysis method: By chemical kinetics (kinetics) analysis, the association rate constant (Ka value) and dissociation rate constant (Kd value) are measured to obtain the dissociation constant (KD value) as an affinity index, etc. As the equipment for SPR analysis, 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), Autolab SPR (trademark) (manufactured by Metrohm), etc. can be exemplified.

[0547] The BLI method (BioLayer Interferometry) is a method of measuring the interaction between biomolecules using biolayer interferometry. As the equipment used when analyzing the interaction using the BLI method, the Octet system (manufactured by Pall ForteBio) etc. can be exemplified.

[0548] The ELISA (Enzyme-linked ImmunoSorbent Assay) method is a method of capturing the target antigen or antibody contained in the sample solution using a specific antibody or antigen and detecting and quantifying it using an enzyme reaction. An antigen or antibody labeled with an enzyme is incorporated into the reaction system, and the enzyme activity is detected. The detection of the enzyme activity uses a substrate whose absorption spectrum changes due to the reaction, and it is converted into a numerical value by absorbance measurement.

[0549] Cell-ELISA is a method that captures the object to be measured located on the cell surface of each cell and uses an enzyme reaction for detection and quantification.

[0550] The RIA method (Radio Immunoassay) uses a radioactive substance to label an antibody and measures the radioactivity from the antibody, thereby enabling quantification of the antibody.

[0551] Flow cytometry analysis is a method of dispersing cells into a fluid, causing the fluid to flow thinly, and optically analyzing each cell. An antibody labeled with a fluorescent dye binds to the cell surface antigen through an antigen-antibody reaction, and the fluorescence intensity of the labeled antibody bound to the cell is measured, thereby quantifying the antigen-binding property of the antibody.

[0552] Examples of antibodies having excellent antigen-binding specificity among the anti-HLA / NY-ESO antibodies and the like of the present invention include: 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.) as anti-HLA / NY-ESO scFv.

[0553] 3. An antibody or its binding fragment that specifically binds to HLA / NY-ESO

[0554] 3-1. Anti-HLA / NY-ESO or its binding fragment

[0555] The present invention provides an antibody or its binding fragment that recognizes and binds to HLA / NY-ESO.

[0556] As described above, HLA / NY-ESO is a complex containing HLA-A2 and the 9mer NY-ESO peptide (SLLMWITQC, SEQ ID NO: 1). The NY-ESO peptide is an intracellular protein and is a peptide derived from the cancer-testis antigen NY-ESO-1 or LAGE-1. HLA / NY-ESO is presented on the surface of cancer cells.

[0557] The anti-HLA / NY-ESO antibody of the present invention and the antigen-binding fragment of the antibody (hereinafter also referred to as the antibody of the present invention, etc.) can be either a monoclonal antibody or a polyclonal antibody. The isotype of the monoclonal antibody of the present invention is not particularly limited, and examples thereof include IgG such as IgG1, IgG2, IgG3, IgG4, IgM, IgA such as IgA1, IgA2, IgD, Ig, etc. The isotype and subclass of the monoclonal antibody can be determined, for example, by the double immunodiffusion method in agar, ELISA method, RIA method, etc. As the monoclonal antibody of the present invention, antibodies from non-human animals (non-human animal antibodies), human antibodies, chimerized antibodies (also called "chimeric antibodies"), humanized antibodies, etc. can be mentioned, and preferably human antibodies can be used. The scope of the antibody of the present invention also includes mutants of the antibody (the "mutant antibody" described later), for example, the scope of human antibodies also includes human mutant antibodies.

[0558] As non-human animal antibodies, antibodies from vertebrates such as mammals and birds can be mentioned. As antibodies from mammals, antibodies from rodents such as mouse antibodies and rat antibodies can be mentioned. As antibodies from birds, chicken antibodies can be mentioned.

[0559] As chimerized antibodies, examples include, but are not limited to, antibodies formed by the binding of the variable region of a non-human animal antibody to the constant region of a human antibody (human immunoglobulin).

[0560] As humanized antibodies, examples 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 (the variable region of human immunoglobulin); antibodies formed by transplanting partial sequences of the framework region of a non-human animal antibody into a human antibody in addition to the CDRs; antibodies formed by substituting one or more amino acids from any of the above non-human animal antibodies with human-type amino acids, etc.

[0561] Antibodies can be produced by various well-known methods. As well-known methods, they can be produced by methods such as using hybridomas and cellular immunization methods, or by genetic recombination methods. In addition, methods for obtaining human antibodies displayed on phage selected from a human antibody library are also known. For example, a phage display method can be used in which the variable region of a human antibody is used as scFv, expressed on the phage surface, and phages that bind to the antigen are selected. By analyzing the genes of the phages selected based on 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 clarified, an expression vector having that sequence can be produced, introduced into an appropriate host, and expressed, whereby a human antibody can be obtained (WO92 / 01047, WO92 / 20791, WO93 / 06213, WO93 / 11236, WO93 / 19172, WO95 / 01438, WO95 / 15388, Annu.Rev.Immunol(1994)12, 433-455).

[0562] The highly active antibody thus obtained can also be used as a lead antibody, and the gene encoding the lead antibody can be mutated to produce a mutant with higher activity (the "mutant antibody" described later).

[0563] As CDRH1 to CDRH3 included in the heavy chain of the anti-HLA / NY-ESO antibody or its antigen-binding fragment of the present invention, the following can be preferably selected: CDRH1 consisting of the amino acid sequence shown by SEQ ID NO:54( Fig.61 ), CDRH2 consisting of the amino acid sequence shown by SEQ ID NO:55( Fig.61 ), and a combination of heavy chain CDRH3 consisting of the amino acid sequence shown by SEQ ID NO:56( Fig.61 ) or an amino acid sequence in which the 6th amino acid in the amino acid sequence shown by SEQ ID NO:56( Fig.61 ) is N(Asn). More preferably, the following combinations of CDRH1 to CDRH3 included in the heavy chain variable region can be cited: the NYA-0001 heavy chain variable region consisting of the amino acid sequence shown by SEQ ID NO:6( Fig.13 ); the NYA-0082 heavy chain variable region consisting of the amino acid sequence shown by SEQ ID NO:18( Fig.25 ); the NYA-2023 heavy chain variable region consisting of the amino acid numbers 21 to 140 of the amino acid sequence shown by SEQ ID NO:27( Fig.34 ); the NYA-2023 heavy chain variable region consisting of the amino acid sequence shown by SEQ ID NO:28( Fig.35The NYA-2027 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence shown in Fig.36 ; the NYA-1143 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence shown in SEQ ID NO:29( Fig.33 ); the NYA-1163 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence shown in SEQ ID NO:26( Fig.34 ); the NYA-2023 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence shown in SEQ ID NO:27( Fig.35 ); 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:28( Fig.43 ); the NYA-2035 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence shown in SEQ ID NO:36( Fig.54 ); the NYA-2044 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence shown in SEQ ID NO:47( Fig.55 ); the NYA-2045 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence shown in SEQ ID NO:48( Fig.57 ); the NYA-2047 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence shown in SEQ ID NO:50( Fig.58 ); the NYA-2048 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence shown in SEQ ID NO:51( Fig.59 ); the NYA-2060 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence shown in SEQ ID NO:52( Fig.60 ); the NYA-2061 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence shown in SEQ ID NO:53( Fig.37 ); or the NYA-2143 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence shown in SEQ ID NO:30(

[0564] In addition, a combination of CDRH1 to CDRH3 included in the NYA-3061 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence shown in SEQ ID NO:156( Fig.152 ) can also be cited.

[0565] As CDRL1 to CDRL3 included in the light chain of the anti-HLA / NY-ESO antibody or its antigen-binding fragment of the present invention, the following can be preferably selected: the amino acid sequence shown by SEQ ID NO:57( Fig.61 ), or CDRL1 composed of an amino acid sequence in which the 7th amino acid is W (Trp) or the 8th amino acid is K (Lys) in the amino acid sequence shown by SEQ ID NO:57( Fig.61 ), CDRL2 composed of the amino acid sequence shown by SEQ ID NO:58( Fig.61 ), and CDRL3 composed of the amino acid sequence shown by SEQ ID NO:59( Fig.61 ), or an amino acid sequence in which the 2nd amino acid is A (Ala) or S (Ser) in the amino acid sequence shown by SEQ ID NO:59( Fig.61 ).

[0566] More preferably, the following combinations of CDRL1 to CDRL3 included in the light chain variable region can be cited: the NYA-0001 light chain variable region composed of the amino acid sequence shown by SEQ ID NO:8( Fig.15 ); the NYA-0082 light chain variable region composed of the amino acid sequence shown by SEQ ID NO:20( Fig. 27 ); the NYA-1143 light chain variable region composed of the amino acid numbers 156 to 266 of the amino acid sequence shown by SEQ ID NO:29( Fig.36 ); the NYA-1163 light chain variable region composed of the amino acid numbers 156 to 266 of the amino acid sequence shown by SEQ ID NO:26( Fig.33 ); the NYA-2023 light chain variable region composed of the amino acid numbers 156 to 266 of the amino acid sequence shown by SEQ ID NO:27( Fig.34 ); the NYA-2027 light chain variable region composed of the amino acid numbers 156 to 266 of the amino acid sequence shown by SEQ ID NO:28( Fig.35 ); the NYA-2035 light chain variable region composed of the amino acid numbers 156 to 266 of the amino acid sequence shown by SEQ ID NO:36( Fig.43 ); the NYA-2044 light chain variable region composed of the amino acid numbers 156 to 266 of the amino acid sequence shown by SEQ ID NO:47( Fig.54 ); the NYA-2045 light chain variable region composed of the amino acid numbers 156 to 266 of the amino acid sequence shown by SEQ ID NO:48( Fig.55 ); the NYA-2045 light chain variable region composed of the amino acid numbers 156 to 266 of the amino acid sequence shown by SEQ ID NO:50( Fig.57The NYA-2047 light chain variable region consisting of amino acid numbers 156 to 266 of the amino acid sequence shown in SEQ ID NO: 51( Fig.58 ) The NYA-2048 light chain variable region consisting of amino acid numbers 156 to 266 of the amino acid sequence shown in SEQ ID NO: 52( Fig.59 ) The NYA-2060 light chain variable region consisting of amino acid numbers 156 to 266 of the amino acid sequence shown in SEQ ID NO: 53( Fig.60 ) The NYA-2061 light chain variable region consisting of amino acid numbers 156 to 266 of the amino acid sequence shown in SEQ ID NO: 53( Fig.37 ) or the NYA-2143 light chain variable region consisting of amino acid numbers 156 to 266 of the amino acid sequence shown in SEQ ID NO: 30(

[0567] In addition, the combination of CDRL1 to CDRL3 included in the NYA-3061 light chain variable region consisting of amino acid numbers 161 to 271 of the amino acid sequence shown in SEQ ID NO: 156( Fig.152 ) can also be cited.

[0568] As CDRH1 to CDRH3 included in the heavy chain of the anti-HLA / NY-ESO antibody or its antigen-binding fragment of the present invention and CDRL1 to CDRL3 included in the light chain, preferably selected are: CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 54( Fig.61 ), CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 55( Fig.61 ), the amino acid sequence shown in SEQ ID NO: 56( Fig.61 ), or the heavy chain CDRH3 consisting of the amino acid sequence in which the 6th amino acid in the amino acid sequence shown in SEQ ID NO: 56( Fig.61 ) is N (Asn), CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 57( Fig.61 ), or the amino acid sequence in which the 7th amino acid and / or the 8th amino acid in the amino acid sequence shown in SEQ ID NO: 57( Fig.61 ) is N (Asn) and / or K (Lys), CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 58( Fig.61 ), and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 59( Fig.61) the amino acid sequence shown, or a combination of CDRL3 consisting of an amino acid sequence in which the second amino acid in the amino acid sequence shown in SEQ ID NO:59 is A (Ala) or S (Ser). More preferably, examples of the combination of CDRH1 to CDRH3 and CDRL1 to CDRL3 respectively included in the following heavy chain variable region and light chain variable region are: 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-1143 heavy chain variable region and light chain variable region consisting of amino acid numbers 21 to 140 and 156 to 266 of the amino acid sequence shown in SEQ ID NO:29( Fig.36 ) ; the NYA-1163 heavy chain variable region and light chain variable region consisting of amino acid numbers 21 to 140 and 156 to 266 of the amino acid sequence shown in SEQ ID NO:26( Fig.33 ) ; the NYA-2023 heavy chain variable region and light chain variable region consisting of amino acid numbers 21 to 140 and 156 to 266 of the amino acid sequence shown in SEQ ID NO:27( Fig.34 ) ; the NYA-2027 heavy chain variable region and light chain variable region consisting of amino acid numbers 21 to 140 and 156 to 266 of the amino acid sequence shown in SEQ ID NO:28( Fig.35 ) ; the NYA-2035 heavy chain variable region and light chain variable region consisting of amino acid numbers 21 to 140 and 156 to 266 of the amino acid sequence shown in SEQ ID NO:36( Fig.43 ) ; the NYA-2044 heavy chain variable region and light chain variable region consisting of amino acid numbers 21 to 140 and 156 to 266 of the amino acid sequence shown in SEQ ID NO:47( Fig.54 ) ; the NYA-2045 heavy chain variable region and light chain variable region consisting of amino acid numbers 21 to 140 and 156 to 266 of the amino acid sequence shown in SEQ ID NO:48( Fig.55 ) ; the NYA-2047 heavy chain variable region and light chain variable region consisting of amino acid numbers 21 to 140 and 156 to 266 of the amino acid sequence shown in SEQ ID NO:50( Fig.57 ) ; the NYA-2048 heavy chain variable region and light chain variable region consisting of amino acid numbers 21 to 140 and 156 to 266 of the amino acid sequence shown in SEQ ID NO:51( Fig.58 ) ; the NYA-2060 heavy chain variable region and light chain variable region consisting of amino acid numbers 21 to 140 and 156 to 266 of the amino acid sequence shown in SEQ ID NO:52( Fig.59 ) ; the NYA-2060 heavy chain variable region and light chain variable region consisting of amino acid numbers 21 to 140 and 156 to 266 of the amino acid sequence shown in SEQ ID NO:53( Fig.60The NYA-2061 heavy chain variable region and light chain variable region composed of amino acid numbers 21 to 140 and 156 to 266 of the amino acid sequence shown in Fig.37 ; or, the NYA-2143 heavy chain variable region and light chain variable region composed of amino acid numbers 21 to 140 and 156 to 266 of the amino acid sequence shown in

[0569] In addition, combinations of CDRH1 to CDRH3 and CDRL1 to CDRL3 respectively contained in the NYA-3061 heavy chain variable region and light chain variable region composed of amino acid numbers 21 to 140 and 161 to 271 of the amino acid sequence shown in SEQ ID NO: 156 Fig.152 can also be cited.

[0570] As the heavy chain variable region of the anti-HLA / NY-ESO antibody or its antigen-binding fragment of the present invention, the above heavy chain CDRs or heavy chain variable regions containing combinations thereof can be cited as preferred examples. More preferably, examples include: 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 as described above.

[0571] As the light chain variable region of the anti-HLA / NY-ESO antibody or its antigen-binding fragment of the present invention, the above light chain CDRs or light chain variable regions containing combinations thereof can be cited as preferred examples. More preferably, examples include: 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.

[0572] As the heavy chain variable region and light chain variable region of the anti-HLA / NY-ESO antibody or its antigen-binding fragment of the present invention, the CDRs of the above heavy chain and light chain or the heavy chain variable region and light chain variable region containing a combination thereof can be cited as preferred examples. More preferably, the following can be exemplified: the combination of the heavy chain variable region and light chain variable region of NYA-0001, the combination of the heavy chain variable region and light chain variable region of NYA-0082, the combination of the heavy chain variable region and light chain variable region of NYA-1143, the combination of the heavy chain variable region and light chain variable region of NYA-1163, the combination of the heavy chain variable region and light chain variable region of NYA-2023, the combination of the heavy chain variable region and light chain variable region of NYA-2027, the combination of the heavy chain variable region and light chain variable region of NYA-2035, the combination of the heavy chain variable region and light chain variable region of NYA-2044, the combination of the heavy chain variable region and light chain variable region of NYA-2045, the combination of the heavy chain variable region and light chain variable region of NYA-2047, the combination of the heavy chain variable region and light chain variable region of NYA-2048, the combination of the heavy chain variable region and light chain variable region of NYA-2060, the combination of the heavy chain variable region and light chain variable region of NYA-2061, and the combination of the heavy chain variable region and light chain variable region of NYA-2143, and the combination of the heavy chain variable region and light chain variable region of NYA-3061.

[0573] As the heavy chain of the anti-HLA / NY-ESO antibody or its antigen-binding fragment of the present invention, the heavy chain containing the above preferred or more preferred heavy chain variable region can be cited as a preferred example. More preferably, the following can be exemplified: the heavy chain of NYA-0001, the heavy chain of NYA-0082, the heavy chain of NYA-1143, the heavy chain of NYA-1163, the heavy chain of NYA-2023, the heavy chain of NYA-2027, the heavy chain of NYA-2035, the heavy chain of NYA-2044, the heavy chain of NYA-2045, the heavy chain of NYA-2047, the heavy chain of NYA-2048, the heavy chain of NYA-2060, the heavy chain of NYA-2061, the heavy chain of NYA-2143, and the heavy chain of NYA-3061.

[0574] As the light chain of the anti-HLA / NY-ESO antibody or its antigen-binding fragment of the present invention, the light chain containing the above preferred or more preferred light chain variable region can be cited as a preferred example. More preferably, the following can be exemplified: the light chain of NYA-0001, the light chain of NYA-0082, the light chain of NYA-1143, the light chain of NYA-1163, the light chain of NYA-2023, the light chain of NYA-2027, the light chain of NYA-2035, the light chain of NYA-2044, the light chain of NYA-2045, the light chain of NYA-2047, the light chain of NYA-2048, the light chain of NYA-2060, the light chain of NYA-2061, the light chain of NYA-2143, and the light chain of NYA-3061.

[0575] As the heavy and light chains of the anti-HLA / NY-ESO antibody or its antigen-binding fragment of the present invention, preferred examples may include heavy and light chains respectively containing the above-mentioned preferred or more preferred heavy-chain variable region and light-chain variable region. More preferably, examples may include: 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 combinations of the heavy and light chains of NYA-3061.

[0576] The antigen-binding fragment of an antibody refers to a fragment or its modification that retains at least the antigen-binding property among the functions of the antibody. As the functions of the antibody, generally, the following may be mentioned: antigen-binding activity, activity of regulating antigen activity, antibody-dependent cytotoxic activity, and complement-dependent cytotoxic activity, etc. As the functions of the antibody, etc. and the multispecific molecules containing the antibody, etc. of the present invention, for example, the following may be mentioned: redirection of T cells, activation of T cells, and cytotoxic activity against cancer cells generated by activation of T cells.

[0577] As the antigen-binding fragment of an antibody, there is no particular limitation as long as it is a fragment of the antibody that retains at least the antigen-binding property among the activities of the antibody. For example, but not limited to: Fab, Fab’, F(ab’) 2 , Fv, single-chain Fv (scFv) formed by connecting the Fv of the heavy and light chains with an appropriate linker, single-domain antibody (sdAb), etc. A molecule containing a part other than the antigen-binding fragment of the antibody of the present invention, such as scFv having a linker part, is also included in the meaning of the antigen-binding fragment of the antibody of the present invention.

[0578] A molecule formed by deleting one or several or several or more amino acids at the amino terminus and / or carboxyl terminus of the antibody protein and retaining at least a part of the functions of the antibody is also included in the meaning of the antigen-binding fragment of the antibody. Such a modification of the antigen-binding fragment of the antibody is also included in the antibody or its antigen-binding fragment, or its modification (described later) of the present invention.

[0579] One form of the antibody or its antigen-binding fragment of the present invention is scFv. ScFv is obtained by connecting the heavy-chain variable region and the light-chain variable region of the antibody with a polypeptide linker (Pluckthun A. The Pharmacology of Monoclonal Antibodies 113, edited by Rosenberg and Moore, Springer Verlag, New York, 269-315 (1994), Nature Biotechnology (2005), 23, 1126-1136). In addition, tandem scFv prepared by connecting two scFvs with a polypeptide linker can also be used as a bispecific molecule. In addition, trisomes composed of three or more scFvs can also be used as bispecific molecules.

[0580] As an scFv specific to HLA / NY-ESO (also called "anti-HLA / NY-ESO scFv"), scFv containing the above CDRH1 to CDRH3 and CDRL1 to CDRL3 can be preferably exemplified; more preferably, scFv containing the above heavy-chain variable region and light-chain variable region can be exemplified; further preferably, NYA-0001 (SEQ ID NO: 70( Fig.71 ), amino acid numbers 21 to 266 of the amino acid sequence shown); NYA-0082 (containing the amino acid sequence shown in SEQ ID NO: 18( Figure 25 ) and the amino acid sequence shown in SEQ ID NO: 20( Figure 27 )); NYA-1143 (SEQ ID NO: 29( Figure 36 ), amino acid numbers 21 to 266 of the amino acid sequence shown); NYA-1163 (SEQ ID NO: 26( Figure 33 ), amino acid numbers 21 to 266 of the amino acid sequence shown); NYA-2023 (SEQ ID NO: 27( Figure 34 ), amino acid numbers 21 to 266 of the amino acid sequence shown); NYA-2027 (SEQ ID NO: 28( Figure 35 ), amino acid numbers 21 to 266 of the amino acid sequence shown); NYA-2035 (SEQ ID NO: 36( Figure 43 ), amino acid numbers 21 to 266 of the amino acid sequence shown); NYA-2044 (SEQ ID NO: 47( Figure 54 ), amino acid numbers 21 to 266 of the amino acid sequence shown); NYA-2045 (SEQ ID NO: 48( Figure 55 ), amino acid numbers 21 to 266 of the amino acid sequence shown); NYA-2047 (SEQ ID NO: 50(Figure 57 ) amino acid numbers 21 to 266 of the amino acid sequence shown in); NYA-2048 (SEQ ID NO:51( Figure 58 ) amino acid numbers 21 to 266 of the amino acid sequence shown in); NYA-2060 (SEQ ID NO:52( Figure 59 ) amino acid numbers 21 to 266 of the amino acid sequence shown in); NYA-2061 (SEQID NO:53( Figure 60 ) amino acid numbers 21 to 266 of the amino acid sequence shown in); and NYA-2143 (SEQ ID NO:30( Figure 37 ) amino acid numbers 21 to 266 of the amino acid sequence shown in). Additionally, NYA-3061 (SEQ ID NO:156( Figure 152 ) amino acid numbers 21 to 271 of the amino acid sequence shown in) can also be exemplified.

[0581] Preferred forms of the anti-HLA / NY-ESO scFv include forms fused with a FLAG-His tag on the carboxyl-terminal side (also simply referred to as "tagged form"). As preferred tagged forms, the following can be cited: NYA-0001 tagged form (SEQID NO:70( Figure 71 ) amino acid numbers 20 to 292); NYA-1143 tagged form (SEQ ID NO:29( Figure 36 ) amino acid numbers 20 to 292); NYA-1163 tagged form (SEQ ID NO:26( Figure 33 ) amino acid numbers 20 to 292); NYA-2023 tagged form (SEQ ID NO:27( Figure 34 ) amino acid numbers 20 to 292); NYA-2027 tagged form (SEQ ID NO:28( Figure 35 ) amino acid numbers 20 to 292); NYA-2035 tagged form (SEQ ID NO:36( Figure 43 ) amino acid numbers 20 to 292); NYA-2044 tagged form (SEQ ID NO:47( Figure 54 ) amino acid numbers 20 to 292); NYA-2045 tagged form (SEQ ID NO:48( Figure 55 ) amino acid numbers 20 to 292); NYA-2047 tagged form (SEQ ID NO:50( Figure 57 ) amino acid numbers 20 to 292); NYA-2048 tagged form (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).

[0582] Among them, in the Fc-added bispecific molecule, NYA-2023 and its tag addition, NYA-2047 and its tag addition, NYA-2048 and its tag addition, NYA-2060 and its tag addition, and NYA-2061 and its tag addition have excellent biological activities, physicochemical properties, etc., and are more preferred.

[0583] In addition, when the anti-HLA / NY-ESO scFv and its tag addition are expressed in a host cell, a signal peptide can be added to its amino terminus. As the amino acid sequence of the anti-HLA / NY-ESO scFv tag addition to which a signal peptide is added, SEQ ID NO:70, SEQ ID NO:29, SEQ ID NO:26 to SEQ ID NO:28, SEQ ID NO:36, SEQ IDNO:47, SEQ ID NO:48, SEQ ID NO:50 to SEQ ID NO:53, and SEQ ID NO:30( Figure 71 , 36 , 33 to 35, 43, 54, 55, 57 to 60, and 37) can be cited.

[0584] An scFv can be obtained by phage display, which uses the variable regions of an antibody as a single-chain antibody (scFv) and expresses it on the surface of a phage, and then selects the phage that binds to the antigen (Nature Biotechnology (2005), 23, (9), p. 1105-1116). By analyzing the genes of the phages selected based on their binding to the antigen, 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 clarified, an expression vector having this sequence can be prepared, introduced into an appropriate host, and expressed, thereby enabling the acquisition of a human antibody (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).

[0585] The antibody of the present invention may also be an antibody having a single heavy-chain variable region and no light-chain sequence. Such an antibody is called a single-domain antibody (sdAb) or a nanobody, and it has been reported that it has 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 of the present invention.

[0586] In addition, the present invention includes a single-chain immunoglobulin formed by connecting 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 a single-chain immunoglobulin, 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.

[0587] In the scFv of the present invention, the heavy-chain variable region and the light-chain variable region may also be bound by a disulfide bond.

[0588] If the anti-HLA / NY-ESO antibody of the present invention binds to HLA / NY-ESO, it may also be an antibody composed of parts from multiple different antibodies. For example, antibodies formed by exchanging heavy chains and / or light chains between multiple different antibodies, antibodies formed by exchanging the full lengths of heavy chains and / or light chains, antibodies formed by exchanging only variable regions or only constant regions, antibodies formed by exchanging all or part of CDRs, etc. The heavy chain variable region and the light chain variable region of a chimeric antibody may also be from different anti-HLA / NY-ESO antibodies of the present invention. The heavy chain CDR1 to heavy chain CDR3 and the light chain CDR1 to light chain CDR3 in the variable regions of the heavy chain and the light chain of a humanized antibody may be from two or more anti-HLA / NY-ESO antibodies of the present invention. The heavy chain CDR1 to heavy chain CDR3 and the light chain CDR1 to light chain CDR3 in the variable regions of the heavy chain and the light chain of a human antibody may also be a combination of CDRs possessed by two or more anti-HLA / NY-ESO antibodies of the present invention.

[0589] The anti-HLA / NY-ESO antibody of the present invention also includes the following antibodies: antibodies that contain an amino acid sequence encoded by a nucleotide sequence of a polynucleotide that hybridizes under stringent conditions with a complementary strand of a polynucleotide containing a nucleotide sequence encoding the amino acid sequence contained in the anti-HLA / NY-ESO antibody of the present invention and that bind to HLA / NY-ESO.

[0590] It may also be the following antibody or its antigen-binding fragment: containing the amino acid sequence contained in the heavy chain variable region of the anti-HLA / NY-ESO antibody or its antigen-binding fragment of the present invention (preferably the amino acid sequence of amino acid numbers 21 to 140 of the amino acid sequence shown in SEQ ID NO: 27, the amino acid sequence shown in SEQ ID NO: 38, the amino acid sequence shown in SEQ ID NO: 39, or the amino acid sequence of amino acid numbers 21 to 140 of the amino acid sequence shown in SEQ ID NO: 160, SEQ ID NO: 197 or SEQ ID NO: 198), and / or, the amino acid sequence contained in the light chain variable region (preferably the amino acid sequence of amino acid numbers 156 to 266 of the amino acid sequence shown in SEQ ID NO: 27, the amino acid sequence of amino acid numbers 156 to 266 of the amino acid sequence shown in SEQ ID NO: 52, the amino acid sequence shown in SEQ ID NO: 40, the amino acid sequence of amino acid numbers 161 to 271 of the amino acid sequence shown in SEQ ID NO: 160, or the amino acid sequence of amino acid numbers 156 to 266 of the amino acid sequence shown in SEQ ID NO: 197 or SEQ ID NO: 198), and having an amino acid sequence with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% identity, and an antibody or its antigen-binding fragment that binds to HLA / NY-ESO.

[0591] Regarding the position and length of the light chain variable region, compared with the case determined according to the definition of IMGT, if a definition different from IMGT (such as Kabat, Chothia, AbM, contact, etc.) is used for determination, one or more amino acids, such as arginine and glycine, may sometimes be contained at the carboxyl terminus of the amino acid sequence of the light chain variable region determined according to the definition of IMGT. An antibody or its binding fragment having such a light chain variable region is also included in the antibody or its binding fragment of the present invention.

[0592] As the antibody of the present invention, etc., it may also be an antibody, etc. that introduces a mutation into the binding fragment of the anti-HLA / NY-ESO antibody of the present invention and optimizes the binding ability to HLA / NY-ESO, particularly HLA / NY-ESO of humans and / or cynomolgus monkeys. As a specific method for introducing a mutation, examples include: random mutagenesis using the mismatch PCR method, site-specific amino acid mutation introduction using an NNK library, site-specific mutation introduction using structural information, and combinations thereof.

[0593] 3-2. Mutant of anti-HLA / NY-ESO antibody (mutated antibody)

[0594] The mutant antibodies of the anti-HLA / NY-ESO antibody of the present invention preferably can have the following functions: reducing the susceptibility to proteolysis or oxidation of proteins; maintaining or improving biological activities and functions, or inhibiting the decline and change of biological activities and functions; improving or regulating antigen-binding ability, or endowing physicochemical properties or functional properties. It is known that when specific amino acid side chains on the surface of a protein change, the function and activity of the protein will change. Examples of this include deamidation of asparagine side chains, isomerization of aspartic acid side chains, etc. Mutant antibodies that substitute other amino acids to prevent such changes in amino acid side chains are also included within the scope of the mutant antibodies of the present invention.

[0595] As an example of the mutant antibody of the present invention, an antibody having an amino acid sequence formed by conservative amino acid substitution in the amino acid sequence possessed by the antibody can be cited. Conservative amino acid substitution means substitution occurring within an amino acid group associated with an amino acid side chain.

[0596] 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-containing group = asparagine and glutamine; aliphatic group = alanine, valine, leucine and isoleucine; and aromatic group = phenylalanine, tryptophan and tyrosine. The amino acid substitution in the mutant antibody is preferably carried out within a range that does not reduce the antigen-binding activity possessed by the original antibody.

[0597] The following are also included in the anti-HLA / NY-ESO antibody, its antigen-binding fragment, its mutant (mutant antibody or its antigen-binding fragment), or the molecule of the present invention: mutant antibodies having an amino acid sequence in which conservative amino acid substitution and / or other mutations have been made in the amino acid sequence possessed by an antibody of the present invention such as NYA-2023 and binding to HLA / NY-ESO, their antigen-binding fragments, molecules containing them, etc.; chimerized antibodies, humanized antibodies, human antibodies having an amino acid sequence in which conservative amino acid substitution and / or other mutations have been made in any of the amino acid sequences of CDRH1 to CDRH3 and CDRL1 to CDRL3 derived from an antibody of the present invention containing NYA-2023 etc. and binding to HLA / NY-ESO, their antigen-binding fragments, molecules containing them, etc.

[0598] 3-3. Binding Fragments of Anti-HLA / NY-ESO Antibodies

[0599] In one form of the present invention, an antigen-binding fragment of the anti-HLA / NY-ESO antibody of the present invention (hereinafter simply referred to as "binding fragment") is provided. Among the binding fragments of the anti-HLA / NY-ESO antibody of the present invention, there are included binding fragments of chimeric antibodies, humanized antibodies or human antibodies. The binding fragment of an antibody refers to a fragment or its modified product that retains at least the antigen-binding property among the functions possessed by the antibody. As the functions of such an antibody, generally, the following can be mentioned: antigen-binding activity, activity for regulating antigen activity (e.g., agonist activity), activity for internalizing the antigen in cells, activity for inhibiting or promoting the interaction with a substance that interacts with the antigen, and the like.

[0600] As the binding fragment of an antibody, there is no particular limitation as long as it is a fragment of the antibody that retains at least the antigen-binding property among the activities possessed by the antibody. As such a binding fragment of an antibody, for example, but not limited to: Fab, Fab’, F(ab’) 2 , single-chain Fab (scFab) formed by connecting the carboxyl terminus of the Fab light chain and the amino terminus of the Fab heavy chain through an appropriate linker, Fv, single-chain Fv (scFv) formed by connecting the Fv of the heavy chain and the light chain through an appropriate linker, single-domain antibody (sdAb) having a single heavy-chain variable region and no light-chain sequence, or also called nanobody (Muyldemans S. et al., Protein Eng., (1994) 7(9), 1129-35, Hamers-Casterman C. et al., Nature (1993) 363(6428), 446-448), etc. Molecules containing parts other than the binding fragment of the antibody of the present invention, such as scFab and scFv having a linker part, are also included in the meaning of the binding fragment of the antibody of the present invention.

[0601] 3-4. Modifiers and complexes of the anti-HLA / NY-ESO antibody or its binding fragment

[0602] The present invention provides a modified product of an antibody or its binding fragment. The modified product of the antibody or its binding fragment of the present invention refers to a modified product formed by chemically or biologically modifying the antibody or its binding fragment of the present invention. Chemical modified products include: chemical bonds leading to chemical moieties of the amino acid backbone, chemical modified products of N-linked or O-linked carbohydrate chains, etc. Biological modified products include: modified products of post-translational modifications (for example, addition of sugar chains at N-linkages or O-linkages, processing of the amino-terminal region or carboxyl-terminal region, deamidation, isomerization of aspartic acid, oxidation of methionine), modified products formed by expressing in a prokaryotic host cell to add a methionine residue at the amino terminus, etc. In addition, substances labeled for detecting or separating the antibody or antigen of the present invention, such as enzyme labels, fluorescent labels, and affinity labels, are also included in the meaning of this modified product. Such a modified product of the antibody or its binding fragment of the present invention can be used to improve the stability and blood retention of the original antibody or its binding fragment of the present invention, reduce antigenicity, detect or separate the antibody or antigen, etc.

[0603] Examples of the chemical moieties included as chemical modified products are: water-soluble polymers such as polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymer, carboxymethyl cellulose, dextran, and polyvinyl alcohol.

[0604] Examples of biological modified products are: modified products obtained by modification through enzyme treatment and cell treatment, etc., fusion products with other peptides such as tags added by genetic recombination, and modified products prepared using cells expressing endogenous or exogenous sugar chain modifying enzymes as hosts.

[0605] This modification can be carried out at any position or at a desired position in the antibody or its binding fragment, and the same modification or two or more different modifications can also be carried out at one or more positions.

[0606] However, deletion of these heavy chain sequences, or modification of the heavy chain or light chain sequences will not affect the antigen-binding ability and effector functions (such as complement activation and antibody-dependent cell cytotoxicity) of the antibody.

[0607] Accordingly, the present invention also encompasses antibodies that have undergone such deletions or modifications. For example, the following can be cited: deletion mutants in which one or two amino acids are deleted from the carboxyl terminus of the heavy chain (Journal of Chromatography A; 705; 129-134 (1995)); deletion mutants in which the two amino acid residues, glycine and lysine, at the carboxyl terminus of the heavy chain are deleted and the new carboxyl-terminal proline residue is amidated (Analytical Biochemistry, 360: 75-83 (2007)); antibodies in which the glutamine or glutamate residue at the amino terminus of the heavy or light chain of the antibody is modified by pyroglutamylation (International Patent Publication No. WO2013 / 147153), etc. (these are collectively referred to as "deletion mutants"). However, as long as the antigen-binding ability and effector function are maintained, the deletion mutants at the carboxyl termini of the heavy and light chains of the antibodies of 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) can be either full-length heavy chains and heavy chains selected from the group consisting of the above deletion mutants, or any combination of two of them. The quantitative ratio or molecular number ratio of each deletion mutant is affected by the type and culture conditions of the mammalian cultured cells that produce the antibody of the present invention, but as the main component of the antibody of the present invention, the case where one amino acid residue is deleted from the carboxyl terminus of both heavy chains can be cited.

[0608] In addition, even if one or several amino acids from an expression vector and / or a signal sequence, etc. are added to the amino terminus and / or carboxyl terminus of the antibody or its antigen-binding fragment of the present invention (and part or all of them are modified as described above), as long as the desired antigen-binding activity can be maintained, it is included within the scope of the modified antibody or its antigen-binding fragment of the present invention, and molecules containing such modified antibodies or antigen-binding fragments are also included within the scope of the molecules of the present invention.

[0609] In the present invention, "antibody or its binding fragment" includes the meaning of "modified antibody or its antigen-binding fragment". In addition, the "antibody or its antigen-binding fragment" contained in the molecules, multispecific molecules, bispecific molecules, etc. of the present invention includes the meaning of the "modified antibody or its antigen-binding fragment" involved.

[0610] In addition, by regulating the sugar chain modification (such as glycosylation, defucosylation, etc.) that binds to the antibody of the present invention, the antibody-dependent cell cytotoxic activity can be enhanced. As techniques for regulating antibody sugar chain modification, but not limited to, International Patent Publications WO99 / 54342, WO00 / 61739, WO02 / 31140, etc. can be cited.

[0611] The present invention also encompasses complexes (immunoconjugates) formed by linking the above antibodies to other molecules via a linker. As an example of an antibody-drug conjugate formed by conjugating an antibody to a radioactive substance or a pharmacologically active compound (drug), an ADC (Antibody-Drug Conjugate) can be cited (Methods Mol Biol. (2013) 1045: 1-27; Nature Biotechnology (2005) 23, p. 1137-1146).

[0612] In addition, the present invention also encompasses complexes formed by linking these antibodies to other functional polypeptides. As an example of such an antibody-peptide complex, a complex of an antibody and an albumin-binding polypeptide can be cited (Protein Eng Des Sel. (2012) (2): 81-8).

[0613] Modifications of the above antibodies, antibodies with regulated sugar chain modifications, and complexes are included in the antibodies of the present invention; binding fragments of the above modifications of antibodies, antibodies with regulated sugar chain modifications, and complexes are included in the binding fragments of the antibodies of the present invention.

[0614] 4. Method for producing antibodies

[0615] The antibodies of the present invention can be produced, for example, by inserting DNA encoding the heavy chain variable region or DNA encoding the light chain variable region into an expression vector, transforming a host cell for expression using the vector, and culturing the host cell, thereby enabling the cell to produce the antibodies of the present invention as recombinant antibodies.

[0616] Regarding the DNA encoding the antibody, by ligating the DNA encoding the heavy chain variable region to the DNA encoding the heavy chain constant region, DNA encoding the heavy chain can be obtained; in addition, by ligating the DNA encoding the light chain variable region to the DNA encoding the light chain constant region, DNA encoding the light chain can be obtained.

[0617] Regarding the anti-HLA / NY-ESO antibody of the present invention, the DNA encoding the heavy chain and the DNA encoding the light chain described above can be inserted into an expression vector, and the host cell can be transformed with this vector and cultured to produce the anti-HLA / NY-ESO antibody of the present invention. At this time, the DNA encoding the heavy chain and the DNA encoding the light chain described above can be introduced into the same expression vector, and the host cell can be transformed with this vector, or the DNA encoding the heavy chain and the DNA encoding the light chain can be introduced into different expression vectors respectively, and the host cell can be transformed with two vectors. At this time, the DNA encoding the heavy chain variable region and the light chain variable region can also be introduced into a vector that has previously introduced the DNA encoding the heavy chain constant region and the DNA encoding the light chain constant region. In addition, this vector may also contain DNA encoding a signal peptide that promotes the secretion of antibodies by host cells. In this case, the DNA encoding the signal peptide is ligated in-frame with the DNA encoding the antibody. After the antibody is produced, the signal peptide is removed, and thus the antibody as a mature protein can be obtained.

[0618] At this time, the DNA encoding the heavy chain variable region, the DNA encoding the light chain variable region, the DNA formed by ligating the DNA encoding the heavy chain variable region and the DNA encoding the heavy chain constant region, and the DNA formed by ligating the DNA encoding the light chain variable region and the DNA encoding the light chain constant region can also be functionally ligated with elements such as a promoter, an enhancer, and a polyadenylation signal. Here, functionally ligating means performing ligation so that the element can achieve its function.

[0619] The expression vector is not particularly limited as long as it can replicate in hosts such as animal cells, bacteria, and yeast. For example, known plasmids, phages, etc. can be cited. As the vector for constructing the expression vector, for example, pcDNA (trademark) (Thermo Fisher Scientific), Flexi (registered trademark) vector (Promega), pUC19, pUEX2 (produced by Amersham), pGEX-4T, pKK233-2 (produced by Pharmacia), pMAM-neo (produced by Clontech), etc. can be cited. As the host cell, prokaryotic cells such as Escherichia coli and Bacillus subtilis can be used, or eukaryotic cells such as yeast and animal cells can be used, and eukaryotic cells are preferably used. 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 known methods to transform the host cell. For example, electroporation, calcium phosphate precipitation method, DEAE-dextran transfection method, etc. can be cited. The produced antibody can be purified using separation and purification methods used for ordinary proteins. For example, affinity chromatography, other chromatography, filter membranes, ultrafiltration membranes, salting out, dialysis, etc. can be appropriately selected and combined.

[0620] 5. Molecules that bind to HLA / NY-ESO

[0621] 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 different epitopes on one molecule or different epitopes on two or more molecules, and comprise a plurality of different antigen-binding fragments. Such multispecific molecules include, but are not limited to: IgG-type multispecific molecules; multispecific molecules having two or more variable regions; for example, antibody fragments such as tandem scFv (taFv), single-chain diabody, diabody and triabody, antibody fragments linked by covalent or non-covalent bonds. The multispecific molecule may also comprise Fc.

[0622] In addition to the anti-HLA / NY-ESO antibodies of the present invention or antigen-binding fragments thereof, the multispecific molecules of the present invention may further comprise one or two or more other antibodies or antigen-binding fragments of such antibodies. As antigen-binding fragments of other antibodies, for example, Fab, F(ab)’, Fv, scFv, sdAb can be cited.

[0623] The preferred multispecific molecules of the present invention further comprise an anti-CD3 antibody or an antigen-binding fragment thereof, and also specifically bind to CD3.

[0624] The anti-CD3 antibody or antigen-binding fragment thereof comprised in the multispecific molecules of the present invention is not particularly limited as long as it is an antibody or a binding fragment thereof that binds to human CD3, and preferably also binds to CD3 of non-human primates such as cynomolgus monkeys. As a more preferred anti-CD3 antibody or antigen-binding fragment thereof, an antibody or antigen-binding fragment thereof containing the following heavy-chain variable region and light-chain variable region can be exemplified: a heavy-chain variable region CDRH1 consisting of the amino acid sequence shown in SEQ ID NO:141; a heavy-chain variable region CDRH2 consisting of the amino acid sequence shown in SEQ ID NO:142; a heavy-chain variable region CDRH3 consisting of the amino acid sequence shown in SEQ ID NO:143; a light-chain variable region CDRL1 consisting of the amino acid sequence shown in SEQ ID NO:144; a light-chain variable region CDRL2 consisting of the amino acid sequence shown in SEQ ID NO:145; and a light-chain variable region CDRL3 consisting of the amino acid sequence shown in SEQ ID NO:146 (above reference Figure 142 )

[0625] As a more preferred antibody or antigen-binding fragment thereof containing CDRH1~CDRH3 and CDRL1~CDRL3, an antibody or antigen-binding fragment thereof containing the following heavy-chain variable region can be exemplified: consisting of SEQ ID NO:136 ( Figure 137) The C3E-7034 heavy chain variable region consisting of the amino acid sequence of amino acid numbers 2 to 119 of the amino acid sequence shown; from SEQ ID NO: 137( Figure 138 ) The C3E-7036 heavy chain variable region consisting of the amino acid sequence of amino acid numbers 2 to 119 of the amino acid sequence shown; from SEQ ID NO: 138( Figure 139 ) The C3E-7085 heavy chain variable region consisting of the amino acid sequence of amino acid numbers 2 to 119 of the amino acid sequence shown; from SEQ ID NO: 139( Figure 140 ) The C3E-7088 heavy chain variable region consisting of the amino acid sequence of amino acid numbers 2 to 119 of the amino acid sequence shown; from SEQ ID NO: 140( Figure 141 ) The C3E-7093 heavy chain variable region consisting of the amino acid sequence of amino acid numbers 2 to 119 of the amino acid sequence shown; from SEQ ID NO: 155( Figure 151 ) The C3E-7096 heavy chain variable region consisting of the amino acid sequence of amino acid numbers 272 to 389 of the amino acid sequence shown; from SEQ IDNO: 156( Figure 152 ) The C3E-7096 heavy chain variable region consisting of the amino acid sequence of amino acid numbers 277 to 394 of the amino acid sequence shown; from SEQ ID NO: 157( Figure 153 ) The C3E-7097 heavy chain variable region consisting of the amino acid sequence of amino acid numbers 277 to 394 of the amino acid sequence shown.

[0626] In addition, as a more preferred antibody or antigen-binding fragment thereof containing CDRH1 to CDRH3 and CDRL1 to CDRL3, an antibody or antigen-binding fragment thereof containing the following light chain variable regions can be exemplified: the C3E-7034 light chain variable region consisting of the amino acid sequence of amino acid numbers 135 to 243 of the amino acid sequence shown by SEQ ID NO: 136( Figure 137 ) The C3E-7036 light chain variable region consisting of the amino acid sequence of amino acid numbers 135 to 241 of the amino acid sequence shown; from SEQ IDNO: 137( Figure 138 ) The C3E-7085 light chain variable region consisting of the amino acid sequence of amino acid numbers 135 to 241 of the amino acid sequence shown; from SEQ ID NO: 138( Figure 139 ) The C3E-7088 light chain variable region consisting of the amino acid sequence of amino acid numbers 135 to 243 of the amino acid sequence shown; from SEQ ID NO: 139( Figure 140 ) The C3E-7088 light chain variable region consisting of the amino acid sequence of amino acid numbers 135 to 243 of the amino acid sequence shown; from SEQ ID NO: 140( Figure 141The C3E-7093 light chain variable region consisting of the amino acid sequence from amino acid number 135 to 243 of the amino acid sequence shown in Figure 151 The C3E-7096 light chain variable region consisting of the amino acid sequence from amino acid number 405 to 511 of the amino acid sequence shown in Figure 152 The C3E-7096 light chain variable region consisting of the amino acid sequence from amino acid number 410 to 516 of the amino acid sequence shown in Figure 153 The C3E-7097 light chain variable region consisting of the amino acid sequence from amino acid number 410 to 516 of the amino acid sequence shown in

[0627] As a more preferred antibody or its antigen-binding fragment containing CDRH1 to CDRH3 and CDRL1 to CDRL3, examples include antibodies or their antigen-binding fragments containing the following combinations of heavy chain variable regions and light chain variable regions: the combination of the C3E-7034 heavy chain variable region and light chain variable region consisting of amino acid numbers 2 to 119 and 135 to 243 shown in SEQ ID NO:136( Figure 137 ); the combination of the C3E-7036 heavy chain variable region and light chain variable region consisting of amino acid numbers 2 to 119 and 135 to 241 shown in SEQ ID NO:137( Figure 138 ); the combination of the C3E-7078 heavy chain variable region and light chain variable region consisting of amino acid numbers 2 to 119 and 135 to 243 shown in SEQ ID NO:147( Figure 143 ); the combination of the C3E-7085 heavy chain variable region and light chain variable region consisting of amino acid numbers 2 to 119 and 135 to 241 shown in SEQ ID NO:138( Figure 139 ); the combination of the C3E-7088 heavy chain variable region and light chain variable region consisting of amino acid numbers 2 to 119 and 135 to 243 shown in SEQ ID NO:139( Figure 140 ); the combination of the C3E-7093 heavy chain variable region and light chain variable region consisting of amino acid numbers 2 to 119 and 135 to 243 shown in SEQ ID NO:140( Figure 141 ); the combination of the C3E-7096 heavy chain variable region and light chain variable region consisting of amino acid numbers 272 to 389 and 405 to 511 shown in SEQ ID NO:155( Figure 151 ); the combination of the C3E-7096 heavy chain variable region and light chain variable region consisting of amino acid numbers 277 to 394 and 410 to 516 shown in SEQ ID NO:156( Figure 152 ); the combination of the C3E-7096 heavy chain variable region and light chain variable region consisting of amino acid numbers 277 to 394 and 410 to 516 shown in SEQ ID NO:157(Figure 153 ) The combination of the heavy chain variable region and the light chain variable region of C3E-7097 composed of amino acid numbers 277 to 394 and 410 to 516 shown in

[0628] In addition, as a more preferred antibody or antigen-binding fragment thereof containing CDRH1 to CDRH3 and CDRL1 to CDRL3, examples include antibodies or antibody-binding fragments containing the following scFv and any one of these scFv: composed of the amino acid sequence of amino acid numbers 2 to 243 of the amino acid sequence shown in SEQ ID NO: 136 ( Figure 137 ) C3E-7034 scFv; composed of the amino acid sequence of amino acid numbers 2 to 241 of the amino acid sequence shown in SEQ ID NO: 137 ( Figure 138 ) C3E-7036 scFv; composed of the amino acid sequence of amino acid numbers 2 to 243 of the amino acid sequence shown in SEQ ID NO: 147 ( Figure 143 ) C3E-7078 scFv; composed of the amino acid sequence of amino acid numbers 2 to 241 of the amino acid sequence shown in SEQ ID NO: 138 ( Figure 139 ) C3E-7085 scFv; composed of the amino acid sequence of amino acid numbers 2 to 243 of the amino acid sequence shown in SEQ ID NO: 139 ( Figure 140 ) C3E-7088 scFv; composed of the amino acid sequence of amino acid numbers 2 to 243 of the amino acid sequence shown in SEQ ID NO: 140 ( Figure 141 ) C3E-7093 scFv. A preferred form of scFv specific to CD3 (also called "anti-CD3 scFv") includes a form with a FLAG-His tag attached to the carboxyl-terminal side (also simply referred to as "tagged form"). As a preferred tagged form, examples 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 ), and more preferably C3E-7085 can be cited.

[0629] Preferred examples of the multispecific molecules of the present invention include bispecific molecules. "Bispecific" means capable of binding to two different epitopes of the same molecule or two different epitopes on two molecules, and includes antibodies or antigen-binding fragments having such bispecificity. The bispecific molecule of the present invention binds to HLA / NY-ESO and also binds to CD3.

[0630] As the bispecific molecule of the present invention, molecules having the following structure (configuration) can be cited.

[0631] In the bispecific molecule of the bis-scFv type, two scFvs that bind to different epitopes are each connected to one Fc of a dimer through a linker, or are directly bound without a linker. Alternatively, two scFvs that bind to different epitopes are each connected to CH and CL through a linker, and then each connected to one Fc of a dimer through a linker. This bispecific molecule is a configuration formed by the association of a mutated Fc with a heterozygote, wherein the introduced mutation forms a heterodimer downstream of each of the two different scFvs. The bispecific molecule of the bis-scFv type is called a bis-type bispecific molecule, or simply called a bis-type ( Figure 6A (b)).

[0632] In the present invention, for example, a bis-type bispecific molecule composed of anti-HLA-A2 / NY-ESO scFv and anti-CD3 scFv can be used.

[0633] Alternatively, as the bispecific molecule of the present invention, it can be a molecule as follows: a bispecific molecule in which a Fab and an scFv that bind to different epitopes, the first antibody Fab is bound to one Fc of a dimer through a linker, and the second antibody scFv is bound to the other Fc through a linker. This bispecific molecule is a configuration formed by the association of a mutated Fc with a heterozygote, wherein the introduced mutation forms a heterodimer downstream of each of the Fab and the scFv. This bispecific molecule is called a heterozygous bispecific molecule or a heterozygous type ( Figure 6A (a)). In the present invention, for example, a heterozygous type composed of anti-HLA-A2 / NY-ESO Fab and anti-CD3 scFv can be used.

[0634] Alternatively, it can also be a bispecific molecule formed by binding the Fab of the first antibody and the scFv of the second antibody to one Fc of the dimer through a linker. In this case, either the Fab can be bound to the Fc and the scFv can be bound to the Fab, or the scFv can be bound to the Fc and the Fab can be bound to the scFv. Preferably, the Fab is bound and the scFv is bound to the Fab. The binding of the Fab and the scFv can bind the scFv to the variable region of the Fab through a linker. This bispecific molecule is a configuration formed by the association of an Fc with introduced mutations, wherein the introduced mutations form a heterodimer downstream of the connection of the scFv and the Fab through a linker. This bispecific molecule is called an scFv-Fab-heterodimer Fc-type bispecific molecule, or scFv-Fab-heterodimer Fc-type( Figure 6A (c)).

[0635] In the present invention, for example, an scFv-Fab-heterodimer Fc-type composed of anti-CD3 scFv and anti-HLA-A2 / NY-ESO Fab can be used.

[0636] In addition, a taFv( Figure 3 (c)) having a configuration in which two scFvs of the first antibody and the second antibody are connected through a linker can be bound to one Fc of the dimer through a linker, or can be directly bound without a linker. This bispecific molecule is called a taFv-heterodimer Fc-type bispecific molecule, or taFv-heterodimer Fc-type( Figure 3 (d)). This bispecific molecule is a configuration formed by the association of an Fc with introduced mutations and a heterozygote, wherein the introduced mutations form a heterodimer downstream of the taFv. There is no limitation on the connection order of the first antibody and the second antibody in the taFv. When the connection order of the first antibody and the second antibody in the taFv is reversed, corresponding to the original bispecific molecule called taFv-heterodimer Fc-type, it is called taFv (reverse)-heterodimer Fc-type (also called taFv (reverse)-Fc-type).

[0637] Figure 6A (a) shows the structure of the hybrid-type bispecific molecule, Figure 6A (b) shows the structure of the dual-type bispecific molecule, Figure 6A (c) shows the structure of the scFv-Fab-heterodimer Fc-type bispecific molecule. In addition, Figure 3 (a) shows the structure of the scFv, Figure 3 (b) shows the structure of the Fab, Figure 3 (c) shows the structure of the taFv, Figure 3 (d) shows the structure of the taFv-heterodimer Fc-type bispecific molecule, Figure 3(e) shows the structure of the taFv-Fab-heterodimeric Fc-type bispecific molecule. Additionally, Figure 6B (a) shows the structure of the taFv-heterodimeric Fc-type bispecific molecule (the same as Figure 3 (d)), Figure 6B (b) shows the structure of the taFv (reverse)-heterodimeric Fc-type bispecific molecule, Figure 6B (c) shows the structure of the first polypeptide contained in the taFv (reverse)-heterodimeric Fc-type bispecific molecule, Figure 6B (d) shows the structure of the second polypeptide contained in the taFv (reverse)-heterodimeric Fc-type bispecific molecule.

[0638] The bispecific molecule of the present invention has a structure formed by the association of multiple polypeptides.

[0639] In the present invention, for example, as the taFv, taFv of anti-HLA-A2 / NY-ESO scFv and anti-CD3 scFv can be used. Regarding the taFv-heterodimeric Fc-type bispecific molecule, preferably, (a) it contains a first polypeptide and a second polypeptide. The first polypeptide sequentially contains, from the N-terminus to the C-terminus, an scFv specifically binding to HLA / NY-ESO, an scFv specifically binding to CD3, and an immunoglobulin Fc region (i); the second polypeptide contains a hinge region and an 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 the taFv-heterodimeric Fc-type bispecific molecule is shown in Figure 3 (d). As shown in Figure 3 (d), the Fc region (i) part of the first polypeptide binds to the Fc region (ii) part of the second polypeptide represented by being blackened, and the first polypeptide and the second polypeptide associate. Figure 3 (f) represents the first polypeptide, Figure 3 (g) represents the second polypeptide. For example, in Figure 3 (d), the scFv represented by white is anti-HLA-A2 / NY-ESO scFv, and the scFv represented by the upper right diagonal line is anti-CD3 scFv.

[0640] The first polypeptide contained in the more preferred taFv-heterodimeric Fc-type bispecific molecule of the present invention comprises the following amino acid sequences: the amino acid sequence from position 21 to position 511 of the amino acid sequence shown in SEQ ID NO:85, the amino acid sequence from position 21 to position 511 of the amino acid sequence shown in SEQ ID NO:87, the amino acid sequence from position 21 to position 511 of the amino acid sequence shown in SEQ ID NO:88, the amino acid sequence from position 21 to position 511 of the amino acid sequence shown in SEQ ID NO:89, the amino acid sequence from position 21 to position 511 of the amino acid sequence shown in SEQ ID NO:90, the amino acid sequence from position 21 to position 511 of the amino acid sequence shown in SEQ ID NO:91, the amino acid sequence from position 21 to position 511 of the amino acid sequence shown in SEQ ID NO:92, the amino acid sequence from position 21 to position 511 of the amino acid sequence shown in SEQ ID NO:93, the amino acid sequence from position 21 to position 511 of the amino acid sequence shown in SEQ ID NO:94, the amino acid sequence from position 21 to position 511 of the amino acid sequence shown in SEQ ID NO:95, the amino acid sequence from position 21 to position 511 of the amino acid sequence shown in SEQ ID NO:96, the amino acid sequence from position 21 to position 511 of the amino acid sequence shown in SEQ ID NO:86, the amino acid sequence from position 21 to position 511 of the amino acid sequence shown in SEQ ID NO:149, the amino acid sequence from position 21 to position 511 of the amino acid sequence shown in SEQ ID NO:150, the amino acid sequence from position 20 to position 511 of the amino acid sequence shown in SEQ ID NO:155, the amino acid sequence from position 20 to position 516 of the amino acid sequence shown in SEQ ID NO:156, or the amino acid sequence from position 20 to position 516 of the amino acid sequence shown in SEQ ID NO:157.The first polypeptide contained in the further preferred taFv-heterodimer Fc-type bispecific molecule comprises the amino acid sequence from position 529 to position 745 of the amino acid sequence shown 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 position 534 to position 750 of the amino acid sequence shown in SEQ ID NO:156 or SEQ ID NO:157. The first polypeptide contained in the even more preferred taFv-heterodimer Fc-type bispecific molecule consists of the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:85; the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:87; the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:88; the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:89; the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:90; the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:91; the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:92; the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:93; the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:94; the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:95; the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:96; the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:86; the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:149; the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:150; or the amino acid sequence from position 20 to position 745 of the amino acid sequence shown in SEQ ID NO:155.Alternatively, it is composed of the amino acid sequence from position 20 to position 750 of the amino acid sequence shown in SEQ ID NO: 156 or the amino acid sequence from position 20 to position 750 of the amino acid sequence shown in SEQ ID NO: 157.

[0641] The second polypeptide contained in the preferred taFv - heterodimer Fc - type bispecific molecule of the present invention comprises a hinge region and a mutant Fc from a human antibody. More preferably, the second polypeptide contained in the taFv - heterodimer Fc - type bispecific molecule comprises the amino acid sequence from position 20 to position 246 of the amino acid sequence shown in SEQ ID NO: 84.

[0642] Among them, the following antibodies can be exemplified as the preferred taFv-heterodimer Fc-type bispecific molecules of the present invention: NYF-0016 formed by the association of a first polypeptide composed of the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:85 and a second polypeptide composed of the amino acid sequence from position 21 to position 246 of the amino acid sequence shown in SEQ ID NO:84; NYF-0022 formed by the association of a first polypeptide composed of the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:87 and a second polypeptide composed of the amino acid sequence from position 21 to position 246 of the amino acid sequence shown in SEQ ID NO:84; NYF-0023 formed by the association of a first polypeptide composed of the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:88 and a second polypeptide composed of the amino acid sequence from position 21 to position 246 of the amino acid sequence shown in SEQ ID NO:84; NYF-0027 formed by the association of a first polypeptide composed of the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:89 and a second polypeptide composed of the amino acid sequence from position 21 to position 246 of the amino acid sequence shown in SEQ ID NO:84; NYF-0035 formed by the association of a first polypeptide composed of the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:90 and a second polypeptide composed of the amino acid sequence from position 21 to position 246 of the amino acid sequence shown in SEQ ID NO:84; NYF-0044 formed by the association of a first polypeptide composed of the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:91 and a second polypeptide composed of the amino acid sequence from position 21 to position 246 of the amino acid sequence shown in SEQ ID NO:84; NYF-0045 formed by the association of a first polypeptide composed of the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:92 and a second polypeptide composed of the amino acid sequence from position 21 to position 246 of the amino acid sequence shown in SEQ ID NO:84; NYF-0047 formed by the association of a first polypeptide composed of the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:93 and a second polypeptide composed of the amino acid sequence from position 21 to position 246 of the amino acid sequence shown in SEQ ID NO:84; NYF-0048 formed by the association of a first polypeptide composed of the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:94 and a second polypeptide composed of the amino acid sequence from position 21 to position 246 of the amino acid sequence shown in SEQ ID NO:84;NYF-0060 formed by the association of a first polypeptide consisting of the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:95 and a second polypeptide consisting of the amino acid sequence from position 21 to position 246 of the amino acid sequence shown in SEQ ID NO:84; NYF-0061 formed by the association of a first polypeptide consisting of the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:96 and a second polypeptide consisting of the amino acid sequence from position 21 to position 246 of the amino acid sequence shown in SEQ ID NO:84; NYF-0019 formed by the association of a first polypeptide consisting of the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:86 and a second polypeptide consisting of the amino acid sequence from position 21 to position 246 of the amino acid sequence shown in SEQ ID NO:84; NYF-0014 formed by the association of a first polypeptide consisting of the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:149 and a second polypeptide consisting of the amino acid sequence from position 21 to position 246 of the amino acid sequence shown in SEQ ID NO:84; and NYF-0082 formed by the association of a first polypeptide consisting of the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:150 and a second polypeptide consisting of the amino acid sequence from position 21 to position 246 of the amino acid sequence shown in SEQ ID NO:84.;

[0643] In addition, the following can be cited as preferred taFv - heterodimer Fc - type bispecific molecules of the present invention: NYZ-0038 formed by the association of a first polypeptide consisting of the amino acid sequence from position 20 to position 745 of the amino acid sequence shown in SEQ ID NO:155 and a second polypeptide consisting of the amino acid sequence from position 20 to position 246 of the amino acid sequence shown in SEQ ID NO:84; NYZ-0082 formed by the association of a first polypeptide consisting of the amino acid sequence from position 20 to position 750 of the amino acid sequence shown in SEQ ID NO:156 and a second polypeptide consisting of the amino acid sequence from position 20 to position 246 of the amino acid sequence shown in SEQ ID NO:84; NYZ-0083 formed by the association of a first polypeptide consisting of the amino acid sequence from position 20 to position 750 of the amino acid sequence shown in SEQ ID NO:157 and a second polypeptide consisting of the amino acid sequence from position 20 to position 246 of the amino acid sequence shown in SEQ ID NO:84.

[0644] Among them, NYF-0023, NYF-0047, NYF-0048, NYF-0060, NYF-0061, NYZ-0038, NYZ-0082 and NYZ-0083 have particularly excellent biological activities, physicochemical properties, etc., and are thus preferred.

[0645] In addition, the first antibody taFv can directly bind to one Fc of the dimer with or without a linker, and the Fab of the first antibody or the second antibody can directly bind to the other Fc with or without a linker. This bispecific molecule has 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 taFv-Fab-heterodimer Fc type ( Figure 3 ).

[0646] In the present invention, as the taFv included in the taFv-Fab-heterodimer Fc type bispecific molecule, for example, taFv of anti-HLA-A2 / NY-ESO scFv and anti-CD3 scFv can be used; as Fab, for example, HLA / NY-ESO Fab can be used.

[0647] Regarding the taFv-Fab-heterodimer Fc type bispecific molecule, preferably, (a) it contains a first polypeptide, a second polypeptide, and a third polypeptide. The first polypeptide sequentially contains scFv specifically binding to human HLA / NY-ESO, scFv specifically binding to CD3, and immunoglobulin Fc region (i) from the N-terminus to the C-terminus; the second polypeptide is composed of an immunoglobulin heavy chain containing Fc region (ii); the third polypeptide is composed of an immunoglobulin light chain; more preferably, (b) the second polypeptide and the third polypeptide associate, and (c) the first polypeptide and the second polypeptide associate in Fc region (i) and Fc region (ii). Examples of the taFv-Fab-heterodimer Fc type bispecific molecule are 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). As Figure 3(e) As shown, the Fc region (i) of the first polypeptide binds to the second polypeptide consisting of an immunoglobulin heavy chain with the Fc region (ii) shown in black at the Fc region (i) of the first polypeptide and the Fc region (ii) of the second polypeptide. Additionally, the immunoglobulin light chain binds to the second polypeptide. This preferred taFv-Fab-heterodimeric Fc-type bispecific molecule can also be formed by the binding of a taFv-heterodimeric Fc-type bispecific molecule containing a taFv and the Fc region of an immunoglobulin to a Fab. As the amino acid sequence contained in the first polypeptide of the preferred taFv-Fab heterodimeric Fc-type bispecific molecule, examples include: the amino acid sequence from position 21 to position 511 of the amino acid sequence shown in SEQ ID NO:85, the amino acid sequence from position 21 to position 511 of the amino acid sequence shown in SEQ ID NO:87, the amino acid sequence from position 21 to position 511 of the amino acid sequence shown in SEQ ID NO:88, the amino acid sequence from position 21 to position 511 of the amino acid sequence shown in SEQ ID NO:89, the amino acid sequence from position 21 to position 511 of the amino acid sequence shown in SEQ ID NO:90, the amino acid sequence from position 21 to position 511 of the amino acid sequence shown in SEQ ID NO:91, the amino acid sequence from position 21 to position 511 of the amino acid sequence shown in SEQ ID NO:92, the amino acid sequence from position 21 to position 511 of the amino acid sequence shown in SEQ ID NO:93, the amino acid sequence from position 21 to position 511 of the amino acid sequence shown in SEQ ID NO:94, the amino acid sequence from position 21 to position 511 of the amino acid sequence shown in SEQ ID NO:95, the amino acid sequence from position 21 to position 511 of the amino acid sequence shown in SEQ ID NO:96, the amino acid sequence from position 21 to position 511 of the amino acid sequence shown in SEQ ID NO:86, the amino acid sequence from position 21 to position 511 of the amino acid sequence shown in SEQ ID NO:149; and the amino acid sequence from position 21 to position 511 of the amino acid sequence shown in SEQ ID NO:150.The first polypeptide contained in the more preferred taFv-Fab heterodimeric Fc-type bispecific molecule consists of the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:85; the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:87; the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:88; the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:89; the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:90; the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:91; the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:92; the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:93; the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:94; the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:95; the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:96; and the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:86; the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:149; or, the amino acid sequence from position 21 to position 745 of the amino acid sequence shown in SEQ ID NO:150.

[0648] The second polypeptide contained in the preferred taFv-Fab-heterodimeric Fc-type bispecific molecule of the present invention comprises the variable region, CH1 region and hinge region of a human antibody or humanized antibody heavy chain, and a mutant Fc; the second polypeptide contained in the more preferred taFv-Fab-heterodimeric Fc-type bispecific molecule comprises the amino acid sequence from position 20 to position 242 of the amino acid sequence shown in SEQ ID NO:99.

[0649] The third polypeptide contained in the preferred taFv-Fab-heterodimeric Fc-type bispecific molecule of the present invention comprises the variable region and constant region of a human antibody or humanized antibody light chain; the third polypeptide contained in the more preferred taFv-Fab-heterodimeric Fc-type bispecific molecule comprises positions 21 to 131 of the amino acid sequence shown in SEQ ID NO:100.

[0650] The variable region, CH1 region in the second polypeptide, and the third polypeptide contained in the preferred taFv-Fab-heterodimeric Fc-type bispecific molecule form a Fab. The preferred Fab is the Fab of an anti-HLA / NY-ESO antibody, such as the Fab of NYA-0001.

[0651] In the present invention, as the scFv contained in the scFv-Fab-heterodimeric Fc-type bispecific molecule, for example, an anti-HLA-A2 / NY-ESO scFv or an anti-CD3 scFv can be used; as the Fab, for example, an HLA / NY-ESO Fab or an anti-CD3 Fab can be used.

[0652] Regarding the scFv-Fab-heterodimeric Fc-type bispecific molecule, preferably, (a) it contains a first polypeptide, a second polypeptide, and a third polypeptide. The first polypeptide sequentially contains, from the N-terminus to the C-terminus, an scFv that specifically binds to human HLA / NY-ESO, the variable region and the constant region CH1 of an antibody heavy chain that specifically binds to CD3, and an immunoglobulin Fc region (i); the second polypeptide contains an immunoglobulin hinge region and an Fc region (ii); 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 antibody light chain of the third polypeptide in the variable region and the 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. Examples of the scFv-Fab-heterodimeric Fc-type bispecific molecule are 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. As Figure 6A (shown in (c), the Fc region (i) part of the first polypeptide associates with the Fc region (ii) part of the second polypeptide represented by being blackened, and the first polypeptide and the third polypeptide associate. For example, in Figure 6A (c), the scFv represented by the upper right diagonal line is an anti-HLA-A2 / NY-ESO scFv, and the Fab represented by white, square patterns, and horizontal lines is an anti-CD3 Fab.

[0653] As the amino acid sequence contained in the first polypeptide of the preferred scFv-Fab-heterodimeric Fc-type bispecific molecule, the amino acid sequence from the 21st to the 394th position of the amino acid sequence shown in SEQ ID NO: 160 can be exemplified, and more preferably, the amino acid sequence from the 20th to the 724th position can be exemplified.

[0654] In addition, as the other amino acid sequence contained in the first polypeptide included in the preferred scFv-Fab heterodimer Fc-type bispecific molecule, the amino acid sequence from position 21 to position 389 of the amino acid sequence shown in SEQ ID NO: 197 can be exemplified, and more preferably, the amino acid sequence from position 20 to position 719 can be exemplified.

[0655] In addition, as the other amino acid sequence contained in the first polypeptide included in the preferred scFv-Fab heterodimer Fc-type bispecific molecule, the amino acid sequence from position 21 to position 389 of the amino acid sequence shown in SEQ ID NO: 198 can be exemplified, and more preferably, the amino acid sequence from position 20 to position 719 can be exemplified.

[0656] The second polypeptide contained in the preferred scFv-Fab heterodimer Fc-type bispecific molecule of the present invention includes a hinge region and a mutant Fc from a human antibody. More preferably, the second polypeptide contained in the scFv-Fab heterodimer Fc-type bispecific molecule includes the amino acid sequence from position 20 to position 246 of the amino acid sequence shown in SEQ ID NO: 84.

[0657] The third polypeptide contained in the preferred scFv-Fab heterodimer Fc-type bispecific molecule of the present invention includes a light chain from a human antibody. As the third polypeptide contained in the more preferred scFv-Fab heterodimer Fc-type bispecific molecule, the amino acid sequence from position 21 to position 127 of the amino acid sequence shown in SEQ ID NO: 161 can be exemplified, and more preferably, the amino acid sequence from position 21 to position 233 can be exemplified.

[0658] Among them, NYZ-1010 formed by the association of the following first polypeptide, second polypeptide, and third polypeptide can be exemplified as the preferred scFv-Fab heterodimer Fc-type bispecific molecule of the present invention: a first polypeptide composed of the amino acid sequence from position 20 to position 724 of the amino acid sequence shown in SEQ ID NO: 160; a second polypeptide composed of the amino acid sequence from position 20 to position 246 of the amino acid sequence shown in SEQ ID NO: 84; a third polypeptide composed of the amino acid sequence from position 21 to position 233 of the amino acid sequence shown in SEQ ID NO: 161.

[0659] In addition, NYZ-1007, which is formed by the association of the following first polypeptide, second polypeptide, and third polypeptide, can also be cited as a preferred scFv-Fab-heterodimer Fc-type bispecific molecule of the present invention: a first polypeptide composed of the amino acid sequence from position 20 to position 719 of the amino acid sequence shown in SEQ ID NO: 197; a second polypeptide composed of the amino acid sequence from position 20 to position 246 of the amino acid sequence shown in SEQ ID NO: 84; a third polypeptide composed of the amino acid sequence from position 21 to position 233 of the amino acid sequence shown in SEQ ID NO: 161.

[0660] In addition, NYZ-1017, which is formed by the association of the following first polypeptide, second polypeptide, and third polypeptide, can also be cited as a preferred scFv-Fab-heterodimer Fc-type bispecific molecule of the present invention: a first polypeptide composed of the amino acid sequence from position 20 to position 719 of the amino acid sequence shown in SEQ ID NO: 198; a second polypeptide composed of the amino acid sequence from position 20 to position 246 of the amino acid sequence shown in SEQ ID NO: 84; a third polypeptide composed of the amino acid sequence from position 21 to position 233 of the amino acid sequence shown in SEQ ID NO: 161.

[0661] One, two, or more than two peptides contained in the bispecific molecule of the present invention can be the aforementioned "deletion mutants", that is, at their carboxyl termini, especially at the carboxyl termini derived from the heavy chain of the antibody, one or two (or more) amino acids have undergone mutations (including deletions). For example, the carboxyl terminus of the amino acid sequence of the first polypeptide contained in NYZ-1010, one of the preferred scFv-Fab-heterodimer Fc-type bispecific molecules of the present invention, can be Lys at position 724 of SEQ ID NO: 160, Gly at position 723 with one amino acid deleted, or any one of the mixtures containing them. Similarly, the carboxyl terminus of the amino acid sequence of the second polypeptide contained in the preferred scFv-Fab-heterodimer Fc-type bispecific molecule of the present invention can be Lys at position 246 of SEQ ID NO: 84, Gly at position 245 with one amino acid deleted, or any one of the mixtures containing them.

[0662] The scFv and Fab contained in the bispecific molecule of the present invention are preferably scFv and Fab of humanized antibodies or human antibodies, and the Fc is preferably Fc of human antibodies.

[0663] In the variable regions contained in the bispecific molecule of the present invention, starting from the amino-terminal side of the antibody, they can be combined in the order of the heavy-chain variable region and the light-chain variable region, or they can also be combined in the order of the light-chain variable region and the heavy-chain variable region. An adapter (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 tandem scFv-type bispecific molecule may also bind an adapter, 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, an example is that, starting from the amino terminus, the heavy-chain variable region, the first adapter, the light-chain variable region, the second adapter, the FLAG tag, and the His tag are sequentially combined.

[0664] The adapter includes a single-chain polypeptide or a single-chain oligopeptide, or may also include synthetic substances such as PEG, nucleotides, sugar chains, and compounds. In addition, as long as two polypeptides are bound, there is no particular limitation, and known adapters can be used.

[0665] As the length of the adapter, for example, the length of the peptide adapter is 5 to 30 amino acids. When the bispecific molecule contains multiple adapters, peptide adapters with the same length or different lengths can be used.

[0666] As the peptide adapter, for example, a cycle of (Gly·Gly·Gly·Gly·Ser) (SEQ ID NO:161) can be cited, but one to several amino acid residues different from Gly and Ser can also be added thereto.

[0667] In summary, among the structures (configurations) that the multispecific antibody of the present invention, especially the bispecific antibody, can adopt, the preferred ones are the taFv-heterodimer Fc type, the taFv-Fab-heterodimer Fc type, and the scFv-Fab-heterodimer Fc type; more preferably the taFv-heterodimer Fc type. The configuration (taFv-heterodimer Fc type) arranged in the order of anti-HLA / NY-ESO scFv and anti-CD3 scFv from the N terminus to the C terminus is further preferred compared to the reverse configuration (taFv (reverse)-heterodimer Fc type) (Example 11, etc.). In addition, another more preferred configuration is the scFv-Fab-heterodimer Fc type.

[0668] The present invention also includes the following molecules: molecules containing the amino acid sequence encoded by the nucleotide sequence of a polynucleotide that hybridizes under stringent conditions with the complementary strand of a polynucleotide containing the nucleotide sequence encoding the amino acid sequence contained in the molecule of the present invention, and that binds to HLA / NY-ESO and preferably further binds to CD3.

[0669] The present invention also includes the following molecules: molecules comprising an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% identity to the amino acid sequence comprised by the molecules of the present invention, and which bind to HLA / NY-ESO and preferably also bind to CD3.

[0670] The antibodies of the present invention, their binding fragments, and multispecific antibodies comprising them possess excellent biological activities, physicochemical properties (hereinafter referred to as "physicochemical properties"), safety, pharmacokinetics and other properties. (a) As biological activities or their indicators, 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 100 nM or less, or 50 nM or less, preferably 20 nM or less, or 10 nM or less, more preferably 5 nM or less. Additionally, for example, for the cell lines U266B1 and / or NCI-H1703 expressing endogenous human NY-ESO, the EC 50 value of the cytotoxic activity exerted using human peripheral blood mononuclear cells as effector cells is 20 nM or less, preferably 10 nM or less, more preferably 5 nM or less (methods for the measurement and calculation of in vitro cytotoxic activity include, but are not limited to, the methods described in Example 8). Additionally, for example, 0.1 mL of a suspension of 6×10 7 cells / mL of the human squamous cell lung cancer cell line NCI-H1703 was transplanted subcutaneously into NOG mice. Four days later, 0.2 mL of 3.75×10 7Cells / mL of the suspension were transplanted into the tail vein. Starting 14 days later, the volume of the tumor was measured once a week for 3 administrations of the antibody. At this time, the tumor growth inhibitory activity was 50% or more, preferably 75% or more, more preferably 90% or more, relative to the control group administered with the solvent (as the method for measuring and calculating the in vivo anti-tumor activity, examples include but are not limited to the method described in Example 9). (b) The impurities contained in biopharmaceuticals are related to the safety of the drug, so it is necessary to appropriately regulate and manage whether there is an increase during manufacturing and storage. Among them, HMWS (aggregates) is one of the main impurities, which can cause immunogenic risk and reduction of drug efficacy, etc., so this item should be strictly managed. Regarding the management of impurities, evaluation must be carried out not only during manufacturing, including the in-process stability (whether there is an increase) during and after the manufacturing process. Since the expiration date of the drug is set based on the results of long-term stability tests, antibodies with good stability over time can be set with a longer expiration date. Therefore, as the indicators for screening suitable antibodies for biopharmaceuticals, that is, the physicochemical properties of the present invention, acid resistance (inhibiting the formation of HMWS, etc.) and solution stability (inhibiting the formation of HMWS, etc.) can be cited. As other indicators, host cells suitable for the production of the antibodies or their binding fragments or molecules containing them of the present invention can be exemplified. For example, recombinant cells obtained by introducing a gene encoding the amino acid sequence they contain into Expi293F cells have a high yield and a high recovery rate in the culture. The preferred antibodies of the present invention with these physicochemical property characteristics, their antigen-binding fragments, and multispecific antibodies containing them can achieve the following: their solutions can be exposed to acidic conditions, and their manufacturing can be carried out or easily carried out, such as chromatography methods such as protein A and ion exchange, and steps such as virus inactivation; the formation of HMWS in their solutions can be inhibited at a low level, so their manufacturing, formulation, circulation, and storage of drugs containing them can be carried out or easily carried out; they can be produced efficiently. Regarding acid resistance, for example, keep at pH 3.5 at room temperature for 1 hour, and then measure and calculate HMWS by size exclusion chromatography. The content of the generated HMWS is 5% or less, preferably 2% or less, more preferably 1% or less (as the method for measuring and calculating the content of HMWS for acid resistance evaluation, examples include but are not limited to the method described in Example 19-1). Regarding solution stability, for example, dissolve in a solution composed of 25 mM histidine and 5% sorbitol, adjust the pH value to 6.0, the concentration is 25 mg / ml, and store at 25°C for 6 days. Then measure and calculate HMWS by size exclusion chromatography. The content of HMWS is 20% or less, preferably 10% or less (as the method for measuring and calculating the content of HMWS for solution stability evaluation, examples include but are not limited to the method described in Example 19-2). As the method for measuring and calculating production efficiency or recovery rate, examples include but are not limited to the methods described in Examples 20 and 21.(c) As the safety or its index, examples include antigen recognition characteristics, observations during administration, etc. For example, it will recognize multiple amino acids on the wild-type NY-ESO peptide and will not bind to homologous peptides with amino acid sequences similar but not identical to the wild-type NY-ESO peptide, and the risk of side effects caused by off-target effects is low. In addition, it can be predicted that in the immunogenicity screening based on the ISPRI network (EpiVax, Inc), the immunogenicity is low and the risk of side effects such as cytokine production caused by antibodies is low. In addition, for example, when administering NYF-0023, NYF-0045, NYF-0047, NYF-0048, NYF-0060, NYF-0061, NYZ-0082 or NYZ-1010 included in the bispecific antibody of the present invention to Balb / c mice, no observations adverse to the half-life in the blood were confirmed, nor were any significant toxic observations such as weight loss confirmed. In addition, when administering NYZ-0082 or NYZ-1010 to cynomolgus monkeys once, no observations adverse to the half-life in the blood were confirmed, and no changes caused by the administration were confirmed in terms of general condition, body weight, food intake, body temperature measurement, and the amount of cytokines in plasma. (d) As the kinetics or its index, examples include the half-life in the blood, etc. For example, when administering several bispecific antibodies obtained in the present invention to Balb / c mice or cynomolgus monkeys, no observations adverse to the half-life in the blood were confirmed. The antibodies, their binding fragments and molecules of the present invention having excellent biological activities, physicochemical properties, safety, in vivo kinetics and other properties can preferably be included in a pharmaceutical composition. Preferred antibodies or their antigen-binding fragments 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, but 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 preferably, but not limited to: NYF-0061, NYZ-0038, NYZ-0082, NYZ-0088, NYZ-1007, NYZ-1010, NYZ-1017.

[0671] In the present invention, the "site" to which the antibody binds, that is, the "site" recognized by the antibody, refers to a partial peptide or partial higher-order structure on the antigen to which the antibody binds or recognizes. In the present invention, this site is also called an epitope or a binding site of the antibody. Examples of the site on HLA / NY-ESO to which the anti-HLA / NY-ESO antibody of the present invention binds or recognizes include: multiple amino acids in the HLA / NY-ESO peptide, partial higher-order structures, and the like.

[0672] Antibodies or their binding fragments that "bind to the same site as the antibody or binding fragment of the present invention" are also included in the present invention. An "antibody that binds to the same site as a certain antibody" refers to another antibody that binds to the site on the antigen molecule recognized by the antibody. If the partial peptide or partial conformation on the antigen molecule bound by the first antibody 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 this second antibody is also included in the present invention. In addition, if an 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 antibody of the present invention, antibodies that compete with the monoclonal antibody of the present invention in binding to HLA / NY-ESO, and their binding fragments preferably have one or more of the in vitro cytotoxic activity and in vivo anti-tumor activity described in (a) and the properties described in (b) to (d), more preferably have three or more properties, and most preferably have all properties.

[0673] 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 as follows: appropriately excise the amino acid sequence of an antigen from the C-terminus or N-terminus, prepare a series of peptides, study the reactivity of the antibody to these peptides, and after determining the approximate recognition site, synthesize short peptides and study the reactivity of the antibody to these short peptides. Additionally, for example, the binding site can also be determined as follows: delete or substitute a specific site or region in the amino acid sequence of an antigen or antigen fragment peptide with another amino acid sequence, or introduce mutations into the amino acid sequence, and study the reactivity of the antibody to these peptides. Antigen fragment peptides can be prepared using techniques such as genetic recombination and peptide synthesis.

[0674] When an antibody binds to or recognizes a partial higher-order structure of an antigen, X-ray crystallography can be used to lock in the amino acid residues on the antigen adjacent to the antibody, thereby determining the binding site of the antibody. For example, the binding site can be determined by binding and crystallizing an antibody or its fragment, and an antigen or its fragment, and performing structural analysis to lock in the amino acid residues on the antigen that have an interaction distance with the antibody. The interaction distance is Hereinafter, preferably Hereinafter, more preferably Hereinafter. One or more such amino acid residues that have an interaction distance with the antibody can constitute the antigen-binding site (epitope) of the antibody. When there are two or more such amino acid residues, the amino acids may not be adjacent to each other in the primary sequence.

[0675] The anti-HLA / NY-ESO antibody or its binding fragment of the present invention can specifically recognize multiple amino acids present in the amino acid sequence of HLA / NY-ESO. Antibodies or their binding fragments that recognize these multiple amino acids, antibodies or their binding fragments that compete with the antibody or its binding fragment of the present invention in binding to HLA / NY-ESO, or antibodies or their binding fragments that have an interaction distance with these multiple amino acids are also included in the present invention. Additionally, multispecific antibodies containing such antibodies or their binding fragments are also included in the present invention.

[0676] 6. Pharmaceutical Composition

[0677] The present invention includes an anticancer agent containing the anti-HLA / NY-ESO antibody of the present invention or a multispecific molecule that binds to HLA / NY-ESO as an active ingredient.

[0678] The anti-cancer agent of the present invention can be used for one or more of the following cancers or tumors: carcinoma, sarcoma, lymphoma, leukemia, myeloma, germ cell tumor, brain tumor, carcinoid tumor, neuroblastoma, retinoblastoma, nephroblastoma. Specifically, for carcinoma, examples include: renal carcinoma, melanoma, squamous cell carcinoma, basal cell carcinoma, conjunctival carcinoma, 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, appendiceal cancer, anal cancer, liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, adrenal cancer, bladder cancer, prostate cancer, uterine cancer, vaginal cancer, etc.; for sarcoma, examples include: 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, etc.; for lymphoma, examples include: B-cell lymphoma, NK / T-cell lymphoma, Hodgkin lymphoma, etc.; for leukemia, examples include: myeloid leukemia, lymphocytic leukemia, myeloproliferative disease, myelodysplastic syndrome, etc.; for myeloma, examples include: multiple myeloma, etc.; for germ cell tumor, examples include: testicular cancer, ovarian cancer, etc.; for brain tumor, examples include: glioma, meningioma, etc.

[0679] The anti-cancer 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, and a pharmaceutically acceptable carrier, diluent, solubilizer, emulsifier, preservative, adjuvant, etc. The "pharmaceutically acceptable carrier", etc. can be appropriately selected from a wide range according to the type of the target disease and the administration form of the medicament. The administration method of the anti-cancer agent of the present invention can be appropriately selected. For example, it can be administered by injection, and local infusion, intraperitoneal administration, selective intravenous infusion, intravenous injection, subcutaneous injection, organ perfusion fluid infusion, etc. can also be adopted. In addition, the solution for injection can be formulated using a saline solution, a glucose solution, or a mixture of saline and glucose solution, a carrier composed of various buffers, etc. In addition, it can also be formulated in a powder state and mixed with the above liquid carrier when in use to prepare an injection solution.

[0680] Regarding other administration methods, they can also be appropriately selected together with the development of the preparation. For example, in the case of oral administration, oral liquid preparations, powders, pills, capsules, tablets, etc. can be applied. In the case of oral liquid preparations, as oral liquid modifiers such as suspensions and syrups, the following substances can be used for manufacturing: water; saccharides such as sucrose, sorbitol, and fructose; glycols such as PEG; oils such as sesame oil and soybean oil; preservatives such as alkyl p - hydroxybenzoates; flavoring agents such as strawberry flavor and mint, etc. Powders, pills, capsules, and tablets can be formulated using the following substances: excipients such as lactose, glucose, sucrose, and mannitol; disintegrants such as starch and sodium arginine; lubricants such as magnesium stearate and talc; binders such as polyvinyl alcohol, hydroxypropyl cellulose, and gelatin; surfactants such as fatty acid esters; plasticizers such as glycerol, etc. Tablets and capsules are preferred unit administration forms in the compositions of the present invention in terms of ease of administration. When manufacturing tablets and capsules, solid manufacturing carriers are used.

[0681] The amount of the anti - HLA / NY - ESO antibody or the multispecific molecule that binds to HLA / NY - ESO and is effectively used for treatment varies according to the nature of the treated condition, the age and status of the patient, and can ultimately be determined by the doctor. For example, the amount per administration is 0.0001 mg to 100 mg per 1 kg body weight. The prescribed dosage can be administered once every 1 to 180 days, or can be divided and administered 2 times, 3 times, 4 times or more per day at appropriate intervals.

[0682] In addition, polynucleotides encoding the amino acid sequences of the anti - HLA / NY - ESO antibodies or multispecific molecules that bind to HLA / NY - ESO of the present invention, vectors containing such polynucleotides, cells containing such polynucleotides or vectors, and pharmaceutical compositions containing any one of such polynucleotides, such vectors, and such cells as active ingredients are also included in the present invention. The pharmaceutical composition is preferably an anticancer agent.

[0683] In addition, the pharmaceutical compositions of the present invention can be used in combination with other agents. The other agents are chemotherapeutic agents, radiotherapy, biological drugs, etc., and there is no particular limitation. They can be administered or applied simultaneously or at different times with the pharmaceutical compositions of the present invention, and can be administered or applied as a single preparation containing the pharmaceutical compositions of the present invention or two or more different preparations or therapies.

[0684] [Examples]

[0685] The present invention will be described in more detail in the following examples, but the present invention is not limited by these examples.

[0686] In addition, for each operation related to gene manipulation in the following examples, unless otherwise specified, it is carried out according to the methods described in "Molecular Cloning" (written by Sambrook, J., Fritsch, E.F. and Maniatis, T., published by Cold Spring Harbor Laboratory Press in 1989) and the methods described in experimental books used by other persons skilled in the art; or, when using commercially available reagents and reagent kits, it is carried out according to the instructions of the commercial products. For the synthesis of primers required for gene synthesis and vector construction, synthesis was commissioned as needed (FASMAC Co., Ltd., Thermo Fisher Scientific Inc., and Eurofins Genomics).

[0687] (Example 1) Obtaining anti-HLA / NY-ESO antibody from a human antibody phage library

[0688] 1)-1 Preparation of HLA / NY-ESO antigen protein

[0689] After refolding by greatly diluting each inclusion body prepared from Escherichia coli (BL21(DE3), Agilent Technologies, Inc.) expressing a truncation of HLA-A*0201 (GenBank: ASA47534.1) (adding a biotin ligase recognition sequence, SEQ ID NO: 33) and β2-microglobulin (UniProtKB - P61769, SEQ ID NO: 34) and the NY-ESO peptide: SLLMWITQC (SEQ ID NO: 1 in the sequence listing), the HLA-A*0201 / β2-microglobulin / NY-ESO peptide complex (hereinafter referred to as HLA / NY-ESO) was prepared by a gel filtration column (Superdex 200 10 / 300, GE Healthcare).

[0690] As a negative control antigen, the same refolding was carried out using the MAGEC-1 peptide: ILFGISLREV (SEQ ID NO: 2 in the sequence listing) to prepare the HLA-A*0201 / β2-microglobulin / MAGEC-1 peptide complex (hereinafter referred to as HLA / MC1). Then, the prepared HLA / NY-ESO and HLA / MC1 were biotinylated by the biotin ligase of Escherichia coli and separated by a gel filtration column (Superdex 200 10 / 300, GE Healthcare) to prepare each biotinylated protein.

[0691] 1)-2 Isolation of scFv with binding ability to HLA / NY-ESO

[0692] Isolate scFv that binds to HLA / NY-ESO from a human antibody phage library. First, add phages to Dynabeads streptavidin M-280 (Thermo Fisher Scientific) immobilized with biotinylated HLA / MC1, and recover the unbound phages. Next, add Dynabeads streptavidin M-280Ag immobilized with biotinylated HLA / NY-ESO, and use a magnetic stand (DynaMag-2, Thermo Fisher Scientific) to remove the unbound phages through washing operations.

[0693] After that, infect Escherichia coli (XL-1Blue, Agilent Technologies) with the phages that bind to HLA / NY-ESO, and recover and amplify the phages that bind to HLA / NY-ESO. After a total of 3 rounds of screening, after switching from polyclonal phagemids to an expression vector for Escherichia coli with a FLAG tag and a His tag added to the carboxyl terminus of the scFv, transform Escherichia coli, and express the scFv in the presence of IPTG (Isopropyl-β-D-thiogalactopyranoside) (Sigma-Aldrich) for ELISA screening.

[0694] 1)-3 Screen for HLA / NY-ESO-binding scFv by ELISA

[0695] To each well of a 384-well Maxi-sorp plate (Black, Nunc), add 50 μL of NeutrAvidin (Life Technologies) diluted to 1 μg / mL with PBS (0.01 M phosphate-buffered saline containing 0.138 M sodium chloride and 0.0027 M potassium chloride, pH 7.4, Sigma-Aldrich). Let it stand overnight at 4°C for immobilization. After washing 3 times with PBS containing 0.05% Tween-20 (Bio-Rad) (ELISA buffer), dilute it to 1 μg / mL with PBS and add the biotinylated HLA / NY-ESO used in Examples 1)-2. Incubate with shaking at room temperature for 1 hour. After washing 3 times with ELISA buffer, block it with Blocker Casein (Thermo Fisher Scientific) and then wash 3 times with ELISA buffer. Subsequently, add the culture solution of Escherichia coli expressing scFv and react at room temperature for 2 hours. After washing 3 times with ELISA buffer, add 50 μL of anti-FLAG antibody (Sigma-Aldrich) labeled with horseradish peroxidase (HRP) diluted 5000-fold with ELISA buffer and react at room temperature for 1 hour. After washing 5 times with ELISA buffer, add SuperSignal Pico ELISA chemiluminescent substrate (Thermo Fisher Scientific) and measure the chemiluminescence after 10 minutes using a microplate reader (Envision 2104 Multilabel Reader, PerkinElmer) to isolate the ELISA-positive clones that bind to HLA / NY-ESO.

[0696] 1)-4 Determination of the nucleotide sequence and amino acid sequence of the ELISA-positive clone NY-R119

[0697] From the ELISA-positive clones obtained in 1)-3, select NY-R119 as the scFv with strong binding ability to HLA / NY-ESO and excellent recognition specificity. The analysis of the nucleotide sequences of the heavy and light chain variable regions of NY-R119 was performed using the DyeTerminator method (BigDye (registered trademark) Terminator v3.1, Thermo Fisher Scientific). The primer sequences for sequence analysis are shown below.

[0698] Primer A: 5’-CTCTTCGCTATTACGCCAGCTGGCGA-3’ (SEQ ID NO:3 in the sequence listing ( Figure 10 ))

[0699] Primer B: 5’-ATAACAATTTCACACAGGAAACAGCTATGA-3’ (SEQ ID NO:4 in the sequence listing( Figure 11 ))

[0700] The nucleotide sequence of the cDNA encoding the heavy chain variable region of NY-R119 that was determined is shown in SEQ ID NO:5( Figure 12 ), and the amino acid sequence is shown in SEQ ID NO:6( Figure 13 ).

[0701] The nucleotide sequence of the cDNA encoding the light chain variable region of NY-R119 that was determined is shown in SEQ ID NO:7( Figure 14 ), and the amino acid sequence is shown in SEQ ID NO:8( Figure 15 ).

[0702] The CDR sequences of NY-R119 in the CDR definitions 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 ), and CDRL3 is shown in SEQ ID NO:59( Figure 61 ).

[0703] 1) Preparation of 1)-5NYA-0001

[0704] 1)-5-1 Construction of the 1)-5NYA-0001 expression vector

[0705] To prepare various samples for evaluation, an expression vector for mammalian cell culture of NY-R119 was constructed. In addition, NY-R119 expressed in mammalian cell culture was named NYA-0001. The scFv portion, heavy chain and light chain variable regions, and CDRH1-3 and CDRL1-3 of NY-R119 and NYA-0001 are composed of the same amino acid sequence. Using the In-Fusion HD Cloning Kit (CLONTECH), the DNA fragment encoding NYA-0001 was inserted into an expression vector for mammalian cells with pcDNA3.3 (Thermo Fisher Scientific) as the backbone to construct the NYA-0001 expression vector.

[0706] The nucleotide sequence of the constructed NYA-0001 expression vector was re-analyzed, and it was confirmed that the full-length nucleotide sequence of NYA-0001 is the nucleotide sequence shown in SEQ ID NO:69 of the sequence listing ( Figure 70 ). Additionally, in the above nucleotide sequence, the full-length amino acid sequence of NYA-0001 encoded by this sequence is the amino acid sequence shown in SEQ ID NO:70 ( Figure 71 ). In this sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 266th is NYA-0001, and the amino acid sequence from the 267th to the 292nd is the Flag-His tag. Additionally, the amino acid sequence from the 46th to the 53rd is CDRH1, the amino acid sequence from the 71st to the 78th is CDRH2, and the amino acid sequence from the 117th to the 129th is CDRH3. Furthermore, the amino acid sequence from the 181st to the 188th is CDRL1, the amino acid sequence from the 206th to the 208th is CDRL2, and the amino acid sequence from the 245th to the 256th is CDRL3.

[0707] 1)-5-2 Expression and purification of NYA-0001

[0708] According to the manual, Expi293F cells (Thermo Fisher Scientific) were subcultured. The culture solution of Expi293F cells in the logarithmic growth phase was diluted to 2.5×10 6 cells / mL with Expi293 expression medium (Thermo Fisher Scientific) for the production of NYA-0001. 0.3 mg of the NYA-0001 expression vector and 0.9 mg of polyethyleneimine (Polyscience #24765) were added to 20 mL of Opti-Pro SFM medium (Thermo Fisher Scientific) and stirred gently, then left for 5 minutes, and then added to Expi293F cells. The cells were cultured with shaking at 135 rpm in an incubator at 37°C and 8% CO 2 for 6 days. The obtained culture supernatant was filtered through a 0.2-μm filter membrane (Millipore) to obtain the culture supernatant of NYA-0001. Purification was carried out as follows: After elution and concentration using Ni Sepharose excel (GE Healthcare), purification was performed through a gel filtration column (Superdex200 Increase, GE Healthcare) equilibrated with 25 mM histidine, 300 mM NaCl, 5% sorbitol, and pH 6.0. The purified protein sample was used for analytical size exclusion chromatography (SEC). After determining the purity and concentration, it was used for various evaluations.

[0709] (Example 2) Preparation of NYA-0001 Mutant

[0710] 2)-1 Obtaining of NYA-0001 Mutant

[0711] Using the NYA-0001 gene as a template, a method of introducing mutations by PCR (Error-prone-based library), or a method of constructing a library by synthesizing oligomers in which 20 kinds of amino acids are randomly mutated for each residue of all residues of the CDR (oligo-based library), a phage library was constructed, and high-binding ability clones were screened. NYA-0060, NYA-0068, and NYA-0082 were obtained as high-binding ability mutants, and the nucleotide sequences of each were determined.

[0712] The nucleotide sequence of the cDNA encoding the heavy chain variable region of NYA-0060 obtained is shown in SEQ ID NO:9( Figure 16 ), and the amino acid sequence is shown in SEQ ID NO:10( Figure 17 ).

[0713] The nucleotide sequence of the cDNA encoding the light chain variable region of NYA-0060 obtained is shown in SEQ ID NO:11( Figure 18 ), and the amino acid sequence is shown in SEQ ID NO:12( Figure 19 ).

[0714] The nucleotide sequence of the cDNA encoding the heavy chain variable region of NYA-0068 obtained is shown in SEQ ID NO:13( Figure 20 ), and the amino acid sequence is shown in SEQ ID NO:14( Figure 21 ).

[0715] The nucleotide sequence of the cDNA encoding the light chain variable region of NYA-0068 obtained is shown in SEQ ID NO:15( Figure 22 ), and the amino acid sequence is shown in SEQ ID NO:16( Figure 23 ).

[0716] The nucleotide sequence of the cDNA encoding the heavy chain variable region of NYA-0082 obtained is shown in SEQ ID NO:17( Figure 24 ), and the amino acid sequence is shown in SEQ ID NO:18( Figure 25 ).

[0717] The nucleotide sequence of the cDNA encoding the light chain variable region of NYA-0082 obtained is shown in SEQ ID NO:19( Figure 26 ), and the amino acid sequence is shown in SEQ ID NO:20(Figure 27 )。

[0718] 2)-2 Using the mutation sites and combinations identified from NYA-0060, NYA-0068, and NYA-0082, high-binding ability mutants were prepared.

[0719] Through the In-Fusion HD Cloning Kit (CLONTECH), DNA fragments of NYA-1163 and NYA-2023 encoding each mutation site of NYA-0060 and NYA-0068 were inserted into an expression vector for mammalian cells with pcDNA3.3 (Thermo Fisher Scientific) as the backbone, and an scFv expression vector for mammalian cells was constructed.

[0720] In addition, as the mutation sites and their combinations identified from NYA-0060, NYA-0068, and NYA-0082, NYA-2027, NYA-1143, and NYA-2143 were designed. Site-directed mutagenesis was introduced into the NYA-0001 expression vector constructed in 1)-5, or DNA fragments encoding the target scFv were inserted into an expression vector for mammalian cells with pcDNA3.3 (Thermo Fisher Scientific) as the backbone through the In-Fusion HD Cloning Kit (CLONTECH), thereby constructing each scFv expression vector for mammalian cells.

[0721] The nucleotide sequence of the constructed scFv expression vector was re-analyzed, and it was confirmed that the full-length nucleotide sequence of NYA-1163 is the nucleotide sequence shown in SEQ ID NO:21 of the sequence listing ( Figure 28 )).

[0722] It was confirmed that the full-length nucleotide sequence of NYA-2023 is the nucleotide sequence shown in SEQ ID NO:22 of the sequence listing ( Figure 29 )).

[0723] It was confirmed that the full-length nucleotide sequence of NYA-2027 is the nucleotide sequence shown in SEQ ID NO:23 of the sequence listing ( Figure 30 )).

[0724] It was confirmed that the full-length nucleotide sequence of NYA-1143 is the nucleotide sequence shown in SEQ ID NO:24 of the sequence listing ( Figure 31 )).

[0725] It was confirmed that the full-length nucleotide sequence of NYA-2143 is the nucleotide sequence shown in SEQ ID NO:25 of the sequence listing ( Figure 32 )).

[0726] In addition, in the above nucleotide sequences, the full-length amino acid sequences of NYA-1163, NYA-2023, NYA-2027, NYA-1143, and NYA-2143 encoded by the sequences were identified.

[0727] The full-length amino acid sequence of NYA-1163 is the amino acid sequence shown in SEQ ID NO:26 of the sequence listing ( Figure 33 ). In this sequence, the amino acid sequence from the 1st to the 19th position is the signal sequence, the amino acid sequence from the 21st to the 266th position is NYA-1163, and the amino acid sequence from the 267th to the 292nd position is the Flag-His tag. Additionally, the amino acid sequence from the 46th to the 53rd position is CDRH1, the amino acid sequence from the 71st to the 78th position is CDRH2, and the amino acid sequence from the 117th to the 129th position is CDRH3. Further, 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-2023 is the amino acid sequence shown in SEQ ID NO:27 of the sequence listing ( Figure 34 ). In this sequence, the amino acid sequence from the 1st to the 19th position is the signal sequence, the amino acid sequence from the 21st to the 266th position is NYA-2023, and the amino acid sequence from the 267th to the 292nd position is the Flag-His tag. Additionally, the amino acid sequence from the 46th to the 53rd position is CDRH1, the amino acid sequence from the 71st to the 78th position is CDRH2, and the amino acid sequence from the 117th to the 129th position is CDRH3. Further, 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-2027 is the amino acid sequence shown in SEQ ID NO:28 of the sequence listing ( Figure 35 ). In this sequence, the amino acid sequence from the 1st to the 19th position is the signal sequence, the amino acid sequence from the 21st to the 266th position is NYA-2027, and the amino acid sequence from the 267th to the 292nd position is the Flag-His tag. Additionally, the amino acid sequence from the 46th to the 53rd position is CDRH1, the amino acid sequence from the 71st to the 78th position is CDRH2, and the amino acid sequence from the 117th to the 129th position is CDRH3. Further, 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-1143 is the amino acid sequence shown in SEQ ID NO:29 of the Sequence Listing ( Figure 36 ). In this sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 266th is NYA-1143, and the amino acid sequence from the 267th to the 292nd is the Flag-His tag. Additionally, the amino acid sequence from the 46th to the 53rd is CDRH1, the amino acid sequence from the 71st to the 78th is CDRH2, and the amino acid sequence from the 117th to the 129th is CDRH3. Also, amino acid numbers 181 to 188 are CDRL1, amino acid numbers 206 to 208 are CDRL2, and amino acid numbers 245 to 256 are CDRL3.

[0731] The full-length amino acid sequence of NYA-2143 is the amino acid sequence shown in SEQ ID NO:30 of the Sequence Listing ( Figure 37 ). In this sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 266th is NYA-2143, and the amino acid sequence from the 267th to the 292nd is the Flag-His tag. Additionally, the amino acid sequence from the 46th to the 53rd is CDRH1, the amino acid sequence from the 71st to the 78th is CDRH2, and the amino acid sequence from the 117th to the 129th is CDRH3. Also, amino acid numbers 181 to 188 are CDRL1, amino acid numbers 206 to 208 are CDRL2, and amino acid numbers 245 to 256 are CDRL3.

[0732] It should be noted that the CDR sequences of NYA-1163 and NYA-0001 are all the same. Additionally, the CDR sequences of NYA-1143, NYA-2143, and NYA-2023 are the same. 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 ), and CDRL3 is shown in SEQ ID NO:59 ( Figure 61 ). Furthermore, 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 ), and CDRH3 is shown in SEQID 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 ).

[0733] In addition, NYA-1154, which combines the binding mutation sites seen when screening for high-binding ability clones, was designed, and site-specific mutations were introduced into the NYA-0001 expression vector, thereby constructing a NYA-1154 expression vector with pcDNA3.3 (Thermo Fisher Scientific) as the backbone. The nucleotide sequence of the constructed scFv expression vector was re-analyzed and confirmed to be the nucleotide sequence shown in SEQ ID NO:31( Figure 38 ). In the above nucleotide sequence, the full-length amino acid sequence of NYA-1154 encoded by this sequence (SEQ ID NO:32( Figure 39 )) was confirmed. In this sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 266th is NYA-1154, and the amino acid sequence from the 267th to the 292nd is the Flag-His tag. In addition, the amino acid sequence from the 46th to the 53rd is CDRH1, the amino acid sequence from the 71st to the 78th is CDRH2, and the amino acid sequence from the 117th to the 129th is CDRH3. 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.

[0734] 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 ).

[0735] By the same method as in 1)-5-2, NYA-1163, NYA-2023, NYA-2027, NYA-1143, NYA-2143, and NYA-1154 were expressed, and each target scFv was purified. The purified protein samples were used for analytical SEC, and after determining the purity and concentration, they were used for various detections.

[0736] 2)-3 Preparation of NYA-1143 Mutants

[0737] 2)-3-1 Preparation of NYA-2035

[0738] As a mutant of NYA-1143, NYA-2035 was designed, and site-specific mutations were introduced into mammalian cells using the NYA-1143 expression vector, thereby constructing the NYA-2035 expression vector. The nucleotide sequence of the constructed scFv expression vector was re-analyzed and confirmed to be the nucleotide sequence shown in SEQ ID NO:35( Figure 42 ). In the above nucleotide sequence, the full-length amino acid sequence of NYA-2035 encoded by this sequence was determined (SEQ ID NO:36( Figure 43 ). In this sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 266th is NYA-2035, and the amino acid sequence from the 267th to the 292nd is the Flag-His tag. Additionally, the amino acid sequence from the 46th to the 53rd is CDRH1, the amino acid sequence from the 71st to the 78th is CDRH2, and the amino acid sequence from the 117th to the 129th is CDRH3. Moreover, 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 as follows: CDRH1 is shown in SEQ ID NO:54( Figure 61 ), CDRH2 is shown in SEQID 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 ), and CDRL3 is shown in SEQ ID NO:59( Figure 61 ).

[0739] 2)-3-2 Preparation of NYA-1143 CDR-Grafted Mutants

[0740] To improve the physicochemical properties, the NYA-1143CDR grafted mutants were designed. The sequences of the framework regions of VH and VL of NYA-1143 were compared with the framework regions of the human subgroup consensus sequence and germline sequence 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 VH, the human germline sequence IGHV3_30*15 and the consensus sequence of human gamma chain subgroup 3 were selected as the receptors with high sequence identity in their framework regions; for VL, the human germline sequence IGLV1-44*01 was selected as the receptor with high sequence identity in its framework region. Next, the amino acid sequences of the framework regions of each receptor were aligned with the amino acid sequence of NYA-1143, and the residues using different amino acids were identified.

[0741] 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)), the framework residues to be transferred to the receptor were selected. By the above method, NYA-1143-VH01, NYA-1143-VH02, and NYA-1143-VH03 were designed as the CDR grafted mutant amino acid sequences of the VH part 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 part of NYA-1143. Each of these amino acid sequences is SEQ ID NO:40( Figure 47 ).

[0742] The designed VH and VL sequences were combined to design various scFvs shown below. The scFv with the VH part of NYA-1143 replaced by the amino acid sequence of NYA-1143-VH01 was named NYA-2044. In addition, the scFv with the VL part of NYA-2044 replaced by the amino acid sequence of NYA-1143-VL01 was named NYA-2045.

[0743] The scFv obtained by replacing the VH portion of NYA-1143 with the amino acid sequence of NYA-1143-VH02 was named NYA-2047. Additionally, the scFv obtained by replacing the VL portion of NYA-2047 with the amino acid sequence of NYA-1143-VL01 was named NYA-2048.

[0744] The scFv obtained by replacing the VH portion of NYA-1143 with the amino acid sequence of NYA-1143-VH03 was named NYA-2060. Additionally, the scFv obtained by replacing the VL portion of NYA-2060 with the amino acid sequence of NYA-1143-VL01 was named NYA-2061.

[0745] For various NYA-1143 CDR grafting mutants designed, DNA fragments were fully synthesized (by FASMAC Co., Ltd.), and the DNA fragments were ligated using the In-Fusion HD Cloning Kit (from CLONTECH Laboratories, Inc.), thereby constructing an scFv expression vector for mammalian cells with pcDNA3.3 (from Thermo Fisher Scientific Inc.) as the backbone.

[0746] The nucleotide sequence of the constructed scFv expression vector was re-analyzed, and it was confirmed that the full-length nucleotide sequence of NYA-2044 is the nucleotide sequence shown in SEQ ID NO:41 of the Sequence Listing ( Figure 48 )

[0747] It was confirmed that the full-length nucleotide sequence of NYA-2045 is the nucleotide sequence shown in SEQ ID NO:42 of the Sequence Listing ( Figure 49 )

[0748] It was confirmed that the full-length nucleotide sequence of NYA-2047 is the nucleotide sequence shown in SEQ ID NO:43 of the Sequence Listing ( Figure 50 )

[0749] It was confirmed that the full-length nucleotide sequence of NYA-2048 is the nucleotide sequence shown in SEQ ID NO:44 of the Sequence Listing ( Figure 51 )

[0750] It was confirmed that the full-length nucleotide sequence of NYA-2060 is the nucleotide sequence shown in SEQ ID NO:45 of the Sequence Listing ( Figure 52 )

[0751] It was confirmed that the full-length nucleotide sequence of NYA-2061 is the nucleotide sequence shown in SEQ ID NO:46 of the Sequence Listing ( Figure 53 )

[0752] In addition, in the above nucleotide sequences, the full-length amino acid sequences of NYA-2044, NYA-2045, NYA-2047, NYA-2048, NYA-2060, and NYA-2061 encoded by the 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 the same as those of NYA-1143.

[0753] The full-length amino acid sequence of NYA-2044 is the amino acid sequence shown in SEQ ID NO:47 of the sequence listing ( Figure 54 ). In this sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 266th is NYA-2044, and the amino acid sequence from the 267th to the 292nd is the Flag-His tag. In addition, the amino acid sequence from the 46th to the 53rd is CDRH1, the amino acid sequence from the 71st to the 78th is CDRH2, and the amino acid sequence from the 117th to the 129th is CDRH3. 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-2045 is the amino acid sequence shown in SEQ ID NO:48 of the sequence listing ( Figure 55 ). In this sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 266th is NYA-2045, and the amino acid sequence from the 267th to the 292nd is the Flag-His tag. In addition, the amino acid sequence from the 46th to the 53rd is CDRH1, the amino acid sequence from the 71st to the 78th is CDRH2, and the amino acid sequence from the 117th to the 129th is CDRH3. 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-2047 is the amino acid sequence shown in SEQ ID NO:50 of the sequence listing ( Figure 57) The amino acid sequence shown. In this sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 266th is NYA-2047, and the amino acid sequence from the 267th to the 292nd is the Flag-His tag. Additionally, the amino acid sequence from the 46th to the 53rd is CDRH1, the amino acid sequence from the 71st to the 78th is CDRH2, and the amino acid sequence from the 117th to the 129th is CDRH3. Also, amino acid numbers 181 to 188 are CDRL1, amino acid numbers 206 to 208 are CDRL2, and amino acid numbers 245 to 256 are CDRL3.

[0756] The full-length amino acid sequence of NYA-2048 is the amino acid sequence shown in SEQ ID NO:51 of the sequence listing ( Figure 58 ) The amino acid sequence shown. In this sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 266th is NYA-2048, and the amino acid sequence from the 267th to the 292nd is the Flag-His tag. Additionally, the amino acid sequence from the 46th to the 53rd is CDRH1, the amino acid sequence from the 71st to the 78th is CDRH2, and the amino acid sequence from the 117th to the 129th is CDRH3. Also, amino acid numbers 181 to 188 are CDRL1, amino acid numbers 206 to 208 are CDRL2, and amino acid numbers 245 to 256 are CDRL3.

[0757] The full-length amino acid sequence of NYA-2060 is the amino acid sequence shown in SEQ ID NO:52 of the sequence listing ( Figure 59 ) The amino acid sequence shown. In this sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 266th is NYA-2060, and the amino acid sequence from the 267th to the 292nd is the Flag-His tag. Additionally, the amino acid sequence from the 46th to the 53rd is CDRH1, the amino acid sequence from the 71st to the 78th is CDRH2, and the amino acid sequence from the 117th to the 129th is CDRH3. Also, amino acid numbers 181 to 188 are CDRL1, amino acid numbers 206 to 208 are CDRL2, and amino acid numbers 245 to 256 are CDRL3.

[0758] The full-length amino acid sequence of NYA-2061 is the amino acid sequence shown in SEQ ID NO:53 of the sequence listing ( Figure 60The amino acid sequence shown in

[0759] 2) Expression and purification of the NYA-1143 mutant

[0760] By the same method as in 1)-5-2, NYA-2035, NYA-2044, NYA-2045, NYA-2047, NYA-2048, NYA-2060, and NYA-2061 prepared in 2)-3-1 and 2)-3-2 were expressed, and each target scFv was purified. The purified protein samples were used for SEC for analysis. After determining the purity and concentration, they were used for various detections.

[0761] (Example 3) Preparation of scFv of anti-HLA / NY-ESO reference antibody

[0762] 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.

[0763] Each DNA fragment of NYC-0003 and NYC-0004 was fully synthesized (Thermo Fisher Scientific), and a mammalian cell scFv expression vector with pcDNA3.3 (Thermo Fisher Scientific) as the backbone was constructed using the In-Fusion HD Cloning Kit (CLONTECH).

[0764] The nucleotide sequences of the constructed scFv expression vectors were re-analyzed, and it was confirmed that the full-length nucleotide sequences of NYC-0003 and NYC-0004 are the nucleotide sequences shown in SEQ ID NO:65 of the sequence listing ( Figure 66 ) and SEQ ID NO:66 ( Figure 67 ). In addition, in the above nucleotide sequences, the full-length amino acid sequence of NYC-0003 encoded by this sequence is SEQ ID NO:67 ( Figure 68) The amino acid sequence shown, and the full-length amino acid sequence of NYC-0004 is SEQ ID NO: 68( Figure 69 ) The amino acid sequence shown.

[0765] By the same method as in 1)-5-2, NYC-0003 and NYC-0004 were expressed, and each target scFv was purified. Each purified protein sample was used for SEC for analysis. After determining the purity and concentration, it was used for various detections.

[0766] (Example 4) Evaluation of the binding ability to HLA / NY-ESO using Biacore

[0767] Using a Biacore T200, the anti-HLA / NY-ESO scFv was captured as a ligand onto an immobilized anti-His antibody, and the antigen was used as the analyte for measurement. 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 onto a sensor chip CM5 (GE Healthcare). Each anti-HLA / NY-ESO scFv to be evaluated, diluted to 0.5 μg / mL with HBS-EP+ (GE Healthcare), was contacted at 10 μL / min for 60 seconds and immobilized. Then, various concentrations of HLA / NY-ESO diluted with HBS-EP+ were used as analytes, and each sample was added at a flow rate of 30 μL / min for 120 seconds. The K was calculated by single-cycle kinetic analysis measured at intervals of 600 seconds D , and the results are shown in Table 1. The results indicate that compared with the parental antibody NYA-0001, its mutants all bind strongly to HLA / NY-ESO. In addition, compared with NYC-0004, the binding of NYA-1143, NYA-2023, NYA-2143, NYA-2044, NYA-2045, NYA-2060, and NYA-2061 is stronger, and among them, the K of NYA-1143, NYA-2044, NYA-2045, and NYA-2143 D is shown to be below 1 nM.

[0768] [Table 1]

[0769] 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

[0770] (Example 5) Analysis of the recognition amino acids in the NY-ESO peptide of anti-HLA / NY-ESO scFv

[0771] Adjust the human lymphoblastoid cell line T2 (ATCC) cells to an appropriate concentration with AIM-V medium (Thermo Fisher Scientific) containing 20% FBS, add 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 IDNO:127( Figure 128 ), SEQ ID NO:128( Figure 129 ), SEQ ID NO:129( Figure 130 )) and gp100 peptide (SEQ ID NO:130( Figure 131 )) (all from Sigma Genosys) and dissolve them in DMSO to a 5 mM solution to make the final concentration 50 μM; or add 1 / 100 volume of DMSO and incubate at 37 °C for 4 hours. After washing twice with AIM-V medium containing 20% FBS, prepare them into an appropriate concentration with PBS containing 5% FBS, add the LIVE / DEAD Fixable Dead Cell Stain Kit (Thermo Fisher Scientific), and let stand at 4 °C for 30 minutes. After washing twice with PBS containing 5% FBS, divide the cells into two groups and use PBS containing 5% FBS at 10 5The cells per 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 with PBS containing 5% FBS was added at a volume of 25 μL / well, and the mixture was left standing at 4°C for 30 minutes. After washing twice with PBS containing 5% FBS, Penta-His Alexa Fluor488 (QIAGEN) diluted with PBS containing 5% FBS was added at a volume of 25 μL / well, and the mixture was left standing at 4°C for 30 minutes. After washing twice with PBS containing 5% FBS, anti-mouse-IgG Alexa Fluor488 (Thermo Fisher Scientific) diluted with PBS containing 5% FBS was added at a volume of 25 μL / well, and the mixture was left standing 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 Corporation) and then resuspended with PBS containing 5% FBS. To normalize by the amount of HLA / peptide complex, for another group of cells, HLA-A2 antibody BB7.2-Alexa Fluor 488 diluted to 10 μg / mL with PBS containing 5% FBS, or mouse IgG2b-Alexa Fluor 488, was added at a volume of 25 μL / well, and the mixture was left standing 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 Corporation) and then resuspended with PBS containing 5% FBS. These cell suspensions were detected using a flow cytometer (CantoII, Becton Dickinson). Data analysis was performed using Flowjo (Treestar), and the geometric mean fluorescence intensity (gMFI) of Alexa Fluor 488 in the dead cell removal fraction of T2 cells was measured. The value representing the binding of each scFv normalized by the amount of HLA / peptide complex on T2 cells was calculated using the following formula for the normalized gMFI.

[0772] Normalized gMFI = (A / B) / ((C / D) / (E / F))

[0773] · A / B = Relative gMFI

[0774] A: gMFI of T2 cells with DMSO added with antibody or with each peptide added

[0775] B: gMFI of T2 cells with DMSO not added with antibody or with each peptide added

[0776] · (C / D) / (E / F) = Correction value of HLA / peptide complex amount of T2 cells with DMSO or with each peptide added

[0777] C: gMFI of T2 cells with DMSO supplemented with HLA-A2 antibody or with each peptide

[0778] D: gMFI of T2 cells with DMSO supplemented with mouse IgG2b antibody or with each peptide

[0779] E: gMFI of T2 cells with DMSO supplemented with HLA-A2 antibody

[0780] F: gMFI of T2 cells with DMSO supplemented with mouse IgG2b antibody

[0781] As Figure 1 shown, compared with the wild-type NY-ESO peptide, the binding of anti-HLA / NY-ESO scFv NYA-0001, 1143, 2044, 2045, 2047, 2048, 2060, 2061 to T2 cells supplemented with peptides with point mutations introduced at amino acids 1, 4, 5, and 7 was reduced to less than half, indicating recognition of amino acids 1, 4, 5, and 7 of the NY-ESO peptide. Similarly, it was also shown that NYA-1154 recognized amino acids 1 and 5, NYA-1163 recognized amino acids 1, 3, 4, 5, 6, and 7, NYA-2023, 2027, 2035 recognized amino acids 1, 4, and 5, and NYA-2143 recognized amino acids 1, 5, and 7. On the other hand, it was also shown that NYC-0003 and 0004 recognized amino acids 4 and 5.

[0782] (Example 6) Evaluation of antigen-binding specificity of anti-HLA / NY-ESO scFv

[0783] To retrieve human peptides (hereinafter referred to as "homologous peptides") in the human proteome (Swiss-Prot) that may bind and are similar but not identical to the amino acid sequence of the NY-ESO peptide: SLLMWITQC (SEQ ID NO:1), 9-mer peptides with the same amino acids 1, 4, and 5 recognized by many of the antibodies of the present invention were retrieved. For the retrieved 9-mer peptides, the binding to HLA-A0201 was predicted using NetMHCPan2.8, and 9-mer peptides with a predicted IC50 of 500 nM or less were selected as homologous peptides for evaluating the binding specificity of anti-HLA / NY-ESO scFv. T2 cells were adjusted to an appropriate concentration with AIM-V medium (Thermo Fisher Scientific) containing 20% FBS, and the NY-ESO peptide (SEQ ID NO:1), homologous peptides DOLPP1, IL20RB, PRKD2, CD163, P2RY8 (SEQ ID NO:131( Figure 2A )、SEQ ID NO:132( 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 this solution was made 50 μM, or 1 / 100 volume of DMSO was added to this solution. The binding of each antibody was evaluated by the same method as in Example 5. The value of the binding of each scFv normalized by the amount of HLA / peptide complex on T2 cells, normalized gMFI, was calculated by the following formula.

[0784] Normalized gMFI = (A / B) / ((C / D) / (E / F))

[0785] ·A / B = Relative gMFI

[0786] A: gMFI of DMSO added with antibody or T2 cells added with each peptide

[0787] B: gMFI of DMSO without added antibody or T2 cells added with each peptide

[0788] ·(C / D) / (E / F) = Correction value of the amount of HLA / peptide complex of DMSO or T2 cells added with each peptide

[0789] C: gMFI of DMSO added with HLA-A2 antibody or T2 cells added with each peptide

[0790] D: gMFI of DMSO added with mouse IgG2b antibody or T2 cells added with each peptide

[0791] E: gMFI of T2 cells added with DMSO added with HLA-A2 antibody

[0792] F: gMFI of T2 cells added with DMSO added with mouse IgG2b antibody

[0793] As Figure 2B shown, anti-HLA / NY-ESO scFv NYA-0001, 1143, 1163, 2023, 2027, 2035, 2044, 2045, 2047, 2048, 2060, 2061, 2143 did not bind to the cells added with homologous peptides, indicating high specificity. On the other hand, it was observed that NYA-1154, NYC-0003, 0004 bound to some of the T2 cells added with homologous peptides.

[0794] (Example 7) Preparation of Fc-added anti-HLA / NY-ESO-anti-CD3 bispecific molecule

[0795] 7)-1 Preparation of expression vector of Fc-added anti-HLA / NY-ESO-anti-CD3 bispecific molecule

[0796] 7)-1-1 Preparation of expression vector of taFv-heterodimeric Fc-type bispecific molecule

[0797] To evaluate the taFv-heterodimeric Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule, expression vectors of each molecule were designed. As the anti-HLA / NY-ESO antibody, NYA-1143, NYA-2143, NYA-1163, NYA-2023, NYA-2027, NYA-2035, NYA-2044, NYA-2045, NYA-2047, NYA-2048, NYA-2060, and NYA-2061 were used. As the anti-CD3 antibody, C3E-7085 (WO2018 / 117237), a humanized anti-CD3 scFv, was used. As the heterodimeric Fc sequence, an Fc sequence (WO2014 / 190441) into which mutations that reduce effector function and form heteropolymers were introduced was used.

[0798] A DNA fragment encoding Fc (HC1 or HC2) into which mutations that reduce effector function and form heteropolymers were introduced was synthesized (FASMAC Co., Ltd.), and a mammalian cell expression vector with pcDNA3.3 (Thermo Fisher Scientific) as the backbone was prepared using the In-Fusion HD Cloning Kit (CLONTECH), and named "p_HC1".

[0799] A mammalian cell expression vector was prepared by introducing a DNA fragment encoding HC2 into the carboxyl terminus of taFv in which NYA-1143 and C3E-7085 were linked with a GGGGS linker, and named "p_NYF-0016-HC2".

[0800] A mammalian cell expression vector was prepared by introducing site-specific mutations into p_NYF-0016-HC2, and by introducing a DNA fragment encoding HC2 into the carboxyl terminus of taFv in which NYA-2143 and C3E-7085 were linked with a GGGGS linker, and named "p_NYF-0019-HC2".

[0801] Using the In-Fusion HD Cloning Kit (CLONTECH), a partial base sequence of NYA-1143 encoding p_NYF-0016-HC2 was replaced with a DNA fragment encoding NYA-1163 to prepare an expression vector for mammalian cells in which a DNA fragment encoding HC2 was introduced into the carboxyl terminus of taFv that links NYA-1163 and C3E-7085 with a GGGGS linker, named "p_NYF-0022-HC2".

[0802] An expression vector for mammalian cells in which a DNA fragment encoding HC2 was introduced into the carboxyl terminus of taFv that links NYA-2023 and C3E-7085 with a GGGGS linker was prepared by introducing site-specific mutations into p_NYF-0016-HC2, named "p_NYF-0023-HC2".

[0803] An expression vector for mammalian cells in which a DNA fragment encoding HC2 was introduced into the carboxyl terminus of taFv that links NYA-2027 and C3E-7085 with a GGGGS linker was prepared by introducing site-specific mutations into an expression vector for mammalian cells in which a DNA fragment encoding HC2 was introduced into the carboxyl terminus of taFv that links NYA-0001 and C3E-7085 with a GGGGS linker, named "p_NYF-0027-HC2".

[0804] An expression vector for mammalian cells in which a DNA fragment encoding HC2 was introduced into the carboxyl terminus of taFv that links NYA-2035 and C3E-7085 with a GGGGS linker was prepared by introducing site-specific mutations into p_NYF-0016-HC2, named "p_NYF-0035-HC2".

[0805] Using the In-Fusion HD Cloning Kit (CLONTECH), a partial base sequence of NYA-1143 encoding p_NYF-0016-HC2 was replaced with a DNA fragment encoding NYA-2044 to prepare an expression vector for mammalian cells in which a DNA fragment encoding HC2 was introduced into the carboxyl terminus of taFv that links NYA-2044 and C3E-7085 with a GGGGS linker, named "p_NYF-0044-HC2".

[0806] Using the In-Fusion HD Cloning Kit (CLONTECH), a partial nucleotide sequence of NYA-1143 encoding p_NYF-0016-HC2 was replaced with a DNA fragment encoding NYA-2045 to produce an expression vector for mammalian cells in which a DNA fragment encoding HC2 was introduced into the carboxyl terminus of taFv linked with NYA-2045 and C3E-7085 by a GGGGS linker, named "p_NYF-0045-HC2".

[0807] Using the In-Fusion HD Cloning Kit (CLONTECH), a partial nucleotide sequence of NYA-1143 encoding p_NYF-0016-HC2 was replaced with a DNA fragment encoding NYA-2047 to produce an expression vector for mammalian cells in which a DNA fragment encoding HC2 was introduced into the carboxyl terminus of taFv linked with NYA-2047 and C3E-7085 by a GGGGS linker, named "p_NYF-0047-HC2".

[0808] Using the In-Fusion HD Cloning Kit (CLONTECH), a partial nucleotide sequence of NYA-1143 encoding p_NYF-0016-HC2 was replaced with a DNA fragment encoding NYA-2048 to produce an expression vector for mammalian cells in which a DNA fragment encoding HC2 was introduced into the carboxyl terminus of taFv linked with NYA-2048 and C3E-7085 by a GGGGS linker, named "p_NYF-0048-HC2".

[0809] By introducing site-specific mutations into p_NYF-0044-HC2, an expression vector for mammalian cells in which a DNA fragment encoding HC2 was introduced into the carboxyl terminus of taFv linked with NYA-2060 and C3E-7085 by a GGGGS linker was produced, named "p_NYF-0060-HC2".

[0810] By introducing site-specific mutations into p_NYF-0045-HC2, an expression vector for mammalian cells in which a DNA fragment encoding HC2 was introduced into the carboxyl terminus of taFv linked with NYA-2061 and C3E-7085 by a GGGGS linker was produced, named "p_NYF-0061-HC2".

[0811] The nucleotide sequence of p_HC1 was re-analyzed, and it was confirmed that the full-length nucleotide sequence of HC1 was the nucleotide sequence shown in SEQ ID NO:71 of the Sequence Listing ( Figure 72 )

[0812] The nucleotide sequence of p_NYF-0016-HC2 was re-analyzed, and it was confirmed that the full-length nucleotide sequence of NYF-0016-HC2 is the nucleotide sequence shown in SEQ ID NO:72 of the sequence listing( Figure 73 ).

[0813] The nucleotide sequence of p_NYF-0019-HC2 was re-analyzed, and it was confirmed that the full-length nucleotide sequence of NYF-0019-HC2 is the nucleotide sequence shown in SEQ ID NO:73 of the sequence listing( Figure 74 ).

[0814] The nucleotide sequence of p_NYF-0022-HC2 was re-analyzed, and it was confirmed that the full-length nucleotide sequence of NYF-0022-HC2 is the nucleotide sequence shown in SEQ ID NO:74 of the sequence listing( Figure 75 ).

[0815] The nucleotide sequence of p_NYF-0023-HC2 was re-analyzed, and it was confirmed that the full-length nucleotide sequence of NYF-0023-HC2 is the nucleotide sequence shown in SEQ ID NO:75 of the sequence listing( Figure 76 ).

[0816] The nucleotide sequence of p_NYF-0027-HC2 was re-analyzed, and it was confirmed that the full-length nucleotide sequence of NYF-0027-HC2 is the nucleotide sequence shown in SEQ ID NO:76 of the sequence listing( Figure 77 ).

[0817] The nucleotide sequence of p_NYF-0035-HC2 was re-analyzed, and it was confirmed that the full-length nucleotide sequence of NYF-0035-HC2 is the nucleotide sequence shown in SEQ ID NO:77 of the sequence listing( Figure 78 ).

[0818] The nucleotide sequence of p_NYF-0044-HC2 was re-analyzed, and it was confirmed that the full-length nucleotide sequence of NYF-0044-HC2 is the nucleotide sequence shown in SEQ ID NO:78 of the sequence listing( Figure 79 ).

[0819] The nucleotide sequence of p_NYF-0045-HC2 was re-analyzed, and it was confirmed that the full-length nucleotide sequence of NYF-0045-HC2 is the nucleotide sequence shown in SEQ ID NO:79 of the sequence listing( Figure 80 ).

[0820] The nucleotide sequence of p_NYF-0047-HC2 was re-analyzed, and it was confirmed that the full-length nucleotide sequence of NYF-0047-HC2 is the nucleotide sequence shown in SEQ ID NO:80 of the sequence listing( Figure 81 ).

[0821] The nucleotide sequence of p_NYF-0048-HC2 was re-analyzed, and it was confirmed that the full-length nucleotide sequence of NYF-0048-HC2 is the nucleotide sequence shown in SEQ ID NO:81 of the sequence listing( Figure 82 ).

[0822] The nucleotide sequence of p_NYF-0060-HC2 was re-analyzed, and it was confirmed that the full-length nucleotide sequence of NYF-0060-HC2 is the nucleotide sequence shown in SEQ ID NO:82 of the sequence listing( Figure 83 ).

[0823] The nucleotide sequence of p_NYF-0061-HC2 was re-analyzed, and it was confirmed that the full-length nucleotide sequence of NYF-0061-HC2 is the nucleotide sequence shown in SEQ ID NO:83 of the sequence listing( Figure 84 ).

[0824] In addition, in the above nucleotide sequences, 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 sequences were confirmed.

[0825] The full-length amino acid sequence of HC1 is the amino acid sequence shown in SEQ ID NO:84 of the sequence listing( Figure 85 ). In this sequence, the amino acid sequence from the 1st to the 19th position is the signal sequence, and the amino acid sequence from the 20th to the 246th position is HC.

[0826] The full-length amino acid sequence of NYF-0016-HC2 is the amino acid sequence shown in SEQ ID NO:85 of the sequence listing( Figure 86 ). In this sequence, the amino acid sequence from the 1st to the 19th position is the signal sequence, the amino acid sequence from the 21st to the 511th position is NYA-1143―C3E-7085taFv, the amino acid sequence from the 512th to the 513th position is the linker, and the amino acid sequence from the 514th to the 745th position is HC2.

[0827] The full-length amino acid sequence of NYF-0019-HC2 is the amino acid sequence shown in SEQ ID NO:86 of the Sequence Listing ( Figure 87 ). In this sequence, the amino acid sequence from position 1 to position 19 is the signal sequence, the amino acid sequence from position 21 to position 511 is NYA-2143―C3E-7085taFv, the amino acid sequence from position 512 to position 513 is the linker, and the amino acid sequence from position 514 to position 745 is HC2.

[0828] The full-length amino acid sequence of NYF-0022-HC2 is the amino acid sequence shown in SEQ ID NO:87 of the Sequence Listing ( Figure 88 ). In this sequence, the amino acid sequence from position 1 to position 19 is the signal sequence, the amino acid sequence from position 21 to position 511 is NYA-1163―C3E-7085taFv, the amino acid sequence from position 512 to position 513 is the linker, and the amino acid sequence from position 514 to position 745 is HC2.

[0829] The full-length amino acid sequence of NYF-0023-HC2 is the amino acid sequence shown in SEQ ID NO:88 of the Sequence Listing ( Figure 89 ). In this sequence, the amino acid sequence from position 1 to position 19 is the signal sequence, the amino acid sequence from position 21 to position 511 is NYA-2023―C3E-7085taFv, the amino acid sequence from position 512 to position 513 is the linker, and the amino acid sequence from position 514 to position 745 is HC2.

[0830] The full-length amino acid sequence of NYF-0027-HC2 is the amino acid sequence shown in SEQ ID NO:89 of the Sequence Listing ( Figure 90 ). In this sequence, the amino acid sequence from position 1 to position 19 is the signal sequence, the amino acid sequence from position 21 to position 511 is NYA-2027―C3E-7085taFv, the amino acid sequence from position 512 to position 513 is the linker, and the amino acid sequence from position 514 to position 745 is HC2.

[0831] The full-length amino acid sequence of NYF-0035-HC2 is the amino acid sequence shown in SEQ ID NO:90 of the Sequence Listing ( Figure 91 ). In this sequence, the amino acid sequence from position 1 to position 19 is the signal sequence, the amino acid sequence from position 21 to position 511 is NYA-2035―C3E-7085taFv, the amino acid sequence from position 512 to position 513 is the linker, and the amino acid sequence from position 514 to position 745 is HC2.

[0832] The full-length amino acid sequence of NYF-0044-HC2 is the amino acid sequence shown in SEQ ID NO:91 of the sequence listing ( Figure 92 ). In this sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 511th is NYA-2044―C3E-7085taFv, the amino acid sequence from the 512th to the 513th is the linker, and the amino acid sequence from the 514th to the 745th is HC2.

[0833] The full-length amino acid sequence of NYF-0045-HC2 is the amino acid sequence shown in SEQ ID NO:92 of the sequence listing ( Figure 93 ). In this sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 511th is NYA-2045―C3E-7085taFv, the amino acid sequence from the 512th to the 513th is the linker, and the amino acid sequence from the 514th to the 745th is HC2.

[0834] The full-length amino acid sequence of NYF-0047-HC2 is the amino acid sequence shown in SEQ ID NO:93 of the sequence listing ( Figure 94 ). In this sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 511th is NYA-2047―C3E-7085taFv, the amino acid sequence from the 512th to the 513th is the linker, and the amino acid sequence from the 514th to the 745th is HC2.

[0835] The full-length amino acid sequence of NYF-0048-HC2 is the amino acid sequence shown in SEQ ID NO:94 of the sequence listing ( Figure 95 ). In this sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 511th is NYA-2048―C3E-7085taFv, the amino acid sequence from the 512th to the 513th is the linker, and the amino acid sequence from the 514th to the 745th is HC2.

[0836] The full-length amino acid sequence of NYF-0060-HC2 is the amino acid sequence shown in SEQ ID NO:95 of the sequence listing ( Figure 96 ). In this sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 511th is NYA-2060―C3E-7085taFv, the amino acid sequence from the 512th to the 513th is the linker, and the amino acid sequence from the 514th to the 745th is HC2.

[0837] The full-length amino acid sequence of NYF-0061-HC2 is the amino acid sequence shown in SEQ ID NO:96 of the Sequence Listing( Figure 97 ). In this sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 511th is NYA-2061―C3E-7085taFv, the amino acid sequence from the 512th to the 513th is the linker, and the amino acid sequence from the 514th to the 745th is HC2.

[0838] 7)- Preparation of the expression vector of the taFv-Fab-heterodimeric Fc-type bispecific molecule

[0839] A vector for expressing the taFv-Fab-heterodimeric Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule was designed. NYA-0001 was used as the anti-HLA / NY-ESO antibody. C3E-7085 (WO2018 / 117237), a humanized anti-CD3 scFv, was used as the anti-CD3 antibody. The heterodimeric Fc sequence used was the Fc sequence (WO2014 / 190441) into which mutations that reduce effector function and form heteropolymers were introduced.

[0840] An expression vector for mammalian cells was prepared by introducing the heavy chain variable region of NYA-0001, the CH1 region from human IgG, and a DNA fragment that reduces effector function and encodes HC1-k delete, and was named "p_NYA-0001-Fab-HC1-k delete". In addition, an expression vector for mammalian cells was prepared by introducing the light chain variable region of NYA-0001 and a DNA fragment encoding the CL region from human IgG, and was named "p_NYA-0001-LC".

[0841] The nucleotide sequence of p_NYA-0001-Fab-HC1-k delete was re-analyzed, and it was confirmed that the full-length nucleotide sequence of NYA-0001-Fab-HC1-k delete is the nucleotide sequence shown in SEQ ID NO:97 of the Sequence Listing( Figure 98 ).

[0842] The nucleotide sequence of p_NYA-0001-LC was re-analyzed, and it was confirmed that the full-length nucleotide sequence of NYA-0001-LC is the nucleotide sequence shown in SEQ ID NO:98 of the Sequence Listing( Figure 99 ).

[0843] In addition, in the above nucleotide sequences, the full-length amino acid sequences of NYA-0001-Fab-HC1-k delete and NYA-0001-LC encoded by the sequences were confirmed.

[0844] The full-length amino acid sequence of NYA-0001-Fab-HC1-k deletion is the amino acid sequence shown in SEQ ID NO:99 of the sequence listing ( Figure 100 ). In this sequence, the amino acid sequence from the 1st to the 19th position is the signal sequence, the amino acid sequence from the 20th to the 139th position is the variable region, and the amino acid sequence from the 140th to the 468th position is the constant region. Additionally, the amino acid sequence from the 45th to the 52nd position is CDRH1 (SEQ ID NO:54( Figure 61 ), the amino acid sequence from the 70th to the 77th position is CDRH2 (SEQID NO:55( Figure 61 ), and the amino acid sequence from the 116th to the 128th position is CDRH3 (SEQ ID NO:56( Figure 61 ).

[0845] The full-length amino acid sequence of NYA-0001-LC is the amino acid sequence shown in SEQ ID NO:100 of the sequence listing ( Figure 101 ). The amino acid sequence from the 1st to the 20th position is the signal sequence, the amino acid sequence from the 21st to the 131st position is the variable region, and the amino acid sequence from the 132nd to the 237th position is the constant region. Additionally, 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 ), and amino acid numbers 110 to 121 are CDRL3 (SEQ ID NO:59( Figure 61 ).

[0846] 7) - Expression of 2Fc-added anti-HLA / NY-ESO-anti-CD3 bispecific molecule

[0847] 7) - 2 - 1taFv - heterodimeric Fc-type bispecific molecule expression

[0848] Subculture Expi293F cells (Thermo Fisher Scientific) according to the manual. In Expi293 expression medium (Thermo Fisher Scientific), dilute the Expi293F cell culture solution in the logarithmic growth phase to 2.5×10 6cells / mL for the production of various bispecific molecules. 0.3 mg of a vector mixture of vector p_NYF-0016-HC2 and p_HC1 mixed at a ratio of 1:1.5, 0.9 mg of polyethyleneimine (Polyscience #24765) were added to 20 mL of Opti-Pro SFM medium (Thermo Fisher Scientific) and stirred gently, then left for 5 minutes, and then added to Expi293F cells. Cultured with shaking at 135 rpm in a 37 °C, 8% CO 2 incubator for 6 days, and the resulting culture supernatant was filtered through a 0.2 μm filter membrane (Millipore) to obtain a culture supernatant of taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0016). The respective vectors constituting NYF-0016 were expressed, and the resulting amino acid sequences are shown in SEQ ID NO:85 of the sequence listing ( Figure 86 ) and SEQ ID NO:84 ( Figure 85 ).

[0849] By the same method, using p_NYF-0019-HC2, p_HC1, a culture supernatant of taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0019) was expressed and prepared. The respective vectors constituting NYF-0019 were expressed, and the resulting amino acid sequences are shown in SEQ ID NO:86 of the sequence listing ( Figure 87 ) and SEQ ID NO:84 ( Figure 85 ).

[0850] Using p_NYF-0022-HC2, p_HC1, a culture supernatant of taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0022) was expressed and prepared. The respective vectors constituting NYF-0022 were expressed, and the resulting amino acid sequences are shown in SEQ ID NO:87 of the sequence listing ( Figure 88 ) and SEQ ID NO:84 ( Figure 85 ).

[0851] Using p_NYF-0023-HC2, p_HC1, a culture supernatant of taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0023) was expressed and prepared. The respective vectors constituting NYF-0023 were expressed, and the resulting amino acid sequences are shown in SEQ ID NO:88 of the sequence listing ( Figure 89 ) and SEQ ID NO:84 ( Figure 85 ).

[0852] Using p_NYF-0027-HC2 and p_HC1, the culture supernatant of the taFv-heterodimeric Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0027) was expressed and prepared. Each vector constituting NYF-0027 was expressed, and the resulting amino acid sequences are shown in SEQ ID NO:89 of the sequence listing ( Figure 90 ) and SEQ ID NO:84 ( Figure 85 ).

[0853] Using p_NYF-0035-HC2 and p_HC1, the culture supernatant of the taFv-heterodimeric Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0035) was expressed and prepared. Each vector constituting NYF-0035 was expressed, and the resulting amino acid sequences are shown in SEQ ID NO:90 of the sequence listing ( Figure 91 ) and SEQ ID NO:84 ( Figure 85 ).

[0854] Using p_NYF-0044-HC2 and p_HC1, the culture supernatant of the taFv-heterodimeric Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0044) was expressed and prepared. Each vector constituting NYF-0044 was expressed, and the resulting amino acid sequences are shown in SEQ ID NO:91 of the sequence listing ( Figure 92 ) and SEQ ID NO:84 ( Figure 85 ).

[0855] Using p_NYF-0045-HC2 and p_HC1, the culture supernatant of the taFv-heterodimeric Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0045) was expressed and prepared. Each vector constituting NYF-0045 was expressed, and the resulting amino acid sequences are shown in SEQ ID NO:92 of the sequence listing ( Figure 93 ) and SEQ ID NO:84 ( Figure 85 ).

[0856] Using p_NYF-0047-HC2 and p_HC1, the culture supernatant of the taFv-heterodimeric Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0047) was expressed and prepared. Each vector constituting NYF-0047 was expressed, and the resulting amino acid sequences are shown in SEQ ID NO:93 of the sequence listing ( Figure 94 ) and SEQ ID NO:84 ( Figure 85 ).

[0857] Using p_NYF-0048-HC2 and p_HC1, the culture supernatant of the taFv-heterodimeric Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0048) was expressed and prepared. Each vector constituting NYF-0048 was expressed, and the resulting amino acid sequences are shown in SEQ ID NO:94 of the sequence listing ( Figure 95 ) and SEQ ID NO:84 ( Figure 85 ).

[0858] Using p_NYF-0060-HC2 and p_HC1, the culture supernatant of the taFv-heterodimeric Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0060) was expressed and prepared. Each vector constituting NYF-0060 was expressed, and the resulting amino acid sequences are shown in SEQ ID NO:95 of the sequence listing ( Figure 96 ) and SEQ ID NO:84 ( ​ ).

[0859] Using p_NYF-0061-HC2 and p_HC1, the culture supernatant of the taFv-heterodimeric Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0061) was expressed and prepared. Each vector constituting NYF-0061 was expressed, and the resulting amino acid sequences are shown in SEQ ID NO:96 of the sequence listing ( ​ ) and SEQ ID NO:84 ( ​ ).

[0860] 7)-2-2 Expression of taFv-Fab-heterodimeric Fc-type bispecific molecule

[0861] By the same method as in 7)-2-1, using a vector mixture of p_NYF-0023-HC2, p_NYA-0001-Fab-HC1-k delete, and p_NYA-0001-LC mixed in a ratio of 1:1:1.5, the culture supernatant of the taFv-Fab-heterodimeric Fc-type bispecific molecule (NYF-0058) was expressed and prepared. Each vector constituting NYF-0058 was expressed, and the resulting amino acid sequences are shown in SEQ ID NO:88 of the sequence listing (amino acid numbers 20 to 745 in 89) Figure, SEQ ID NO:99 ( ​ ) amino acid numbers 20 to 468, and SEQ ID NO:100 ( ​ ) amino acid numbers 21 to 237.

[0862] 7) Purification of the Fc-added anti-HLA / NY-ESO-anti-CD3 bispecific molecule

[0863] Various bispecific molecules were purified from the culture supernatant obtained in 7)-2 by a two-stage procedure of protein A affinity chromatography and gel filtration chromatography.

[0864] The culture supernatant was loaded onto a MabSelect SuRe column (GE Healthcare Bioscience, also simply referred to as "GE Healthcare") equilibrated with PBS at pH 7.4 to adsorb the target bispecific molecule. After removing the non-adsorbed components with PBS, the adsorbed components were eluted with an acetic acid buffer at pH 3.5. After adjusting the pH of the eluted fraction to neutral with a Tris buffer at pH 9.5, the eluted fraction was concentrated and applied to a gel filtration column Superdex 200 10 / 300 (GE Healthcare Bioscience) pre-equilibrated with 25 mM histidine, 300 mM NaCl, 5% sorbitol, and pH 5.5. From the peak fraction obtained by gel filtration chromatography, the fraction corresponding to the target heterodimer was recovered, and it was confirmed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) that the target anti-HLA / NY-ESO-anti-CD3 bispecific molecule had been formed. The purified protein sample was used for analytical SEC, and after determining the purity and concentration, it was used for various evaluations.

[0865] (Example 8) Evaluation of the cytotoxic activity of the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule

[0866] 8)-1 Preparation of target cells

[0867] In RPMI1640 medium (FUJIFILM Wako Pure Chemical Corporation) containing 10% FBS, cell lines expressing endogenous human NY-ESO (U266B1 and NCI-H1703) and cell lines not expressing endogenous human NY-ESO (AGS and CFPAC-1) were prepared at a concentration of 1×10 6 cells / mL, and chromium-51 radionuclide (PerkinElmer) was added in an amount of 100 μL per 1 mL of each cell suspension, and the cells were cultured at 37°C and 5% CO 2 for 2 hours. After washing twice with RPMI1640 medium containing 10% FBS, the cells were resuspended again with RPMI1640 medium containing 10% FBS to make the concentration 1×10 5 cells / mL, and this product was used as target cells.

[0868] 8)-2 Preparation of effector cells

[0869] Commercially available cryopreserved human PBMC (Cellular Technology Limited) was thawed at 37°C and transferred to a solution supplemented with Anti-aggregate Wash reagent (Cellular Technology Limited) in RPMI 1640 medium containing 10% FBS, washed twice, and then prepared into 1×10 6 cells / mL in RPMI 1640 medium containing 10% FBS as effector cells.

[0870] 8)-3 Cytotoxicity assay

[0871] To each well of a 96-well U-bottom microplate, add the respective target cells obtained in 8)-1 in an amount of 50 μL / well. To this, add various Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules prepared at various concentrations in Example 7 in an amount of 50 μL / well, add the effector cells prepared in Example 8)-2 in an amount of 100 μL / well, centrifuge at 1000 rpm for 1 minute at room temperature, and then culture at 37°C and 5% CO 2 for 20 to 24 hours. Recover 50 μL of the supernatant into a LumaPlate (PerkinElmer), dry at approximately 50°C for about 2 hours, and then measure using a microplate reader (TopCount, PerkinElmer). The experiment was repeated three times, and the cell lysis rate was calculated using the following formula.

[0872] Cell lysis rate (%) = (A - B) / (C - B) × 100

[0873] A: Count of the sample well.

[0874] B: Average value of the counts of the background (wells without added antibody) (n = 3). When adding the antibody, 50 μL of the detection medium was added. Otherwise, the same operations as the sample well were performed. C: Average value of the counts of the maxim...

Claims

1. A solution preparation, which contains an antibody or its binding fragment that specifically binds to human HLA / NY-ESO, as well as saccharides and a buffer. The antibody or its binding fragment that specifically binds to human HLA / NY-ESO comprises: 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 A light-chain CDRL3 consisting of the amino acid sequence shown in SEQ ID NO:

59.

2. The solution preparation according to claim 1, wherein the solution preparation further contains a surfactant.

3. The solution preparation according to claim 1, wherein, The saccharides are selected from the group consisting of sucrose, sorbitol, and fructose.

4. The solution preparation according to claim 1, wherein, The buffer is selected from the group consisting of acetate buffer, phosphate buffer, and histidine buffer.

5. The solution preparation according to claim 2, wherein, The surfactant is a fatty acid ester.

6. The solution preparation according to claim 1, wherein, The antibody or its binding fragment comprises: a heavy-chain variable region consisting of the amino acid sequence from the 21st to the 140th amino acid sequence shown in SEQ ID NO:53, and a light-chain variable region consisting of the amino acid sequence from the 156th to the 266th amino acid sequence shown in SEQ ID NO:53; or, A heavy-chain variable region consisting of the amino acid sequence from the 21st to the 140th amino acid sequence shown in SEQ ID NO:156, and a light-chain variable region consisting of the amino acid sequence from the 161st to the 271st amino acid sequence shown in SEQ ID NO:

156.

7. The solution preparation according to claim 1, wherein, The antibody or its binding fragment is an scFv.

8. The solution preparation according to claim 7, wherein, The antibody or its binding fragment is an scFv consisting of the amino acid sequence from the 21st to the 266th amino acid sequence shown in SEQ ID NO:53; or, An scFv consisting of the amino acid sequence from the 21st to the 271st amino acid sequence shown in SEQ ID NO:

156.

9. A pharmaceutical composition, which contains the solution preparation according to any one of claims 1 to 8 as an active ingredient.

10. A solution preparation, which contains a molecule that specifically binds to human HLA / NY-ESO, as well as saccharides and a buffer. The molecule comprises the antibody or its binding fragment according to any one of claims 1, 6 to 8.

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