Dual-specific antibodies

Abstract

To provide a novel antibody that can be used as an antitumor agent, and an antitumor agent containing a molecule containing the antibody as an active ingredient. [Solution] Heavy chain CDRH1 consisting of the amino acid sequence represented by Sequence ID No. 54, The heavy chain CDRH2 consists of the amino acid sequence represented by SEQ ID NO: 55. The heavy chain CDRH3 consists of the amino acid sequence represented by SEQ ID NO: 56. A light chain CDRL1 consisting of the amino acid sequence represented by Sequence ID No. 57, or a light chain CDRL1 consisting of the amino acid sequence represented by Sequence ID No. 57 in which the 7th amino acid is W and / or the 8th amino acid is K, A light chain CDRL2 consisting of an amino acid sequence represented by DNN, and A light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 59, or a light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 57, in which the second amino acid is A or S. An antibody containing or a conjugated fragment thereof.

Description

[Technical Field]

[0001] This invention relates to antibodies, bispecific antibodies, and the like that are useful for treating cancer. [Background technology]

[0002] NY-ESO-1 is a molecule identified from esophageal cancer by serological analysis of recombinant cDNA expression libraries (SEREX) (Non-Patent Literature 1), and LAGE-1, also known as NY-ESO-2, is a molecule identified by representational difference analysis of tumor cDNA libraries (Non-Patent Literature 2). Both molecules are known to be expressed only in the testes in normal tissue, but their functions remain unclear. Since NY-ESO-1 and LAGE-1 have been reported to be expressed in a wide range of cancer types, including melanoma, lung cancer, bladder cancer, ovarian cancer, soft tissue sarcoma, and myeloma (Non-Patent Literature 3), a link to cancer has been suggested. Furthermore, regarding the correlation with malignancy, there are reports that NY-ESO-1 is expressed more in metastatic lesions than in primary melanomas (Non-Patent Literature 4), NY-ESO-1 and LAGE-1 are expressed more in advanced urothelial carcinomas compared to early-stage cancers (Non-Patent Literature 5), and NY-ESO-1 is expressed more in high-risk myelomas with chromosomal abnormalities than in myelomas with normal chromosomes (Non-Patent Literature 6). Based on this information, NY-ESO-1 and LAGE-1 have attracted attention as molecules with high cancer specificity, and much research and development is being done as drug targets for cancer vaccine therapy, but no drugs have been approved to date.

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

[0004] One application of binding molecules to cancer target molecules is CD3 bispecific antibodies, which utilize T-cell redirection—a mechanism that induces T cells to become cancer cells and exhibits antitumor effects through cytotoxicity (Non-Patent Literature 9, 10). Currently, a CD3 bispecific antibody drug on the market is blinatumomab, a bispecific T-cell engager (BiTE) targeting CD19. It has been approved for acute lymphoblastic leukemia (ALL), and clinical trials targeting other hematological cancers are underway. However, the bispecific antibody format is tandem scFv (taFv), which lacks an Fc region, and its half-life in the blood after administration to a patient is very short compared to IgG-type antibodies commonly used as therapeutic antibodies (Non-Patent Literature 11).

[0005] As bispecific antibodies having a heterodimer Fc region with a blood half-life comparable to that of IgG-type antibodies, various antibody formats such as knobs-into-holes, CrossMAb, DuoBody® (Patent Documents 4, 5, 6, 7) are being used in the research and clinical trials of CD3 bispecific antibodies, and CD3 bispecific antibodies using antibodies against HLA / NY-ESO peptide complexes have been reported (Non-Patent Document 12).

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Patent Document 7

Patent Document 8

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

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

[0009] The inventors of this invention have diligently conducted research to solve the above problems and have 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.

[0010] In other words, the present invention encompasses the following inventions. [1] Heavy chain CDRH1 consisting of the amino acid sequence represented by Sequence ID No. 54, The heavy chain CDRH2 consists of the amino acid sequence represented by SEQ ID NO: 55. The heavy chain CDRH3 consists of the amino acid sequence represented by SEQ ID NO: 56. A light chain CDRL1 consisting of the amino acid sequence represented by Sequence ID No. 57, or a light chain CDRL1 consisting of the amino acid sequence represented by Sequence ID No. 57 in which the 7th amino acid is W and / or the 8th amino acid is K, A light chain CDRL2 consisting of an amino acid sequence represented by DNN, and A light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 59, or a light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 59 in which the second amino acid is A or S. An antibody or its conjugated fragment that specifically binds to human HLA / NY-ESO, including the above.

[0011] [2] (i) Heavy chain CDRH1 consisting of the amino acid sequence represented by Sequence ID No. 54, The heavy chain CDRH2 consists of the amino acid sequence represented by SEQ ID NO: 55. The heavy chain CDRH3 consists of the amino acid sequence represented by SEQ ID NO: 56. The light chain CDRL1 consists of the amino acid sequence represented by sequence number 57. A light chain CDRL2 consisting of an amino acid sequence represented by DNN, and CDRL3, a light chain consisting of the amino acid sequence represented by SEQ ID NO: 59. (ii) Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 54, The heavy chain CDRH2 consists of the amino acid sequence represented by SEQ ID NO: 55. The heavy chain CDRH3 consists of the amino acid sequence represented by SEQ ID NO: 56. In the amino acid sequence represented by Sequence ID No. 57, the light chain CDRL1 consists of an amino acid sequence in which the 7th amino acid is W. A light chain CDRL2 consisting of an amino acid sequence represented by DNN, and CDRL3, a light chain consisting of the amino acid sequence represented by SEQ ID NO: 59. (iii) Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 54, The heavy chain CDRH2 consists of the amino acid sequence represented by SEQ ID NO: 55. The heavy chain CDRH3 consists of the amino acid sequence represented by SEQ ID NO: 56. The light chain CDRL1 consists of the amino acid sequence represented by sequence number 57. A light chain CDRL2 consisting of an amino acid sequence represented by DNN, and In the amino acid sequence represented by Sequence ID No. 59, the light chain CDRL3 consists of an amino acid sequence in which the second amino acid is A. (iv) Heavy chain CDRH1 consisting of the amino acid sequence represented by Sequence ID No. 54, The heavy chain CDRH2 consists of the amino acid sequence represented by SEQ ID NO: 55. The heavy chain CDRH3 consists of the amino acid sequence represented by SEQ ID NO: 56. The light chain CDRL1 consists of the amino acid sequence represented by sequence number 57. A light chain CDRL2 consisting of an amino acid sequence represented by DNN, and The light chain CDRL3, which consists of an amino acid sequence in which the second amino acid is S in the amino acid sequence represented by Sequence ID No. 59, (v) Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 54, The heavy chain CDRH2 consists of the amino acid sequence represented by SEQ ID NO: 55. The heavy chain CDRH3 consists of the amino acid sequence represented by SEQ ID NO: 56. In the amino acid sequence represented by Sequence ID No. 57, the light chain CDRL1 consists of an amino acid sequence in which the 7th amino acid is W and the 8th amino acid is K. A light chain CDRL2 consisting of an amino acid sequence represented by DNN, and The light chain CDRL3 consists of the amino acid sequence represented by sequence number 59. An antibody or conjugated fragment thereof from [1] comprising one or more groups of CDRH1-CDRH3 and CDRL1-CDRL3 selected from the group consisting of (i)-(v) of [1].

[0012] [3] The antibody or its conjugated fragment according to [1], comprising a heavy chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence of amino acid numbers 21 to 140 of the amino acid sequence represented by SEQ ID NO: 27, or the amino acid sequence represented by 38 or 39, and a light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence of amino acid numbers 156 to 266 of the amino acid sequence represented by SEQ ID NO: 27 or SEQ ID NO: 52, or the amino acid sequence represented by SEQ ID NO: 40.

[0013] [4] (H1) Heavy chain variable region consisting of the amino acid sequence represented by Sequence ID No. 6 (H2) Heavy chain variable region consisting of the amino acid sequence represented by Sequence ID No. 18, (H3) Heavy chain variable region consisting of amino acid sequences 21 to 140 of the amino acid sequence represented by Sequence ID No. 29, (H4) The heavy chain variable region consisting of amino acid sequences 21 to 140 of the amino acid sequence represented by Sequence ID No. 26, (H5) The heavy chain variable region consisting of amino acid sequences 21 to 140 of the amino acid sequence represented by Sequence ID No. 27, (H6) The heavy chain variable region consisting of amino acid sequences 21 to 140 of the amino acid sequence represented by Sequence ID No. 28, (H7) The heavy chain variable region consisting of amino acid sequences 21 to 140 of the amino acid sequence represented by Sequence ID No. 36, (H8) The heavy chain variable region consisting of amino acid sequences 21 to 140 of the amino acid sequence represented by Sequence ID No. 47, (H9) The heavy chain variable region consisting of amino acid sequences 21 to 140 of the amino acid sequence represented by Sequence ID No. 48, (H10) The heavy chain variable region consisting of amino acid sequences 21 to 140 of the amino acid sequence represented by Sequence ID No. 50, (H11) The heavy chain variable region consisting of amino acid sequences 21 to 140 of the amino acid sequence represented by Sequence ID No. 51, (H12) The heavy chain variable region consisting of amino acid sequences 21 to 140 of the amino acid sequence represented by Sequence ID No. 52, (H13) The heavy chain variable region consisting of amino acid sequences 21 to 140 of the amino acid sequence represented by Sequence ID No. 53, (H14) The heavy chain variable region consisting of amino acids 21 to 140 of the amino acid sequence represented by Sequence ID No. 30, or (H15) The heavy chain variable region consisting of amino acid sequences 21 to 140 of the amino acid sequence represented by Sequence ID No. 156, and, (L1) Light chain variable region consisting of the amino acid sequence represented by Sequence ID No. 8, (L2) Light chain variable region consisting of the amino acid sequence represented by Sequence ID No. 20, (L3) Light chain variable region consisting of amino acid sequences 156 to 266 of the amino acid sequence represented by SEQ ID NO: 29, (L4) Light chain variable region consisting of amino acid sequences 156 to 266 of the amino acid sequence represented by Sequence ID No. 26, (L5) Light chain variable region consisting of amino acid sequences 156 to 266 of the amino acid sequence represented by Sequence ID No. 27, (L6) The light chain variable region consisting of amino acid sequences 156 to 266 of the amino acid sequence represented by Sequence ID No. 28, (L7) The light chain variable region consisting of amino acid sequences 156 to 266 of the amino acid sequence represented by SEQ ID NO: 36, (L8) The light chain variable region consisting of amino acid sequences 156 to 266 of the amino acid sequence represented by SEQ ID NO: 47, (L9) The light chain variable region consisting of amino acid sequences 156 to 266 of the amino acid sequence represented by SEQ ID NO: 48, (L10) The light chain variable region consisting of amino acid sequences 156 to 266 of the amino acid sequence represented by SEQ ID NO: 50. (L11) The light chain variable region consisting of amino acid sequences 156 to 266 of the amino acid sequence represented by SEQ ID NO: 51, (L12) The light chain variable region consisting of amino acid sequences 156 to 266 of the amino acid sequence represented by Sequence ID No. 52, (L13) The light chain variable region consisting of amino acid sequences 156 to 266 of the amino acid sequence represented by SEQ ID NO: 53, (L14) The light chain variable region consisting of amino acid sequences 156 to 266 of the amino acid sequence represented by SEQ ID NO: 30, or (L15) Light chain variable region consisting of amino acid sequences 161 to 271 of the amino acid sequence represented by SEQ ID NO: 156 An antibody or its conjugated fragment containing [1].

[0014] [5] (H1L1) Heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 6 and light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 8, (H2L2) Heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 18 and light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 20, (H3L3) Heavy chain variable region consisting of amino acid sequences 21 to 140 of the amino acid sequence represented by SEQ ID NO: 29 and light chain variable region consisting of amino acid sequences 156 to 266 of the amino acid sequence represented by SEQ ID NO: 29, (H4L4) Heavy chain variable region consisting of amino acid sequences 21 to 140 of the amino acid sequence represented by SEQ ID NO: 26 and light chain variable region consisting of amino acid sequences 156 to 266 of the amino acid sequence represented by SEQ ID NO: 26, (H5L5) Heavy chain variable region consisting of amino acid sequences 21 to 140 of the amino acid sequence represented by SEQ ID NO: 27 and light chain variable region consisting of amino acid sequences 156 to 266 of the amino acid sequence represented by SEQ ID NO: 27, (H6L6) Heavy chain variable region consisting of amino acid sequences 21 to 140 of the amino acid sequence represented by SEQ ID NO: 28 and light chain variable region consisting of amino acid sequences 156 to 266 of the amino acid sequence represented by SEQ ID NO: 28, (H7L7) Heavy chain variable region consisting of amino acid sequences 21 to 140 of the amino acid sequence represented by SEQ ID NO: 36 and light chain variable region consisting of amino acid sequences 156 to 266 of the amino acid sequence represented by SEQ ID NO: 36, (H8L8) Heavy chain variable region consisting of amino acid sequences 21 to 140 of the amino acid sequence represented by SEQ ID NO: 47 and light chain variable region consisting of amino acid sequences 156 to 266 of the amino acid sequence represented by SEQ ID NO: 47, (H9L9) Heavy chain variable region consisting of amino acid sequences 21 to 140 of the amino acid sequence represented by SEQ ID NO: 48 and light chain variable region consisting of amino acid sequences 156 to 266 of the amino acid sequence represented by SEQ ID NO: 48, (H10L10) Heavy chain variable region consisting of amino acid sequences 21 to 140 of the amino acid sequence represented by SEQ ID NO: 50 and light chain variable region consisting of amino acid sequences 156 to 266 of the amino acid sequence represented by SEQ ID NO: 50, (H11L11) Heavy chain variable region consisting of amino acid sequences 21 to 140 of the amino acid sequence represented by SEQ ID NO: 51 and light chain variable region consisting of amino acid sequences 156 to 266 of the amino acid sequence represented by SEQ ID NO: 51, (H12L12) Heavy chain variable region consisting of amino acid sequences 21 to 140 of the amino acid sequence represented by SEQ ID NO: 52 and light chain variable region consisting of amino acid sequences 156 to 266 of the amino acid sequence represented by SEQ ID NO: 52, (H13L13) Heavy chain variable region consisting of amino acid sequences 21 to 140 of the amino acid sequence represented by SEQ ID NO: 53 and light chain variable region consisting of amino acid sequences 156 to 266 of the amino acid sequence represented by SEQ ID NO: 53, (H14L14) A heavy chain variable region consisting of amino acid sequences 21 to 140 of the amino acid sequence represented by SEQ ID NO: 30 and a light chain variable region consisting of amino acid sequences 156 to 266 of the amino acid sequence represented by SEQ ID NO: 30, or (H15L14) Heavy chain variable region consisting of amino acid sequences 21 to 140 of the amino acid sequence represented by SEQ ID NO: 156 and light chain variable region consisting of amino acid sequences 161 to 271 of the amino acid sequence represented by SEQ ID NO: 156 The antibody or its conjugated fragment, including [4].

[0015] [6] An antibody or its conjugated fragment that is an scFv of any of [1] to [5]. [7] (s1) scFv consisting of amino acid sequences 21-266 of the amino acid sequence represented by sequence number 70, (s2) scFv including a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 18 and a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 20, (s3) scFv consisting of amino acid sequences 21 to 266 of the amino acid sequence represented by SEQ ID NO: 29, (s4) scFv consisting of amino acid sequences 21 to 266 of the amino acid sequence represented by SEQ ID NO: 26, (s5) scFv consisting of amino acid sequences 21 to 266 of the amino acid sequence represented by Sequence ID No. 27, (s6) scFv consisting of amino acid sequences 21 to 266 of the amino acid sequence represented by SEQ ID NO: 28, (s7) scFv consisting of amino acid sequences 21 to 266 of the amino acid sequence represented by sequence number 36, (s8) scFv consisting of amino acid sequences 21 to 266 of the amino acid sequence represented by sequence number 47, (s9) scFv consisting of amino acid sequences 21 to 266 of the amino acid sequence represented by sequence number 48, (s10) scFv consisting of amino acid sequences 21 to 266 of the amino acid sequence represented by sequence number 50, (s11) scFv consisting of amino acid sequences 21 to 266 of the amino acid sequence represented by sequence number 51, (s12) scFv consisting of amino acid sequences 21 to 266 of the amino acid sequence represented by sequence number 52, (s13) scFv consisting of amino acid sequences 21 to 266 of the amino acid sequence represented by sequence number 53, (s14) scFv consisting of the amino acid sequence from position 21 to 266 of the amino acid sequence represented by SEQ ID NO: 30, or (s15) scFv consisting of amino acid sequences 21 to 271 of the amino acid sequence represented by sequence number 156, The antibody or its conjugated fragment of [6].

[0016] [8] A polynucleotide encoding one of the antibodies [1] to [7] or a conjugated fragment thereof. A vector containing the polynucleotides [9] [8].

[10] Host cells containing the polynucleotides of [8] or the vector of [9].

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

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

[12] An antibody or its conjugated fragment obtained by the method of

[11] that specifically binds to human HLA / NY-ESO.

[13] An antibody or its conjugate fragment having the properties described in (i) or (ii) below and binding to HLA-A2 / NY-ESO: (i) The antibody or its binding fragment described in [7] binds to a site on HLA-A2 / NY-ESO recognized by the antibody; (ii) The antibody or its binding fragment described in [7] competes with LHLA-A2 / NY-ESO for binding.

[14] A pharmaceutical composition containing an antibody or its conjugated fragment from any of [1] to [7],

[12] and

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

[10] as an active ingredient. A molecule that specifically binds to human HLA / NY-ESO, comprising any of the antibodies or their binding fragments from [1] to [7],

[12] , and

[13] .

[16] A multispecific antibody molecule of

[15] .

[17] A bispecific antibody molecule of

[15] .

[18] Any molecule from

[15] to

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

[0017]

[19] An antibody or a conjugated fragment thereof that specifically binds to CD3, (CCH1) Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 141, (CCH2) Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 142, or heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 142 in which the third amino acid is N or S, (CCH3) Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 143, (CCL1) Light chain CDRL1 consisting of the amino acid sequence represented by sequence number 144, (CCL2) Light chain CDRL2 consisting of an amino acid sequence represented by RDD, or a light chain CDRL2 consisting of an amino acid sequence in which the second amino acid is N, and (CCL3) Light chain CDRL3 consisting of the amino acid sequence represented by sequence number 146. A molecule of

[18] which is an antibody or a conjugate fragment thereof that specifically binds to CD3.

[0018]

[20] An antibody or its binding fragment that specifically binds to CD3, (CH1) The heavy chain variable region consisting of the amino acid sequence from the 2nd to the 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 136, (CH2) The heavy chain variable region consisting of the amino acid sequence from the 2nd to the 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 137, (CH3) The heavy chain variable region consisting of the amino acid sequence from the 2nd to the 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 147, (CH4) The heavy chain variable region consisting of the amino acid sequence from the 2nd to the 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 138, (CH5) The heavy chain variable region consisting of the amino acid sequence from the 2nd to the 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 139, (CH6) The heavy chain variable region consisting of the amino acid sequence from the 2nd to the 119th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 140, (CH7) The heavy chain variable region consisting of amino acid sequences 272-389 of the amino acid sequence represented by SEQ ID NO: 155, (CH8) The heavy chain variable region consisting of amino acid sequences 277 to 394 of the amino acid sequence represented by SEQ ID NO: 156, or (CH9) The heavy chain variable region consisting of amino acid sequences 277 to 394 of the amino acid sequence represented by SEQ ID NO: 157, and (CL1) The light chain variable region consisting of amino acid sequences 135 to 243 of the amino acid sequence represented by SEQ ID NO: 136. (CL2) The light chain variable region consisting of amino acid sequences 135 to 241 of the amino acid sequence represented by SEQ ID NO: 137, (CL3) Light chain variable region consisting of amino acid sequences 135 to 243 of the amino acid sequence represented by SEQ ID NO: 147, (CL4) Light chain variable region consisting of amino acid sequences 135 to 241 of the amino acid sequence represented by SEQ ID NO: 138, (CL5) The light chain variable region consisting of amino acid sequences 135 to 243 of the amino acid sequence represented by sequence number 139, (CL6) The light chain variable region consisting of amino acid sequences 135 to 243 of the amino acid sequence represented by SEQ ID NO: 140, (CL7) Light chain variable region consisting of amino acid sequences 405 to 511 of the amino acid sequence represented by SEQ ID NO: 155, (CL8) The light chain variable region consisting of amino acid sequences 410 to 516 of the amino acid sequence represented by SEQ ID NO: 156, or (CL9) Light chain variable region consisting of amino acid sequences 410 to 516 of the amino acid sequence represented by SEQ ID NO: 157, A molecule containing

[19] .

[0019]

[21] An antibody or a conjugated fragment thereof that specifically binds to CD3, (CH1CL1) Heavy chain variable region consisting of amino acid sequences 2 to 119 of the amino acid sequence represented by SEQ ID NO: 136 and light chain variable region consisting of amino acid sequences 135 to 243 of the amino acid sequence represented by SEQ ID NO: 136, (CH2CL2) Heavy chain variable region consisting of amino acid sequences 2 to 119 of the amino acid sequence represented by SEQ ID NO: 137 and light chain variable region consisting of amino acid sequences 135 to 241 of the amino acid sequence represented by SEQ ID NO: 137, (CH3CL3) Heavy chain variable region consisting of amino acid sequences 2 to 119 of the amino acid sequence represented by SEQ ID NO: 147 and light chain variable region consisting of amino acid sequences 135 to 243 of the amino acid sequence represented by SEQ ID NO: 147, (CH4CL4) Heavy chain variable region consisting of amino acid sequences 2 to 119 of the amino acid sequence represented by SEQ ID NO: 138 and light chain variable region consisting of amino acid sequences 135 to 241 of the amino acid sequence represented by SEQ ID NO: 138, (CH5CL5) Heavy chain variable region consisting of amino acid sequences 2 to 119 of the amino acid sequence represented by SEQ ID NO: 139 and light chain variable region consisting of amino acid sequences 135 to 243 of the amino acid sequence represented by SEQ ID NO: 139, (CH6CL6) Heavy chain variable region consisting of amino acid sequences 2 to 119 of the amino acid sequence represented by SEQ ID NO: 140 and light chain variable region consisting of amino acid sequences 135 to 243 of the amino acid sequence represented by SEQ ID NO: 140, (CH7CL7) Heavy chain variable region consisting of amino acid sequences 272-389 of the amino acid sequence represented by SEQ ID NO: 155 and light chain variable region consisting of amino acid sequences 272-389 of the amino acid sequence represented by SEQ ID NO: 155, (CH8CL8) A heavy chain variable region consisting of amino acid sequences 277 to 394 of the amino acid sequence represented by SEQ ID NO: 156 and a light chain variable region consisting of amino acid sequences 410 to 516 of the amino acid sequence represented by SEQ ID NO: 156, or (CH9CL9) A molecule of

[20] comprising a heavy chain variable region consisting of amino acid sequences 277 to 394 of the amino acid sequence represented by SEQ ID NO: 157 and a light chain variable region consisting of amino acid sequences 410 to 516 of the amino acid sequence represented by SEQ ID NO: 157.

[22] Any molecule from

[18] to

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

[0020]

[23] scFv, (CS1) scFv consisting of the amino acid sequence from the 2nd to the 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 136, (CS2) scFv consisting of the amino acid sequence from the 2nd to the 241st amino acid sequence of the amino acid sequence represented by sequence number 137, (CS3) scFv consisting of the amino acid sequence from the 2nd to the 243rd amino acid sequence of the amino acid sequence represented by sequence number 147, (CS4) scFv consisting of the amino acid sequence from the 2nd to the 241st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 138, (CS5) scFv consisting of the amino acid sequence from the 2nd to the 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 139, (CS6) scFv consisting of the amino acid sequence from the 2nd to the 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 140, (CS7) scFv consisting of amino acid sequences 272-511 of the amino acid sequence represented by sequence number 155, (CS8) scFv consisting of amino acid sequences 277 to 516 of the amino acid sequence represented by SEQ ID NO: 156, or (CS9) scFv consisting of amino acid sequences 277-516 of the amino acid sequence represented by sequence number 157, A molecule containing

[22] .

[0021]

[24] A first polypeptide comprising, in that order 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); and, A molecule comprising a second polypeptide containing an Fc region (ii), preferably any of the molecules

[18] to

[23] , which associates at the Fc region (i) and the Fc region (ii).

[25] The molecule of

[24] , which is an antibody or a conjugate fragment thereof that specifically binds to human HLA / NY-ESO, which is the scFv of [1].

[26] The molecule of

[24] , which is an antibody or a conjugate fragment thereof that specifically binds to human HLA / NY-ESO, and is an scFv that specifically binds to human HLA / NY-ESO [2].

[27] The molecule of

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

[28] The molecule of

[24] , which is an antibody or a conjugate fragment thereof that specifically binds to human HLA / NY-ESO, and which is an scFv that specifically binds to human HLA / NY-ESO [4].

[29] The molecule of

[24] , which is an antibody or a conjugate fragment thereof that specifically binds to human HLA / NY-ESO, and which is an scFv that specifically binds to human HLA / NY-ESO [5].

[0022]

[30] Any molecule from

[19] to

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

[18] , which is an scFv.

[31] Any molecule from

[19] to

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

[19] , which is an scFv.

[32] Any molecule from

[19] to

[24] in which the scFv that specifically binds to CD3 is an antibody that specifically binds to CD3 of scFv

[20] or

[21] or a conjugate fragment thereof.

[33] Any molecule from

[19] to

[24] in which the scFv that specifically binds to CD3 is an antibody that specifically binds to CD3 of scFv

[23] or a conjugate fragment thereof.

[0023]

[34] The amino acid sequences from the 21st to the 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 85, the amino acid sequences from the 21st to the 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 87, the amino acid sequences from the 21st to the 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 88, the amino acid sequences from the 21st to the 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 89, the amino acid sequences from the 21st to the 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 90, the amino acid sequences from the 21st to the 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 91, the amino acid sequences from the 21st to the 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 92, and the 21st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 93 Any molecule from

[24] to

[33] containing an amino acid sequence selected from the group consisting of the amino acid sequence from position 1 to 511, the amino acid sequence from position 21 to 511 of the amino acid sequence represented by SEQ ID NO: 94, the amino acid sequence from position 21 to 511 of the amino acid sequence represented by SEQ ID NO: 95, the amino acid sequence from position 21 to 511 of the amino acid sequence represented by SEQ ID NO: 96, the amino acid sequence from position 21 to 511 of the amino acid sequence represented by SEQ ID NO: 86, the amino acid sequence from position 21 to 511 of the amino acid sequence represented by SEQ ID NO: 149, and the amino acid sequence from position 21 to 511 of the amino acid sequence represented by SEQ ID NO: 150.

[35] Any molecule from

[24] to

[33] comprising an amino acid sequence selected from the group consisting of the amino acid sequences from the 21st to the 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 155, the amino acid sequences from the 21st to the 516th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 156, and the amino acid sequences from the 21st to the 516th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 157.

[36] A molecule in any of the

[24] -

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

[37] A molecule of any of the following

[24] -

[33] and

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

[0024]

[38] The first polypeptide is the amino acid sequence from the 20th to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 85, the amino acid sequence from the 20th to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 87, the amino acid sequence from the 20th to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 88, the amino acid sequence from the 20th to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 89, the amino acid sequence from the 20th to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 90, the amino acid sequence from the 20th to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 91, the amino acid sequence from the 20th to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 92, and the amino acid sequence represented by SEQ ID NO: 93 A molecule of

[34] or

[36] comprising an amino acid sequence selected from the group consisting of the amino acid sequences of positions 20 to 745 of an acid sequence, the amino acid sequences of positions 20 to 745 of the amino acid sequence represented by SEQ ID NO: 94, the amino acid sequences of positions 20 to 745 of the amino acid sequence represented by SEQ ID NO: 95, the amino acid sequences of positions 20 to 745 of the amino acid sequence represented by SEQ ID NO: 96, the amino acid sequences of positions 20 to 745 of the amino acid sequence represented by SEQ ID NO: 86, the amino acid sequences of positions 20 to 745 of the amino acid sequence represented by SEQ ID NO: 149, and the amino acid sequences of positions 20 to 745 of the amino acid sequence represented by SEQ ID NO: 150.

[39] A molecule of

[35] or

[37] wherein the first polypeptide consists of an amino acid sequence selected from the group consisting of the amino acid sequences from the 20th to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 155, an amino acid sequence selected from the group consisting of the amino acid sequences from the 20th to the 750th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 156, or an amino acid sequence selected from the group consisting of the amino acid sequences from the 20th to the 750th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 157.

[40] Any of the molecules

[24] to

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

[0025]

[41] comprising a first polypeptide, a second polypeptide, and a third polypeptide, The first polypeptide comprises, in that order 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 consists of an immunoglobulin heavy chain containing the Fc region (ii), The third polypeptide consists of an immunoglobulin light chain. Preferably, the second polypeptide and the third polypeptide are associated, The first polypeptide and the second peptide are associated in their respective Fc regions. A molecule from

[18] to

[23] .

[42] The molecule of

[41] , which is an antibody or a conjugate fragment thereof that specifically binds to human HLA / NY-ESO, and is an scFv that specifically binds to human HLA / NY-ESO, [1].

[43] The molecule of

[41] , which is an antibody or a conjugate fragment thereof that specifically binds to human HLA / NY-ESO, which is the scFv of [2].

[44] The molecule of

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

[45] The molecule of

[41] , which is an antibody or a conjugate fragment thereof that specifically binds to human HLA / NY-ESO, and which is an scFv that specifically binds to human HLA / NY-ESO [4].

[46] The molecule of

[41] , which is an antibody or a conjugate fragment thereof that specifically binds to human HLA / NY-ESO, and which is an scFv that specifically binds to human HLA / NY-ESO [5].

[0026]

[47] Any molecule from

[42] to

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

[18] , which is an scFv.

[48] ​​Any molecule

[42] -

[46] in which the scFv specifically binds to CD3 is an antibody that specifically binds to the CD3 of the scFv

[19] or a conjugate thereof.

[49] Any molecule from

[42] to

[46] , wherein the scFv that specifically binds to CD3 is an antibody that specifically binds to CD3 as described in

[20] or

[21] , or a conjugate fragment thereof.

[50] Any molecule

[42] -

[46] in which the scFv specifically binds to CD3 is an antibody that specifically binds to the CD3 of scFv

[23] or a conjugate thereof.

[51] A molecule in which the second polypeptide contains the amino acid sequence of amino acid numbers 20-242 of the amino acid sequence represented by Sequence ID No. 99, one of the molecules in

[41] -

[50] .

[52] Any molecule from

[41] to

[51] , the third polypeptide having the amino acid sequence represented by SEQ ID NO: 100.

[0027]

[53] The first polypeptide is The amino acid sequences from the 21st to the 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 85, the amino acid sequences from the 21st to the 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 87, the amino acid sequences from the 21st to the 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 88, the amino acid sequences from the 21st to the 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 89, the amino acid sequences from the 21st to the 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 90, the amino acid sequences from the 21st to the 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 91, the amino acid sequences from the 21st to the 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 92, and the 21st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 93 Any molecule from

[41] to

[52] containing an amino acid sequence selected from the group consisting of the amino acid sequence from the 1st to the 511th amino acid sequence, the amino acid sequence from the 21st to the 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 94, the amino acid sequence from the 21st to the 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 95, the amino acid sequence from the 21st to the 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 96, the amino acid sequence from the 21st to the 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 86, the amino acid sequence from the 21st to the 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 149, and the amino acid sequence from the 21st to the 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 150.

[0028]

[54] The first polypeptide is the amino acid sequence from the 20th to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 85, the amino acid sequence from the 20th to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 87, the amino acid sequence from the 20th to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 88, the amino acid sequence from the 20th to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 89, the amino acid sequence from the 20th to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 90, the amino acid sequence from the 20th to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 91, the amino acid sequence from the 20th to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 92, and the amino acid sequence represented by SEQ ID NO: 93 A molecule consisting of an amino acid sequence selected from the group comprising the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 94, the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 95, the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 96, the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 86, the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 149, and the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 150, any of the molecules

[41] to

[53] .

[0029]

[55] A molecule according to any one of

[18] to

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

[56] The molecule of

[55] , wherein the scFv that specifically binds to human HLA / NY-ESO is the scFv [1], which is an antibody or a conjugate fragment thereof that specifically binds to human HLA / NY-ESO.

[57] A molecule of

[55] which is an antibody or a conjugate fragment thereof that specifically binds to human HLA / NY-ESO, and which is an scFv that specifically binds to human HLA / NY-ESO [2].

[58] The molecule of

[55] , which is an antibody or a conjugate fragment thereof that specifically binds to human HLA / NY-ESO, which is the scFv of [3].

[59] A molecule of

[55] which is an antibody or a conjugate fragment thereof that specifically binds to human HLA / NY-ESO, and which is an scFv that specifically binds to human HLA / NY-ESO [4].

[60] A molecule of

[55] which is an antibody or a conjugate fragment thereof that specifically binds to human HLA / NY-ESO, and which is an scFv that specifically binds to human HLA / NY-ESO, [5].

[61] Any molecule of

[18] to

[21] in which an antibody or a conjugated fragment thereof that specifically binds to CD3 is Fab.

[62] Any molecule from

[56] to

[61] , wherein the Fab that specifically binds to CD3 is an antibody or a conjugate fragment thereof that specifically binds to the CD3 of the Fab

[18] .

[63] Any molecule from

[56] to

[61] , wherein the Fab that specifically binds to CD3 is an antibody or a conjugate fragment thereof that specifically binds to the CD3 of the Fab

[19] .

[64] Any molecule from

[56] to

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

[20] or

[21] which is the Fab.

[65] Any molecule of

[55] to

[64] wherein the first polypeptide comprises the amino acid sequence from position 21 to 394 of the amino acid sequence represented by SEQ ID NO: 160.

[66] The first polypeptide is a molecule from any of

[55] to

[65] containing the amino acid sequence described in any one of (i) to (iii) below: (i) The amino acid sequence from position 20 to 724 of the amino acid sequence represented by SEQ ID NO: 160; (ii) The amino acid sequence from position 20 to 719 of the amino acid sequence represented by SEQ ID NO: 197; (iii) The amino acid sequence from position 20 to 719 of the amino acid sequence represented by SEQ ID NO: 198. .

[67] Any of the molecules

[55] to

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

[68] Any of the molecules

[56] to

[67] wherein the third polypeptide comprises the amino acid sequence from the 21st to the 127th amino acid sequence of the amino acid sequence represented by Sequence ID No. 161.

[69] Any of the molecules

[56] to

[68] wherein the third polypeptide comprises the amino acid sequence from position 21 to 233 of the amino acid sequence represented by SEQ ID NO: 161. A molecule according to any of

[70]

[15] ~

[69] , wherein one or more polypeptides contained in the molecule have one or more amino acids deleted from the carboxyl terminus of the amino acid sequence. A polynucleotide containing a nucleotide sequence that encodes an amino acid sequence found in any of the molecules

[71]

[15] ~

[70] . A vector containing the polynucleotides

[72] and

[71] .

[73] A host cell containing the polynucleotides of

[71] or the vector of

[72] .

[74] A method for producing molecules that specifically bind to human HLA / NY-ESO and human CD3, comprising the steps of (i) culturing host cells of

[73] and (ii) purifying an antibody or its binding fragment from the culture obtained in step (i). A molecule obtained by the methods of

[75]

[74] that specifically binds to human HLA / NY-ESO and human CD3. A pharmaceutical composition comprising any of the molecules from

[76]

[15] to

[70] and

[75] , the polynucleotide of

[71] , the vector of

[72] , or the host cell of

[73] as an active ingredient.

[77] A pharmaceutical composition of

[14] or

[76] which is an anticancer agent.

[78] Cancers include kidney cancer, melanoma, squamous cell carcinoma, basal cell carcinoma, conjunctival cancer, oral cancer, laryngeal cancer, pharyngeal cancer, thyroid cancer, lung cancer (non-small cell lung cancer (adenocarcinoma, squamous cell carcinoma, large cell carcinoma), small cell lung cancer), breast cancer, esophageal cancer, stomach cancer, duodenal cancer, small intestine cancer, colorectal cancer, rectal cancer, appendiceal cancer, anal cancer, liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, adrenal cancer, bladder cancer, prostate cancer, uterine cancer, vaginal cancer, liposarcoma, angiosarcoma, and chondrosarcoma. A pharmaceutical composition

[77] comprising one or more selected from the group consisting of tumors, rhabdomyosarcoma, Ewing's sarcoma, osteosarcoma, undifferentiated pleomorphic sarcoma, myxoid fibrosarcoma, malignant peripheral schwannoma, retroperitoneal sarcoma, synovial sarcoma, uterine sarcoma, gastrointestinal stromal tumor, leiomyosarcoma, epithelioid sarcoma, B-cell lymphoma, T-NK cell lymphoma, Hodgkin lymphoma, myeloid leukemia, lymphocytic leukemia, myeloproliferative disorders, myelodysplastic syndromes, multiple myeloma, testicular cancer, and ovarian cancer.

[79] Any pharmaceutical composition of

[76] to

[78] used in combination with other drugs. This specification includes the disclosures of Japanese Patent Application No. 2020-061476, which forms the basis of the priority of this application. [Effects of the Invention]

[0030] According to the present invention, an antibody that binds to HLA-A2 / NY-ESO, and a novel bispecific antibody (bispecific molecule) that binds to both HLA-A2 / NY-ESO and CD3 can be obtained. Furthermore, a novel pharmaceutical composition containing such an antibody (molecule) as an active ingredient can be obtained. The antibody, or the molecule, has cytotoxic activity and is useful as a therapeutic or prophylactic agent for cancer and the like. [Brief explanation of the drawing]

[0031] [Figure 1]Figure 1 is a table showing the standardized gMFI for various point mutation peptide-added T2 cells of anti-HLA / NY-ESO scFv NYA-0001, 1143, 1154, 1163, 2023, 2027, 2035, 2044, 2045, 2047, 2048, 2060, 2061, 2143, NYC-0003, and 0004. *Underlined values ​​indicate that the standardized gMFI for each scFv is less than half that of NY-ESO peptide-added T2 cells. [Figure 2A] Figure 2A is a table showing the information of the selected homologous peptides. Binding affinity to HLA-A0201 was predicted using NetMHCPan2.8 and is shown as the 50% inhibitory concentration (IC50). [Figure 2B] Figure 2B is a table showing the standardized gMFI values ​​for various homologous peptide-treated T2 cells of anti-HLA / NY-ESO scFv NYA-0001, 1143, 1154, 1163, 2023, 2027, 2035, 2044, 2045, 2047, 2048, 2060, 2061, 2143, NYC-0003, and 0004. *Underlined values ​​indicate that each scFv has a higher standardized gMFI value compared to T2 cells treated with DMSO. [Figure 3]Figure 3 shows the antibody formats used in this embodiment. (a) scFv: A format in which the antibody H chain variable region (VH, described later) and the antibody L chain variable region (VL, described later) (both white) are linked together with a linker. In this embodiment, anti-HLA-A2 / NY-ESO and CD3 scFv were used for evaluation. (b) Fab: A format consisting of VH (white), the antibody H chain constant region (CH1: checkerboard pattern), VL (white), and the antibody L chain constant region (horizontal line). In this embodiment, anti-HLA-A2 / NY-ESO Fab, etc., were used for evaluation. (c) taFv: A format in which two types of scFv (white and upper right diagonal line) are linked together with a linker. In this embodiment, taFv containing anti-HLA-A2 / NY-ESO scFv and anti-CD3 scFv were used for evaluation. (d) taFv-heterodimer Fc type: This format involves adding an Fc molecule (upper left diagonal) containing a heterodimer-forming mutation to the C-terminus of taFv (also called the first polypeptide), which hetero-associates with another Fc molecule (blacked out: also called the second polypeptide). In this example, taFv-heterodimer Fc containing anti-HLA-A2 / NY-ESO scFv and anti-CD3 scFv was used for evaluation. (e) taFv-Fab-heterodimer Fc type: This format involves adding Fab to the above taFv-heterodimer Fc type. In this example, taFv-Fab-heterodimer Fc using taFv containing anti-HLA-A2 / NY-ESO scFv and anti-CD3 scFv, and HLA-A2 / NY-ESO Fab was used for evaluation. (f) The first polypeptide common to both taFv-heterodimer Fc type and taFv-Fab-heterodimer Fc type is shown. The first polypeptide comprises, in order from the N-terminus to the C-terminus, an scFv that specifically binds to human HLA / NY-ESO, an scFv that specifically binds to CD3, and an Fc region (i). (g) Shows the second polypeptide of the taFv-heterodimer Fc type. The second polypeptide comprises a hinge region and an Fc region (ii). (h) Shows the second polypeptide of the taFv-Fab-heterodimer Fc type. The second polypeptide comprises an immunoglobulin heavy chain containing a hinge region and an Fc region (ii). (i) Shows the third polypeptide of the taFv-Fab-heterodimer Fc type.The third polypeptide comprises an immunoglobulin light chain. [Figure 4A] Figure 4A 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, exhibit cytotoxic activity against endogenous human NY-ESO-expressing U266B1 cells in the presence of human PBMCs. Error bars in the figure indicate the standard deviation (n=3). [Figure 4B] Figure 4B shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules, NYF-0016 and NYF-0058, exhibit cytotoxic activity against endogenous human NY-ESO-expressing U266B1 cells in the presence of human PBMCs. Error bars in the figure indicate the standard deviation (n=3). [Figure 4C] Figure 4C shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules, NYF-0023, NYF-0045, NYF-0048, NYF-0060, and NYF-0061, exhibit cytotoxic activity against endogenous human NY-ESO-expressing U266B1 cells in the presence of human PBMCs. The error bars in the figure represent the standard deviation (n=3). [Figure 4D] Figure 4D 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, exhibit cytotoxic activity against endogenous human NY-ESO-expressing NCI-H1703 cells in the presence of human PBMCs. Error bars in the figure indicate the standard deviation (n=3). [Figure 4E] Figure 4E shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules, NYF-0016 and NYF-0058, exhibit cytotoxic activity against endogenous human NY-ESO-expressing NCI-H1703 cells in the presence of human PBMCs. Error bars in the figure indicate the standard deviation (n=3). [Figure 4F]Figure 4F shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules, NYF-0023, NYF-0045, NYF-0048, NYF-0060, and NYF-0061, exhibit cytotoxic activity against endogenous human NY-ESO-expressing NCI-H1703 cells in the presence of human PBMCs. The error bars in the figure represent the standard deviation (n=3). [Figure 4G] Figure 4G 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, do not exhibit cytotoxic activity against endogenous human NY-ESO-nonexpressing AGS cells in the presence of human PBMCs. The error bars in the figure represent the standard deviation (n=3). [Figure 4H] Figure 4H shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules, NYF-0016 and NYF-0058, do not exhibit cytotoxic activity against endogenous human NY-ESO-nonexpressing AGS cells in the presence of human PBMCs. The error bars in the figure indicate the standard deviation (n=3). [Figure 4I] Figure 4I 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, do not exhibit cytotoxic activity against endogenous human NY-ESO-nonexpressing AGS cells in the presence of human PBMCs. The error bars in the figure indicate the standard deviation (n=3). [Figure 4J] Figure 4J 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, do not exhibit cytotoxic activity against endogenous human NY-ESO-nonexpressing CFPAC-1 cells in the presence of human PBMCs. The error bars in the figure represent the standard deviation (n=3). [Figure 4K]Figure 4K shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules, NYF-0016 and NYF-0058, do not exhibit cytotoxic activity against endogenous human NY-ESO-nonexpressing CFPAC-1 cells in the presence of human PBMCs. The error bars in the figure indicate the standard deviation (n=3). [Figure 4L] Figure 4L 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, do not exhibit cytotoxic activity against endogenous human NY-ESO-nonexpressing CFPAC-1 cells in the presence of human PBMCs. The error bars in the figure represent the standard deviation (n=3). [Figure 5A] Figure 5A shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules, NYF-0016, NYF-0019, NYF-0044, and NYF-0047, exhibit antitumor activity in a human PBMC transfer model. The error bars in the figure indicate the standard error (n=4 for NYF-0044 Day 32 only, n=5 for the others). [Figure 5B] Figure 5B 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, exhibit antitumor activity in a human PBMC transfer model. The error bars in the figure indicate the standard error (n=5, n=6 only for the vehicle control group). [Figure 5C] Figure 5C 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, exhibit antitumor activity in a human PBMC transfer model. The error bars in the figure indicate the standard error (n=5). [Figure 6A]Figure 6A shows the antibody formats used in this embodiment. (a) Hybrid type: This format involves adding a heterodimer-forming mutation (shaded and blacked out) to the C-terminus of both Fab and scFv, resulting in the association of the two Fc molecules. In this embodiment, a Hybrid type containing anti-HLA-A2 / NY-ESO Fab and anti-CD3 scFv was used for evaluation. (b) Dual type: This format involves adding a heterodimer-forming mutation (shaded and blacked out in the upper left) to the C-terminus of two different types of scFv, resulting in the association of the two Fc heterodimers. In this embodiment, a Dual type containing anti-HLA-A2 / NY-ESO scFv and anti-CD3 scFv was used for evaluation. (c) scFv-Fab-heterodimerFc type: This format involves adding a heterodimer-forming mutation (shaded and blacked out in the upper left) to the C-terminus of scFv and Fab linked by a linker, resulting in the association of the two Fc molecules with another Fc molecule (blacked out). In this example, the scFv-Fab heterodimer Fc type containing anti-CD3 scFv (upper right diagonal) and anti-HLA-A2 / NY-ESO Fab was used for evaluation. Additionally, the scFv-Fab heterodimer Fc type containing anti-HLA-A2 / NY-ESO scFv (upper right diagonal) and anti-CD3 Fab was also used for evaluation. [Figure 6B]Figure 6B shows the antibody format shown in this embodiment. (a) taFv-heterodimer Fc type: Same as Figure 3(d). This format has an Fc (upper left diagonal line) with a mutation that forms a heterodimer added to the C-terminus of taFv, and heteroassociates with another Fc (blacked out). In this embodiment, the taFv-heterodimer Fc type consisting of anti-HLA-A2 / NY-ESO scFv and anti-CD3 scFv was used for evaluation. (b) taFv(inversed)-heterodimer Fc type: The order of the two scFv in the above taFv-heterodimer Fc type is reversed. In this embodiment, the taFv(inversed)-heterodimer Fc type consisting of anti-CD3 scFv and anti-HLA-A2 / NY-ESO scFv was used for evaluation. (c) The first polypeptide of the taFv(inversed)-heterodimer Fc type is shown. The first polypeptide comprises, in order from the N-terminus to the C-terminus, an scFv that specifically binds to CD3, an scFv that specifically binds to human HLA / NY-ESO, and an Fc region (i). (d) The second polypeptide is a taFv(inversed)-heterodimer Fc type. The second polypeptide comprises a hinge region and an Fc region (ii). [Figure 7A] Figure 7A shows the cytotoxic activity of various anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules—Hybrid (NYG-3143), Dual (NYG-2143), and taFv-heterodimer Fc (NYF-0011)—against endogenous human NY-ESO-expressing U266B1 cells in the presence of human PBMCs. Error bars in the figure indicate the standard deviation (n=3). [Figure 7B] Figure 7B shows the cytotoxic activity of various anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules—scFv-Fab-heterodimer Fc (NYF-0003), taFv-heterodimer Fc (NYF-0010), and taFv(inversed)-heterodimer Fc (NYF-0004)—against endogenous human NY-ESO-expressing U266B1 cells in the presence of human PBMCs. Error bars in the figure indicate the standard deviation (n=3). [Figure 8]Amino acid sequence of the peptide in NY-ESO (SEQ ID NO: 1) [Figure 9] Amino acid sequence of peptide in MAGEC-1 (SEQ ID NO: 2) [Figure 10] Primer 1 for scFv sequencing analysis (SEQ ID NO: 3) [Figure 11] Primer 2 for scFv sequencing analysis (SEQ ID NO: 4) [Figure 12] Nucleotide sequence of the heavy chain variable region of NYA-0001 (SEQ ID NO: 5) [Figure 13] Amino acid sequence of the heavy chain variable region of NYA-0001 (SEQ ID NO: 6) [Figure 14] Nucleotide sequence of the light chain variable region of NYA-0001 (SEQ ID NO: 7) [Figure 15] Amino acid sequence of the light chain variable region of NYA-0001 (SEQ ID NO: 8) [Figure 16] Nucleotide sequence of the heavy chain variable region of NYA-0060 (SEQ ID NO: 9) [Figure 17] Amino acid sequence of the heavy chain variable region of NYA-0060 (SEQ ID NO: 10) [Figure 18] Nucleotide sequence of the light chain variable region of NYA-0060 (SEQ ID NO: 11) [Figure 19] Amino acid sequence of the light chain variable region of NYA-0060 (SEQ ID NO: 12) [Figure 20] Nucleotide sequence of the heavy chain variable region of NYA-0068 (SEQ ID NO: 13) [Figure 21] Amino acid sequence of the heavy chain variable region of NYA-0068 (SEQ ID NO: 14) [Figure 22] Nucleotide sequence of the light chain variable region of NYA-0068 (SEQ ID NO: 15) [Figure 23] Amino acid sequence of the light chain variable region of NYA-0068 (SEQ ID NO: 16) [Figure 24] Nucleotide sequence of the heavy chain variable region of NYA-0082 (SEQ ID NO: 17) [Figure 25] Amino acid sequence of the heavy chain variable region of NYA-0082 (SEQ ID NO: 18) [Figure 26]Nucleotide sequence of the light chain variable region of NYA-0082 (SEQ ID NO: 19) [Figure 27] Amino acid sequence of the light chain variable region of NYA-0082 (SEQ ID NO: 20) [Figure 28] Nucleotide sequence of the NYA-1163 tag adduct (SEQ ID NO: 21) [Figure 29] Nucleotide sequence of the NYA-2023 tag adduct (SEQ ID NO: 22) [Figure 30] Nucleotide sequence of the NYA-2027 tag adduct (SEQ ID NO: 23) [Figure 31] Nucleotide sequence of the NYA-1143 tag adduct (SEQ ID NO: 24) [Figure 32] Nucleotide sequence of the NYA-2143 tag adduct (SEQ ID NO: 25) [Figure 33] The amino acid sequence of the NYA-1163 tag adduct (SEQ ID NO: 26) shows that NYA-1163 consists of amino acids 21-266. [Figure 34] The amino acid sequence of the NYA-2023 tag adduct (SEQ ID NO: 27). NYA-2023 consists of amino acids 21-266. [Figure 35] The amino acid sequence of the NYA-2027 tag adduct (SEQ ID NO: 28). NYA-2027 consists of amino acids 21-266. [Figure 36] The amino acid sequence of the NYA-1143 tag adduct (SEQ ID NO: 29), NYA-1143 consists of amino acids 21-266. [Figure 37] The amino acid sequence of the NYA-2143 tag adduct (SEQ ID NO: 30), NYA-2143 consists of amino acids 21-266. [Figure 38] Nucleotide sequence of the NYA-1154 tag adduct (SEQ ID NO: 31) [Figure 39] The amino acid sequence of the NYA-1154 tag adduct (SEQ ID NO: 32), NYA-1154 consists of amino acids 21-266. [Figure 40] Amino acid sequence of the truncated form of HLA-A*0201 (GenBank: ASA47534.1) (SEQ ID NO: 33) [Figure 41]Amino acid sequence of β2-microglobin (SEQ ID NO: 34) [Figure 42] Nucleotide sequence of the NYA-2035 tag adduct (SEQ ID NO: 35) [Figure 43] The amino acid sequence of the NYA-2035 tag adduct (SEQ ID NO: 36) shows that NYA-2035 contains amino acids 21-266. [Figure 44] Amino acid sequence of NYA-1143-VH01 (SEQ ID NO: 37) [Figure 45] Amino acid sequence of NYA-1143-VH02 (SEQ ID NO: 38) [Figure 46] Amino acid sequence of NYA-1143-VH03 (SEQ ID NO: 39) [Figure 47] Amino acid sequence of NYA-1143-VL01 (SEQ ID NO: 40) [Figure 48] Nucleotide sequence of the NYA-2044 tag adduct (SEQ ID NO: 41) [Figure 49] Nucleotide sequence of the NYA-2045 tag adduct (SEQ ID NO: 42) [Figure 50] Nucleotide sequence of the NYA-2047 tag adduct (SEQ ID NO: 43) [Figure 51] Nucleotide sequence of the NYA-2048 tag adduct (SEQ ID NO: 44) [Figure 52] Nucleotide sequence of NYA-2060 tag adduct (SEQ ID NO: 45) [Figure 53] Nucleotide sequence of the NYA-2061 tag adduct (SEQ ID NO: 46) [Figure 54] The amino acid sequence of the NYA-2044 tag adduct (SEQ ID NO: 47), NYA-2044 consists of amino acids 21-266. [Figure 55] The amino acid sequence of the NYA-2045 tag adduct (SEQ ID NO: 48), NYA-2045 consists of amino acids 21-266. [Figure 56] Amino acid sequence of NYA-0082 (SEQ ID NO: 49) [Figure 57] The amino acid sequence of the NYA-2047 tag adduct (SEQ ID NO: 50), NYA-2047 consists of amino acids 21-266. [Figure 58] The amino acid sequence of the NYA-2048 tag adduct (SEQ ID NO: 51). NYA-2048 consists of amino acids 21-266. [Figure 59] The amino acid sequence of the NYA-2060 tag adduct (SEQ ID NO: 52). NYA-2060 consists of amino acids 21-266. [Figure 60] The amino acid sequence of the NYA-2061 tag adduct (SEQ ID NO: 53), NYA-2061 consists of amino acids 21-266. [Figure 61] Amino acid sequences of CDRH1-CDRH3 in the heavy chain and CDRL1-L3 in the light chain of NYA-0001 (SEQ ID NOs. 54-57 and 59) [Figure 62] Amino acid sequence of CDRL1 in NYA-2023 (SEQ ID NO: 60) [Figure 63] Amino acid sequence of CDRL3 in NYA-2027 (SEQ ID NO: 61) [Figure 64] Amino acid sequences of CDRH3 and CDRL3 of NYA-1154 (SEQ ID NOs. 62 and 63) [Figure 65] Amino acid sequence of CDRL1 in NYA-0035 (SEQ ID NO: 64) [Figure 66] Nucleotide sequence of the NYC-0003 tag adduct (SEQ ID NO: 65) [Figure 67] Nucleotide sequence of the NYC-0004 tag adduct (SEQ ID NO: 66) [Figure 68] The amino acid sequence of the NYC-0003 tag adduct (SEQ ID NO: 67), where NYC-0003 consists of amino acids 21-263. [Figure 69] The amino acid sequence of the NYC-0004 tag adduct (SEQ ID NO: 68), where NYC-0004 corresponds to amino acids 21-263. [Figure 70] Nucleotide sequence of the NYA-0001 tag adduct (SEQ ID NO: 69) [Figure 71] The amino acid sequence of the NYA-0001 tag adduct (SEQ ID NO: 70), NYA-0001 consists of amino acids 21-266. [Figure 72] HC1 nucleotide sequence (SEQ ID NO: 71) [Figure 73]Nucleotide sequence of NYF-0016-HC2 (SEQ ID NO: 72) [Figure 74] Nucleotide sequence of NYF-0019-HC2 (SEQ ID NO: 73) [Figure 75] Nucleotide sequence of NYF-0022-HC2 (SEQ ID NO: 74) [Figure 76] Nucleotide sequence of NYF-0023-HC2 (SEQ ID NO: 75) [Figure 77] Nucleotide sequence of NYF-0027-HC2 (SEQ ID NO: 76) [Figure 78] Nucleotide sequence of NYF-0035-HC2 (SEQ ID NO: 77) [Figure 79] Nucleotide sequence of NYF-0044-HC2 (SEQ ID NO: 78) [Figure 80] Nucleotide sequence of NYF-0045-HC2 (SEQ ID NO: 79) [Figure 81] Nucleotide sequence of NYF-0047-HC2 (SEQ ID NO: 80) [Figure 82] Nucleotide sequence of NYF-0048-HC2 (SEQ ID NO: 81) [Figure 83] Nucleotide sequence of NYF-0060-HC2 (SEQ ID NO: 82) [Figure 84] Nucleotide sequence of NYF-0061-HC2 (SEQ ID NO: 83) [Figure 85] Amino acid sequence of HC1 (SEQ ID NO: 84) [Figure 86] The amino acid sequence of NYF-0016-HC2 (SEQ ID NO: 85), NYA-1143 is amino acid numbers 21-266, and C3E-7085 is amino acid numbers 272-511. [Figure 87] The amino acid sequence of NYF-0019-HC2 (SEQ ID NO: 86), NYA-2143 is amino acid numbers 21-266, and C3E-7085 is amino acid numbers 272-511. [Figure 88] The amino acid sequence of NYF-0022-HC2 (SEQ ID NO: 87), NYA-1163 is amino acid numbers 21-266, and C3E-7085 is amino acid numbers 272-511. [Figure 89]The amino acid sequence of NYF-0023-HC2 (SEQ ID NO: 88), NYA-2023 is amino acid numbers 21-266, and C3E-7085 is amino acid numbers 272-511. [Figure 90] The amino acid sequence of NYF-0027-HC2 (SEQ ID NO: 89), NYA-2027 is amino acid numbers 21-266, and C3E-7085 is amino acid numbers 272-511. [Figure 91] The amino acid sequence of NYF-0035-HC2 (SEQ ID NO: 90), NYA-2035 is amino acid numbers 21-266, and C3E-7085 is amino acid numbers 272-511. [Figure 92] The amino acid sequence of NYF-0044-HC2 (SEQ ID NO: 91), NYA-2044 is amino acid numbers 21-266, and C3E-7085 is amino acid numbers 272-511. [Figure 93] The amino acid sequence of NYF-0045-HC2 (SEQ ID NO: 92), NYA-2045 is amino acid numbers 21-266, and C3E-7085 is amino acid numbers 272-511. [Figure 94] The amino acid sequence of NYF-0047-HC2 (SEQ ID NO: 93), NYA-2047 is amino acid numbers 21-266, and C3E-7085 is amino acid numbers 272-511. [Figure 95] The amino acid sequence of NYF-0048-HC2 (SEQ ID NO: 94), NYA-2048 is amino acid numbers 21-266, and C3E-7085 is amino acid numbers 272-511. [Figure 96] The amino acid sequence of NYF-0060-HC2 (SEQ ID NO: 95), NYA-2060 is amino acid numbers 21-266, and C3E-7085 is amino acid numbers 272-511. [Figure 97] The amino acid sequence of NYF-0061-HC2 (SEQ ID NO: 96), NYA-2061 is amino acid numbers 21-266, and C3E-7085 is amino acid numbers 272-511. [Figure 98] Nucleotide sequence of NYA-0001-Fab-HC1-k delete (SEQ ID NO: 97) [Figure 99] Nucleotide sequence of NYA-0001-LC (SEQ ID NO: 98) [Figure 100] The amino acid sequence of NYA-0001-Fab-HC1-k delete (SEQ ID NO: 99) shows that the heavy chain variable region of NYA-0001 consists of amino acids 20-139. [Figure 101] The amino acid sequence of NYA-0001-LC (SEQ ID NO: 100) shows that the light chain variable region of NYA-0001 consists of amino acids 21-131. [Figure 102] Nucleotide sequence of NYA-1143-Fab-HC1-k delete (SEQ ID NO: 101) [Figure 103] Nucleotide sequence of NYA-1143-LC (SEQ ID NO: 102) [Figure 104] Nucleotide sequence of C3E-7085-HC2-k deleteC (SEQ ID NO: 103) [Figure 105] The amino acid sequence of NYA-1143-Fab-HC1-k delete (SEQ ID NO: 104) shows that the heavy chain variable region of NYA-1143 is amino acid numbers 20-139. [Figure 106] The amino acid sequence of NYA-1143-LC (SEQ ID NO: 105) shows that the light chain variable region of NYA-1143 consists of amino acids 21-131. [Figure 107] The amino acid sequence of C3E-7085-HC2-k delete (SEQ ID NO: 106), where C3E-7085 is amino acid number 21-260. [Figure 108] Nucleotide sequence of NYA-1143-HC1-k delete (SEQ ID NO: 107) [Figure 109] The amino acid sequence of NYA-1143-HC1-k delete (SEQ ID NO: 108), NYA-1143 is amino acid number 21-266. [Figure 110] Nucleotide sequence of C3E-7085-NYA-1154-Fab-HC2-k delete (SEQ ID NO: 109) [Figure 111] Nucleotide sequence of NYA-1154-LC (SEQ ID NO: 110) [Figure 112] Nucleotide sequence of OAA-HC1-k delete (SEQ ID NO: 111) [Figure 113]The amino acid sequence of C3E-7085-NYA-1154-Fab-HC2-k delete (SEQ ID NO: 112) is shown. C3E-7085 consists of amino acids 21-260, and the heavy chain variable region of NYA-1154 consists of amino acids 266-285. [Figure 114] The amino acid sequence of NYA-1154-LC (SEQ ID NO: 113) shows that the light chain variable region of NYA-1154 consists of amino acids 21-131. [Figure 115] The amino acid sequence of OAA-HC1-k delete (SEQ ID NO: 114) [Figure 116] Nucleotide sequence of NYF-0010-HC2-k delete (SEQ ID NO: 115) [Figure 117] Nucleotide sequence of NYF-0004-HC2-k delete (SEQ ID NO: 116) [Figure 118] Nucleotide sequence of NYF-0011-HC2-k delete (SEQ ID NO: 117) [Figure 119] The amino acid sequence of NYF-0010-HC2-k delete (SEQ ID NO: 18), NYA-1154 is amino acid numbers 21-266, and C3E-7085 is amino acid numbers 272-511. [Figure 120] The amino acid sequence of NYF-0004-HC2-k delete (SEQ ID NO: 119) C3E-7085 has amino acid numbers 21-260, and NYA-1154 has amino acid numbers 272-511. [Figure 121] The amino acid sequence of NYF-0011-HC2-k delete (SEQ ID NO: 120), NYA-1143 is amino acid numbers 21-266, and C3E-7085 is amino acid numbers 272-511. [Figure 122] Amino acid sequence of point mutation NY-ESO peptide 1F (SEQ ID NO: 121) [Figure 123] Amino acid sequence of point mutation NY-ESO peptide 2M (SEQ ID NO: 122) [Figure 124] Amino acid sequence of point mutation NY-ESO peptide 3A (SEQ ID NO: 123) [Figure 125] Amino acid sequence of point mutation NY-ESO peptide 4A (SEQ ID NO: 124) [Figure 126] Amino acid sequence of point mutation NY-ESO peptide 5A (SEQ ID NO: 125) [Figure 127] Amino acid sequence of point mutation NY-ESO peptide 6L (SEQ ID NO: 126) [Figure 128] Amino acid sequence of point mutation NY-ESO peptide 7F (SEQ ID NO: 127) [Figure 129] Amino acid sequence of point mutation NY-ESO peptide 8A (SEQ ID NO: 128) [Figure 130] Amino acid sequence of point mutation NY-ESO peptide 9A (SEQ ID NO: 129) [Figure 131] Amino acid sequence of gp100 peptide (SEQ ID NO: 130) [Figure 132] Amino acid sequence of homologous peptide DOLPP1 (SEQ ID NO: 131) [Figure 133] Amino acid sequence of homologous peptide IL20RB (SEQ ID NO: 132) [Figure 134] Amino acid sequence of homologous peptide PRKD2 (SEQ ID NO: 133) [Figure 135] Amino acid sequence of homologous peptide CD163 (SEQ ID NO: 134) [Figure 136] Amino acid sequence of homologous peptide P2RY8 (SEQ ID NO: 135) [Figure 137] Amino acid sequence of C3E-7034 (SEQ ID NO: 136) [Figure 138] Amino acid sequence of C3E-7036 (SEQ ID NO: 137) [Figure 139] Amino acid sequence of C3E-7085 (SEQ ID NO: 138) [Figure 140] Amino acid sequence of C3E-7088 (SEQ ID NO: 139) [Figure 141] Amino acid sequence of C3E-7093 (SEQ ID NO: 140) [Figure 142] Amino acid sequences of CDRH1-CDRH3 of the heavy chain and CDRL1-CDRL3 of the light chain of C3E-7085 (Sequence IDs 141-144 and 146) [Figure 143] Amino acid sequence of C3E-7078 (SEQ ID NO: 147) [Figure 144] Nucleotide sequence of NYF-0014-HC2 (SEQ ID NO: 148) [Figure 145] The amino acid sequence of NYF-0014-HC2 (SEQ ID NO: 149), NYA-0001 is amino acid numbers 21-266, and C3E-7085 is amino acid numbers 272-511. [Figure 146] The amino acid sequence of NYF-0082-HC2 (SEQ ID NO: 150), NYA-0082 is amino acid numbers 21-266, and C3E-7085 is amino acid numbers 272-511. [Figure 147] Amino acid sequence of human CD3ε (SEQ ID NO: 151) [Figure 148] Full-length nucleotide sequence of NYZ-0038-HC2 (SEQ ID NO: 152) [Figure 149] Full-length nucleotide sequence of NYZ-0082-HC2 (SEQ ID NO: 153) [Figure 150] Full-length nucleotide sequence of NYZ-0083-HC2 (SEQ ID NO: 154) [Figure 151] The full amino acid sequence of NYZ-0038-HC2 (SEQ ID NO: 155), NYA-2061 is amino acid numbers 21-266, and C3E-7096 is amino acid numbers 272-511. [Figure 152] The full amino acid sequence of NYZ-0082-HC2 (SEQ ID NO: 156), NYA-3061 is amino acid numbers 21-271, and C3E-7096 is amino acid numbers 277-516. [Figure 153] The full amino acid sequence of NYZ-0083-HC2 (SEQ ID NO: 157), NYA-3061 is amino acid numbers 21-271, and C3E-7097 is amino acid numbers 277-516. [Figure 154] Full-length nucleotide sequence of NYZ-1010-HC2 (SEQ ID NO: 158) [Figure 155] Full-length nucleotide sequence of C3E-7085-LC (SEQ ID NO: 159) [Figure 156] The full amino acid sequence of NYZ-1010-HC2 (SEQ ID NO: 160) is shown, NYA-3061 consists of amino acids 21-271, and the C3E-7085 heavy chain variable region consists of amino acids 277-394. [Figure 157] Full-length amino acid sequence of C3E-7085-LC (SEQ ID NO: 161) [Figure 158A] Figure 158A is a table showing the standardized gMFI for various point mutation peptide-added CD3e knockout T2 cells for the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules NYZ-0038, 0082, 0083, and 1010. *Underlined text indicates that the gMFI for each Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule is less than half, and *bold text indicates less than one-quarter, compared to the standardized gMFI for NY-ESO peptide-added CD3e knockout T2 cells. [Figure 158B] Figure 158B is a table showing the standardized gMFI for various homologous peptide-added CD3e knockout T2 cells for the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules NYZ-0038, 0082, 0083, and 1010. *Underlined values ​​indicate that each Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule has a higher standardized gMFI compared to DMSO-added CD3e knockout T2 cells. [Figure 159A] Figure 159A shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules, NYZ-0038, 0082, and 0083, exhibit cytotoxic activity against endogenous human NY-ESO-expressing U266B1 cells in the presence of human PBMCs. Error bars in the figure indicate the standard deviation (n=3). [Figure 159B] Figure 159B shows that NYZ-1010, an Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, exhibits cytotoxic activity against endogenous human NY-ESO-expressing U266B1 cells in the presence of human PBMCs. Error bars in the figure indicate the standard deviation (n=3). [Figure 159C] Figure 159C shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules, NYZ-0082 and 1010, exhibit cytotoxic activity against endogenous human NY-ESO-expressing NCI-H1703 cells in the presence of human PBMCs. Error bars in the figure indicate the standard deviation (n=3). [Figure 159D]Figure 159D shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules, NYZ-0038 and 0083, exhibit cytotoxic activity against endogenous human NY-ESO-expressing NCI-H1703 cells in the presence of human PBMCs. Error bars in the figure indicate the standard deviation (n=3). [Figure 159E] Figure 159E shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules, NYZ-0038, 0082, and 0083, do not exhibit cytotoxic activity against endogenous human NY-ESO-nonexpressing AGS cells in the presence of human PBMCs. The error bars in the figure indicate the standard deviation (n=3). [Figure 159F] Figure 159F shows that NYZ-1010, an Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, does not exhibit cytotoxic activity against endogenous human NY-ESO-nonexpressing AGS cells in the presence of human PBMCs. The error bars in the figure indicate the standard deviation (n=3). [Figure 159G] Figure 159G shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules, NYZ-0082 and 1010, do not exhibit cytotoxic activity against endogenous human NY-ESO-nonexpressing CFPAC-1 cells in the presence of human PBMCs. The error bars in the figure indicate the standard deviation (n=3). [Figure 159H] Figure 159H shows that the Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules, NYZ-0038 and 0083, do not exhibit cytotoxic activity against endogenous human NY-ESO-nonexpressing CFPAC-1 cells in the presence of human PBMCs. The error bars in the figure indicate the standard deviation (n=3). [Figure 160A] Figure 160A shows that NYZ-0038, an Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, exhibits antitumor activity in a human PBMC transfer model. Error bars in the figure indicate the standard error (n=5). [Figure 160B]Figure 160B shows that NYZ-0082, an Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, exhibits antitumor activity in a human PBMC transfer model. Error bars in the figure indicate the standard error (n=5). [Figure 160C] Figure 160C shows that NYZ-0083, an Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, exhibits antitumor activity in a human PBMC transfer model. Error bars in the figure indicate the standard error (n=5). [Figure 160D] Figure 160D shows that NYZ-1010, an Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule, exhibits antitumor activity in a human PBMC transfer model. Error bars in the figure indicate the standard error (n=5). [Figure 161] Amino acid sequence of the peptide linker (SEQ ID NO: 162) [Figure 162] This figure shows the percentage of polymer content when various Fc-added anti-HLA / NY-ESO-anti-CD3 bispecific molecules are treated with acid. [Figure 163] This figure shows the results of the solution stability evaluation of the anti-HLA / NY-ESO scFv heterodimer Fc. [Figure 164] Full-length nucleotide sequence of NYA-3061 (SEQ ID NO: 163) [Figure 165] Full-length amino acid sequence of NYA-3061 (SEQ ID NO: 164) [Figure 166] Full-length nucleotide sequence of NYC-0005 (SEQ ID NO: 165) [Figure 167] Full-length amino acid sequence of NYC-0005 (SEQ ID NO: 166) [Figure 168] Full-length nucleotide sequence of NYC-0006 (SEQ ID NO: 167) [Figure 169] Full-length amino acid sequence of NYC-0006 (SEQ ID NO: 168) [Figure 170] Full-length nucleotide sequence of NYC-0007 (SEQ ID NO: 169) [Figure 171] Full-length amino acid sequence of NYC-0007 (SEQ ID NO: 170) [Figure 172] Full-length nucleotide sequence of NYC-0008 (SEQ ID NO: 171) [Figure 173] Full-length amino acid sequence of NYC-0008 (SEQ ID NO: 172) [Figure 174] Full-length nucleotide sequence of NYC-0009 (SEQ ID NO: 173) [Figure 175] Full-length amino acid sequence of NYC-0009 (SEQ ID NO: 174) [Figure 176] Full-length nucleotide sequence of NYC-0010 (SEQ ID NO: 175) [Figure 177] Full-length amino acid sequence of NYC-0010 (SEQ ID NO: 176) [Figure 178] Full-length HC-h nucleotide sequence (SEQ ID NO: 177) [Figure 179] Full-length amino acid sequence of HC-h (SEQ ID NO: 178) [Figure 180] NYD-2047-HC-k full-length nucleotide sequence (SEQ ID NO: 179) [Figure 181] Full-length amino acid sequence of NYD-2047-HC-k (SEQ ID NO: 180) [Figure 182] NYD-2061-HC-k full-length nucleotide sequence (SEQ ID NO: 181) [Figure 183] Full-length amino acid sequence of NYD-2061-HC-k (SEQ ID NO: 182) [Figure 184] Full-length nucleotide sequence of NYD-3061-HC-k (SEQ ID NO: 183) [Figure 185] Full-length amino acid sequence of NYD-3061-HC-k (SEQ ID NO: 184) [Figure 186] Full-length nucleotide sequence of NYC-0011-HC-k (SEQ ID NO: 185) [Figure 187] Full-length amino acid sequence of NYC-0011-HC-k (SEQ ID NO: 186) [Figure 188] Full-length nucleotide sequence of NYC-0012-HC-k (SEQ ID NO: 187) [Figure 189] Full-length amino acid sequence of NYC-0012-HC-k (SEQ ID NO: 188) [Figure 190] Full-length nucleotide sequence of NYC-0013-HC-k (SEQ ID NO: 189) [Figure 191] Full-length amino acid sequence of NYC-0013-HC-k (SEQ ID NO: 190) [Figure 192] NYC-0014-HC-k full-length nucleotide sequence (SEQ ID NO: 191) [Figure 193] Full-length amino acid sequence of NYC-0014-HC-k (SEQ ID NO: 192) [Figure 194] Full-length nucleotide sequence of NYC-0015-HC-k (SEQ ID NO: 193) [Figure 195] Full-length amino acid sequence of NYC-0015-HC-k (SEQ ID NO: 194) [Figure 196] Full-length nucleotide sequence of NYC-0016-HC-k (SEQ ID NO: 195) [Figure 197] Full-length amino acid sequence of NYC-0016-HC-k (SEQ ID NO: 196) [Figure 198] The full amino acid sequence of NYZ-1007-HC2 (SEQ ID NO: 197) is shown, NYA-2061 consists of amino acids 21-266, and the C3E-7085 heavy chain variable region consists of amino acids 272-389. [Figure 199] The full amino acid sequence of NYZ-1017-HC2 (SEQ ID NO: 198) is shown, NYA-2047 consists of amino acids 21-266, and the C3E-7085 heavy chain variable region consists of amino acids 277-389. [Modes for carrying out the invention]

[0032] The present invention will be described in detail below. 1.Definition In this invention, "gene" means a nucleotide chain or its complementary chain containing a base sequence that codes for the amino acids of a protein. For example, polynucleotides, oligonucleotides, DNA, mRNA, cDNA, cRNA, etc., which are nucleotide chains or their complementary chains containing a base sequence that codes for the amino acids of a protein, are included in the meaning of "gene." Such genes are single-stranded, double-stranded, or triple-stranded or more nucleotides. The meaning of "gene" is also included in associations of DNA and RNA chains, those in which ribonucleotides (RNA) and deoxyribonucleotides (DNA) are mixed on a single nucleotide chain, and double-stranded or triple-stranded or more nucleotides containing such nucleotide chains. In this invention, base sequence and nucleotide sequence are synonymous.

[0033] In this invention, "polynucleotide," "nucleotide chain," "nucleic acid," and "nucleic acid molecule" are synonymous, and for example, DNA, RNA, probes, oligonucleotides, primers, etc., are also included in the meaning of "polynucleotide." Such polynucleotides are single-stranded, double-stranded, or polynucleotides consisting of three or more strands, and the meaning of "polynucleotide" is also included in the meaning of "polynucleotide," as well as aggregates of DNA and RNA strands, polynucleotides on a single polynucleotide chain in which ribonucleotides (RNA) and deoxyribonucleotides (DNA) are mixed, and aggregates of double-stranded or triple-stranded polynucleotides containing such polynucleotide chains.

[0034] In this invention, "polypeptide," "peptide," and "protein" are synonymous. In this invention, the term "antigen" may be used to mean "immunogen." In this invention, "cells" include various cells derived from individual animals, subcultured cells, primary cultured cells, cell lines, recombinant cells, and microorganisms.

[0035] In this invention, "antibody" is synonymous with "immunoglobulin." However, in the case of the anti-HLA / NY-ESO antibody of this invention, "antibody" is used to mean an immunoglobulin having a constant region and a variable region. The antibody is not particularly limited to being naturally occurring or an immunoglobulin produced by partial or complete synthesis. The anti-HLA / NY-ESO antibody of this invention is included in the "molecule" described below.

[0036] In this invention, "NY-ESO peptide" refers to the peptide (SLLMWITQC: Sequence ID No. 1) consisting of NY-ESO-1 and the 9 amino acids from position 157 to 165 of LAGE-1.

[0037] In this invention, "HLA-A2 / NY-ESO" refers to a complex of NY-ESO peptide and Histocompatibility Leukocyte Antigen-A2 (HLA-A2), and is also written as "HLA / NY-ESO".

[0038] In this invention, "anti-HLA-A2 / NY-ESO antibody" means an antibody that binds to HLA-A2 / NY-ESO, in other words, an antibody that recognizes HLA-A2 / NY-ESO. Similarly, "anti-HLA-A2 / NY-ESO scFv" means 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 written as "anti-HLA / NY-ESO antibody" and "anti-HLA / NY-ESO scFv," respectively.

[0039] The basic structure of a four-chain antibody consists of two identical light chains (L chains) and two identical heavy chains (H chains). The light chains are bound to the heavy chains by a single covalent disulfide bond. The two heavy chains are linked to each other by one or more disulfide bonds, depending on the isotype of the heavy chains. Each light and heavy chain has intrachain disulfide bonds at regular intervals. The heavy and light chains have a constant region with very similar amino acid sequences and a variable region with low similarity in amino acid sequences. The light chain has a variable region (VL) at its amino terminus that is followed by a constant region (CL). The heavy chain has a variable region (VH) at its amino terminus that is followed by three constant regions (CH1 / CH2 / CH3). VL and VH are paired, and CL is aligned with the first constant region (CH1) of the heavy chain. VL and VH are paired to form a single antigen-binding site.

[0040] The constant region of the antibody of the present invention is not particularly limited, but for the antibody of the present invention to treat or prevent human diseases, a human antibody is preferably used. Examples of heavy chain constant regions of human antibodies include Cγ1, Cγ2, Cγ3, Cγ4, Cμ, Cδ, Cα1, Cα2, Cε, etc. Examples of light chain constant regions of human antibodies include Cκ, Cλ, etc.

[0041] Fab consists of a heavy chain VH followed by CH1, and a light chain VL followed by CL. VH and VL contain a complementarity-determining region (CDR).

[0042] Fc (also referred to as the Fc region) is the carboxyl-terminal region of the constant region of the heavy chain, includes CH2 and CH3, and is a dimer. The Fc of the present invention may be a natural sequence Fc or a mutant Fc (referred to as "mutant Fc") in which mutations are made to the natural sequence. In the multispecific molecules and bispecific molecules of the present invention, a preferred Fc region is a mutant Fc, and more preferably a pair of Fcs capable of forming a heterodimer. Examples of a pair of Fcs include the combination of Fc(i) contained in the first polypeptide and Fc(ii) contained in the second polypeptide described later, but are not limited thereto as long as a pair of Fcs can associate (form a heterodimer).

[0043] Examples of mutant Fc include a modified Fc region (including a heterodimeric Fc region) contained in a heteromultimer having improved stability disclosed in WO2013 / 063702, an Fc containing a CH3 region of an immunoglobulin derived from an IgG antibody having "protrusions" and "voids" contained in a heteromultimer disclosed in WO1996 / 27011, an Fc containing a CH3 domain contained in a heterodimer that is electrostatically advantageous by substituting one or more amino acid residues with charged amino acids disclosed in WO2009 / 089004, a heterodimeric Fc region contained in a heterodimer using a conformational mutation and / or pI (isoelectric point) mutation disclosed in WO2014 / 110601, a heterodimeric Fc containing a CH3 domain including a modification that eliminates or reduces binding to protein A disclosed in WO2010 / 151792, etc., but are not limited thereto.

[0044] The variable region consists of a region with extreme variability called the hypervariable region (HVR) and a relatively invariant region called the framework region (FR) separated by that region. The variable regions of the natural heavy and light chains include four FRs connected by three hypervariable regions, and the hypervariable regions of each chain are held in close proximity to the hypervariable regions of the other chain by the FRs, contributing to the formation of the antigen-binding site of the antibody.

[0045] It is known that antibody molecules have three complementarity determining regions (CDRs) in both the heavy and light chains. These CDRs, also known as hypervariable regions, are located within the variable regions of the antibody's heavy and light chains and exhibit particularly high variability in the primary structure. They are typically separated into three locations on the primary structure of the heavy and light chain polypeptide chains. In this invention, the complementarity determining regions of the antibody are denoted as CDRH1, CDRH2, and CDRH3 from the amino-terminal end of the heavy chain amino acid sequence, and as CDRL1, CDRL2, and CDRL3 from the amino-terminal end of the light chain amino acid sequence. These regions are in close proximity to each other in terms of three-dimensional structure and determine the specificity for the antigen to which they bind.

[0046] In this invention, the position and length of the CDR were determined according to the definition of IMGT (Developmental and Comparative Immunology 27 (2003) 55-77).

[0047] FRs are variable regions other than CDR residues. Variable regions generally have four FRs: FR1, FR2, FR3, and FR4.

[0048] The CDRs and FRs contained in the heavy and light chains are arranged in the order FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4 and FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4, respectively, from the amino terminus to the carboxyl terminus.

[0049] The positions of CDR and FR can also be determined by various definitions well known in the art, such as those for Kabat, Chothia, AbM, and contact, in addition to IMGT.

[0050] In the present invention, "antibody antigen-binding fragment" means a partial fragment of an antibody having antigen-binding activity, composed of a heavy chain variable region and a light chain variable region. Examples of "antibody antigen-binding fragments" include, but are not limited to, antigen-binding fragments such as Fab, F(ab')2, scFv, Fab', Fv, and single-domain antibody (sdAb). Such antibody antigen-binding fragments may be obtained by treating the full-length molecule of the antibody protein with an enzyme such as papain or pepsin, or they may be recombinant proteins produced in a suitable host cell using a recombinant gene. In the present invention, "antibody binding fragment" is synonymous with "antibody antigen-binding fragment."

[0051] In the present invention, the "site" to which the antibody binds, that is, the "site" recognized by the antibody, means a partial peptide or partial higher-order structure on an antigen to which the antibody binds or recognizes.

[0052] In the present invention, such a site is also called an epitope or antibody binding site. In the present invention, a "mutant antibody" means a polypeptide that has an amino acid sequence in which amino acids are substituted, deleted, or added (addition includes insertion) (hereinafter collectively referred to as "mutation") in the amino acid sequence of the original antibody, and that binds to HLA / NY-ESO of the present invention. The number of mutant amino acids in such a mutant antibody is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, or 50. Such a mutant antibody is also included in the "antibody" of the present invention.

[0053] In this invention, "several" in "one or several" refers to 2 to 10 items. In this specification, "molecule" refers to a molecule containing the antibody or an antigen-binding fragment of an antibody, and further includes a multispecific molecule formed from an antibody or multiple antigen-binding fragments derived therefrom.

[0054] In this specification, "multispecific molecule," "multispecific molecule," and "multispecific molecule" are synonymous and are not particularly limited to any molecule capable of binding to multiple distinct epitopes on one molecule and / or to distinct epitopes on two or more molecules. Antibodies containing heavy chain variable regions (VH) and light chain variable regions (VL) are also included as multispecific molecules. Such multispecific molecules include, but are not limited to, full-length antibody molecules having two or more different heavy and light chains, i.e., IgG-type multispecific molecules, and molecules consisting of antigen-binding fragments having two or more VLs and VHs, i.e., molecules derived by combining Fab, Fab', Fv, scFv, sdAb, etc., i.e., tandem scFv, diabody, single-stranded diabody, triabody, etc. Other molecules produced by genetically or chemically linking an antigen-binding protein that does not have an immunoglobulin backbone to an antigen-binding fragment are also included as multispecific molecules. In the present invention, such multispecific molecules may also be referred to as "multispecific antibodies," "multispecific antibodies," etc., unless they do not contain antibodies or their antigen-binding fragments.

[0055] The anti-HLA / NY-ESO antibody of the present invention, or the antigen-binding fragment of said antibody, or the molecule of the present invention, exhibits activities and properties such as biological activity and physicochemical properties (also called physical properties). Specifically, these include various biological activities such as cytotoxic activity, ADCC activity, and antitumor activity (all described later), as well as binding activity to antigens and epitopes, and physical properties such as stability during manufacturing and storage, and thermal stability.

[0056] In the present invention, "hybridizing under stringent conditions" means performing hybridization in a solution containing 5×SSC at 65°C, and then hybridizing under washing conditions or equivalent conditions in an aqueous solution containing 2×SSC-0.1%SDS at 65°C for 20 minutes, in an aqueous solution containing 0.5×SSC-0.1%SDS at 65°C for 20 minutes, and in an aqueous solution containing 0.2×SSC-0.1%SDS at 65°C for 20 minutes. SSC is an aqueous solution of 150mMNaCl-15mM sodium citrate, and n×SSC means SSC at n times the concentration.

[0057] In this invention, "cytotoxicity" refers to any pathological change in a cell, and is not limited to direct trauma, but includes any structural or functional damage to cells, such as DNA breakage, base dimerization, chromosome breakage, damage to the cell division apparatus, and decreased activity of various enzymes. In this invention, "cytotoxic activity" means causing the above-mentioned cytotoxicity.

[0058] In this invention, "antibody-dependent cellular cytotoxicity (ADCC) activity" refers to the activity by which NK cells damage target cells such as tumor cells via antibodies.

[0059] In this invention, "cytotoxic activity by T cell redirection" means inducing the above-mentioned cytotoxicity through a multispecific molecule containing an antitumor antigen antibody and an anti-HLA / NY-ESO antibody. That is, the antitumor antigen antibody binds to target tumor cells, and the anti-HLA / NY-ESO antibody binds to T cells, thereby bringing the target tumor cells and T cells closer together and inducing cytotoxicity through T cell activation. This molecule can be included in a pharmaceutical composition.

[0060] 2. Antigen 2-1. HLA / NY-ESO antigen In this invention, "HLA / NY-ESO" is used interchangeably with "HLA / NY-ESO protein".

[0061] HLA / NY-ESO is a ternary complex of HLA-A2, β2-Microglobulin, and NY-ESO peptide. HLA-A2 is a type of HLA allele and is known as the most frequent allele 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, FIG. 8) consists of 9 amino acids from positions 157 to 165 of NY-ESO-1 and LAGE-1, and has been reported to be presented by HLA-A2.

[0062] 2-2. CD3 antigen In the present invention, "CD3" is used in the same meaning as the CD3 protein. CD3 is expressed on T cells as part of the multi-molecular T cell receptor complex and is a complex of five types of polypeptides, the γ chain, δ chain, ε chain, ζ chain, and η chain (the molecular weights are 25,000 - 28,000, 21,000, 20,000, 16,000, and 22,000 in order).

[0063] Examples of the CD3 complex include the γ, δ, ε, ζ, and η chains. These are also referred to as subunits. When an anti-CD3 antibody binds to T cells, it induces cytotoxicity through T cell activation. Many anti-CD3 antibodies bind to CD3ε.

[0064] The nucleotide sequence of the cDNA encoding human CD3ε is registered in NCBI / GenBank with the accession number: NM_000733 (NM_000733.3), and 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 (FIG. 147) of the sequence list.

[0065] 2-3. Preparation of Antigen The antigen proteins used in the present invention, HLA / NY-ESO and CD3 (hereinafter, HLA / NY-ESO and CD3 will also be collectively referred to as the antigen proteins), can be prepared by purification and isolation from animal tissue (including body fluids), cells derived from said tissue, or cell cultures, genetic recombination, in vitro translation, chemical synthesis, etc.

[0066] The cDNA of the antigen protein can be obtained, for example, by the so-called PCR method, which involves using a cDNA library of an organ expressing the mRNA of the antigen protein as a template and performing a polymerase chain reaction (hereinafter referred to as "PCR") using primers that specifically amplify the cDNA of the antigen protein (Saiki, RK, et al., Science (1988) 239, 487-49).

[0067] The cDNA of the antigen protein also includes polynucleotides that hybridize under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence encoding the antigen protein expressed in humans or rats, and which encode a protein having equivalent biological activity to the antigen protein.

[0068] Furthermore, the cDNA of the antigen protein also includes splicing variants transcribed from the antigen protein gene locus expressed in humans or rats, or polynucleotides that hybridize thereto under stringent conditions, and which encode proteins having biological activity equivalent to that of the antigen protein.

[0069] The nucleotide sequence of the antigen protein gene also includes the amino acid sequence of the human or rat antigen protein, or the amino acid sequence obtained by removing the signal sequence from these sequences, in which one to several amino acids are substituted, deleted, or added, and which encodes a protein having the same biological activity as the antigen protein.

[0070] The antigen protein also includes proteins that consist of an amino acid sequence encoded by a splicing variant transcribed from the gene locus of the antigen protein in humans or rats, or an amino acid sequence in which one or more amino acids are substituted, deleted, or added, and which have the same biological activity as the antigen protein.

[0071] 2-4 Binding specificity to antigen proteins The anti-HLA / NY-ESO antibody and its antigen-binding fragment of the present invention recognize HLA / NY-ESO; that is, they bind to the HLA / NY-ESO antigen. The presence of HLA / NY-ESO is not known in non-human animals such as mice, rats, and cynomolgus monkeys.

[0072] The anti-CD3 antibody and its binding fragments contained in the multispecific molecule of the present invention recognize, i.e., bind to the CD3 antigen. Such anti-CD3 antibodies preferably bind to human CD3, monkey CD3, etc., and more preferably bind to human CD3 and cynomolgus monkey CD3. On the other hand, such preferred anti-CD3 antibodies do not bind to rat and / or mouse CD3.

[0073] The antitumor activity of the multispecific molecule of the present invention can be evaluated, for example, by (i) transplanting human cancer cells, human cancer tissue, etc., into non-human animals transplanted with human peripheral blood lymphocytes, preferably rats or mice, more preferably rats or mice with impaired endogenous effector function (e.g., immunodeficient rats or mice), or (ii) transplanting mouse cancer cells, etc., into non-human animals, preferably rats or mice, that have had the human CD3 gene knocked in, preferably rats or mice. By performing evaluations using such immunodeficient animals or knock-in animals, various assays, immunohistochemistry, etc., can be carried out using living mice and / or rats, which is advantageous in the development of pharmaceuticals and nonclinical studies containing the multispecific molecule of the present invention. In the present invention, "recognition", that is, "binding", means a binding that is not non-specific adsorption. As a criterion for determining whether recognition has occurred, that is, whether binding has occurred, for example, the dissociation constant (hereinafter referred to as "KD") can be cited. The KD value of the HLA / NY-ESO or CD3 for a preferred antibody of the present invention is 1×10 -5 M or less, 5×10 -6 M or less, 2×10 -6 M or less, 1×10 -6 M or less, and a 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 -9 M or less. Examples of the anti-HLA / NY-ESO scFv of the present invention having excellent antigen-binding activity include NYA-1143, NYA-2023, NYA-2143, NYA-2044, NYA-2045, NYA-2060, NYA-2061, and NYA-3061. The KD values of HLA / NY-ESO such as NYA-1143, NYA-2044, NYA-2045, and NYA-2143 are 1×10 -9 M or less (Example 4, etc.).

[0074] 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 the SPR method or the BLI method, or by the ELISA method, the RIA method, etc. The binding between the antigen expressed on the cell surface and the antibody can be measured by the flow cytometry method or the like.

[0075] The Surface Plasmon Resonance (SPR) method is an analytical technique used to determine the dissociation constant (KD value), which is an indicator of affinity, by measuring the binding rate constant (Ka value) and dissociation rate constant (Kd value) through reaction kinetic analysis. Examples of instruments used for SPR analysis include Biacore™ (manufactured by GE Healthcare), ProteOn™ (manufactured by BioRad), SPR-Navi™ (manufactured by BioNavis), Spreeta™ (manufactured by Texas Instruments), SPRi-PlexII™ (manufactured by Horiba Corporation), and Autolab SPR™ (manufactured by Metrohm).

[0076] BioLayer Interferometry (BLI) is a method for measuring biomolecular interactions using biolayer interference. Examples of instruments used for interaction analysis using BLI include the Octet system (manufactured by Pall ForteBio).

[0077] ELISA (Enzyme-linked ImmunoSorbent Assay) is a method for detecting and quantifying a target antigen or antibody in a sample solution by capturing it with a specific antibody or antigen and utilizing an enzymatic reaction. Enzyme-labeled antigens or antibodies are incorporated into the reaction system, and enzyme activity is detected. For enzyme activity detection, a substrate whose absorbance spectrum changes during the reaction is used, and the activity is quantified by absorbance measurement.

[0078] Cell-ELISA is a method that captures the target organism on the cell surface along with the cell itself, and then detects and quantifies it using enzymatic reactions.

[0079] In the RIA (Radio Immunoassay) method, antibodies can be quantified by labeling them with a radioactive substance and measuring the radioactivity of the antibodies.

[0080] Flow cytometry is a technique that disperses cells in a fluid and flows the fluid in a narrow stream to optically analyze individual cells. Antibodies labeled with fluorescent dyes bind to cell surface antigens through an antigen-antibody reaction, and the antigen-binding ability of the antibody is quantified by measuring the fluorescence intensity produced by the labeled antibody bound to the cell.

[0081] Among the anti-HLA / NY-ESO antibodies of the present invention, examples of those having excellent antigen-binding specificity include the anti-HLA / NY-ESO scFvs NYA-0001, NYA-1143, NYA-1163, NYA-2023, NYA-2027, NYA-2035, NYA-2044, NYA-2045, NYA-2047, NYA-2048, NYA-2060, NYA-2061, NYA-2143, and NYA-3061 (Example 6, etc.).

[0082] 3. Antibodies or their binding fragments that specifically bind to HLA / NY-ESO. 3-1 Anti-HLA / NY-ESO or its binding fragments This invention provides an antibody or a binding fragment thereof that recognizes and binds to HLA / NY-ESO.

[0083] As mentioned above, HLA / NY-ESO is a complex containing HLA-A2 and a 9-mer NY-ESO peptide (SLLMWITQC: SEQ ID NO: 1). The NY-ESO peptide is an intracellular protein and is derived from NY-ESO-1 or LAGE-1, which are Cancer-Testis antigens. HLA / NY-ESO is presented on the surface of cancer cells.

[0084] The anti-HLA / NY-ESO antibody and the antigen-binding fragment of the antibody (hereinafter also referred to as "the antibody of the present invention") may be either a monoclonal antibody or a polyclonal antibody. The isotype of the monoclonal antibody of the present invention is not particularly limited and can include, for example, IgG such as IgG1, IgG2, IgG3, IgG4, IgM, IgA such as IgA1, IgA2, IgD, Ig, etc. The isotype and subclass of the monoclonal antibody can be determined by, for example, the octerony method, ELISA method, RIA method, etc. Examples of the monoclonal antibody of the present invention include antibodies derived from non-human animals (non-human animal antibodies), human antibodies, chimeric antibodies (also referred to as "chimeric antibodies"), and humanized antibodies, and preferably human antibodies can be used. The range of antibodies of the present invention also includes antibody variants ("mutant antibodies" described later), and for example, the range of human antibodies also includes human mutant antibodies.

[0085] Examples of non-human animal antibodies include antibodies derived from vertebrates such as mammals and birds. Examples of mammalian-derived antibodies include rodent-derived antibodies such as mouse antibodies and rat antibodies. Examples of bird-derived antibodies include chicken antibodies.

[0086] Examples of chimeric antibodies include, but are not limited to, antibodies formed by binding a variable region derived from a non-human animal antibody to a constant region of a human antibody (human immunoglobulin).

[0087] Examples of humanized antibodies include, but are not limited to, those obtained by transplanting the CDR (Chronic Derived Range) from the variable region of a non-human animal antibody into a human antibody (the variable region of human immunoglobulin), those obtained by transplanting part of the framework region sequence of a non-human animal antibody in addition to the CDR into a human antibody, and those obtained by substituting one or more amino acids derived from any of these non-human animal antibodies with human-type amino acids.

[0088] Antibodies can be produced by various known methods. Known methods include using hybridomas, cell-mediated immunoassay, and genetic recombination. Methods for obtaining human antibodies from phage display selected from human antibody libraries are also known. For example, phage display can be used, where the variable region of a human antibody is expressed as scFv on the phage surface, and phages that bind to the antigen are selected. By analyzing the genes of the phages selected by binding to the antigen, the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined. Once the DNA sequence of the scFv that binds to the antigen is identified, an expression vector containing that sequence can be constructed and introduced into a suitable host to express it, thereby obtaining human antibodies (WO92 / 01047, WO92 / 20791, WO93 / 06213, WO93 / 11236, WO93 / 19172, WO95 / 01438, WO95 / 15388, Annu. Rev. Immunol (1994) 12, 433-455).

[0089] The highly active antibody obtained in this way can be used as a lead antibody, and the gene encoding the lead antibody can be mutated to create a more highly active mutant (a "mutant antibody," described later).

[0090] Preferably, the CDRH1 to CDRH3 contained in the heavy chain of the anti-HLA / NY-ESO antibody or its antigen-binding fragment of the present invention include a combination of CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 54 (Figure 61), CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 55 (Figure 61), and a heavy chain CDRH3 consisting of the amino acid sequence represented by sequence number 56 (Figure 61), or an amino acid sequence in which the 6th amino acid in the amino acid sequence represented by SEQ ID NO: 56 (Figure 61) is N (Asn).More preferably, the NYA-0001 heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 6 (Figure 13), the NYA-0082 heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 18 (Figure 25), the NYA-2023 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence represented by SEQ ID NO: 27 (Figure 34), the NYA-2027 heavy chain variable region consisting of amino acid numbers 21 to 140 of the amino acid sequence represented by SEQ ID NO: 28 (Figure 35), and the amino acid sequence represented by SEQ ID NO: 29 (Figure 36) The NYA-1143 heavy chain variable region consisting of amino acid numbers 21-140, the NYA-1163 heavy chain variable region consisting of amino acid numbers 21-140 of the amino acid sequence represented by SEQ ID NO: 26 (Figure 33), the NYA-2023 heavy chain variable region consisting of amino acid numbers 21-140 of the amino acid sequence represented by SEQ ID NO: 27 (Figure 34), the NYA-2027 heavy chain variable region consisting of amino acid numbers 21-140 of the amino acid sequence represented by SEQ ID NO: 28 (Figure 35), and the amino acid sequence represented by SEQ ID NO: 36 (Figure 43) The NYA-2035 heavy chain variable region consisting of amino acid numbers 21-140, the NYA-2044 heavy chain variable region consisting of amino acid numbers 21-140 of the amino acid sequence represented by SEQ ID NO: 47 (Figure 54), the NYA-2045 heavy chain variable region consisting of amino acid numbers 21-140 of the amino acid sequence represented by SEQ ID NO: 48 (Figure 55), the NYA-2047 heavy chain variable region consisting of amino acid numbers 21-140 of the amino acid sequence represented by SEQ ID NO: 50 (Figure 57), and the amino acid numbers of the amino acid sequence represented by SEQ ID NO: 51 (Figure 58) Examples of combinations of CDRH1 to CDRH3 included in the NYA-2048 heavy chain variable region consisting of amino acids 21 to 140, the NYA-2060 heavy chain variable region consisting of amino acids 21 to 140 of the amino acid sequence represented by SEQ ID NO: 52 (Figure 59), the NYA-2061 heavy chain variable region consisting of amino acids 21 to 140 of the amino acid sequence represented by SEQ ID NO: 53 (Figure 60), or the NYA-2143 heavy chain variable region consisting of amino acids 21 to 140 of the amino acid sequence represented by SEQ ID NO: 30 (Figure 37). Furthermore, we can list combinations of CDRH1 to CDRH3 contained in the NYA-3061 heavy chain variable region, consisting of amino acid numbers 21 to 140 of the amino acid sequence represented by sequence number 156 (Figure 152).

[0091] Preferably, the CDRL1 to CDRL3 included in the light chain of the anti-HLA / NY-ESO antibody or its antigen-binding fragment of the present invention include a combination of the following: CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 57 (Figure 61), or an amino acid sequence in which the 7th amino acid is W (Trp) or the 8th amino acid is K (Lys); CDRL2 consisting of the amino acid sequence represented by DNN (Figure 61); and CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 59 (Figure 61), or an amino acid sequence in which the 2nd amino acid is A (Ala) or S (Ser).

[0092] More preferably, the NYA-0001 light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 8 (Figure 15), the NYA-0082 light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 20 (Figure 27), the NYA-1143 light chain variable region consisting of amino acid numbers 156-266 of the amino acid sequence represented by SEQ ID NO: 29 (Figure 36), the NYA-1163 light chain variable region consisting of amino acid numbers 156-266 of the amino acid sequence represented by SEQ ID NO: 26 (Figure 33), the NYA-2023 light chain variable region consisting of amino acid numbers 156-266 of the amino acid sequence represented by SEQ ID NO: 27 (Figure 34), the NYA-2027 light chain variable region consisting of amino acid numbers 156-266 of the amino acid sequence represented by SEQ ID NO: 28 (Figure 35), the NYA-2035 light chain variable region consisting of amino acid numbers 156-266 of the amino acid sequence represented by SEQ ID NO: 36 (Figure 43), and the amino acid sequence represented by SEQ ID NO: 47 (Figure 54) Examples of combinations of CDRL1 to CDRL3 included in the NYA-2044 light chain variable region consisting of amino acids 156 to 266, the NYA-2045 light chain variable region consisting of amino acids 156 to 266 of the amino acid sequence represented by SEQ ID NO: 48 (Figure 55), the NYA-2047 light chain variable region consisting of amino acids 156 to 266 of the amino acid sequence represented by SEQ ID NO: 50 (Figure 57), the NYA-2048 light chain variable region consisting of amino acids 156 to 266 of the amino acid sequence represented by SEQ ID NO: 51 (Figure 58), the NYA-2060 light chain variable region consisting of amino acids 156 to 266 of the amino acid sequence represented by SEQ ID NO: 52 (Figure 59), the NYA-2061 light chain variable region consisting of amino acids 156 to 266 of the amino acid sequence represented by SEQ ID NO: 53 (Figure 60), or the NYA-2143 light chain variable region consisting of amino acids 156 to 266 of the amino acid sequence represented by SEQ ID NO: 30 (Figure 37). Furthermore, we can list combinations of CDRL1 to CDRL3 contained in the NYA-3061 light chain variable region, which consists of amino acid numbers 161 to 271 of the amino acid sequence represented by Sequence ID No. 156 (Figure 152).

[0093] Preferably, the CDRH1-CDRH3 contained in the heavy chain and CDRL1-CDRL3 contained in the light chain of the anti-HLA / NY-ESO antibody or its antigen-binding fragment of the present invention include combinations of the following: CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 54 (Figure 61), CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 55 (Figure 61), heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 56 (Figure 61), or the amino acid sequence in which the 6th amino acid is N (Asn) in the amino acid sequence represented by SEQ ID NO: 56 (Figure 61), CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 57 (Figure 61), or the amino acid sequence in which the 7th amino acid is N (Asn) and / or the 8th amino acid is K (Lys), CDRL2 consisting of the amino acid sequence represented by DNN (Figure 61), and CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 59 (Figure 61), or the amino acid sequence in which the 2nd amino acid is A (Ala) or S (Ser).More preferably, the NYA-0001 heavy chain variable region and light chain variable region consisting of the amino acid sequences represented by SEQ ID NOs. 6 and 8, respectively; the NYA-1143 heavy chain variable region and light chain variable region consisting of amino acid numbers 21-140 and 156-266 of the amino acid sequence represented by SEQ ID NOs. 29 (Figure 36); the NYA-1163 heavy chain variable region and light chain variable region consisting of amino acid numbers 21-140 and 156-266 of the amino acid sequence represented by SEQ ID NOs. 26 (Figure 33); and the NYA-1163 heavy chain variable region consisting of amino acid numbers 21-140 and 156-266 of the amino acid sequence represented by SEQ ID NOs. 27 (Figure 34). The NYA-2023 heavy chain variable region and light chain variable region consisting of 266 amino acids, the NYA-2027 heavy chain variable region and light chain variable region consisting of amino acid numbers 21-140 and 156-266 of the amino acid sequence represented by SEQ ID NO: 28 (Figure 35), the NYA-2035 heavy chain variable region and light chain variable region consisting of amino acid numbers 21-140 and 156-266 of the amino acid sequence represented by SEQ ID NO: 36 (Figure 43), and the NYA-2044 heavy chain variable region and light chain variable region consisting of amino acid numbers 21-140 and 156-266 of the amino acid sequence represented by SEQ ID NO: 47 (Figure 54). The regions, the NYA-2045 heavy chain variable region and light chain variable region consisting of amino acid numbers 21-140 and 156-266 of the amino acid sequence represented by SEQ ID NO: 48 (Figure 55), the NYA-2047 heavy chain variable region and light chain variable region consisting of amino acid numbers 21-140 and 156-266 of the amino acid sequence represented by SEQ ID NO: 50 (Figure 57), the NYA-2048 heavy chain variable region and light chain variable region consisting of amino acid numbers 21-140 and 156-266 of the amino acid sequence represented by SEQ ID NO: 51 (Figure 58), and the amino acid sequence represented by SEQ ID NO: 52 (Figure 59) Examples of combinations of CDRH1-CDRH3 and CDRL1-CDRL3 included in the NYA-2060 heavy chain variable region and light chain variable region consisting of amino acid numbers 21-140 and 156-266, the NYA-2061 heavy chain variable region and light chain variable region consisting of amino acid numbers 21-140 and 156-266 of the amino acid sequence represented by Sequence ID No. 53 (Figure 60), or the NYA-2143 heavy chain variable region and light chain variable region consisting of amino acid numbers 21-140 and 156-266 of the amino acid sequence represented by Sequence ID No. 30 (Figure 37) are as follows. Furthermore, combinations of CDRH1-CDRH3 and CDRL1-CDRL3 included in the NYA-3061 heavy chain variable region and light chain variable region, which consist of amino acid numbers 21-140 and 161-271, respectively, of the amino acid sequence represented by Sequence ID No. 156 (Figure 152), can be listed.

[0094] As examples of the heavy chain variable regions of the anti-HLA / NY-ESO antibody or its antigen-binding fragment of the present invention, those containing the above-mentioned heavy chain CDRs or combinations thereof can be cited, and more preferably, the NYA-0001 heavy chain variable region, NYA-0082 heavy chain variable region, NYA-1143 heavy chain variable region, NYA-1163 heavy chain variable region, NYA-2023 heavy chain variable region, NYA-2027 heavy chain variable region, NYA-2035 heavy chain variable region, NYA-2044 heavy chain variable region, NYA-2045 heavy chain variable region, NYA-2047 heavy chain variable region, NYA-2048 heavy chain variable region, NYA-2060 heavy chain variable region, NYA-2061 heavy chain variable region, NYA-2143 heavy chain variable region, and NYA-3061 heavy chain variable region can be cited. The amino acid sequences of each heavy chain variable region are as described above.

[0095] As examples of the light chain variable regions of the anti-HLA / NY-ESO antibody or its antigen-binding fragment of the present invention, those containing the above-mentioned light chain CDRs or combinations thereof can be cited, and more preferably, the NYA-0001 light chain variable region, NYA-0082 light chain variable region, NYA-1143 light chain variable region, NYA-1163 light chain variable region, NYA-2023 light chain variable region, NYA-2027 light chain variable region, NYA-2035 light chain variable region, NYA-2044 light chain variable region, NYA-2045 light chain variable region, NYA-2047 light chain variable region, NYA-2048 light chain variable region, NYA-2060 light chain variable region, NYA-2061 light chain variable region, NYA-2143 light chain variable region, and NYA-3061 light chain variable region can be cited. The amino acid sequences of each light chain variable region are as described above.

[0096] As examples of 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, those containing the above heavy chain and light chain CDRs or combinations thereof can be cited, and more preferably, NYA-0001 heavy chain variable region and light chain variable region, NYA-0082 heavy chain variable region and light chain variable region, NYA-1143 heavy chain variable region and light chain variable region, NYA-1163 heavy chain variable region and light chain variable region, NYA-2023 heavy chain variable region and light chain variable region, NYA-2027 heavy chain variable region and light chain variable region Examples include the NYA-2035 heavy chain variable region and light chain variable region, the NYA-2044 heavy chain variable region and light chain variable region, the NYA-2045 heavy chain variable region and light chain variable region, the NYA-2047 heavy chain variable region and light chain variable region, the NYA-2048 heavy chain variable region and light chain variable region, the NYA-2060 heavy chain variable region and light chain variable region, the NYA-2061 heavy chain variable region and light chain variable region, and combinations of the NYA-2143 heavy chain variable region and light chain variable region, as well as combinations of the NYA-3061 heavy chain variable region and light chain variable region.

[0097] As examples of the heavy chain of the anti-HLA / NY-ESO antibody or its antigen-binding fragment of the present invention, those containing the above-mentioned preferred or more preferred heavy chain variable region can be cited, and more preferably, examples include the NYA-0001 heavy chain, NYA-0082 heavy chain, NYA-1143 heavy chain, NYA-1163 heavy chain, NYA-2023 heavy chain, NYA-2027 heavy chain, NYA-2035 heavy chain, NYA-2044 heavy chain, NYA-2045 heavy chain, NYA-2047 heavy chain, NYA-2048 heavy chain, NYA-2060 heavy chain, NYA-2061 heavy chain, NYA-2143 heavy chain, and NYA-3061 heavy chain.

[0098] As examples of the light chain of the anti-HLA / NY-ESO antibody or its antigen-binding fragment of the present invention, those containing the above-mentioned preferred or more preferred light chain variable region can be cited, and more preferably, examples include the NYA-0001 light chain, NYA-0082 light chain, NYA-1143 light chain, NYA-1163 light chain, NYA-2023 light chain, NYA-2027 light chain, NYA-2035 light chain, NYA-2044 light chain, NYA-2045 light chain, NYA-2047 light chain, NYA-2048 light chain, NYA-2060 light chain, NYA-2061 light chain, NYA-2143 light chain, and NYA-3061 light chain.

[0099] As examples of the heavy chain and light chain of the anti-HLA / NY-ESO antibody or its antigen-binding fragment of the present invention, those containing the above-mentioned preferred or more preferred heavy chain variable region and light chain variable region, respectively, can be cited. More preferably, examples include combinations of the heavy chain and light chain of NYA-0001, NYA-1143, NYA-1163, NYA-2023, NYA-2027, NYA-2035, NYA-2044, NYA-2045, NYA-2047, NYA-2048, NYA-2060, NYA-2061, NYA-2143, and NYA-3061.

[0100] An antigen-binding fragment of an antibody refers to a fragment or modification thereof that retains at least the antigen-binding function of the antibody. Generally, such antibody functions include antigen-binding activity, activity that modulates antigen activity, antibody-dependent cytotoxic activity, and complement-dependent cytotoxic activity. Functions of the antibody of the present invention and multispecific molecules containing the antibody of the present invention include, for example, T cell redirection, T cell activation, and cytotoxic activity of cancer cells by activating T cells.

[0101] The antigen-binding fragment of the antibody is not particularly limited as long as it is a fragment of the antibody that retains at least antigen-binding activity among the activities of the antibody. Examples include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv (scFv) obtained by linking the heavy chain and light chain Fv with an appropriate linker, and single-domain antibodies (sdAb). Molecules that include parts other than the antigen-binding fragment of the antibody of the present invention, such as scFv possessing a linker portion, are also included in the meaning of the antigen-binding fragment of the antibody of the present invention.

[0102] Molecules that have one or more amino acids deleted from the amino-terminus and / or carboxyl-terminus of an antibody protein, and that retain at least some of the functions of the antibody, are also included in the meaning of antibody antigen-binding fragments. Modified versions of such antibody antigen-binding fragments are also included in the antibodies or their antigen-binding fragments, or their modifications (described later) of the present invention.

[0103] One embodiment of the antibody or antigen-binding fragment thereof according to the present invention is an scFv. An scFv is obtained by linking the heavy chain variable region and the light chain variable region of an antibody with a polypeptide linker (Pluckthun A. The Pharmacology of Monoclonal Antibodies 113, Rosenburg and Moore eds., Springer Verlag, New York, 269-315 (1994), Nature Biotechnology (2005), 23, 1126-1136). Furthermore, a tandem scFv, prepared by linking two scFvs with a polypeptide linker, can also be used as a bispecific molecule. In addition, a triabody consisting of three or more scFvs can also be used as a multispecific molecule.

[0104] HLA / NY-ESO specific scFv (also called "anti-HLA / NY-ESO scFv") preferably include those containing the above CDRH1-CDRH3 and CDRL1-CDRL3, more preferably those containing the above heavy chain variable region and light chain variable region, even more preferably NYA-0001 (amino acid numbers 21-266 of the amino acid sequence represented by SEQ ID NO: 70 (Figure 71)), NYA-0082 (including the amino acid sequence represented by SEQ ID NO: 18 (Figure 25) and SEQ ID NO: 20 (including the amino acid sequence represented by Figure 27)), NYA-1143 (sequence number (amino acid numbers 21-266 of the amino acid sequence represented by Sequence ID No. 29 (Figure 36)), NYA-1163 (amino acid numbers 21-266 of the amino acid sequence represented by Sequence ID No. 26 (Figure 33)), NYA-2023 (amino acid numbers 21-266 of the amino acid sequence represented by Sequence ID No. 27 (Figure 34)), NYA-2027 (amino acid numbers 21-266 of the amino acid sequence represented by Sequence ID No. 28 (Figure 35)), NYA-2035 (amino acid numbers 21-266 of the amino acid sequence represented by Sequence ID No. 36 (Figure 43)) NYA-2044 (amino acid numbers 21-266 of the amino acid sequence represented by SEQ ID NO: 47 (Figure 54)), NYA-2045 (amino acid numbers 21-266 of the amino acid sequence represented by SEQ ID NO: 48 (Figure 55)), NYA-2047 (amino acid numbers 21-266 of the amino acid sequence represented by SEQ ID NO: 50 (Figure 57)), NYA-2048 (amino acid numbers 21-266 of the amino acid sequence represented by SEQ ID NO: 51 (Figure 58)), NYA-2060 (sequence number Examples include amino acid numbers 21-266 of the amino acid sequence represented by sequence number 52 (Figure 59), NYA-2061 (amino acid numbers 21-266 of the amino acid sequence represented by sequence number 53 (Figure 60)), and NYA-2143 (amino acid numbers 21-266 of the amino acid sequence represented by sequence number 30 (Figure 37)). Furthermore, NYA-3061 (amino acid numbers 21-271 of the amino acid sequence represented by sequence number 156 (Figure 152)) can also be given as an example.

[0105] Preferred embodiments of anti-HLA / NY-ESO scFv include those in which a FLAG-His tag is fused to the carboxyl terminus (also simply called "tagged adducts"). Preferred tagged adducts include the NYA-0001 tagged adduct (amino acid numbers 20-292 of SEQ ID NO: 70 (Figure 71)), the NYA-1143 tagged adduct (amino acid numbers 20-292 of SEQ ID NO: 29 (Figure 36)), the NYA-1163 tagged adduct (amino acid numbers 20-292 of SEQ ID NO: 26 (Figure 33)), the NYA-2023 tagged adduct (amino acid numbers 20-292 of SEQ ID NO: 27 (Figure 34)), the NYA-2027 tagged adduct (amino acid numbers 20-292 of SEQ ID NO: 28 (Figure 35)), the NYA-2035 tagged adduct (amino acid numbers 20-292 of SEQ ID NO: 36 (Figure 43)), and the NYA-2044 tagged adduct (SEQ ID NO: 47 Examples include the NYA-2045 tag adduct (amino acid numbers 20-292 in SEQ ID NO: 48 (Figure 55)), the NYA-2047 tag adduct (amino acid numbers 20-292 in SEQ ID NO: 50 (Figure 57)), the NYA-2048 tag adduct (amino acid numbers 20-292 in SEQ ID NO: 51 (Figure 58)), the NYA-2060 tag adduct (amino acid numbers 20-292 in SEQ ID NO: 52 (Figure 59)), the NYA-2061 tag adduct (amino acid numbers 20-292 in SEQ ID NO: 53 (Figure 60)), and the NYA-2143 tag adduct (amino acid numbers 20-292 in SEQ ID NO: 30 (Figure 37)).

[0106] Of these, NYA-2023 and its tag adduct, NYA-2047 and its tag adduct, NYA-2048 and its tag adduct, NYA-2060 and its tag adduct, and NYA-2061 and its tag adduct are more preferable as Fc-added bispecific molecules because they possess excellent biological activity, physical properties, etc.

[0107] Furthermore, when anti-HLA / NY-ESO scFv and its tagged adducts are expressed in host cells, a signal peptide can be added to the amino terminus. Examples of amino acid sequences of anti-HLA / NY-ESO scFv tagged adducts with added signal peptides include SEQ ID NOs. 70, 29, 26-28, 36, 47, 48, 50-53, and 30 (Figures 71, 36, 33-35, 43, 54, 55, 57-60, and 37).

[0108] scFv can be obtained by phage display (Nature Biotechnology (2005), 23, (9), pp. 1105-1116), which involves expressing the variable region of an antibody as a single-chain antibody (scFv) on the phage surface and selecting phages that bind to the antigen. By analyzing the genes of the phages selected by binding to the antigen, the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined. Once the DNA sequence of the scFv that binds to the antigen is identified, an expression vector containing that sequence can be constructed and introduced into a suitable host to express it, thereby obtaining human antibodies (WO92 / 01047, WO92 / 20791, WO93 / 06213, WO93 / 11236, WO93 / 19172, WO95 / 01438, WO95 / 15388, Annu. Rev. Immunol (1994) 12, p. 433-455, Nature Biotechnology (2005) 23(9), p. 1105-1116).

[0109] The antibody of the present invention may be an antibody having a single heavy chain variable region and lacking a light chain sequence. Such antibodies are called single-domain antibodies (sdAb) or nanobodies, and have been reported to retain antigen-binding ability (Muyldemans S. et.al., Protein Eng., (1994) 7(9), 1129-35, Hamers-Casterman C. et.al., Nature (1993) 363(6428), 446-448). These antibodies are also included in the meaning of antigen-binding fragment of an antibody in the present invention.

[0110] Furthermore, the present invention includes single-chain immunoglobulins in which the full-length sequences of the heavy and light chains of an antibody are linked using an appropriate linker (Lee, HS, et.al., Molecular Immunology (1999) 36, 61-71; Shirrmann, T. et.al., mAbs (2010), 2(1), 1-4). Such single-chain immunoglobulins can maintain a structure and activity similar to antibodies, which are originally tetramers, by dimerizing. The anti-HLA / NY-ESO antibody of the present invention may also be a single-chain immunoglobulin. In the scFv of the present invention, the heavy chain variable region and the light chain variable region may be bonded by disulfide bonds.

[0111] The anti-HLA / NY-ESO antibody of the present invention may be composed of portions derived from multiple different antibodies when bound to HLA / NY-ESO. Examples include antibodies in which the heavy chain and / or light chain have been exchanged between multiple different antibodies, antibodies in which the entire length of the heavy chain and / or light chain has been exchanged, antibodies in which only the variable region or only the constant region has been exchanged, or antibodies in which all or part of the CDR has been exchanged. The heavy chain variable region and light chain variable region of the chimeric antibody may be derived from different anti-HLA / NY-ESO antibodies of the present invention. The heavy chain CDR1 to CDR3 and light chain CDR1 to CDR3 in the variable regions of the heavy chain and light chain of the humanized antibody may be derived from two or more anti-HLA / NY-ESO antibodies of the present invention. The heavy chain CDR1 to CDR3 and light chain CDR1 to CDR3 in the variable regions of the heavy chain and light chain of the human antibody may be a combination of CDRs possessed by two or more anti-HLA / NY-ESO antibodies of the present invention.

[0112] The anti-HLA / NY-ESO antibody of the present invention includes an amino acid sequence encoded by a nucleotide sequence contained in a polynucleotide that hybridizes under stringent conditions with a complementary polynucleotide containing a nucleotide sequence encoding the amino acid sequence contained in the anti-HLA / NY-ESO antibody of the present invention, and also includes an antibody that binds to HLA / NY-ESO.

[0113] 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 acids 21-140 of the amino acid sequence represented by SEQ ID NO: 27, the amino acid sequence represented by SEQ ID NO: 38, the amino acid sequence represented by SEQ ID NO: 39, or the amino acid sequence of amino acids 21-140 of the amino acid sequence represented by 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 acids 156-266 of the amino acid sequence represented by SEQ ID NO: 27) It may also be an antibody or antigen-binding fragment thereof that contains 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of the same amino acid sequence as the acid sequence, the amino acid sequence from amino acid numbers 156 to 266 of the amino acid sequence represented by SEQ ID NO: 52, the amino acid sequence represented by SEQ ID NO: 40, the amino acid sequence from amino acid numbers 161 to 271 of the amino acid sequence represented by SEQ ID NO: 160, or the amino acid sequence from amino acid numbers 156 to 266 of the amino acid sequence represented by SEQ ID NO: 197 or SEQ ID NO: 198, and that binds to HLA / NY-ESO.

[0114] When the position and length of the light chain variable region are determined using a definition different from that of IMGT (e.g., Kabat, Chothia, AbM, contact, etc.), the carboxyl terminus of the light chain variable region amino acid sequence determined by the IMGT definition may contain one or more additional amino acids, such as arginine or glycine. Antibodies or their conjugated fragments having such light chain variable regions are also included in the antibodies or their conjugated fragments of the present invention.

[0115] The antibody of the present invention may be one in which a mutation has been introduced into the binding fragment of the anti-HLA / NY-ESO antibody of the present invention to optimize its binding ability to HLA / NY-ESO, particularly human and / or cynomolgus monkey HLA / NY-ESO. Specific methods for introducing mutations include random mutagenesis using error-prone PCR, site-directed amino acid mutation introduction using an NNK library, site-directed mutagenesis using structural information, and combinations thereof.

[0116] 3-2. Mutants (mutant antibodies) of anti-HLA / NY-ESO antibodies The mutant antibodies of the anti-HLA / NY-ESO antibody of the present invention may preferably have reduced sensitivity to protein degradation or oxidation, maintain, improve, or suppress the reduction or change of biological activity or function, improve or regulate antigen-binding ability, or confer physicochemical or functional properties. It is known that the function and activity of a protein can change when certain amino acid side chains on its surface are altered, and such examples include deamidation of the asparagine side chain and isomerization of the aspartic acid side chain. Mutant antibodies in which amino acids are replaced with other amino acids to prevent such changes in amino acid side chains are also included in the scope of the mutant antibodies of the present invention.

[0117] An example of a mutant antibody of the present invention is an antibody having an amino acid sequence in which a conservative amino acid substitution has been made in the amino acid sequence of the antibody. A conservative amino acid substitution is a substitution that occurs within an amino acid group related to the amino acid side chain.

[0118] The 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 family = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Other preferred amino acid groups are as follows: aliphatic hydroxyl group = serine and threonine; amide-containing group = asparagine and glutamine; aliphatic group = alanine, valine, leucine and isoleucine; and aromatic group = phenylalanine, tryptophan and tyrosine. It is preferable that amino acid substitutions in such mutant antibodies be carried out within a range that does not reduce the antigen-binding activity of the original antibody.

[0119] Mutant antibodies that have an amino acid sequence in which a conservative amino acid substitution and / or other mutation has been made in the amino acid sequence of an antibody of the present invention such as NYA-2023, and that bind to HLA / NY-ESO, their antigen-binding fragments, molecules containing them, etc., are also included in the anti-HLA / NY-ESO antibodies of the present invention, their antigen-binding fragments, their variants (mutant antibodies or their antigen-binding fragments), or molecules of the present invention.

[0120] 3-3. Binding fragments of anti-HLA / NY-ESO antibodies One aspect of the present invention provides an antigen-binding fragment of an anti-HLA / NY-ESO antibody (hereinafter simply referred to as "binding fragment"). The binding fragment of an anti-HLA / NY-ESO antibody of the present invention includes a chimeric antibody, a humanized antibody, or a binding fragment of a human antibody. An antibody-binding fragment means a fragment or modification thereof that retains at least the antigen-binding function of the antibody. The functions of such an antibody generally include antigen-binding activity, activity that modulates the activity of the antigen (e.g., agonist activity), activity that internalizes the antigen into cells, and activity that inhibits or promotes the interaction of the antigen with substances that interact with the antigen.

[0121] The antibody binding fragment is not particularly limited as long as it is a fragment of the antibody that retains at least antigen-binding activity among the activities of the antibody. Examples of such antibody binding fragments include, but are not limited to, Fab, Fab', F(ab')2, single-chain Fab (scFab) in which the carboxyl terminus of the Fab light chain and the amino terminus of the Fab heavy chain are linked by a suitable linker, Fv, single-chain Fv (scFv) in which the heavy chain and light chain of Fv are linked by a suitable linker, single-domain antibodies (sdAb) that have a single heavy chain variable region and no light chain sequence, or nanobodies. (Muyldemans S.et.al., Protein Eng., (1994) 7(9), 1129-35, Hamers-Casterman C.et.al., Nature (1993) 363(6428), 446-448) Molecules that contain parts other than the antibody binding fragment of the present invention, such as scFab and scFv which possess a linker portion, are also included in the meaning of the antibody binding fragment of the present invention.

[0122] 3-4. Modified or complex anti-HLA / NY-ESO antibodies or their binding fragments. This invention provides modified antibodies or their binding fragments. Modified antibodies or their binding fragments refer to antibodies or their binding fragments that have been chemically or biologically modified. Chemical modifications include the attachment of chemical moieties to the amino acid backbone, and chemical modifications of N-linked or O-linked carbohydrate chains. Biological modifications include those that have been post-translationally modified (e.g., glycosylation of N-linked or O-linked bonds, processing of the amino-terminal or carboxyl-terminal region, deamidation, isomerization of aspartic acid, oxidation of methionine), and those that have had a methionine residue added to the amino terminus by expression using prokaryotic host cells. Furthermore, labeled antibodies or antigens of the present invention, such as enzyme-labeled, fluorescently labeled, or affinity-labeled antibodies, are also included in the definition of such modifications. Such modified antibodies or their binding fragments are useful for improving the stability and blood retention of the original antibody or its binding fragment, reducing antigenicity, and for detecting or isolating such antibodies or antigens.

[0123] Examples of chemical parts included in chemically modified products include water-soluble polymers such as polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymer, carboxymethylcellulose, dextran, and polyvinyl alcohol.

[0124] Examples of biologically modified substances include those modified by enzymatic treatment or cell treatment, fusion products to which tags or other peptides have been added by genetic recombination, and those prepared using cells expressing endogenous or exogenous glycosylation enzymes as hosts.

[0125] Such modifications may be made at any position or a desired position on the antibody or its binding fragment, and one or more identical or two or more different modifications may be made at one or more positions.

[0126] However, deletions of these heavy chain sequences, or modifications of heavy or light chain sequences, do not significantly affect the antigen-binding ability or effector function (such as complement activation or antibody-dependent cytotoxicity) of antibodies.

[0127] Therefore, the present invention also includes antibodies that have undergone such deletions or modifications. For example, examples include a deletion in which one or two amino acids are deleted at the heavy chain carboxyl terminus (Journal of Chromatography A;705;129-134 (1995)), a deletion in which two amino acid residues, glycine and lysine, are deleted at the heavy chain carboxyl terminus, and a proline residue newly located at the carboxyl terminus is amidated (Analytical Biochemistry, 360:75-83 (2007)), and an antibody in which a glutamine or glutamic acid residue at the amino terminus of the heavy or light chain of the antibody is pyroglutamylated (International Patent Publication WO2013 / 147153), etc. (These are collectively referred to as "deletion bodies"). However, as long as antigen-binding ability and effector function are maintained, the deletions of the carboxyl terminus of the heavy and light chains of the antibody according to the present invention are not limited to the above types. If the antibody according to the present invention contains two or more chains (e.g., heavy chains), these two or more chains (e.g., heavy chains) may be one of the heavy chains selected from the group consisting of full-length and the above-mentioned deletions, or they may be a combination of any two of them. The ratio of the amount or number of molecules of each deletion may be affected by the type of mammalian cultured cell that produces the antibody according to the present invention and the culture conditions, but a possible example of the main component of the antibody according to the present invention is the case in which one amino acid residue at the carboxyl terminus is deleted in both of the two heavy chains.

[0128] Furthermore, even if one to several amino acids derived from an expression vector and / or signal sequence are added to the amino terminus and / or carboxyl terminus of the antibody or its antigen-binding fragment (including those included in the molecule, multispecific molecule, bispecific molecule, etc. of the present invention) (and some or all of them are modified as described above), as long as the desired antigen-binding activity is maintained, it is included in the range of modified antibodies or modified antigen-binding fragments of the present invention, and molecules containing such modified antibodies or antigen-binding fragments are also included in the range of molecules of the present invention.

[0129] In the present invention, "antibody or its binding fragment" also includes "modified antibody or its antigen-binding fragment." Furthermore, "antibody or its antigen-binding fragment" contained in the molecule, multispecific molecule, bispecific molecule, etc. of the present invention also includes such "modified antibody or its antigen-binding fragment."

[0130] Furthermore, antibody-dependent cytotoxic activity can be enhanced by regulating the glycosylation (glycosylation, defucoseation, etc.) of the antibody bound to the antibody of the present invention. Known techniques for regulating antibody glycosylation include, but are not limited to, International Patent Publications WO99 / 54342, WO00 / 61739, and WO02 / 31140.

[0131] The present invention also includes immunoconjugates, which are complexes (Immunoconjugates) in which the aforementioned antibodies are linked to other molecules by a linker. An example of an antibody-drug conjugate in which the antibody is bound to a radioactive substance or a compound (drug) with pharmacological activity is the Antibody-Drug Conjugate (ADC) ((Methods Mol Biol. (2013) 1045:1-27; Nature Biotechnology (2005) 23, p.1137-1146).

[0132] Furthermore, the present invention also includes complexes in which these antibodies are linked to other functional polypeptides. An example of such an antibody-peptide complex is one in which the antibody is complexed with an albumin-binding polypeptide (Protein Eng Des Sel. (2012)(2):81-8).

[0133] The modified antibodies, glycosylated antibodies, and complexes described above are included in the antibody of the present invention, and the binding fragments of the modified antibodies, glycosylated antibodies, and complexes described above are included in the binding fragments of the antibody of the present invention.

[0134] 4. Methods for producing antibodies The antibodies of the present invention can be produced in cells as recombinant antibodies by, for example, inserting DNA encoding a heavy chain variable region or DNA encoding a light chain variable region into an expression vector, transforming host cells for expression with the vector, and culturing the host cells.

[0135] Antibody-coding DNA is obtained by ligating the DNA encoding the heavy chain variable region with the DNA encoding the heavy chain constant region, and then obtaining the DNA encoding the light chain by ligating the DNA encoding the light chain variable region with the DNA encoding the light chain constant region.

[0136] The anti-HLA / NY-ESO antibody of the present invention can be produced by inserting the DNA encoding the heavy chain and the DNA encoding the light chain into an expression vector, transforming host cells with the vector, and culturing the host cells. In this case, the DNA encoding the heavy chain and the DNA encoding the light chain may be introduced into the same expression vector and the host cells may be transformed using this vector, or the DNA encoding the heavy chain and the DNA encoding the light chain may be inserted into separate vectors and the host cells may be transformed using the two vectors. In this case, the DNA encoding the heavy chain variable region and the DNA encoding the light chain variable region may be introduced into a vector into which the DNA encoding the heavy chain constant region and the DNA encoding the light chain constant region have been introduced beforehand. Furthermore, the vector may contain DNA encoding a signal peptide that promotes antibody secretion from host cells. In this case, the DNA encoding the signal peptide and the DNA encoding the antibody are linked in-frame. After the antibody is produced, the signal peptide can be removed to obtain the antibody as a mature protein.

[0137] In this case, DNA encoding the heavy chain variable region, DNA encoding the light chain variable region, DNA ligated with DNA encoding the heavy chain variable region and DNA encoding the heavy chain constant region, and DNA ligated with DNA encoding the light chain variable region and DNA encoding the light chain constant region may be functionally linked to elements such as promoters, enhancers, and polyadenylation signals. Functional linking here means linking the elements so that they perform their functions.

[0138] Expression vectors are not particularly limited as long as they can replicate in a host such as animal cells, bacteria, or yeast; for example, known plasmids and phages are examples. Examples of vectors used to construct expression vectors include pcDNA® (ThermoFissher SCIENTIFIC), Flexi® vector (Promega), pUC19, pUEX2 (Amersham), pGEX-4T, pKK233-2 (Pharmacia), and pMAM-neo (Clontech). As host cells, prokaryotic cells such as Escherichia coli and Bacillus subtilis, as well as eukaryotic cells such as yeast and animal cells, can be used, but the use of eukaryotic cells is preferred. For example, as animal cells, human embryonic kidney cell lines such as HEK293 cells and Chinese hamster ovary (CHO) cells can be used. Expression vectors are introduced into host cells by known methods, and the host cells are transformed. Examples include electroporation, calcium phosphate precipitation, and DEAE-dextran transfection. The produced antibodies can be purified using the same separation and purification methods used for conventional proteins. For example, affinity chromatography, other chromatography methods, filtration, ultrafiltration, salting out, dialysis, etc., can be selected and combined as appropriate.

[0139] 5. Molecules that bind to HLA / NY-ESO The molecule of the present invention comprises the anti-HLA / NY-ESO antibody of the present invention or its antigen-binding fragment. Preferably, the molecule of the present invention is a multispecific molecule having two or more antigen-binding sites. That is, it is a molecule capable of binding to two or more different epitopes on one molecule or to two or more different epitopes on two or more molecules, and encapsulates multiple 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, such as tandem scFv (taFv), antibody fragments such as single-stranded diabodies, diabodies and triabodies, and antibody fragments linked by covalent or non-covalent bonds. The multispecific molecule may contain Fc.

[0140] The multispecific molecule of the present invention may include, in addition to the anti-HLA / NY-ESO antibody of the present invention or its antigen-binding fragment, one or more further antibodies or antigen-binding fragments of said antibody. Examples of further antibody antigen-binding fragments include Fab, F(ab)', Fv, scFv, and sdAb.

[0141] A preferred multispecific molecule of the present invention further comprises an anti-CD3 antibody or its antigen-binding fragment, and specifically binds to CD3.

[0142] The anti-CD3 antibody or its antigen-binding fragment included in the multispecific molecule of the present invention is not particularly limited as long as it is an antibody or its binding fragment that binds to human CD3, but preferably it also binds to CD3 of non-human primates such as cynomolgus monkeys. More preferred anti-CD3 antibodies or their antigen-binding fragments include an antibody or its antigen-binding fragment comprising the following amino acid sequences: CDRH1, CDRH2, CDRH3, CDRH1, CDRL1, CDRL2, CDRL2, and CDRL3 (all shown in Figure 142).

[0143] A more preferable antibody or antigen-binding fragment containing CDRH1-CDRH3 and CDRL1-CDRL3 is the C3E-7034 heavy chain variable region consisting of amino acid sequences from amino acid numbers 2 to 119 of the amino acid sequence represented by SEQ ID NO: 136 (Figure 137), the C3E-7036 heavy chain variable region consisting of amino acid sequences from amino acid numbers 2 to 119 of the amino acid sequence represented by SEQ ID NO: 137 (Figure 138), the C3E-7085 heavy chain variable region consisting of amino acid sequences from amino acid numbers 2 to 119 of the amino acid sequence represented by SEQ ID NO: 138 (Figure 139), and the C3E-708 Examples of antibodies or antigen-binding fragments thereof include an 8-chain variable region, a C3E-7093 heavy chain variable region consisting of amino acid sequences from amino acid numbers 2 to 119 of the amino acid sequence represented by SEQ ID NO: 140 (Figure 141), a C3E-7096 heavy chain variable region consisting of amino acid sequences from amino acid numbers 272 to 389 of the amino acid sequence represented by SEQ ID NO: 155 (Figure 151), a C3E-7096 heavy chain variable region consisting of amino acid sequences from amino acid numbers 277 to 394 of the amino acid sequence represented by SEQ ID NO: 156 (Figure 152), and a C3E-7097 heavy chain variable region consisting of amino acid sequences from amino acid numbers 277 to 394 of the amino acid sequence represented by SEQ ID NO: 157 (Figure 153).

[0144] Furthermore, a more preferable antibody or antigen-binding fragment thereof containing CDRH1-CDRH3 and CDRL1-CDRL3 is the C3E-7034 light chain variable region consisting of amino acid sequences from amino acid numbers 135-243 of the amino acid sequence represented by SEQ ID NO: 136 (Figure 137), the C3E-7036 light chain variable region consisting of amino acid sequences from amino acid numbers 135-241 of the amino acid sequence represented by SEQ ID NO: 137 (Figure 138), the C3E-7085 light chain variable region consisting of amino acid sequences from amino acid numbers 135-241 of the amino acid sequence represented by SEQ ID NO: 138 (Figure 139), and the C3E-7085 light chain variable region consisting of amino acid sequences from amino acid numbers 135-243 of the amino acid sequence represented by SEQ ID NO: 139 (Figure 140). Examples of antibodies or antigen-binding fragments thereof include the 3E-7088 light chain variable region, the C3E-7093 light chain variable region consisting of amino acid sequences from amino acid numbers 135 to 243 of the amino acid sequence represented by SEQ ID NO: 140 (Figure 141), the C3E-7096 light chain variable region consisting of amino acid sequences from amino acid numbers 405 to 511 of the amino acid sequence represented by SEQ ID NO: 155 (Figure 151), the C3E-7096 light chain variable region consisting of amino acid sequences from amino acid numbers 410 to 516 of the amino acid sequence represented by SEQ ID NO: 156 (Figure 152), and the C3E-7097 light chain variable region consisting of amino acid sequences from amino acid numbers 410 to 516 of the amino acid sequence represented by SEQ ID NO: 157 (Figure 153).

[0145] Furthermore, more preferable antibodies or antigen-binding fragments containing CDRH1-CDRH3 and CDRL1-CDRL3 include: a combination of C3E-7034 heavy chain variable region and light chain variable region consisting of amino acids 2-119 and 135-243 represented by SEQ ID NO: 136 (Figure 137); a combination of C3E-7036 heavy chain variable region and light chain variable region consisting of amino acids 2-119 and 135-241 represented by SEQ ID NO: 137 (Figure 138); a combination of C3E-7078 heavy chain variable region and light chain variable region consisting of amino acids 2-119 and 135-243 represented by SEQ ID NO: 147 (Figure 143); a combination of C3E-7085 heavy chain variable region and light chain variable region consisting of amino acids 2-119 and 135-241 represented by SEQ ID NO: 138 (Figure 139); and an antibody with amino acid number 2 Examples of antibodies or antigen-binding fragments thereof include a combination of C3E-7088 heavy chain variable region and light chain variable region consisting of amino acids ~119 and 135~243, a combination of C3E-7093 heavy chain variable region and light chain variable region consisting of amino acid numbers 2~119 and 135~243 represented by SEQ ID NO: 140 (Figure 141), a combination of C3E-7096 heavy chain variable region and light chain variable region consisting of amino acid numbers 272~389 and 405~511 represented by SEQ ID NO: 155 (Figure 151), a combination of C3E-7096 heavy chain variable region and light chain variable region consisting of amino acid numbers 277~394 and 410~516 represented by SEQ ID NO: 156 (Figure 152), and a combination of C3E-7097 heavy chain variable region and light chain variable region consisting of amino acid numbers 277~394 and 410~516 represented by SEQ ID NO: 157 (Figure 153).

[0146] Furthermore, more preferable antibodies or antigen-binding fragments containing CDRH1-CDRH3 and CDRL1-CDRL3 include C3E-7034scFv, consisting of amino acid sequences from amino acid numbers 2-243 of the amino acid sequence represented by SEQ ID NO: 136 (Figure 137); C3E-7036scFv, consisting of amino acid sequences from amino acid numbers 2-241 of the amino acid sequence represented by SEQ ID NO: 137 (Figure 138); and C3E-7078, consisting of amino acid sequences from amino acid numbers 2-243 of the amino acid sequence represented by SEQ ID NO: 147 (Figure 143). Examples include scFv, C3E-7085scFv consisting of amino acid sequences from amino acid numbers 2 to 241 of the amino acid sequence represented by SEQ ID NO: 138 (Figure 139), C3E-7088scFv consisting of amino acid sequences from amino acid numbers 2 to 243 of the amino acid sequence represented by SEQ ID NO: 139 (Figure 140), C3E-7093scFv consisting of amino acid sequences from amino acid numbers 2 to 243 of the amino acid sequence represented by SEQ ID NO: 140 (Figure 141), and antibodies or antibody conjugate fragments containing any one of these scFvs. Preferred embodiments of CD3-specific scFv (also called "anti-CD3 scFv") include those with a FLAG-His tag attached to the carboxyl terminus (also simply called "tag adducts"). Suitable tag adducts 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), with C3E-7085 being a more preferred example.

[0147] A preferred example of the multispecific molecule of the present invention is a bispecific molecule. "Bispecificity" means the ability to bind to two different epitopes on the same molecule or to 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 further binds to CD3.

[0148] Examples of bispecific molecules of the present invention include those having the following structure (format).

[0149] In dual scFv type bispecific molecules, two scFv molecules that bind to different epitopes are linked to one of the dimer's Fc atoms by linkers, or directly linked without linkers. Alternatively, two scFv molecules that bind to different epitopes are linked to CH and CL respectively by linkers, and further linked to one of the dimer's Fc atoms by linkers. This bispecific molecule is a format in which Fc atoms, each containing a mutation that forms a heterodimer, are heteroassociated downstream of the two different scFv molecules. Dual scFv type bispecific molecules are called dual-type bispecific molecules, or simply dual-type molecules (Figure 6A(b)).

[0150] In the present invention, for example, a dual-type bispecific molecule consisting of anti-HLA-A2 / NY-ESO scFv and anti-CD3 scFv may be used.

[0151] Alternatively, the bispecific molecule of the present invention may be a bispecific molecule in which Fab and scFv, which bind to different epitopes, are linked via a linker to one side of a dimer Fc, with the Fab of the first antibody and the scFv of the second antibody linked to the other side. This bispecific molecule is in a format in which Fc containing mutations that form heterodimers are heteroassociated downstream of Fab and scFv, respectively. Such a bispecific molecule is called a hybrid bispecific molecule or hybrid type (Figure 6A(a)). In the present invention, for example, a hybrid type consisting of anti-HLA-A2 / NY-ESO Fab and anti-CD3scFv can be used.

[0152] Furthermore, the molecule may be a bispecific molecule in which the Fab of the first antibody and the scFv of the second antibody are linked to one of the dimer Fc via a linker. In this case, Fab may be linked to Fc and scFv may be linked to Fab, or scFv may be linked to Fc and Fab may be linked to scFv. Preferably, Fab is linked and scFv is linked to Fab. The linkage between Fab and scFv can be achieved by linking scFv to the variable region of Fab via a linker. The bispecific molecule is in a format in which an Fc molecule containing a mutation that forms a heterodimer is associated downstream of the linkage between scFv and Fab. Such a bispecific molecule is called an scFv-Fab-heterodimerFc type bispecific molecule or scFv-Fab-heterodimerFc type (Figure 6A(c)).

[0153] In the present invention, for example, an scFv-Fab-heterodimer Fc type consisting of anti-CD3scFv and anti-HLA-A2 / NY-ESO Fab may be used.

[0154] Furthermore, a taFv (Figure 3(c)) having a format in which two types of scFv, a first antibody and a second antibody, are linked by a linker may be directly bound to one of the dimer Fc molecules, either via a linker or without a linker. Such a bispecific molecule is called a taFv-heterodimer Fc type bispecific molecule or taFv-heterodimer Fc type (Figure 3(d)). This bispecific molecule is a format in which Fc molecules with mutations that form a heterodimer are heteroassociated downstream of the taFv. The linking order of the first antibody and the second antibody in the taFv is not limited, but if the linking order of the first antibody and the second antibody in the taFv is reversed, the first bispecific molecule is called a taFv(inversed)-heterodimer Fc type (also called taFv(inversed)-Fc type), whereas the first bispecific molecule is called a taFv-heterodimer Fc type.

[0155] Figure 6A(a) shows the structure of the Hybrid bispecific molecule, Figure 6A(b) shows the structure of the Dual bispecific molecule, and Figure 6A(c) shows the structure of the scFv-Fab-heterodimer Fc bispecific molecule. Additionally, Figure 3(a) shows the structure of scFv, Figure 3(b) shows the structure of Fab, Figure 3(c) shows the structure of taFv, Figure 3(d) shows the structure of the taFv-heterodimer Fc bispecific molecule, and Figure 3(e) shows the structure of the taFv-Fab-heterodimer Fc bispecific molecule. Furthermore, Figure 6B(a) shows the structure of the taFv-heterodimer Fc-type bispecific molecule (same as in Figure 3(d)), Figure 6B(b) shows the structure of the taFv(inversed)-heterodimer Fc-type bispecific molecule, Figure 6B(c) shows the structure of the first polypeptide contained in the taFv(inversed)-heterodimer Fc-type bispecific molecule, and Figure 6(d) shows the structure of the second polypeptide contained in the taFv(inversed)-heterodimer Fc-type bispecific molecule. The bispecific molecule of the present invention has a structure in which multiple polypeptides are associated.

[0156] In the present invention, for example, anti-HLA-A2 / NY-ESOscFv and anti-CD3scFv taFv may be used as taFv. The taFv-heterodimer Fc-type bispecific molecule preferably comprises (a) a first polypeptide comprising, in that order from the N-terminus to the C-terminus, an scFv that specifically binds to HLA / NY-ESO, an scFv that specifically binds to CD3, and an immunoglobulin Fc region (i), and a second polypeptide comprising an immunoglobulin hinge region and an Fc region (ii), and more preferably (b) the first polypeptide and the second polypeptide associated at the Fc region (i) and Fc region (ii). The Fc regions of the first polypeptide and the second polypeptide may contain mutations for heterodimer formation. An example of a taFv-heterodimer Fc-type bispecific molecule is shown in Figure 3(d). As shown in Figure 3(d), the Fc region (i) of the first polypeptide and the Fc region (ii) of the second polypeptide, shown in black, are bound together, indicating association between the first and second polypeptides. Figure 3(f) shows the first polypeptide, and Figure 3(g) shows the second polypeptide. For example, in Figure 3(d), the scFv shown in white is anti-HLA-A2 / NY-ESO scFv, and the scFv shown with a diagonal line to the upper right is anti-CD3 scFv.

[0157] The first polypeptide contained in the more preferred taFv-heterodimer Fc-type bispecific molecule of the present invention is the amino acid sequence from the 21st to the 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 85, the amino acid sequence from the 21st to the 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 87, the amino acid sequence from the 21st to the 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 88, the amino acid sequence from the 21st to the 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 89, the amino acid sequence from the 21st to the 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 90, the amino acid sequence from the 21st to the 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 91 The 511th amino acid sequence, the 21st to 511th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 92, the 21st to 511th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 93, the 21st to 511th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 94, the 21st to 511th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 95, the 21st to 511th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 96, the 21st to 511th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 86, the 21st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 149 The first polypeptide included in the taFv-heterodimer Fc-type bispecific molecule, which includes the amino acid sequence from the 1st to the 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 150, the amino acid sequence from the 20th to the 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 155, the amino acid sequence from the 20th to the 516th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 156, or the amino acid sequence from the 20th to the 516th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 157, is more preferable than SEQ ID NOs: 85, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, The first polypeptide contained in the taFv-heterodimer Fc-type bispecific molecule, which includes the amino acid sequence from position 529 to 745 of the amino acid sequence represented by 86, 149, 150, or 155, or the amino acid sequence from position 534 to 750 of the amino acid sequence represented by SEQ ID NO: 156 or 157, and which is even more preferable, includes the amino acid sequence from position 21 to 745 of the amino acid sequence represented by SEQ ID NO: 85, the amino acid sequence from position 21 to 745 of the amino acid sequence represented by SEQ ID NO: 87, and the amino acid sequence from position 21 to 745 of the amino acid sequence represented by SEQ ID NO: 88.It consists of the amino acid sequences from the 21st to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 89, the amino acid sequences from the 21st to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 90, the amino acid sequences from the 21st to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 91, the amino acid sequences from the 21st to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 92, the amino acid sequences from the 21st to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 93, the amino acid sequences from the 21st to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 94, the amino acid sequences from the 21st to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 95, the amino acid sequences from the 21st to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 96, the amino acid sequences from the 21st to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 86, the amino acid sequences from the 21st to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 149, the amino acid sequences from the 21st to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 150, or the amino acid sequences from the 20th to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 155. Alternatively, it consists of the amino acid sequence from position 20 to 750 of the amino acid sequence represented by SEQ ID NO: 156, or the amino acid sequence from position 20 to 750 of the amino acid sequence represented by SEQ ID NO: 157.

[0158] A preferred taFv-heterodimer Fc-type bispecific molecule of the present invention comprises a hinge region and mutant Fc derived from a human antibody, and an even more preferred taFv-heterodimer Fc-type bispecific molecule comprises the amino acid sequence from position 20 to 246 of the amino acid sequence shown in SEQ ID NO: 84. Among these, NYF-0016 is formed by the association of a first polypeptide consisting of amino acids 21 to 745 of the amino acid sequence represented by SEQ ID NO: 85 and a second polypeptide consisting of amino acids 21 to 246 of the amino acid sequence shown by SEQ ID NO: 84; NYF-0022 is formed by the association of a first polypeptide consisting of amino acids 21 to 745 of the amino acid sequence represented by SEQ ID NO: 87 and a second polypeptide consisting of amino acids 21 to 246 of the amino acid sequence shown by SEQ ID NO: 84; NYF-0023 is formed by the association of a first polypeptide consisting of amino acids 21 to 745 of the amino acid sequence represented by SEQ ID NO: 88 and a second polypeptide consisting of amino acids 21 to 246 of the amino acid sequence shown by SEQ ID NO: 84; NYF-0027 is formed by the association of a first polypeptide consisting of amino acids 21 to 745 of the amino acid sequence represented by SEQ ID NO: 89 and a second polypeptide consisting of amino acids 21 to 246 of the amino acid sequence shown by SEQ ID NO: 84; and a first polypeptide consisting of amino acids 21 to 745 of the amino acid sequence represented by SEQ ID NO: 90 and a second polypeptide NYF-0035 is formed by the association of the second polypeptide consisting of amino acids 21 to 246 of the amino acid sequence shown in sequence number 84, the first polypeptide consisting of amino acids 21 to 745 of the amino acid sequence shown in SEQ ID NO: 91, and the second polypeptide consisting of amino acids 21 to 246 of the amino acid sequence shown in SEQ ID NO: 84, the first polypeptide consisting of amino acids 21 to 745 of the amino acid sequence shown in SEQ ID NO: 92, and the second polypeptide consisting of amino acids 21 to 246 of the amino acid sequence shown in SEQ ID NO: 84 NYF-0045 is formed by the association of a second polypeptide consisting of the following: NYF-0045, NYF-0047 is formed by the association of a first polypeptide consisting of amino acids from the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 93 and a second polypeptide consisting of amino acids from the 21st to 246th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 84, NYF-0048 is formed by the association of a first polypeptide consisting of amino acids from the 21st to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 94 and a second polypeptide consisting of amino acids from the 21st to 246th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 84,NYF-0060 is formed by the association of a first polypeptide consisting of amino acids 21 to 745 of the amino acid sequence represented by SEQ ID NO: 95 and a second polypeptide consisting of amino acids 21 to 246 of the amino acid sequence shown by SEQ ID NO: 84, NYF-0061 is formed by the association of a first polypeptide consisting of amino acids 21 to 745 of the amino acid sequence represented by SEQ ID NO: 96 and a second polypeptide consisting of amino acids 21 to 246 of the amino acid sequence shown by SEQ ID NO: 84, NYF-0019, formed by the association of a second polypeptide consisting of 1, 2, 3, 4, 4, 5, 6, 7, 8, 9

[0159] Furthermore, NYZ-0038, formed by the association of a first polypeptide consisting of amino acids from the 20th to 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 155 and a second polypeptide consisting of amino acids from the 20th to 246th amino acid sequence of the amino acid sequence shown by SEQ ID NO: 84; NYZ-0082, formed by the association of a first polypeptide consisting of amino acids from the 20th to 750th amino acid sequence of the amino acid sequence of the amino acid sequence shown by SEQ ID NO: 156 and a second polypeptide consisting of amino acids from the 20th to 246th amino acid sequence of the amino acid sequence shown by SEQ ID NO: 84; and NYZ-0083, formed by the association of a first polypeptide consisting of amino acids from the 20th to 750th amino acid sequence of the amino acid sequence of the amino acid sequence shown by SEQ ID NO: 157 and a second polypeptide consisting of amino acids from the 20th to 246th amino acid sequence of the amino acid sequence shown by SEQ ID NO: 84, can be exemplified as preferred taFv-heterodimer Fc-type bispecific molecules of the present invention.

[0160] Of these, NYF-0023, NYF-0047, NYF-0048, NYF-0060, NYF-0061, NYZ-0038, NYZ-0082, and NYZ-0083 are particularly suitable due to their excellent biological activity, physical properties, etc.

[0161] Furthermore, the taFv of the first antibody may be directly bound to one of the Fc segments of the dimer, either via a linker or without a linker, and the Fab of either the first or second antibody may be directly bound to the other Fc segment, either via a linker or without a linker. This bispecific molecule is a format in which Fab is added upstream of the Fc region (ii) (blacked out) side of the taFv-heterodimer Fc type second polypeptide. Such a bispecific molecule is called a taFv-Fab-heterodimer Fc type bispecific molecule or taFv-Fab-heterodimer Fc type (Figure 3).

[0162] In the present invention, the taFv contained in the taFv-Fab heterodimer Fc-type bispecific molecule may be, for example, anti-HLA-A2 / NY-ESO scFv and anti-CD3 scFv taFv, and the Fab may be, for example, HLA / NY-ESO Fab.

[0163] The taFv-Fab-heterodimer Fc-type bispecific molecule preferably comprises (a) a first polypeptide comprising, in that order from the N-terminus to the C-terminus, scFv that specifically binds to human HLA / NY-ESO, scFv that specifically binds to CD3, and an immunoglobulin Fc region (i); a second polypeptide comprising an immunoglobulin heavy chain containing an Fc region (ii); and a third polypeptide comprising an immunoglobulin light chain. More preferably, (b) the second polypeptide and the third polypeptide are associated, and (c) the first polypeptide and the second polypeptide are associated at the Fc region (i) and Fc region (ii). An example of the taFv-Fab-heterodimer Fc-type bispecific molecule is shown in Figure 3(e), the first polypeptide in Figure 3(f), the second polypeptide in Figure 3(h), and the third polypeptide in Figure 3(i). As shown in Figure 3(e), a second polypeptide, consisting of an immunoglobulin heavy chain containing the Fc region (i) of the first polypeptide and the Fc region (ii) shown in black, is bound to the Fc region (i) of the first polypeptide and the Fc region (ii) of the second polypeptide, and further, an immunoglobulin light chain is bound to the second polypeptide. Such a suitable taFv-Fab-heterodimer Fc-type bispecific molecule can also be described as a taFv-heterodimer Fc-type bispecific molecule containing taFv and the Fc region of immunoglobulin, to which Fab is bound. Suitable amino acid sequences for the first polypeptide contained in the taFv-Fab heterodimer Fc-type bispecific molecule include the amino acid sequences from the 21st to the 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, and SEQ ID NO: 94.Examples include the amino acid sequences from the 21st to the 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 95, the amino acid sequences from the 21st to the 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 96, the amino acid sequences from the 21st to the 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 86, the amino acid sequences from the 21st to the 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 149, and the amino acid sequences from the 21st to the 511th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 150. More preferably, the first polypeptide contained in the taFv-Fab heterodimer Fc-type bispecific molecule is the amino acid sequence from the 21st to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 85, the amino acid sequences from the 21st to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 87, the amino acid sequences from the 21st to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 88, and the amino acid sequences from the 21st to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 89. It consists of the following amino acid sequences: the amino acid sequences from the 21st to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 90, the amino acid sequences from the 21st to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 91, the amino acid sequences from the 21st to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 92, the amino acid sequences from the 21st to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 93, the amino acid sequences from the 21st to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 94, the amino acid sequences from the 21st to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 95, the amino acid sequences from the 21st to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 96 and the amino acid sequences from the 21st to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 86, the amino acid sequences from the 21st to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 149, or the amino acid sequences from the 21st to the 745th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 150.

[0164] The second polypeptide contained in the preferred taFv-Fab-heterodimer Fc-type bispecific molecule of the present invention comprises a variable region, a CH1 region, and a hinge region of a human antibody or humanized antibody heavy chain, as well as a mutant Fc. A more preferred second polypeptide contained in the taFv-Fab-heterodimer Fc-type bispecific molecule comprises the amino acid sequence from the 20th to the 242nd amino acid sequence represented by SEQ ID NO: 99.

[0165] A preferred third polypeptide contained in the taFv-Fab-heterodimer Fc-type bispecific molecule of the present invention comprises a variable region and a constant region of a human antibody or a humanized antibody light chain, and a more preferred third polypeptide contained in the taFv-Fab-heterodimer Fc-type bispecific molecule comprises amino acids 21 to 131 of the amino acid sequence represented by SEQ ID NO: 100.

[0166] In such a suitable taFv-Fab-heterodimer Fc-type bispecific molecule, the variable region and CH1 region of the second polypeptide, as well as the third polypeptide, constitute Fab, and the suitable Fab is the Fab of an anti-HLA / NY-ESO antibody, for example, the Fab of NYA-0001.

[0167] In the present invention, the scFv contained in the scFv-Fab heterodimer Fc-type bispecific molecule may be, for example, anti-HLA-A2 / NY-ESO scFv or anti-CD3 scFv, and the Fab may be, for example, HLA / NY-ESO Fab or anti-CD3 Fab.

[0168] The scFv-Fab-heterodimer Fc-type bispecific molecule preferably comprises (a) a first polypeptide comprising, in that order from the N-terminus to the C-terminus, scFv that specifically binds to human HLA / NY-ESO, a variable region and constant region CH1 of an antibody heavy chain that specifically binds to CD3, and an immunoglobulin Fc region (i); a second polypeptide comprising an immunoglobulin hinge region and an Fc region (ii); and a third polypeptide comprising an antibody light chain consisting of a variable region and a constant region. More preferably, (b) the first polypeptide and the second polypeptide associate at the Fc region (i) and Fc region (ii), and the first polypeptide associates with the third polypeptide (and its antibody light chain) at the variable region and constant region CH1 of the antibody heavy chain. The Fc regions of the first and second polypeptides may be wild-type or may contain mutations that form a heterodimer. An example of an scFv-Fab-heterodimer Fc-type bispecific molecule is shown in Figure 6A(c). The right half of Figure 6A(c) shows the first and third polypeptides, and the left half shows the second polypeptide. As shown in Figure 6A(c), the Fc region (i) of the first polypeptide and the Fc region (ii) of the second polypeptide, shown in black, associate, and the first and third polypeptides associate. For example, in Figure 6A(c), the scFv represented by the upper right diagonal line is anti-HLA-A2 / NY-ESO scFv, and the Fab represented by white, checkerboard, and horizontal lines is anti-CD3 Fab.

[0169] Suitable examples of amino acid sequences included in the first polypeptide of a scFv-Fab-heterodimer Fc-type bispecific molecule include the amino acid sequences from positions 21 to 394 of the amino acid sequence represented by SEQ ID NO: 160, and more preferably, the amino acid sequences from positions 20 to 724.

[0170] Furthermore, other amino acid sequences included in the first polypeptide contained in a suitable scFv-Fab-heterodimer Fc-type bispecific molecule include the amino acid sequences from positions 21 to 389 of the amino acid sequence represented by Sequence ID No. 197, and more preferably, the amino acid sequences from positions 20 to 719. Furthermore, other amino acid sequences included in the first polypeptide contained in a suitable scFv-Fab-heterodimer Fc-type bispecific molecule include the amino acid sequences from positions 21 to 389 of the amino acid sequence represented by SEQ ID NO: 198, and more preferably, the amino acid sequences from positions 20 to 719.

[0171] A preferred scFv-Fab-heterodimer Fc-type bispecific molecule of the present invention comprises a hinge region derived from a human antibody and a mutant Fc, and an even more preferred scFv-Fab-heterodimer Fc-type bispecific molecule comprises the amino acid sequence from position 20 to 246 of the amino acid sequence shown in SEQ ID NO: 84.

[0172] The third polypeptide contained in the preferred scFv-Fab-heterodimer Fc-type bispecific molecule of the present invention comprises a light chain derived from a human antibody. More preferred examples of the third polypeptide contained in the scFv-Fab-heterodimer Fc-type bispecific molecule include the amino acid sequence from position 21 to 127 of the amino acid sequence shown in SEQ ID NO: 161, and more preferably the amino acid sequence from position 21 to 233.

[0173] Of these, NYZ-1010, formed by the association of a first polypeptide consisting of amino acids from positions 20 to 724 of the amino acid sequence represented by SEQ ID NO: 160, a second polypeptide consisting of amino acids from positions 20 to 246 of the amino acid sequence shown by SEQ ID NO: 84, and a third polypeptide consisting of amino acids from positions 21 to 233 of the amino acid sequence shown by SEQ ID NO: 161, can be exemplified as a preferred scFv-Fab-heterodimer Fc-type bispecific molecule of the present invention.

[0174] Furthermore, NYZ-1007, formed by the association of a first polypeptide consisting of amino acids 20 to 719 of the amino acid sequence represented by SEQ ID NO: 197, a second polypeptide consisting of amino acids 20 to 246 of the amino acid sequence shown by SEQ ID NO: 84, and a third polypeptide consisting of amino acids 21 to 233 of the amino acid sequence shown by SEQ ID NO: 161, can also be exemplified as a preferred scFv-Fab-heterodimer Fc-type bispecific molecule of the present invention.

[0175] Furthermore, NYZ-1017, formed by the association of a first polypeptide consisting of amino acids 20 to 719 of the amino acid sequence represented by SEQ ID NO: 198, a second polypeptide consisting of amino acids 20 to 246 of the amino acid sequence shown by SEQ ID NO: 84, and a third polypeptide consisting of amino acids 21 to 233 of the amino acid sequence shown by SEQ ID NO: 161, can also be exemplified as a preferred scFv-Fab-heterodimer Fc-type bispecific molecule of the present invention.

[0176] One, two, or three or more peptides contained in the bispecific molecules of the present invention may be the aforementioned "deletion molecules," that is, they may have mutations (including deletions) of one or two (or more) amino acids at their carboxyl terminus, particularly at the carboxyl terminus derived from the antibody heavy chain. 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, may be the 724th Lys of SEQ ID NO: 160, the 723rd Gly with one amino acid deleted, or a mixture containing these. Similarly, the carboxyl terminus of the amino acid sequence of the second polypeptide contained in the preferred scFv-Fab-heterodimer Fc-type bispecific molecules of the present invention may be the 246th Lys of SEQ ID NO: 84, the 245th Gly with one amino acid deleted, or a mixture containing these.

[0177] The scFv and Fab contained in the bispecific molecule of the present invention are preferably scFv and Fab of a humanized antibody or a human antibody, and Fc is preferably Fc of a human antibody.

[0178] In the variable regions included in the bispecificity molecule of the present invention, the heavy chain variable region and the light chain variable region may be bound in that order from the amino-terminal side of the antibody, or the light chain variable region and the heavy chain variable region may be bound in that order. A linker may be present between the two variable regions (optional). A glycine residue may also be present at the amino terminus of the variable region on the amino-terminal side (optional). In the tandem scFv type bispecificity molecule, a linker, a FLAG tag, and / or a His tag may be bound to the carboxyl terminus of the variable region on the carboxyl-terminal side (optional). As one preferred embodiment, an example can be given in which the heavy chain variable region, the first linker, the light chain variable region, the second linker, the FLAG tag, and the His tag are bound in that order from the amino terminus.

[0179] The linker may include single-chain polypeptides or single-chain oligopeptides, or synthetic products such as PEG, nucleotides, sugar chains, or compounds. Furthermore, any known linker that connects two polypeptides is not particularly limited and can be used.

[0180] Linker lengths are typically 5 to 30 amino acids, for example, in the case of peptide linkers. When a bispecific molecule contains multiple linkers, peptide linkers of the same length may be used for all of them, or peptide linkers of different lengths may be used.

[0181] Examples of peptide linkers include the repetition of (Gly·Gly·Gly·Gly·Ser) (SEQ ID NO: 161), but these may also have one to several amino acid residues different from Gly and Ser attached to them.

[0182] Of the structures (formats) that the multispecific antibodies, particularly bispecific antibodies, of the present invention can adopt as described above, the preferred ones are the taFv-heterodimer Fc type, the taFv-Fab-heterodimer Fc type, and the scFv-Fab-heterodimer Fc type, with the taFv-heterodimer Fc type being more preferred. The type in which anti-HLA / NY-ESO scFv and anti-CD3 scFv are located in that order from the N-terminus to the C-terminus (taFv-heterodimer Fc type) is even more preferred than the type in which they are located in the reverse order (taFv(inversed)-heterodimer Fc type) (Example 11, etc.). Another more preferred type is the scFv-Fab-heterodimer Fc type.

[0183] The present invention also includes molecules that contain an amino acid sequence encoded by a nucleotide sequence contained in a polynucleotide that hybridizes under stringent conditions with a complementary chain of a polynucleotide containing a nucleotide sequence encoding an amino acid sequence contained in the molecule of the present invention, and that bind to HLA / NY-ESO, and preferably further bind to CD3.

[0184] The present invention also includes molecules that contain an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to the amino acid sequence contained in the molecule of the present invention, and that bind to HLA / NY-ESO, and preferably further bind to CD3.

[0185] The antibodies of the present invention, their conjugated fragments, and multispecific antibodies containing them possess excellent biological activity, physicochemical properties (hereinafter referred to as "physical properties"), safety, and pharmacokinetics. (a) Examples of biological activity or its indicators include antigen-binding activity, in vitro cytotoxic activity, and in vivo antitumor activity. For example, the dissociation constant (KD value) for HLA / NY-ESO is 100 nM or less or 50 nM or less, preferably 20 nM or less or 10 nM or less, and more preferably 5 nM or less. Also, for example, the cytotoxic activity exhibited against the endogenous human NY-ESO expressing cell line U266B1 and / or NCI-H1703 using human peripheral blood mononuclear cells as effector cells is EC 50 The value is 20 nM or less, preferably 10 nM or less, and more preferably 5 nM or less (the method described in Example 8 can be used as an example for measuring and calculating in vitro cytotoxic activity, but is not limited thereto). Furthermore, for example, 6 × 10⁶ human squamous cell lung cancer cell line NCI-H1703 7 0.1 mL of cell / mL suspension was transplanted subcutaneously into NOG mice, and after 4 days, 3.75 × 10⁶ human peripheral blood mononuclear cells were obtained. 7When a cell / mL suspension was transplanted into the tail vein at a dose of 0.2 mL, and the antibody was administered once a week for three doses starting 14 days later, the tumor volume was measured. The tumor growth inhibitory activity compared to the control group administered with the solvent was 50% or more, preferably 75% or more, and more preferably 90% or more (the method described in Example 9 can be used as an example for measuring and calculating in vivo antitumor activity, but is not limited thereto). (b) Impurities contained in biopharmaceuticals are related to the safety of the drug, so it is necessary to appropriately specify and control whether or not they increase during manufacturing and storage. Among these, HMWS (aggregates) is one of the major impurities and is related to immunogenicity risk and decreased drug efficacy, so it is an item that should be controlled particularly strictly. Impurity control should be evaluated not only during manufacturing, but also during the manufacturing process and over time (whether or not they increase) after manufacturing. Since the expiration date of the drug is set based on the results of long-term stability tests, antibodies that are stable over time can be set to have a longer expiration date. Therefore, the physicochemical properties in the present invention that serve as indicators when selecting suitable antibodies for biopharmaceuticals include acid resistance (inhibition of HMWS production, etc.) and solution stability (inhibition of HMWS production, etc.). Other indicators include high yield in cultures of recombinant cells obtained by introducing genes encoding the amino acid sequences contained therein into host cells suitable for the production of the antibodies of the present invention, their binding fragments, or molecules containing them, such as Expi293F cells. Suitable antibodies of the present invention having these physicochemical properties, their antigen-binding fragments, and multispecific antibodies containing them can: expose their solutions to acidic conditions, making it possible or easy to carry out their manufacture, such as chromatography including protein A and ion exchange, and virus inactivation; since the generation of HMWS is kept low even when they are in solution, it is possible or easy to carry out their manufacture, formulation, distribution and storage of pharmaceuticals containing them; and they can be produced efficiently.Regarding acid resistance, for example, the HMWS content calculated by measuring HMWS by size exclusion chromatography after keeping the solution at pH 3.5, room temperature, and incubation for 1 hour is 5% or less, preferably 2% or less, and more preferably 1% or less (the method described in Example 19-1 can be used as an example of measuring and calculating the HMWS content for acid resistance evaluation, but is not limited thereto). Regarding solution stability, for example, the HMWS content calculated by dissolving the solution in pH 6.0 consisting of 25 mM histidine and 5% sorbitol to a concentration of 25 mg / ml, storing it at 25°C for 6 days, and then measuring HMWS by size exclusion chromatography is 20% or less, preferably 10% or less (the method described in Example 19-2 can be used as an example of measuring and calculating the HMWS content for solution stability evaluation, but is not limited thereto). Regarding the measurement and calculation of production efficiency or yield, the methods described in Examples 20 and 21 can be used as examples, but is not limited thereto. (c) Examples of safety or its indicators include antigen recognition characteristics, findings at administration, etc. For example, it recognizes multiple amino acids on the wild-type NY-ESO peptide and does not bind to homologous peptides that have amino acid sequences similar to but not identical to the wild-type NY-ESO peptide, resulting in a low risk of side effects due to off-target effects. Furthermore, it exhibits low immunogenicity in ISPRI web-based immunogenicity screening (EpiVax, Inc.), suggesting a low risk of side effects such as cytokine production caused by anti-antibodies. Additionally, when NYF-0023, NYF-0045, NYF-0047, NYF-0048, NYF-0060, NYF-0061, NYZ-0082, or NYZ-1010, which are included in the bispecific antibodies of the present invention, were administered to Balb / c mice, no problematic findings were observed regarding the blood half-life, and no weight loss or other significant toxic findings were observed. Furthermore, when NYZ-0082 or NYZ-1010 was administered as a single dose to cynomolgus monkeys, no problematic findings were observed regarding the blood half-life, and no administration-related changes were observed in general condition, body weight, food intake, body temperature, or plasma cytokine levels. (d) Examples of pharmacokinetics or their indicators include the blood half-life.For example, when several bispecific antibodies obtained in this invention were administered to Balb / c mice or cynomolgus monkeys, no problematic findings were observed regarding the blood half-life. The antibodies, their conjugated fragments, and molecules of this invention, possessing such excellent biological activity, physicochemical properties, safety, and pharmacokinetics, can be suitably incorporated into pharmaceutical compositions. Suitable antibodies or antigen-binding fragments of the present invention having the antigen-binding activity described in (a) and the properties described in (b) include, but are not limited to, NYA-1143, NYA-1163, NYA-2023, NYA-2027, NYA-2035, NYA-2044, NYA-2045, NYA-2047, NYA-2048, NYA-2060, NYA-2031, NYA-2047, NYA-2061, NYA-2143, and NYA-3061, and more preferably NYA-2047, NYA-2061, NYA-2143, and NYA-3061. Furthermore, suitable multispecific antibodies of the present invention having the properties described in (a) to (d) include NYF-0016, NYF-0019, NYF-0022, NYF-0023, NYF-0027, NYF-0035, NYF-0044, NYF-0045, NYF-0047, NYF-0048, NYF00058, NYF-0060, NYF-0061, NYZ-0038, NYZ-0082, NYZ-0088, and NYZ-1010, and more preferably NYF-0061, NYZ-0038, NYZ-0082, NYZ-0088, NYZ-1007, NYZ-1010, and NYZ-1017, but are not limited to these.

[0186] In the present invention, the "site" to which an antibody binds, that is, the "site" recognized by the antibody, refers to a partial peptide or partial higher-order structure on an antigen to which the antibody binds or recognizes. In the present invention, such a site is also called an epitope or antibody binding site. Examples of sites 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, a partial higher-order structure, and the like.

[0187] The present invention also includes "antibodies or their binding fragments that bind to the same site as the antibody or binding fragment of the present invention." "Antibodies that bind to the same site as a given antibody" means other antibodies that bind to the site on the antigen molecule recognized by that antibody. If a second antibody binds to a partial peptide or partial three-dimensional structure on the antigen molecule to which the first antibody binds, it can be determined that the first and second antibodies bind to the same site. If the first antibody of the present invention has the antigen-binding activity described in (a) above, there is a very high probability that a second antibody that binds to the same site on HLA / NY-ESO will have similar activity, and such a second antibody is also included in the present invention. Furthermore, antibodies that compete with the first antibody of the present invention for binding to HLA / NY-ESO are also included in the present invention if they have the antigen-binding activity described in (a). Antibodies that bind to a site on HLA / NY-ESO recognized by such monoclonal antibodies of the present invention, antibodies that compete with the monoclonal antibodies of the present invention for binding to HLA / NY-ESO, and their binding fragments preferably have one or more of the in vitro cytotoxic activity and in vivo antitumor activity described in (a) and the properties described in (b) to (d), more preferably three or more of them, and optimally all of them.

[0188] The antibody binding site can be determined by methods well known to those skilled in the art, such as immunoassays. For example, a series of peptides can be prepared by appropriately removing the C-terminus or N-terminus of the amino acid sequence of an antigen, and the reactivity of the antibody to these peptides can be examined to determine the general recognition site. After that, the binding site can be determined by synthesizing even shorter peptides and examining the reactivity of the antibody to these peptides. Alternatively, for example, the binding site can be determined by deleting a specific site or region from the amino acid sequence of an antigen or antigen fragment peptide, substituting it with another amino acid sequence, or introducing a mutation into the amino acid sequence, and then examining the reactivity of the antibody to these peptides. Antigen fragment peptides can be prepared using techniques such as genetic engineering and peptide synthesis.

[0189] When an antibody binds to or recognizes a partial higher-order structure of an antigen, the binding site of such antibody can be determined by identifying amino acid residues on the antigen adjacent to the antibody using X-ray crystallography. For example, by binding an antibody or fragment thereof to an antigen or fragment thereof, crystallizing the crystals, and performing structural analysis, amino acid residues on the antigen that have an interaction distance with the antibody can be identified. The interaction distance is 8 Å or less, preferably 6 Å or less, and more preferably 4 Å or less. One or more amino acid residues having such an interaction distance with the antibody can constitute the antigen-binding site (epitope) of the antibody. If there are two or more such amino acid residues, each amino acid does not have to be adjacent to one another in its primary sequence.

[0190] The anti-HLA / NY-ESO antibody or its binding fragment of the present invention specifically recognizes multiple amino acids present in the amino acid sequence of HLA / NY-ESO. Antibodies or their binding fragments that recognize these multiple amino acids, compete with the antibody or its binding fragment of the present invention for binding to HLA / NY-ESO, or have an interaction distance with these multiple amino acids are also included in the present invention. Furthermore, multispecific antibodies containing such antibodies or their binding fragments are also included in the present invention.

[0191] 6. Pharmaceutical Compositions The present invention encompasses anticancer agents that include the anti-HLA / NY-ESO antibody or a multispecific molecule that binds to HLA / NY-ESO as an active ingredient.

[0192] The anticancer agent of the present invention can be used against one or more types of cancer selected from carcinomas, sarcomas, lymphomas, leukemia, myeloma, germ cell tumors, brain tumors, carcinoid tumors, neuroblastomas, retinoblastomas, and nephroblastomas. Specifically, carcinomas include kidney cancer, melanoma, squamous cell carcinoma, basal cell carcinoma, conjunctival cancer, oral cancer, laryngeal cancer, pharyngeal cancer, thyroid cancer, lung cancer (non-small cell lung cancer (adenocarcinoma, squamous cell carcinoma, large cell carcinoma), small cell lung cancer), breast cancer, esophageal cancer, 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, and vaginal cancer, while sarcomas include liposarcoma, angiosarcoma, chondrosarcoma, rhabdomyosarcoma, and Ewing's sarcoma. Examples of uterine tumors include osteosarcoma, undifferentiated pleomorphic sarcoma, myxoid fibrosarcoma, malignant peripheral schwannoma, retroperitoneal sarcoma, synovial sarcoma, uterine sarcoma, gastrointestinal stromal tumor, leiomyosarcoma, and epithelioid sarcoma. Lymphomas include B-cell lymphoma, T- and NK-cell lymphoma, and Hodgkin lymphoma. Leukemias include myeloid leukemia, lymphocytic leukemia, myeloproliferative disorders, and myelodysplastic syndromes. Myelomas include multiple myeloma. Germ cell tumors include testicular cancer and ovarian cancer. Brain tumors include glioma and meningioma.

[0193] The anticancer agent of the present invention may include a therapeutically effective amount of 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 depending on the type of target disease and the form of drug administration. The method of administration of the anticancer agent of the present invention can be appropriately selected, but for example, it can be administered by injection, and local injection, intraperitoneal administration, selective intravenous injection, intravenous injection, subcutaneous injection, organ perfusion fluid injection, etc., can be employed. Furthermore, the injection solution can be formulated using a carrier consisting of a salt solution, glucose solution, a mixture of saline and glucose solution, various buffers, etc. Alternatively, it may be formulated in powder form and mixed with the liquid carrier at the time of use to prepare the injection solution.

[0194] Other administration methods can be appropriately selected along with the development of the formulation. For example, in the case of oral administration, oral liquids, powders, pills, capsules, and tablets can be used. In the case of oral liquids, oral liquid preparations such as suspensions and syrups can be manufactured using water, sugars such as sucrose, sorbitol, and fructose, glycols such as PEG, oils such as sesame oil and soybean oil, preservatives such as alkyl parahydroxybenzoate, and flavors such as strawberry and peppermint. Powders, pills, capsules, and tablets can be formulated using excipients such as lactose, glucose, sucrose, and mannitol, disintegrants such as starch and sodium arginate, lubricants such as magnesium stearate and talc, binders such as polyvinyl alcohol, hydroxypropyl cellulose, and gelatin, surfactants such as fatty acid esters, and plasticizers such as glycerin. Tablets and capsules are preferred unit administration forms in the composition of this invention because they are easy to administer. Solid manufacturing carriers are used when manufacturing tablets and capsules.

[0195] The amount of anti-HLA / NY-ESO antibody or multispecific molecule that binds to HLA / NY-ESO, which is effective for treatment, should be adjusted according to the nature of the condition being treated, the patient's age and condition, and ultimately determined by the physician. For example, it may be 0.0001 mg to 100 mg per kg of body weight per dose. The prescribed dosage may be administered once every 1 to 180 days, or in divided doses of 2, 3, 4 or more times per day at appropriate intervals.

[0196] The present invention also encompasses polynucleotides containing amino acid sequences included in the anti-HLA / NY-ESO antibody or multispecific molecule that binds to HLA / NY-ESO, vectors containing the polynucleotide, cells containing the polynucleotide or vector, and pharmaceutical compositions containing any of the polynucleotide, vector, or cells as active ingredients. Such pharmaceutical compositions are preferably anticancer agents.

[0197] Furthermore, the pharmaceutical composition of the present invention can be used in combination with other agents. These other agents are not limited to chemotherapeutic agents, radiotherapy agents, biopharmaceuticals, etc., and may be administered or applied simultaneously with or on a different schedule from the pharmaceutical composition of the present invention, as a single formulation containing the pharmaceutical composition of the present invention, or as two or more different formulations or therapies. [Examples]

[0198] The present invention will be described in more detail below in the following examples, but the present invention is not limited to these examples.

[0199] In the following examples, unless otherwise specified, each gene manipulation procedure was performed according to the methods described in "Molecular Cloning" (by Sambrook, J., Fritsch, EF, and Maniatis, T., published by Cold Spring Harbor Laboratory Press in 1989) and other experimental manuals used by those skilled in the art, or, if commercially available reagents or kits were used, according to the instructions of the commercially available product. Primer synthesis necessary for gene synthesis and vector construction was outsourced as needed (FASMAC Corporation, Thermo Fisher Scientific, and Eurofins Genomics).

[0200] (Example 1) Acquisition of anti-HLA / NY-ESO antibodies derived from a human antibody phage library 1)-1 Preparation of HLA / NY-ESO antigen proteins HLA-A *Each inclusion body prepared from Escherichia coli (BL21(DE3), Agilent Technologies) expressing the truncated form of 0201 (GenBank:ASA47534.1) (with added biotin ligase recognition sequence: SEQ ID NO: 33) and β2-microglobin (UniProtKB-P61769: SEQ ID NO: 34), along with NY-ESO peptide: SLLMWITQC (SEQ ID NO: 1 in the sequence listing), was refolded at high dilution, and then HLA-A was detected using a gel filtration column (Superdex 200 10 / 300, GE Healthcare). * We prepared the 0201 / β2-microglobin / NY-ESO peptide complex (hereinafter referred to as HLA / NY-ESO).

[0201] As a negative control antigen, MAGEC-1 peptide:ILFGISLREV (sequence number 2 in the sequence listing) was similarly refolded and HLA-A * The 0201 / β2-microglobin / MAGEC-1 peptide complex (hereinafter referred to as HLA / MC1) was prepared. Furthermore, the prepared HLA / NY-ESO and HLA / MC1 were biotinylated with E. coli biotin ligase, and then fractionated using a gel filtration column (Superdex 200 10 / 300, GE Healthcare) to prepare each biotinylated protein.

[0202] 1)-2 Isolation of scFv with binding ability to HLA / NY-ESO scFv that bind to HLA / NY-ESO were isolated from a human antibody phage library. First, phages were added to Dynabeads Streptavidin M-280 (Thermo Fisher Scientific) with biotinylated HLA / MC1 immobilized on the phase, and phages that did not bind were collected. Next, Dynabeads Streptavidin M-280Ag with biotinylated HLA / NY-ESO immobilized on the phase was added, and phages that did not bind were removed by washing using a magnetic stand (DynaMag-2, Thermo Fisher Scientific).

[0203] Subsequently, phages bound to HLA / NY-ESO were used to infect Escherichia coli (XL-1 Blue, Agilent Technologies), and the phages bound to HLA / NY-ESO were recovered and amplified. After a total of three panning cycles, the polyclonal phagemides were transferred to an E. coli expression vector to which FLAG tags and His tags were added to the carboxyl terminus of scFv. The E. coli were then transformed, and scFv was expressed in the presence of IPTG (Isopropyl-β-D-thiogalactopyranoside) (Sigma-Aldrich), and the cells were subjected to ELISA screening.

[0204] 1)-3 Screening of HLA / NY-ESO-conjugated scFv by ELISA 50 μL of NeutrAvidin (Life Technologies), diluted to 1 μg / mL in PBS (0.01 M phosphate-buffered saline containing 0.138 M sodium chloride and 0.0027 M potassium chloride (pH 7.4), Sigma-Aldrich), was added to each 384-well Maxi-sorp plate (Black, Nunc) and allowed to stand overnight at 4°C to solidify. After washing three times with PBS (ELISA buffer) containing 0.05% Tween-20 (Bio-Rad), biotinylated HLA / NY-ESO, also used in Example 1)-2, diluted to 1 μg / mL in PBS, was added and shaken at room temperature for 1 hour. After washing three times with ELISA buffer, the mixture was blocked with Blocker Casein (Thermo Fisher Scientific) and washed three times with ELISA buffer. Subsequently, a culture of E. coli expressing scFv was added and reacted at room temperature for 2 hours. After washing three times with ELISA buffer, 50 μL of horseradish peroxidase (HRP)-labeled anti-FLAG antibody (Sigma-Aldrich), diluted 5000-fold with ELISA buffer, was added and the mixture was reacted at room temperature for 1 hour. After washing five times with ELISA buffer, SuperSignal Pico ELISA Chemiluminescent substrate (Thermo Fisher Scientific) was added, and chemiluminescence after 10 minutes was measured using a plate reader (Envision 2104 Multilabel Reader, Perkin Elmer) to isolate ELISA-positive clones that bound to HLA / NY-ESO.

[0205] 1)-4 Determination of the nucleotide and amino acid sequences of ELISA-positive clone NY-R119 From the ELISA-positive clones obtained in 1)-3, NY-R119 was selected as an scFv with strong binding ability to HLA / NY-ESO and excellent recognition specificity. The nucleotide sequences of the heavy chain and light chain variable region of NY-R119 were analyzed using the Dye Terminator method (BigDye® Terminator v3.1, Thermo Fisher Scientific). The primer sequences used for sequence analysis are as follows. Primer A:5'-CTCTTCGCTATTACGCCAGCTGGCGA-3' (Sequence ID 3 in the sequence listing (Figure 10)) Primer B:5'-ATAACAATTTCACACAGGAAACAGCTATGA-3' (Sequence ID 4 in the sequence listing (Figure 11))

[0206] The nucleotide sequence of the cDNA encoding the variable region of the heavy chain of NY-R119, which 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).

[0207] The nucleotide sequence of the cDNA encoding the variable region of the light chain of NY-R119, which 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).

[0208] The CDR sequences of NY-R119 in the IMGT CDR definition are as follows: CDRH1 is sequence number 54 (Figure 61), CDRH2 is sequence number 55 (Figure 61), CDRH3 is sequence number 56 (Figure 61), CDRL1 is sequence number 57 (Figure 61), CDRL2 is shown as a DNN (Figure 61), and CDRL3 is sequence number 59 (Figure 61).

[0209] 1)-5 Production of NYA-0001 1)-5-1 Construction of NYA-0001 expression vector An expression vector for NY-R119 for mammalian cell culture was constructed for the preparation of various evaluation samples. The NY-R119 expressed in mammalian cell cultures was named NYA-0001. The scFv portion, variable regions of the heavy and light chains, and CDRH1-3 and CDRL1-3 of NY-R119 and NYA-0001 have identical amino acid sequences. The DNA fragment encoding NYA-0001 was inserted into a mammalian cell expression vector with a pcDNA3.3 (Thermo Fisher Scientific) backbone using the In-Fusion HD Cloning Kit (CLONTECH) to construct the NYA-0001 expression vector.

[0210] 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 sequence number 69 (Figure 70) of the sequence listing. Furthermore, from the above nucleotide sequence, the full-length amino acid sequence of NYA-0001 encoded by this sequence was the amino acid sequence shown in sequence number 70 (Figure 71). In this sequence, amino acid sequences 1 to 19 are the signal sequence, amino acid sequences 21 to 266 are NYA-0001, and amino acid sequences 267 to 292 are the Flag-His tag. In addition, amino acid sequences 46 to 53 are CDRH1, amino acid sequences 71 to 78 are CDRH2, and amino acid sequences 117 to 129 are CDRH3. Furthermore, the amino acid sequence from position 181 to 188 is CDRL1, the amino acid sequence from position 206 to 208 is CDRL2, and the amino acid sequence from position 245 to 256 is CDRL3.

[0211] 1)-5-2 NYA-0001 Expression and Purification Expi293F cells (Thermo Fisher Scientific) were subcultured and cultured according to the manual. Expi293F cell culture medium in the logarithmic growth phase was placed in 2.5 × 10⁻⁶ cells. 6The NYA-0001 expression vector was diluted with Expi293 Expression Medium (Thermo Fisher Scientific) to a concentration of cells / mL and used for NYA-0001 production. 0.3 mg of NYA-0001 expression vector and 0.9 mg of Polyethyleneimine (Polyscience #24765) were added to 20 mL of Opti-Pro SFM medium (Thermo Fisher Scientific), gently mixed, and allowed to stand for 5 minutes before being added to Expi293F cells. The culture supernatant was obtained by culturing at 37°C in an 8% CO2 incubator for 6 days with shaking at 135 rpm, and filtered through a 0.2 μm filter (Millipore) to obtain the NYA-0001 culture supernatant. Purification was performed by eluting with Ni Sepharose excel (GE Healthcare), followed by concentration, and then filtration using a gel filtration column (Superdex 200 Increase, GE Healthcare) equilibrated with 25 mM Histidine, 300 mM NaCl, 5% Sorbitor, and pH 6.0. The purified protein sample was subjected to analytical size exclusion chromatography (SEC) to determine purity and concentration, and then used for various evaluations.

[0212] (Example 2) Production of the NYA-0001 mutant 2)-1 Acquisition of the NYA-0001 variant Phage libraries were constructed using either an error-prone-based library method (introducing mutations using PCR with the NYA-0001 gene as a template) or an oligo-based library method (synthesizing oligomers in which each residue of the CDR was randomly mutated into 20 different amino acids). High-binding-ability clones were screened, and NYA-0060, NYA-0068, and NYA-0082 were obtained as high-binding-ability mutants, and their nucleotide sequences were determined.

[0213] The nucleotide sequence of the cDNA encoding the variable region of the heavy chain 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).

[0214] The nucleotide sequence of the cDNA encoding the variable region of the light chain 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).

[0215] The nucleotide sequence of the cDNA encoding the variable region of the heavy chain of NYA-0068 is shown in SEQ ID NO: 13 (Figure 20), and the amino acid sequence is shown in SEQ ID NO: 14 (Figure 21).

[0216] The nucleotide sequence of the cDNA encoding the variable region of the light chain 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).

[0217] The nucleotide sequence of the cDNA encoding the variable region of the heavy chain of NYA-0082 is shown in SEQ ID NO: 17 (Figure 24), and the amino acid sequence is shown in SEQ ID NO: 18 (Figure 25).

[0218] The nucleotide sequence of the cDNA encoding the variable region of the light chain 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).

[0219] 2)-2 Creation of high-binding-ability mutants based on mutation sites and combinations identified from NYA-0060, NYA-0068, and NYA-0082 DNA fragments encoding NYA-1163 and NYA-2023, each possessing the mutation sites of NYA-0060 and NYA-0068 respectively, were inserted into a mammalian cell expression vector with pcDNA3.3 (Thermo Fisher Scientific) as the backbone using the In-Fusion HD Cloning Kit (CLONTECH), thereby constructing a mammalian cell scFv expression vector.

[0220] Furthermore, NYA-2027, NYA-1143, and NYA-2143 were designed using mutant sites and combinations thereof identified from NYA-0060, NYA-0068, and NYA-0082. 1)-5 Site-directed mutagenesis was introduced into the NYA-0001 expression vector constructed in step 5, or the DNA fragment encoding the target scFv was inserted into a mammalian cell expression vector with pcDNA3.3 (Thermo Fisher Scientific) as the backbone using the In-Fusion HD Cloning Kit (CLONTECH) to construct each mammalian cell scFv expression vector.

[0221] 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 sequence number 21 of the sequence listing (Figure 28).

[0222] We confirmed that the full-length nucleotide sequence of NYA-2023 is the nucleotide sequence shown in sequence number 22 of the sequence listing (Figure 29).

[0223] We confirmed that the full-length nucleotide sequence of NYA-2027 is the nucleotide sequence shown in sequence number 23 of the sequence listing (Figure 30).

[0224] We confirmed that the full-length nucleotide sequence of NYA-1143 is the nucleotide sequence shown in sequence number 24 of the sequence listing (Figure 31).

[0225] We confirmed that the full-length nucleotide sequence of NYA-2143 is the nucleotide sequence shown in sequence number 25 of the sequence listing (Figure 32).

[0226] Furthermore, the full-length amino acid sequences of NYA-1163, NYA-2023, NYA-2027, NYA-1143, and NYA-2143 encoded by the nucleotide sequence were confirmed.

[0227] The full-length amino acid sequence of NYA-1163 is the amino acid sequence shown as sequence number 26 (Figure 33) in the sequence listing. In this sequence, amino acids 1 through 19 are the signal sequence, amino acids 21 through 266 are NYA-1163, and amino acids 267 through 292 are the Flag-His tag. Furthermore, amino acids 46 through 53 are CDRH1, amino acids 71 ​​through 78 are CDRH2, and amino acids 117 through 129 are CDRH3. Additionally, amino acids 181 through 188 are CDRL1, amino acids 206 through 208 are CDRL2, and amino acids 245 through 256 are CDRL3.

[0228] The full-length amino acid sequence of NYA-2023 is the amino acid sequence shown as sequence number 27 in the sequence listing (Figure 34). In this sequence, amino acids 1 through 19 are the signal sequence, amino acids 21 through 266 are NYA-2023, and amino acids 267 through 292 are the Flag-His tag. Furthermore, amino acids 46 through 53 are CDRH1, amino acids 71 ​​through 78 are CDRH2, and amino acids 117 through 129 are CDRH3. Additionally, amino acids 181 through 188 are CDRL1, amino acids 206 through 208 are CDRL2, and amino acids 245 through 256 are CDRL3.

[0229] The full-length amino acid sequence of NYA-2027 is the amino acid sequence shown as sequence number 28 (Figure 35) in the sequence listing. In this sequence, amino acids 1 through 19 are the signal sequence, amino acids 21 through 266 are NYA-2027, and amino acids 267 through 292 are the Flag-His tag. Furthermore, amino acids 46 through 53 are CDRH1, amino acids 71 ​​through 78 are CDRH2, and amino acids 117 through 129 are CDRH3. Additionally, amino acids 181 through 188 are CDRL1, amino acids 206 through 208 are CDRL2, and amino acids 245 through 256 are CDRL3.

[0230] The full-length amino acid sequence of NYA-1143 is the amino acid sequence shown as sequence number 29 (Figure 36) in the sequence listing. In this sequence, amino acids 1 through 19 are the signal sequence, amino acids 21 through 266 are NYA-1143, and amino acids 267 through 292 are the Flag-His tag. Furthermore, amino acids 46 through 53 are CDRH1, amino acids 71 ​​through 78 are CDRH2, and amino acids 117 through 129 are CDRH3. Additionally, amino acids 181 through 188 are CDRL1, amino acids 206 through 208 are CDRL2, and amino acids 245 through 256 are CDRL3.

[0231] The full-length amino acid sequence of NYA-2143 is the amino acid sequence shown as sequence number 30 (Figure 37) in the sequence listing. In this sequence, amino acids 1 through 19 are the signal sequence, amino acids 21 through 266 are NYA-2143, and amino acids 267 through 292 are the Flag-His tag. Additionally, amino acids 46 through 53 are CDRH1, amino acids 71 ​​through 78 are CDRH2, and amino acids 117 through 129 are CDRH3. Furthermore, amino acids 181 through 188 are CDRL1, amino acids 206 through 208 are CDRL2, and amino acids 245 through 256 are CDRL3.

[0232] Furthermore, the CDR sequences of NYA-1163 are all identical to those of NYA-0001. Also, the CDR sequences of NYA-1143, NYA-2143, and NYA-2023 are identical, with CDRH1 shown as sequence number 54 (Figure 61), CDRH2 as sequence number 55 (Figure 61), CDRH3 as sequence number 56 (Figure 61), CDRL1 as sequence number 60 (Figure 62), CDRL2 as DNN (Figure 61), and CDRL3 as sequence number 59 (Figure 61). In addition, the CDR sequences of NYA-2027 are shown as follows: CDRH1 as sequence number 54 (Figure 61), CDRH2 as sequence number 55 (Figure 61), CDRH3 as sequence number 56 (Figure 61), CDRL1 as sequence number 57 (Figure 61), CDRL2 as DNN (Figure 61), and CDRL3 as sequence number 61 (Figure 63).

[0233] Furthermore, NYA-1154 was designed by combining binding mutation sites observed during the screening of high-binding-ability clones, and an NYA-1154 expression vector with a pcDNA3.3 (Thermo Fisher Scientific) backbone was constructed by introducing site-directed mutagenesis into an NYA-0001 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: 31 (Figure 38). From the above nucleotide sequence, the full-length amino acid sequence of NYA-1154 encoded by this sequence was confirmed (SEQ ID NO: 32 (Figure 39)). In this sequence, amino acid sequences 1 to 19 are the signal sequence, amino acid sequences 21 to 266 are NYA-1154, and amino acid sequences 267 to 292 are the Flag-His tag. In addition, amino acid sequences 46 to 53 are CDRH1, amino acid sequences 71 to 78 are CDRH2, and amino acid sequences 117 to 129 are CDRH3. Furthermore, 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.

[0234] The CDR sequences of NYA-1154 are shown as follows: CDRH1 is sequence number 54 (Figure 61), CDRH2 is sequence number 55 (Figure 61), CDRH3 is sequence number 62 (Figure 64), CDRL1 is sequence number 57 (Figure 61), CDRL2 is shown as a DNN (Figure 61), and CDRL3 is sequence number 63 (Figure 64).

[0235] NYA-1163, NYA-2023, NYA-2027, NYA-1143, NYA-2143, and NYA-1154 were expressed using the same method as in 1)-5-2, and the target scFv was purified. The purified protein samples were subjected to analytical SEC to determine purity and concentration, and then used in various assays.

[0236] 2)-3 Creation of the NYA-1143 mutant 2)-3-1 Preparation of NYA-2035 NYA-2035 was designed as a mutant of NYA-1143, and an NYA-2035 expression vector was constructed by introducing site-directed mutagenesis into a mammalian cell NYA-1143 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). From 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, amino acid sequences 1 to 19 are the signal sequence, amino acid sequences 21 to 266 are NYA-2035, and amino acid sequences 267 to 292 are the Flag-His tag. In addition, amino acid sequences 46 to 53 are CDRH1, amino acid sequences 71 to 78 are CDRH2, and amino acid sequences 117 to 129 are CDRH3. Furthermore, 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 according to IMGT's CDR definition are shown as follows: CDRH1 is sequence number 54 (Figure 61), CDRH2 is sequence number 55 (Figure 61), CDRH3 is sequence number 56 (Figure 61), CDRL1 is sequence number 64 (Figure 65), CDRL2 is shown as a DNN (Figure 61), and CDRL3 is sequence number 59 (Figure 61).

[0237] 2)-3-2 Construction of NYA-1143CDR graft mutants To improve physical properties, we designed NYA-1143CDR graft mutants. The sequences of the framework regions of the VH and VL regions of NYA-1143 were compared with the framework regions of human subgroup consensus sequences and germline sequences as defined by 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 γ chain subgroup 3 were selected as acceptors with high sequence identity in their respective framework regions, and for VL, the human germline sequence IGLV1-44*01 was selected as an acceptor with high sequence identity in its framework region. Next, the amino acid residues of the framework regions of each acceptor were aligned with the amino acid residues of NYA-1143, and residues using different amino acids were identified.

[0238] Next, using a three-dimensional model of NYA-1143, framework residues to be transplanted onto the acceptor were selected, referencing criteria such as those provided by Queen et al. (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)). Using the above method, NYA-1143-VH01, NYA-1143-VH02, and NYA-1143-VH03 were designed as CDR graft mutant amino acid sequences for the VH portion of NYA-1143. These amino acid sequences are number sequences 37 (Figure 44), 38 (Figure 45), and 39 (Figure 46). NYA-1143-VL01 was designed as a CDR graft mutant amino acid sequence for the VL portion of NYA-1143. These amino acid sequences are number sequence 40 (Figure 47).

[0239] By combining the designed VH and VL sequences, we created the following various scFv sequences. The scFv created by replacing the VH portion of NYA-1143 with the amino acid sequence of NYA-1143-VH01 was named NYA-2044. The scFv created by replacing the VL portion of NYA-2044 with the amino acid sequence of NYA-1143-VL01 was named NYA-2045.

[0240] The scFv obtained by replacing the VH portion of NYA-1143 with the amino acid sequence of NYA-1143-VH02 was named NYA-2047. Furthermore, the scFv obtained by replacing the VL portion of NYA-2047 with the amino acid sequence of NYA-1143-VL01 was named NYA-2048.

[0241] The scFv obtained by replacing the VH portion of NYA-1143 with the amino acid sequence NYA-1143-VH03 was named NYA-2060. Furthermore, the scFv obtained by replacing the VL portion of NYA-2060 with the amino acid sequence NYA-1143-VL01 was named NYA-2061.

[0242] The various NYA-1143CDR graft mutants we designed were synthesized as total DNA fragments (by Fasmac), and these DNA fragments were then fused using the In-Fusion HD Cloning Kit (CLONTECH) to construct a mammalian cell scFv expression vector using pcDNA3.3 (Thermo Fisher Scientific) as the backbone.

[0243] 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 sequence number 41 of the sequence listing (Figure 48).

[0244] We confirmed that the full-length nucleotide sequence of NYA-2045 is the nucleotide sequence shown in sequence number 42 of the sequence listing (Figure 49).

[0245] We confirmed that the full-length nucleotide sequence of NYA-2047 is the nucleotide sequence shown in sequence number 43 of the sequence listing (Figure 50).

[0246] We confirmed that the full-length nucleotide sequence of NYA-2048 is the nucleotide sequence shown in sequence number 44 of the sequence listing (Figure 51).

[0247] The full-length nucleotide sequence of NYA-2060 was confirmed to be the nucleotide sequence shown in sequence number 45 of the sequence listing (Figure 52).

[0248] The full-length nucleotide sequence of NYA-2061 was confirmed to be the nucleotide sequence shown in sequence number 46 of the sequence listing (Figure 53).

[0249] Furthermore, the full-length amino acid sequences of NYA-2044, NYA-2045, NYA-2047, NYA-2048, NYA-2060, and NYA-2061 encoded by the above nucleotide sequences were determined. Note that the CDR sequences of NYA-2044, NYA-2045, NYA-2047, NYA-2048, NYA-2060, and NYA-2061 are identical to those of NYA-1143.

[0250] The full-length amino acid sequence of NYA-2044 is the amino acid sequence shown as sequence number 47 (Figure 54) in the sequence listing. In this sequence, amino acids 1 through 19 are the signal sequence, amino acids 21 through 266 are NYA-2044, and amino acids 267 through 292 are the Flag-His tag. Furthermore, amino acids 46 through 53 are CDRH1, amino acids 71 ​​through 78 are CDRH2, and amino acids 117 through 129 are CDRH3. Additionally, amino acids 181 through 188 are CDRL1, amino acids 206 through 208 are CDRL2, and amino acids 245 through 256 are CDRL3.

[0251] The full-length amino acid sequence of NYA-2045 is the amino acid sequence shown as sequence number 48 (Figure 55) in the sequence listing. In this sequence, amino acids 1 through 19 are the signal sequence, amino acids 21 through 266 are NYA-2045, and amino acids 267 through 292 are the Flag-His tag. Furthermore, amino acids 46 through 53 are CDRH1, amino acids 71 ​​through 78 are CDRH2, and amino acids 117 through 129 are CDRH3. Additionally, amino acids 181 through 188 are CDRL1, amino acids 206 through 208 are CDRL2, and amino acids 245 through 256 are CDRL3.

[0252] The full-length amino acid sequence of NYA-2047 is the amino acid sequence shown as sequence number 50 (Figure 57) in the sequence listing. In this sequence, amino acids 1 through 19 are the signal sequence, amino acids 21 through 266 are NYA-2047, and amino acids 267 through 292 are the Flag-His tag. Additionally, amino acids 46 through 53 are CDRH1, amino acids 71 ​​through 78 are CDRH2, and amino acids 117 through 129 are CDRH3. Furthermore, amino acids 181 through 188 are CDRL1, amino acids 206 through 208 are CDRL2, and amino acids 245 through 256 are CDRL3.

[0253] The full-length amino acid sequence of NYA-2048 is the amino acid sequence shown in sequence number 51 (Figure 58) of the sequence listing. In this sequence, amino acids 1 through 19 are the signal sequence, amino acids 21 through 266 are NYA-2048, and amino acids 267 through 292 are the Flag-His tag. Furthermore, amino acids 46 through 53 are CDRH1, amino acids 71 ​​through 78 are CDRH2, and amino acids 117 through 129 are CDRH3. Additionally, amino acids 181 through 188 are CDRL1, amino acids 206 through 208 are CDRL2, and amino acids 245 through 256 are CDRL3.

[0254] The full-length amino acid sequence of NYA-2060 is the amino acid sequence shown as sequence number 52 (Figure 59) in the sequence listing. In this sequence, amino acids 1 through 19 are the signal sequence, amino acids 21 through 266 are NYA-2060, and amino acids 267 through 292 are the Flag-His tag. Additionally, amino acids 46 through 53 are CDRH1, amino acids 71 ​​through 78 are CDRH2, and amino acids 117 through 129 are CDRH3. Furthermore, amino acids 181 through 188 are CDRL1, amino acids 206 through 208 are CDRL2, and amino acids 245 through 256 are CDRL3.

[0255] The full-length amino acid sequence of NYA-2061 is the amino acid sequence shown as sequence number 53 (Figure 60) in the sequence listing. In this sequence, amino acids 1 through 19 are the signal sequence, amino acids 21 through 266 are NYA-2061, and amino acids 267 through 292 are the Flag-His tag. Furthermore, amino acids 46 through 53 are CDRH1, amino acids 71 ​​through 78 are CDRH2, and amino acids 117 through 129 are CDRH3. Additionally, amino acids 181 through 188 are CDRL1, amino acids 206 through 208 are CDRL2, and amino acids 245 through 256 are CDRL3.

[0256] 2)-3-3 Expression and purification of the NYA-1143 mutant 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 using the same method as in 1)-5-2, and the target scFv was purified. The purified protein samples were subjected to analytical SEC to determine purity and concentration, and then used in various assays.

[0257] (Example 3) Production of scFv of anti-HLA / NY-ESO reference antibody We designed scFvs for antibodies 3M4E5 and T1 (WO2010 / 106431) that exhibit high binding affinity to HLA / NY-ESO, and named the scFv for 3M4E5 NYC-0003 and the scFv for T1 NYC-0004.

[0258] The DNA fragments NYC-0003 and NYC-0004 were totally 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).

[0259] 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 sequence number 65 (Figure 66) and sequence number 66 (Figure 67) of the sequence listing. Furthermore, the amino acid sequence of the full-length NYC-0003 encoded by these nucleotide sequences is the amino acid sequence shown in sequence number 67 (Figure 68), and the amino acid sequence of the full-length NYC-0004 is the amino acid sequence shown in sequence number 68 (Figure 69).

[0260] Using the same method as in 1)-5-2, NYC-0003 and NYC-0004 were expressed, and the respective target scFv were purified. Each purified protein sample was subjected to analytical SEC to determine purity and concentration, and then used in various assays.

[0261] (Example 4) Evaluation of binding ability to HLA / NY-ESO by Biacore Using a Biacore T200, anti-HLA / NY-ESO scFv was captured as a ligand on immobilized anti-His antibody, and the antigen was measured as an analyte. The HLA / NY-ESO prepared in 1)-1 was used as the antigen. The anti-His antibody (His Capture kit, GE Healthcare) was immobilized on a Sensor Chip CM5 (GE Healthcare) according to the kit instructions. Each anti-HLA / NY-ESO scFv to be evaluated, diluted to 0.5 μg / mL with HBS-EP+ (GE Healthcare), was contacted at 10 μL / min for 60 seconds to immobilize. Subsequently, multiple concentrations of HLA / NY-ESO diluted with HBS-EP+ were added as analytes to each sample at a flow rate of 30 μL / min for 120 seconds, and dissociation was measured for 600 seconds using single-cycle kinetic analysis. DThe results were calculated and are shown in Table 1. Compared to the parent antibody NYA-0001, all of its variants showed stronger binding to HLA / NY-ESO. Furthermore, compared to NYC-0004, NYA-1143, NYA-2023, NYA-2143, NYA-2044, NYA-2045, NYA-2060, and NYA-2061 showed stronger binding, and among these, NYA-1143, NYA-2044, NYA-2045, and NYA-2143 showed stronger binding. D It showed a value of 1 nM or less.

[0262] [Table 1]

[0263] (Example 5) Recognition amino acid analysis within the NY-ESO peptide of anti-HLA / NY-ESO scFv Human lymphoblast fusion cell line T2 (ATCC) cells were adjusted to appropriate concentrations in AIM-V medium (Thermo Fisher Scientific) containing 20% ​​FBS, and NY-ESO peptide (SEQ ID NO: 1), point mutation NY-ESO peptides 1F, 2M, 3A, 4A, 5A, 6L, 7F, 8A, 9A (SEQ ID NOs: 121 (Figure 122), 122 (Figure 123), 123 (Figure 124), 124 (Figure 125), 125 (Figure 126), 126 (Figure 127), 127 (Figure 128), 128 (Figure 129), 129 (Figure 130)), and gp100 peptide (SEQ ID NO: 130 (Figure 131)) (all Sigma A solution of Genosys (FBS) dissolved in DMSO at 5 mM was incubated at 37°C for 4 hours, either to a final concentration of 50 μM or by adding 1 / 100th the amount of DMSO. After washing twice with 20% FBS-containing AIM-V medium, the cells were prepared to an appropriate concentration with 5% FBS-containing PBS, and LIVE / DEAD Fixable DeadCell Stain Kit (ThermoFisher Scientific) was added, followed by standing at 4°C for 30 minutes. After washing twice with 5% FBS-containing PBS, the cells were divided into two groups and sterilized with 5% FBS-containing PBS for 10 minutes. 5Cells were seeded at a rate of one cell / well in a 96-well U-bottom microplate, and the supernatant was removed after centrifugation. For one cell group, 25 μL / well of anti-HLA / NY-ESO scFv diluted to 100 nM in 5% FBS-containing PBS was added, and the cells were allowed to stand at 4°C for 30 minutes. After washing twice with 5% FBS-containing PBS, 25 μL / well of Penta-His Alexa Fluor488 (QIAGEN) diluted in 5% FBS-containing PBS was added, and the cells were allowed to stand at 4°C for 30 minutes. After washing twice with 5% FBS-containing PBS, 25 μL / well of Anti-mouse-IgG Alexa Fluor488 (Thermo Fisher Scientific) diluted in 5% FBS-containing PBS was added, and the cells were allowed to stand at 4°C for 30 minutes. After washing twice with PBS containing 5% FBS, the cells were fixed overnight with Mildform 10N (Fujifilm Wako Pure Chemical Industries, Ltd.) and then resuspended in PBS containing 5% FBS. To standardize by HLA / peptide complex amount, 25 μL / well of either HLA-A2 antibody BB7.2-Alexa Fluor 488 or mouse IgG2b-Alexa Fluor 488, diluted to 10 μg / mL in PBS containing 5% FBS, was added to the other cell sample and allowed to stand at 4°C for 30 minutes. After washing twice with PBS containing 5% FBS, the cells were fixed overnight with Mildform 10N (Fujifilm Wako Pure Chemical Industries, Ltd.) and then resuspended in PBS containing 5% FBS. These cell suspensions were detected using a flow cytometer (Canto II, Becton Dickinson). Data analysis was performed using Flowjo (Treestar), and the geometric mean fluorescence intensity (gMFI) of Alexa Fluor 488 in the dead cell-removed fraction of T2 cells was measured. The standardized gMFI, a value indicating the binding affinity of each scFv standardized by the amount of HLA / peptide complex on T2 cells, was calculated using the following formula.

[0264] Standardized gMFI=(A / B)×((C / D) / (E / F)) A / B = relative gMFI A: gMFI of T2 cells treated with antibody-added DMSO or various peptides. B: gMFI of T2 cells treated with DMSO or various peptides without antibodies. (C / D) / (E / F) = Corrected value for HLA / peptide complex levels in T2 cells treated with DMSO or each peptide. gMFI of T2 cells treated with DMSO or various peptides containing C:HLA-A2 antibody D: gMFI of T2 cells treated with DMSO or various peptides containing mouse IgG2b antibody E: gMFI of DMSO-treated T2 cells with added HLA-A2 antibody F: gMFI of DMSO-treated T2 cells with mouse IgG2b antibody added.

[0265] As shown in Figure 1, anti-HLA / NY-ESO scFv NYA-0001, 1143, 2044, 2045, 2047, 2048, 2060, and 2061 showed less than half the binding affinity to T2 containing point mutations at amino acids 1, 4, 5, and 7 of the NY-ESO peptide compared to the wild-type NY-ESO peptide, suggesting that they recognize amino acids 1, 4, 5, and 7 of the NY-ESO peptide. Similarly, NYA-1154 is suggested to recognize amino acids 1 and 5, NYA-1163 recognizes amino acids 1, 3, 4, 5, 6, and 7, NYA-2023, 2027, and 2035 recognize amino acids 1, 4, and 5, and NYA-2143 recognizes amino acids 1, 5, and 7. On the other hand, NYC-0003 and 0004 are suggested to recognize amino acids 4 and 5.

[0266] (Example 6) Evaluation of antigen binding specificity of anti-HLA / NY-ESO scFv To search for human peptides (hereinafter referred to as "homologous peptides") within the human proteome (Swiss-Prot) that have a similar but not identical amino acid sequence to the NY-ESO peptide: SLLMWITQC (SEQ ID NO: 1) and are likely to bind, we searched for 9-mer peptides with matching 1st, 4th, and 5th positions, which are suggested to be recognized by many of this antibody. For the searched 9-mer peptides, we predicted their binding affinity to HLA-A0201 using NetMHCPan2.8, and selected the 9-mer peptide shown in Figure 2A, which was predicted to have an IC50 of 500 nM or less, as the homologous peptide for evaluating the binding specificity of anti-HLA / NY-ESO scFv. T2 cells were prepared to an appropriate concentration in AIM-V medium containing 20% ​​FBS (Thermo Fisher Scientific). NY-ESO peptide (SEQ ID NO: 1), homologous peptide DOLPP1, IL20RB, PRKD2, CD163, P2RY8 (SEQ ID NO: 131 (Figure 132), 132 (Figure 133), 133 (Figure 134), 134 (Figure 135), 135 (Figure 136)), and gp100 peptide (SEQ ID NO: 130 (Figure 131)) (all from Sigma Genosys) were dissolved in 5 mM DMSO. The binding affinity of each antibody was evaluated in the same manner as in Example 5, either by adding 1 / 100th the amount of DMSO to a final concentration of 50 μM. The standardized gMFI, a value indicating the binding affinity of each scFv standardized by the amount of HLA / peptide complex on T2 cells, was calculated using the following formula.

[0267] Standardized gMFI=(A / B)×((C / D) / (E / F)) A / B = relative gMFI A: gMFI of T2 cells treated with antibody-added DMSO or various peptides. B: gMFI of T2 cells treated with DMSO or various peptides without antibodies. (C / D) / (E / F) = Corrected value for HLA / peptide complex levels in T2 cells treated with DMSO or each peptide. gMFI of T2 cells treated with DMSO or various peptides containing C:HLA-A2 antibody D: gMFI of T2 cells treated with DMSO or various peptides containing mouse IgG2b antibody E: gMFI of DMSO-treated T2 cells with added HLA-A2 antibody F: gMFI of DMSO-treated T2 cells with mouse IgG2b antibody added.

[0268] As shown in Figure 2B, anti-HLA / NY-ESO scFv NYA-0001, 1143, 1163, 2023, 2027, 2035, 2044, 2045, 2047, 2048, 2060, 2061, and 2143 did not bind to any of the homologous peptide-treated cells, suggesting high specificity. On the other hand, NYA-1154, NYC-0003, and 0004 showed binding to some homologous peptide-treated T2 cells.

[0269] (Example 7) Preparation of Fc-added anti-HLA / NY-ESO-anti-CD3 bispecific molecules 7)-1 Construction of an Fc-added anti-HLA / NY-ESO-anti-CD3 bispecificity molecule expression vector 7)-1-1 Construction of a taFv-heterodimer Fc-type bispecific molecule expression vector To evaluate the taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule, expression vectors for each molecule were designed. The anti-HLA / NY-ESO antibodies used were NYA-1143, NYA-2143, NYA-1163, NYA-2023, NYA-2027, NYA-2035, NYA-2044, NYA-2045, NYA-2047, NYA-2048, NYA-2060, and NYA-2061. The anti-CD3 antibody used was the humanized anti-CD3scFv C3E-7085 (WO2018 / 117237). For the heterodimer Fc sequence, an Fc sequence (WO2014 / 190441) was used, which introduced a mutation that reduced effector function and formed a heteromultimer.

[0270] DNA fragments encoding Fc (HC1 or HC2) with mutations that reduce effector function and induce heteromultimer formation were synthesized (by Fasmac). An expression vector for mammalian cells was then constructed using the In-Fusion HD Cloning Kit (CLONTECH) with pcDNA3.3 (ThermoFisher Scientific) as the backbone, and named "p_HC1".

[0271] We created a mammalian cell expression vector by linking NYA-1143 and C3E-7085 with a GGGGS linker, and then incorporating a DNA fragment encoding HC2 into the carboxyl terminus of taFv. This vector was named "p_NYF-0016-HC2".

[0272] A mammalian cell expression vector was created by linking NYA-2143 and C3E-7085 with a GGGGS linker, incorporating a DNA fragment encoding HC2 into the carboxyl terminus of taFv. This vector was then constructed by introducing site-directed mutagenesis into p_NYF-0016-HC2 and named "p_NYF-0019-HC2".

[0273] A mammalian cell expression vector was created by linking NYA-1163 and C3E-7085 with a GGGGS linker, and incorporating a DNA fragment encoding HC2 into the carboxyl terminus of taFv. This vector was prepared by replacing the base sequence portion encoding NYA-1143 of p_NYF-0016-HC2 with a DNA fragment encoding NYA-1163 using the In-Fusion HD cloning kit (CLONTECH), and was named "p_NYF-0022-HC2".

[0274] A mammalian cell expression vector was created by linking NYA-2023 and C3E-7085 with a GGGGS linker, incorporating a DNA fragment encoding HC2 into the carboxyl terminus of taFv, and was named "p_NYF-0023-HC2" by introducing site-directed mutagenesis into p_NYF-0016-HC2.

[0275] A mammalian cell expression vector was created by linking NYA-2027 and C3E-7085 with a GGGGS linker and incorporating an HC2-encoding DNA fragment into the carboxyl terminus of a taFv, and was named "p_NYF-0027-HC2".

[0276] A mammalian cell expression vector was created by linking NYA-2035 and C3E-7085 with a GGGGS linker, incorporating a DNA fragment encoding HC2 into the carboxyl terminus of taFv. This vector was then constructed by introducing site-directed mutagenesis into p_NYF-0016-HC2 and named "p_NYF-0035-HC2".

[0277] A mammalian cell expression vector was created by linking NYA-2044 and C3E-7085 with a GGGGS linker, and incorporating an HC2-encoding DNA fragment into the carboxyl terminus of taFv. This vector was prepared by replacing the base sequence portion encoding NYA-1143 of p_NYF-0016-HC2 with a DNA fragment encoding NYA-2044 using the In-Fusion HD cloning kit (CLONTECH), and was named "p_NYF-0044-HC2".

[0278] A mammalian cell expression vector was created by linking NYA-2045 and C3E-7085 with a GGGGS linker, and incorporating a DNA fragment encoding HC2 into the carboxyl terminus of taFv. This vector was prepared by replacing the base sequence portion encoding NYA-1143 of p_NYF-0016-HC2 with a DNA fragment encoding NYA-2045 using the In-Fusion HD cloning kit (CLONTECH), and was named "p_NYF-0045-HC2".

[0279] A mammalian cell expression vector was created by linking NYA-2047 and C3E-7085 with a GGGGS linker, and incorporating an HC2-encoding DNA fragment into the carboxyl terminus of taFv. This vector was prepared by replacing the nucleotide sequence portion encoding NYA-1143 of p_NYF-0016-HC2 with a DNA fragment encoding NYA-2047 using the In-Fusion HD cloning kit (CLONTECH), and was named "p_NYF-0047-HC2".

[0280] A mammalian cell expression vector was created by linking NYA-2048 and C3E-7085 with a GGGGS linker, and incorporating a DNA fragment encoding HC2 into the carboxyl terminus of taFv. This vector was constructed by replacing the base sequence portion encoding NYA-1143 of p_NYF-0016-HC2 with a DNA fragment encoding NYA-2048 using the In-Fusion HD Cloning Kit (CLONTECH), and was named "p_NYF-0048-HC2".

[0281] A mammalian cell expression vector was created by linking NYA-2060 and C3E-7085 with a GGGGS linker, incorporating a DNA fragment encoding HC2 into the carboxyl terminus of taFv. This vector was then constructed by introducing site-directed mutagenesis into p_NYF-0044-HC2 and named "p_NYF-0060-HC2".

[0282] A mammalian cell expression vector was created by linking NYA-2061 and C3E-7085 with a GGGGS linker, incorporating a DNA fragment encoding HC2 into the carboxyl terminus of taFv. This vector was constructed by introducing site-directed mutagenesis into p_NYF-0045-HC2 and named "p_NYF-0061-HC2".

[0283] The nucleotide sequence of p_HC1 was re-analyzed, and it was confirmed that the full-length nucleotide sequence of HC1 is the nucleotide sequence shown in sequence number 71 of the sequence listing (Figure 72).

[0284] 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 sequence number 72 of the sequence listing (Figure 73).

[0285] 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 sequence number 73 of the sequence listing (Figure 74).

[0286] 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 sequence number 74 of the sequence listing (Figure 75).

[0287] 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 sequence number 75 of the sequence listing (Figure 76).

[0288] 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 sequence number 76 of the sequence listing (Figure 77).

[0289] 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 sequence number 77 of the sequence listing (Figure 78).

[0290] 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 sequence number 78 of the sequence listing (Figure 79).

[0291] 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 sequence number 79 of the sequence listing (Figure 80).

[0292] 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 sequence number 80 of the sequence listing (Figure 81).

[0293] 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 sequence number 81 of the sequence listing (Figure 82).

[0294] 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 sequence number 82 (Figure 83) of the sequence listing.

[0295] 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 sequence number 83 (Figure 84) of the sequence listing.

[0296] Furthermore, from 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.

[0297] The full-length amino acid sequence of HC1 is the amino acid sequence shown in sequence number 84 (Figure 85) of the sequence listing. In this sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, and the amino acid sequence from the 20th to the 246th is HC.

[0298] The full-length amino acid sequence of NYF-0016-HC2 is the amino acid sequence shown in sequence number 85 (Figure 86) of the sequence listing. 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-1143-C3E-7085taFv, the amino acid sequence from the 512th to the 513th is the linker, and the amino acid sequence from the 514th to the 745th is HC2.

[0299] The full-length amino acid sequence of NYF-0019-HC2 is the amino acid sequence shown in sequence number 86 (Figure 87) of the sequence listing. 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-2143-C3E-7085taFv, the amino acid sequence from the 512th to the 513th is the linker, and the amino acid sequence from the 514th to the 745th is HC2.

[0300] The full-length amino acid sequence of NYF-0022-HC2 is the amino acid sequence shown in sequence number 87 (Figure 88) of the sequence listing. 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-1163-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.

[0301] The full-length amino acid sequence of NYF-0023-HC2 is the amino acid sequence shown as sequence number 88 (Figure 89) in the sequence listing. 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-2023-C3E-7085taFv, the amino acid sequence from the 512th to the 513th is the linker, and the amino acid sequence from the 514th to the 745th is HC2.

[0302] The full-length amino acid sequence of NYF-0027-HC2 is the amino acid sequence shown as sequence number 89 (Figure 90) in the sequence listing. 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-2027-C3E-7085taFv, the amino acid sequence from the 512th to the 513th is the linker, and the amino acid sequence from the 514th to the 745th is HC2.

[0303] The full-length amino acid sequence of NYF-0035-HC2 is the amino acid sequence shown as sequence number 90 (Figure 91) in the sequence listing. 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-2035-C3E-7085taFv, the amino acid sequence from the 512th to the 513th is the linker, and the amino acid sequence from the 514th to the 745th is HC2.

[0304] The full-length amino acid sequence of NYF-0044-HC2 is the amino acid sequence shown in sequence number 91 (Figure 92) of the sequence listing. 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.

[0305] The full-length amino acid sequence of NYF-0045-HC2 is the amino acid sequence shown as sequence number 92 (Figure 93) in the sequence listing. 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.

[0306] The full-length amino acid sequence of NYF-0047-HC2 is the amino acid sequence shown as sequence number 93 (Figure 94) in the sequence listing. 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.

[0307] The full-length amino acid sequence of NYF-0048-HC2 is the amino acid sequence shown as sequence number 94 (Figure 95) in the sequence listing. 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.

[0308] The full-length amino acid sequence of NYF-0060-HC2 is the amino acid sequence shown as sequence number 95 (Figure 96) in the sequence listing. 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.

[0309] The full-length amino acid sequence of NYF-0061-HC2 is the amino acid sequence shown as sequence number 96 (Figure 97) in the sequence listing. In this sequence, amino acids 1 through 19 are the signal sequence, amino acids 21 through 511 are NYA-2061-C3E-7085taFv, amino acids 512 through 513 are the linker, and amino acids 514 through 745 are HC2.

[0310] 7)-1-2 Construction of a taFv-Fab-heterodimer Fc-type bispecific molecule expression vector A vector for expressing a taFv-Fab heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule was designed. NYA-0001 was used as the anti-HLA / NY-ESO antibody. The humanized anti-CD3 antibody C3E-7085 (WO2018 / 117237) was used. For the heterodimer Fc sequence, an Fc sequence (WO2014 / 190441) was used, which introduced a mutation that reduces effector function and forms a heteromultimer.

[0311] We created a mammalian cell expression vector incorporating the heavy chain variable region of NYA-0001, the human IgG-derived CH1 region, and a DNA fragment that reduces effector function and encodes HC1-k delete. This vector was named "p_NYA-0001-Fab-HC1-k delete". We also created a mammalian cell expression vector incorporating the NYA-0001 light chain variable region and a DNA fragment that encodes the human IgG-derived CL region. This vector was named "p_NYA-0001-LC".

[0312] 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 sequence number 97 of the sequence listing (Figure 98).

[0313] 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 sequence number 98 of the sequence listing (Figure 99).

[0314] Furthermore, the amino acid sequences of NYA-0001-Fab-HC1-k delete and the full-length NYA-0001-LC encoded by the nucleotide sequence were confirmed.

[0315] The full-length amino acid sequence of NYA-0001-Fab-HC1-k is the amino acid sequence shown in sequence number 99 (Figure 100) of the sequence listing. In this sequence, amino acid sequences 1 through 19 are the signal sequence, amino acid sequences 20 through 139 are the variable region, and amino acid sequences 140 through 468 are the constant region. In addition, amino acid sequences 45 through 52 are CDRH1 (sequence number 54 (Figure 61)), amino acid sequences 70 through 77 are CDRH2 (sequence number 55 (Figure 61)), and amino acid sequences 116 through 128 are CDRH3 (sequence number 56 (Figure 61)).

[0316] The full-length amino acid sequence of NYA-0001-LC is the amino acid sequence shown in sequence number 100 (Figure 101) of the sequence listing. The amino acid sequence from the 1st to the 20th is the signal sequence, the amino acid sequence from the 21st to the 131st is the variable region, and the amino acid sequence from the 132nd to the 237th is the constant region. In addition, amino acid numbers 46 to 53 are CDRL1 (sequence number 57 (Figure 61)), amino acid numbers 71 to 73 are CDRL2 (DNN (Figure 61)), and amino acid numbers 110 to 121 are CDRL3 (sequence number 59 (Figure 61)).

[0317] 7)-2 Expression of Fc-added anti-HLA / NY-ESO-anti-CD3 bispecific molecules 7)-2-1 Expression of taFv-heterodimer Fc-type bispecific molecules Expi293F cells (Thermo Fisher Scientific) were subcultured and cultured according to the manual. Expi293F cell culture medium in the logarithmic growth phase was placed in 2.5 × 10⁻⁶ cells. 6The mixture was diluted with Expi293 Expression medium (Thermo Fisher Scientific) to a concentration of cells / mL and used for the production of various bispecific molecules. A vector mixture of vectors p_NYF-0016-HC2 and p_HC1 in a 1:1.5 ratio was added to 20 mL of Opti-Pro SFM medium (Thermo Fisher Scientific), along with 0.3 mg and 0.9 mg of Polyethyleneimine (Polyscience #24765). The mixture was gently mixed and allowed to stand for 5 minutes before being added to Expi293F cells. The culture supernatant was obtained by culturing the cells in an 8% CO2 incubator at 37°C for 6 days with shaking at 135 rpm. The supernatant was filtered through a 0.2 μm filter (Millopore) to obtain the culture supernatant of the taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0016). The amino acid sequences obtained by expressing each vector constituting NYF-0016 are shown in sequence numbers 85 (Figure 86) and 84 (Figure 85) of the sequence listing.

[0318] Using a similar method, the culture supernatant of the taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0019) was prepared for expression using p_NYF-0019-HC2 and p_HC1. The amino acid sequences obtained by expressing each vector constituting NYF-0019 are shown in sequence numbers 86 (Figure 87) and 84 (Figure 85) of the sequence listing.

[0319] The culture supernatant of the taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0022) was prepared using p_NYF-0022-HC2 and p_HC1. The amino acid sequences obtained by expressing each vector constituting NYF-0022 are shown in sequence numbers 87 (Figure 88) and 84 (Figure 85) of the sequence listing.

[0320] The culture supernatant of the taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0023) was prepared for expression using p_NYF-0023-HC2 and p_HC1. The amino acid sequences obtained by expressing each vector constituting NYF-0023 are shown in sequence numbers 88 (Figure 89) and 84 (Figure 85) of the sequence listing.

[0321] The culture supernatant of the taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0027) was prepared for expression using p_NYF-0027-HC2 and p_HC1. The amino acid sequences obtained by expressing each vector constituting NYF-0027 are shown in sequence numbers 89 (Figure 90) and 84 (Figure 85) of the sequence listing.

[0322] The culture supernatant of the taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0035) was prepared using p_NYF-0035-HC2 and p_HC1. The amino acid sequences obtained by expressing each vector constituting NYF-0035 are shown in sequence numbers 90 (Figure 91) and 84 (Figure 85) of the sequence listing.

[0323] The culture supernatant of the taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0044) was prepared using p_NYF-0044-HC2 and p_HC1. The amino acid sequences obtained by expressing each vector constituting NYF-0044 are shown in sequence numbers 91 (Figure 92) and 84 (Figure 85) of the sequence listing.

[0324] The culture supernatant of the taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0045) was prepared for expression using p_NYF-0045-HC2 and p_HC1. The amino acid sequences obtained by expressing each vector constituting NYF-0045 are shown in sequence numbers 92 (Figure 93) and 84 (Figure 85) of the sequence listing.

[0325] The culture supernatant of the taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0047) was prepared for expression using p_NYF-0047-HC2 and p_HC1. The amino acid sequences obtained by expressing each vector constituting NYF-0047 are shown in sequence numbers 93 (Figure 94) and 84 (Figure 85) of the sequence listing.

[0326] The culture supernatant of the taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0048) was prepared for expression using p_NYF-0048-HC2 and p_HC1. The amino acid sequences obtained by expressing each vector constituting NYF-0048 are shown in sequence numbers 94 (Figure 95) and 84 (Figure 85) of the sequence listing.

[0327] The culture supernatant of the taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0060) was prepared for expression using p_NYF-0060-HC2 and p_HC1. The amino acid sequences obtained by expressing each vector constituting NYF-0060 are shown in sequence numbers 95 (Figure 96) and 84 (Figure 85) of the sequence listing.

[0328] The culture supernatant of the taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0061) was prepared using p_NYF-0061-HC2 and p_HC1. The amino acid sequences obtained by expressing each vector constituting NYF-0061 are shown in sequence numbers 96 (Figure 97) and 84 (Figure 85) of the sequence listing.

[0329] 7)-2-2 Expression of taFv-Fab-heterodimer Fc-type bispecific molecule Using the same method as in 7)-2-1, a vector mixture of p_NYF-0023-HC2, p_NYA-0001-Fab-HC1-k delete, and p_NYA-0001-LC in a ratio of 1:1:1.5 was used to prepare the culture supernatant of the taFv-Fab heterodimer Fc-type bispecific molecule (NYF-0058) for expression. The amino acid sequences obtained by expressing each vector constituting NYF-0058 were taken from sequence number 88 of the sequence listing (Figure).89 These are shown as amino acid numbers 20-745 of ) and amino acid numbers 20-468 of SEQ ID NO: 99 (Figure 100) and amino acid numbers 21-237 of SEQ ID NO: 100 (Figure 101).

[0330] 7)-3 Purification of Fc-added anti-HLA / NY-ESO-anti-CD3 bispecific molecules 7)-2 The culture supernatant obtained in this procedure was purified of various bispecific molecules using a two-step process: Protein A affinity chromatography and gel filtration chromatography.

[0331] The culture supernatant was applied to a MabSelectSuRe column (GE Healthcare Bioscience; also simply referred to as "GE Healthcare") equilibrated with PBS pH 7.4, and the target bispecific molecule was adsorbed. After removing non-adsorbed components with PBS, the adsorbed components were eluted with acetate buffer pH 3.5. The eluted fraction was adjusted to neutral pH with Tris buffer pH 9.5, concentrated, and subjected to gel filtration on a Superdex 200 10 / 300 (GE Healthcare Bioscience) column pre-equilibriumized with 25 mM Histidine, 300 mM NaCl, 5% Sorbitor, pH 5.5. From the peak fraction obtained by gel filtration chromatography, the fraction corresponding to the target heterodimer was recovered, and the formation of the target anti-HLA / NY-ESO-anti-CD3 bispecific molecule was confirmed by SDS-polyacrylamide electrophoresis (SDS-PAGE). The purified protein sample was subjected to analytical SEC to determine purity and concentration, and then used for various evaluations.

[0332] (Example 8) Evaluation of cytotoxic activity of Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule 8)-1 Preparation of target cells Endogenous human NY-ESO-expressing cell lines (U266B1 and NCI-H1703) and endogenous human NY-ESO-non-expressing cell lines (AGS and CFPAC-1) were cultured in RPMI1640 medium (Fujifilm Wako Pure Chemical Industries, Ltd.) containing 10% FBS at a rate of 1 × 10⁶ 6Cells were prepared to a concentration of cells / mL, and 100 μL of Chromium-51 Radionuclide (PerkinElmer) was added per 1 mL of each cell suspension. The cells were incubated at 37°C under 5% CO2 conditions for 2 hours. After washing twice with RPMI1640 medium containing 10% FBS, 1 × 10⁶ cells were incubated in RPMI1640 medium containing 10% FBS. 5 The target cells were used after the solution was resuspended to a cell / mL concentration.

[0333] 8)-2 Preparation of effector cells Commercially available frozen human PBMCs (Cellular Technology Limited) were thawed at 37°C, transferred to a solution of RPMI1640 medium containing 10% FBS and an anti-aggregate wash reagent (Cellular Technology Limited), washed twice, and then rinsed in RPMI1640 medium containing 10% FBS for 1 × 10⁶ times. 6 The cells were prepared to a concentration of cells / mL and used as effector cells.

[0334] 8)-3 Cell injury assay Each target cell obtained in 8)-1 was added to a 96-well U-bottom microplate at 50 μL / well. Various Fc-adding anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules prepared in Example 7, prepared at various concentrations, were added at 50 μL / well, and effector cells prepared in Example 8)-2 were added at 100 μL / well. The cells were centrifuged at 1000 rpm for 1 minute at room temperature, and then cultured at 37°C under 5% CO2 conditions for 20-24 hours. 50 μL of the supernatant was collected on a LumaPlate (PerkinElmer), dried at 50°C for approximately 2 hours, and then measured using a plate reader (TopCount: PerkinElmer). The test was performed using a triplicate, and the cell lysis rate was calculated using the following formula. Cell lysis rate (%)=(AB) / (CB)×100 A: Counting the sample wells. B: Average count of background (antibody-free wells) (n=3). 50 μL of assay medium was added when the antibody was added. Otherwise, the procedure was the same as for the sample wells. C: Average count of maximum release (wells in which target cells were lysed with surfactant) (n=3). 50 μL of assay medium was added when the antibody was added. 100 μL of surfactant was added, and 50 μL was transferred to the LumaPlate and measured, similar to the sample wells.

[0335] As shown in Figures 4A-F, cytotoxic activity of various anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules was demonstrated against endogenous human NY-ESO-expressing cell lines (U266B1 and NCI-H1703). The EC was calculated using the Four Parameter Logistics Curve formula of the analysis software (Sigmaplot, version 12.0). 50 The correlation coefficient (R) was calculated, and the results for the U266B1 cell line are shown in Table 2, while the results for the NCI-H1703 cell line are shown in Table 3. Not Applicable (NA) indicates that the correlation coefficient R value could not be calculated and that curve fitting was not possible. On the other hand, as shown in Figure 4G-L, no cytotoxic activity was observed against endogenous human NY-ESO non-expressing cell lines (AGS and CFPAC-1).

[0336] [Table 2]

[0337] [Table 3]

[0338] (Example 9) Evaluation of in vivo activity of Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecule in a human PBMC transfer model. Human squamous cell lung cancer cell line NCI-H1703 (ATCC) was incubated in PBS containing 50% Matrigel (CORNING) in 6 x 10⁶ cells. 7The solution was prepared to the desired cell / mL ratio and 0.1 mL was transplanted subcutaneously into NOG mice (female, 6-7 weeks old) (Day 0). On Day 4, human PBMCs were incubated in PBS at a ratio of 3.75 × 10⁶. 7 The solution was prepared to a cell / mL ratio and 0.2 mL was transplanted into the tail vein. Approximately one week later (Day 6-7), the longest (mm) and shortest (mm) diameters of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the formula shown below. Estimated Tuner Volume (mm 3 ) = Average value of the estimated tumor volume for each individual Estimated tumor volume for each individual = long diameter × short diameter 2 / 2

[0339] On Day 14, patients were divided into groups based on tumor volume, with n=5-6 in each group. Various anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules were administered intravenously via tail vein (1 mg / kg, except for NYF-0058, which was administered at 1.5 mg / kg for comparison). Administration was performed on Day 14, Day 21, and Day 28. Antitumor effects were confirmed in the groups administered with various anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules (Figure 5A-C). Tumor Growth Inhibition (%) on Days 31-32 was calculated using the formula shown below and is listed in Table 4.

[0340] Tumor Growth Inhibition (%) = 100 - (Estimated Tumor Volume of each group / Estimated Tumor Volume of Vehicle Control × 100)

[0341] [Table 4]

[0342] (Example 10) Preparation of various Fc-addition format anti-HLA / NY-ESO-anti-CD3 bispecific molecules 10)-1 Construction of various Fc-addition format anti-HLA / NY-ESO-anti-CD3 bispecificity molecule expression vectors 10)-1-1 Construction of Hybrid Bispecific Molecular Expression Vectors To evaluate the hybrid anti-HLA / NY-ESO-anti-CD3 bispecific molecule, expression vectors for each molecule were designed. NYA-1143 was used as the anti-HLA / NY-ESO antibody. C3E-7085 (PCT / JP2017 / 046006), a humanized anti-CD3 scFv, was used as the anti-CD3 antibody. For the heterodimer Fc sequence, an Fc sequence (WO2014 / 190441) was used, which introduced a mutation that reduced effector function and formed a heteromultimer.

[0343] We created a mammalian cell expression vector incorporating DNA fragments encoding the heavy chain variable region of NYA-1143, the human IgG-derived CH1 region, and the Fc region with a mutation that reduces effector function and forms a heteromultimer. This vector was named "p_NYA-1143-Fab-HC1-k delete". We also created a mammalian cell expression vector incorporating DNA fragments encoding the NYA-1143 light chain variable region and the human IgG-derived CL region, which was named "p_NYA-1143-LC". Furthermore, we created a mammalian cell expression vector, "p_C3E-7085-HC2-k delete", incorporating a DNA fragment encoding the Fc region at the carboxyl terminus of humanized anti-CD3scFv(C3E-7085) with a mutation that reduces effector function and forms a heteromultimer.

[0344] The nucleotide sequence of p_NYA-1143-Fab-HC1-k delete was re-analyzed, and it was confirmed that the full-length nucleotide sequence of NYA-1143-Fab-HC1-k delete is the nucleotide sequence shown in sequence number 101 of the sequence listing (Figure 102).

[0345] The nucleotide sequence of p_NYA-1143-LC was re-analyzed, and it was confirmed that the full-length nucleotide sequence of NYA-1143-LC is the nucleotide sequence shown in sequence number 102 (Figure 103) of the sequence listing.

[0346] The nucleotide sequence of p_C3E-7085-HC2-k delete was re-analyzed, and it was confirmed that the full-length nucleotide sequence of C3E-7085-HC2-k delete is the nucleotide sequence shown in sequence number 103 of the sequence listing (Figure 104).

[0347] Furthermore, the full-length amino acid sequences of NYA-1143-Fab-HC1-k delete, NYA-1143-LC, and C3E-7085-HC2-k delete encoded by the above nucleotide sequence were confirmed.

[0348] The full-length amino acid sequence of NYA-1143-Fab-HC1-k is the amino acid sequence shown in sequence number 104 (Figure 105) of the sequence listing. In this sequence, amino acid sequences 1 through 19 are the signal sequence, amino acid sequences 21 through 139 are the variable region, and amino acid sequences 140 through 468 are the constant region. In addition, amino acid sequences 45 through 52 are CDRH1 (sequence number 54 (Figure 61)), amino acid sequences 70 through 77 are CDRH2 (sequence number 55 (Figure 61)), and amino acid sequences 116 through 128 are CDRH3 (sequence number 56 (Figure 61)).

[0349] The full-length amino acid sequence of NYA-1143-LC is the amino acid sequence shown in sequence number 105 (Figure 106) of the sequence listing. The sequence of amino acids from position 1 to 20 is the signal sequence, the sequence of amino acids from position 21 to 131 is the variable region, and the sequence of amino acids from position 132 to 237 is the constant region. In addition, amino acids 46 to 53 are CDRL1 (sequence number 60 (Figure 62)), amino acids 71 ​​to 73 are CDRL2 (DNN (Figure 61)), and amino acids 110 to 121 are CDRL3 (sequence number 59 (Figure 61)).

[0350] The full-length amino acid sequence of C3E-7085-HC2-k delete is the amino acid sequence shown in sequence number 106 (Figure 107) of the sequence listing. The amino acid sequence from the 1st to the 19th is the signal sequence, the amino acid sequence from the 21st to the 260th is C3E-7085, the amino acid sequence from the 261st to the 262nd is the linker, and the amino acid sequence from the 263rd to the 493rd is Fc, which has been modified to reduce effector function and form a heterodimer.

[0351] 10)-1-2 Preparation of Dual-Type Bispecific Molecular Expression Vectors To evaluate the dual-type anti-HLA / NY-ESO-anti-CD3 bispecific molecule, the following expression vector was designed. A mammalian cell expression vector, "p_NYA-1143-HC1-k delete," was created by incorporating a DNA fragment encoding an Fc region in which a mutation was introduced at the carboxyl terminus of NYA-1143 that reduces effector function and forms a heteromultimer.

[0352] The nucleotide sequence of p_NYA-1143-HC1-k delete was re-analyzed, and it was confirmed that the full-length nucleotide sequence of NYA-1143-HC1-k delete is the nucleotide sequence shown in sequence number 107 (Figure 108) of the sequence listing. Furthermore, the full-length amino acid sequence of NYA-1143-HC1-k delete encoded by the above nucleotide sequence was confirmed. The full-length amino acid sequence of NYA-1143-HC1-k delete is the amino acid sequence shown in sequence number 108 (Figure 109) of the sequence listing. 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, the amino acid sequence from the 267th to the 268th is the linker, and the amino acid sequence from the 269th to the 499th is Fc, which has a mutation introduced that reduces effector function and forms a heterodimer.

[0353] 10)-1-3 Construction of scFv-Fab-heterodimer Fc-type bispecific molecule expression vector To evaluate the scFv-Fab heterodimer Fc type anti-HLA / NY-ESO-anti-CD3 bispecific molecule, expression vectors for each molecule were designed. NYA-1154 was used as the anti-HLA / NY-ESO antibody, and C3E-7085 as the anti-CD3 antibody. For the heterodimer Fc sequence, an Fc sequence was used that introduced a mutation that reduced effector function and formed a heteromultimer.

[0354] An expression vector for mammalian cells was created by incorporating DNA fragments encoding the heavy chain variable region of NYA-1154, the human IgG-derived CH1 region, and the Fc region with a mutation that reduces effector function and forms a heteromultimer at the carboxyl terminus of C3E-7085. This vector was named "p_C3E-7085-NYA-1154-Fab-HC2-k delete". An expression vector for mammalian cells was also created by incorporating DNA fragments encoding the NYA-1154 light chain variable region and the human IgG-derived CL region. This vector was named "p_NYA-1154-LC". Furthermore, an expression vector for mammalian cells was created by incorporating a DNA fragment encoding HC1-k delete. This vector was named "p_OAA-HC1-k delete".

[0355] The nucleotide sequence of p_C3E-7085-NYA-1154-Fab-HC2-k delete was re-analyzed, and it was confirmed that the full-length nucleotide sequence of C3E-7085-NYA-1154-Fab-HC2-k delete is the nucleotide sequence shown in sequence number 109 of the sequence listing (Figure 110).

[0356] The nucleotide sequence of p_NYA-1154-LC was re-analyzed, and it was confirmed that the full-length nucleotide sequence of NYA-1154-LC is the nucleotide sequence shown in sequence number 110 (Figure 111) of the sequence listing.

[0357] The nucleotide sequence of p_OAA-HC1-k delete was re-analyzed, and it was confirmed that the full-length nucleotide sequence of OAA-HC1-k delete is the nucleotide sequence shown in sequence number 111 (Figure 112) of the sequence listing.

[0358] Furthermore, the full-length amino acid sequences of C3E-7085-NYA-1154-Fab-HC2-k delete, NYA-1154-LC, and OAA-HC1-k delete encoded by the above nucleotide sequence were confirmed.

[0359] The full-length amino acid sequence of C3E-7085-NYA-1154-Fab-HC2-k delete is the amino acid sequence shown as sequence number 112 (Figure 113) in the sequence listing. The amino acid sequence from position 1 to 19 is the signal sequence, the amino acid sequence from position 21 to 260 is C3E-7085, the amino acid sequence from position 261 to 385 is the linker, and the amino acid sequence from position 266 to 385 is the NYA-1154-heavy chain variable region. In addition, the amino acid sequence from amino acid number 386 to 714 is the HC2-k delete.

[0360] The full-length amino acid sequence of NYA-1154-LC is the sequence shown in sequence number 113 (Figure 114) of the sequence listing. The sequence of amino acids from position 1 to 20 is the signal sequence, the sequence of amino acids from position 21 to 131 is the variable region, and the sequence of amino acids from position 132 to 237 is the constant region. In addition, amino acids 46 to 53 are CDRL1 (sequence number 57 (Figure 61)), amino acids 71 ​​to 73 are CDRL2 (DNN (Figure 61)), and amino acids 110 to 121 are CDRL3 (sequence number 63 (Figure 64)).

[0361] The full-length amino acid sequence of OAA-HC1-k delete is the amino acid sequence shown in sequence number 114 (Figure 115) of the sequence listing. In this sequence, the amino acid sequence from the 1st to the 19th is the signal sequence, and the amino acid sequence from the 20th to the 245th is the OAA-HC1-k delete.

[0362] 10)-1-4 Preparation of taFv-heterodimer Fc-type bispecific molecule expression vectors for evaluation of various formats To evaluate taFv-heterodimer Fc-type anti-HLA / NY-ESO-anti-CD3 bispecific molecules for various format evaluations, expression vectors for each molecule were designed. NYA-1143 and NYA-1154 were used as anti-HLA / NY-ESO antibodies. C3E-7085, a humanized anti-CD3scFv, was used as the anti-CD3 antibody. For the heterodimer Fc sequence, an Fc sequence was used that introduced a mutation that reduced effector function and formed a heteromultimer.

[0363] We created a mammalian cell expression vector by linking NYA-1154 and C3E-7085 with a GGGGS linker, and then incorporating a DNA fragment encoding HC2-k delete into the carboxyl terminus of taFv. This vector was named "p_NYF-0010-HC2-k delete".

[0364] We created a mammalian cell expression vector by linking C3E-7085 and NYA-1154 with a GGGGS linker, and then incorporating a DNA fragment encoding HC2-k delete into the carboxyl terminus of taFv. This vector was named "p_NYF-0004-HC2-k delete".

[0365] We created a mammalian cell expression vector by linking NYA-1143 and C3E-7085 with a GGGGS linker, and then incorporating a DNA fragment encoding HC2-k delete into the carboxyl terminus of taFv. This vector was named "p_NYF-0011-HC2-k delete".

[0366] The nucleotide sequence of p_NYF-0010-HC2-k delete was re-analyzed, and it was confirmed that the full-length nucleotide sequence of NYF-0010-HC2-k delete is the nucleotide sequence shown in sequence number 115 of the sequence listing (Figure 116).

[0367] The nucleotide sequence of p_NYF-0004-HC2-k delete was re-analyzed, and it was confirmed that the full-length nucleotide sequence of NYF-0004-HC2-k delete is the nucleotide sequence shown in sequence number 116 of the sequence listing (Figure 117).

[0368] The nucleotide sequence of p_NYF-0011-HC2-k delete was re-analyzed, and it was confirmed that the full-length nucleotide sequence of NYF-0011-HC2-k delete is the nucleotide sequence shown in sequence number 117 of the sequence listing (Figure 118).

[0369] Furthermore, the full-length amino acid sequences of NYF-0010-HC2-k delete, NYF-0004-HC2-k delete, and NYF-0011-HC2-k delete encoded by the above nucleotide sequences were confirmed.

[0370] The full-length amino acid sequence of NYF-0010-HC2-k delete is the amino acid sequence shown in sequence number 118 (Figure 119) of the sequence listing. In this sequence, the amino acid sequence from the 1st to the 19th amino acid sequence is the signal sequence, the amino acid sequence from the 21st to the 511th amino acid sequence is NYA-1154-C3E-7085taFv, the amino acid sequence from the 512th to the 513th amino acid sequence is the linker, and the amino acid sequence from the 514th to the 744th amino acid sequence is the HC2-k delete.

[0371] The full-length amino acid sequence of NYF-0004-HC2-k delete is the amino acid sequence shown as sequence number 119 in the sequence listing (Figure 120). In this sequence, amino acid sequences 1 through 19 are the signal sequence, amino acid sequences 21 through 511 are C3E-7085-NYA-1154taFv, amino acid sequences 512 through 513 are the linker, and amino acid sequences 514 through 744 are the HC2-k delete.

[0372] The full-length amino acid sequence of NYF-0011-HC2-k delete is the amino acid sequence shown as sequence number 120 (Figure 121) in the sequence listing. 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-1143-C3E-7085taFv, the amino acid sequence from the 512th to the 513th is the linker, and the amino acid sequence from the 514th to the 745th is the HC2-k delete.

[0373] 10)-2 Expression of various format Fc-added anti-HLA / NY-ESO-anti-CD3 bispecific molecules 10)-2-1 Expression of Hybrid Bispecific Molecules Using the same method as in 7)-2-1, a vector mixture was prepared by mixing p_NYA-1143-Fab-HC1-k delete, p_C3E-7085-HC2-k delete, and p_NYA-1143-LC in a ratio of 1:1:1.5 to prepare the culture supernatant for expression of the hybrid bispecific molecule (NYG-3143). The amino acid sequences obtained by expressing each vector constituting NYG-3143 are shown in sequence numbers 104 (Figure 105), 105 (Figure 106), and 106 (Figure 107) of the sequence listing.

[0374] 10)-2-2 Expression of Dual-Type Bispecific Molecules Using the same method as in 7)-2-1, a vector mixture of p_NYA-1143-HC1-k delete and p_C3E-7085-HC2-k delete in a 2:1 ratio was used to prepare the culture supernatant for the dual-type bispecific molecule (NYG-2143). The amino acid sequences obtained by expressing each vector constituting NYG-2143 are shown in sequence numbers 108 (Figure 109) and 106 (Figure 107) of the sequence listing.

[0375] 10)-2-3 Expression of scFv-Fab heterodimer Fc-type bispecific molecule Using the same method as in 7)-2-1, a vector mixture of p_C3E-7085-NYA-1154-Fab-HC2-k delete, p_OAA-HC1-k delete, and p_NYA-1154-LC in a ratio of 1:1:1.5 was used to prepare the culture supernatant for expression of the scFv-Fab heterodimer Fc type (scFv-Fab-Fc type) bispecific molecule (NYF-0003). The amino acid sequences obtained by expressing each vector constituting NYF-0003 are shown in sequence numbers 112 (Figure 113), 113 (Figure 114), and 114 (Figure 115) of the sequence listing.

[0376] 10)-2-4 Expression of taFv-heterodimer Fc-type bispecific molecules for evaluation of various formats Using the same method as in 7)-2-1, the culture supernatant of the taFv-heterodimer Fc type (taFv-Fc type) anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0010) was prepared for expression using p_NYF-0010-HC2-k delete and p_OAA-HC1-k delete. The amino acid sequences obtained by expressing each vector constituting NYF-0010 are shown in sequence numbers 118 (Figure 119) and 114 (Figure 115) of the sequence listing.

[0377] The culture supernatant of the taFv(inversed)-heterodimer Fc-type (taFv(inversed)-Fc-type) anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0004) was prepared for expression using p_NYF-0004-HC2-k delete and p_OAA-HC1-k delete. The amino acid sequences obtained by expressing each vector constituting NYF-0004 are shown in sequence numbers 119 (Figure 120) and 114 (Figure 115) of the sequence listing.

[0378] The culture supernatant of the taFv-Fc type anti-HLA / NY-ESO-anti-CD3 bispecific molecule (NYF-0011) was prepared for expression using p_NYF-0011-HC2-k delete and p_OAA-HC1-k delete. The amino acid sequences obtained by expressing each vector constituting NYF-0011 are shown in sequence numbers 120 (Figure 121) and 114 (Figure 115) of the sequence listing.

[0379] 10)-3 Purification of various format Fc-added anti-HLA / NY-ESO-anti-CD3 bispecific molecules From the culture supernatants obtained in 10)-2-1 and 10)-2-2, various bispecific molecules were purified using a two-step process involving Protein A affinity chromatography and ceramic hydroxyapatite.

[0380] The culture supernatant was applied to a MabSelectSuRe column (GE Healthcare Bioscience) equilibrated with PBS pH 7.4, and the target bispecific molecule was adsorbed. After removing non-adsorbed components with PBS, the adsorbed components were eluted with acetate buffer pH 3.6. The eluted fraction was neutralized with Tris buffer pH 9.5, and the buffer was changed to 25 mM Histidine, 150 mM NaCl, 5% Sorbitor, pH 5.5. The target fraction solution, diluted 5-fold with 10 mM potassium phosphate / 50 mM MES / pH 6.5 buffer, was applied to a ceramic hydroxyapatite column (Bio-Scale CHT Type-1 Hydroxyapatite Column: Bio-Rad Japan) equilibrated with 10 mM potassium phosphate / 50 mM MES / pH 6.5 buffer. Linear concentration gradient elution with sodium chloride was performed, and the fraction corresponding to the target heterodimer was collected. The samples were subjected to gel filtration on a Superdex 200 10 / 300 (GE Healthcare Bioscience) column pre-equilibriumated with 25 mM Histidine, 300 mM NaCl, 5% Sorbitor, and pH 6.0. From the peak fractions obtained by gel filtration chromatography, the fraction corresponding to the target heterodimer was recovered and the buffer was changed to 25 mM Histidine, 150 mM NaCl, 5% Sorbitor, and pH 5.5. The formation of the target anti-HLA / NY-ESO-anti-CD3 bispecific molecule was confirmed by SDS-polyacrylamide electrophoresis (SDS-PAGE). The purified protein samples were subjected to analytical SEC to determine purity and concentration, and then used for various evaluations.

[0381] Furthermore, various bispecific molecules were purified from the culture supernatants obtained in 10)-2-3 and 10)-2-4 using the same method as in 7)-3, in a two-step process of Protein A affinity chromatography and gel filtration chromatography. The formation of the target anti-HLA / NY-ESO-anti-CD3 bispecific molecules was confirmed by SDS-polyacrylamide electrophoresis (SDS-PAGE). The purified protein samples were subjected to analytical SEC to determine purity and concentration, and then used for various evaluations.

[0382] (Example 11) In vitro activity evaluation of various format Fc-added anti-HLA-A2 / NY-ESO-anti-CD3 bispecific molecules 11)-1 In vitro activity evaluation of Hybrid, Dual, and taFv-Fc anti-HLA-A2 / NY-ESO-anti-CD3...

Claims

1. A bispecific molecule comprising an antibody or its binding fragment that binds to human HLA-A2 / NY-ESO, and an antibody or its binding fragment that specifically binds to CD3, An antibody or its binding fragment that binds to human HLA-A2 / NY-ESO has the properties described in (i) or (ii) below, (i) Heavy chain CDRH1 consisting of the amino acid sequence represented by Sequence ID No. 54, The heavy chain CDRH2 consists of the amino acid sequence represented by SEQ ID NO:

55. A heavy chain CDRH3 consisting of the amino acid sequence represented by Sequence ID No. 56, In the amino acid sequence represented by Sequence ID No. 57, the light chain CDRL1 consists of an amino acid sequence in which the 7th amino acid is W. A light chain CDRL2 consisting of an amino acid sequence represented by DNN, and Light chain CDRL3 consisting of the amino acid sequence represented by Sequence ID No. 59 An antibody or its binding fragment that specifically binds to human HLA / NY-ESO, including the above, binds to a site on HLA-A2 / NY-ESO recognized by the antibody, and recognizes the 1st, 4th, 5th, and 7th amino acids of the NY-ESO peptide represented by SEQ ID NO: 1; (ii) Heavy chain CDRH1 consisting of the amino acid sequence represented by Sequence ID No. 54, The heavy chain CDRH2 consists of the amino acid sequence represented by SEQ ID NO:

55. A heavy chain CDRH3 consisting of the amino acid sequence represented by Sequence ID No. 56, In the amino acid sequence represented by Sequence ID No. 57, the light chain CDRL1 consists of an amino acid sequence in which the 7th amino acid is W. A light chain CDRL2 consisting of an amino acid sequence represented by DNN, and Light chain CDRL3 consisting of the amino acid sequence represented by Sequence ID No. 59 An antibody or its binding fragment that specifically binds to human HLA / NY-ESO, and which competes for binding to HLA-A2 / NY-ESO, and recognizes the 1st, 4th, 5th and 7th amino acids of the NY-ESO peptide represented by SEQ ID NO: 1, An antibody or its binding fragment that specifically binds to CD3, (CCH1) Heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 141, (CCH2) Heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 142, or heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 142 in which the third amino acid is N or S, (CCH3) Heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 143, (CCL1) Light chain CDRL1 consisting of the amino acid sequence represented by sequence number 144, (CCL2) Light chain CDRL2 consisting of an amino acid sequence represented by RDD, or a light chain CDRL2 consisting of an amino acid sequence in which the second amino acid is N, and (CCL3) Light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 146 An antibody or its conjugated fragment that specifically binds to CD3, Bispecific molecules.

2. A bispecific molecule in which an antibody or its binding fragment that binds to human HLA-A2 / NY-ESO has any of the properties described in (i) to (iv) below: (i) Heavy chain variable region consisting of amino acid sequences 21 to 140 of the amino acid sequence represented by SEQ ID NO: 53 and light chain variable region sequence consisting of amino acid sequences 156 to 266 of the amino acid sequence represented by SEQ ID NO: 53 An antibody or its binding fragment that specifically binds to human HLA / NY-ESO, including the above, binds to a site on HLA-A2 / NY-ESO recognized by the antibody, and recognizes the 1st, 4th, 5th, and 7th amino acids of the NY-ESO peptide represented by SEQ ID NO: 1; (ii) Heavy chain variable region consisting of amino acid sequences 21 to 140 of the amino acid sequence represented by SEQ ID NO: 53 and light chain variable region sequence consisting of amino acid sequences 156 to 266 of the amino acid sequence represented by SEQ ID NO: 53 An antibody or its binding fragment that specifically binds to human HLA / NY-ESO, including, competes for binding to HLA-A2 / NY-ESO and recognizes the 1st, 4th, 5th, and 7th amino acids of the NY-ESO peptide represented by SEQ ID NO: 1; (iii) Heavy chain variable region consisting of amino acid sequences 21 to 140 of the amino acid sequence represented by SEQ ID NO: 156 and light chain variable region consisting of amino acid sequences 161 to 271 of the amino acid sequence represented by SEQ ID NO: 156 An antibody or its binding fragment that specifically binds to human HLA / NY-ESO, including the above, binds to a site on HLA-A2 / NY-ESO recognized by the antibody, and recognizes the 1st, 4th, 5th, and 7th amino acids of the NY-ESO peptide represented by SEQ ID NO: 1; (iv) Heavy chain variable region consisting of amino acid sequences 21 to 140 of the amino acid sequence represented by SEQ ID NO: 156 and light chain variable region consisting of amino acid sequences 161 to 271 of the amino acid sequence represented by SEQ ID NO: 156 It contains an antibody or its binding fragment that specifically binds to human HLA / NY-ESO, which competes with HLA-A2 / NY-ESO for binding and recognizes the 1st, 4th, 5th, and 7th amino acids of the NY-ESO peptide represented by SEQ ID NO:

1.

3. The bispecific molecule according to claim 1 or 2, wherein the antibody or binding fragment thereof that binds to human HLA-A2 / NY-ESO is scFv.

4. A bispecific molecule according to any one of claims 1 to 3, wherein the HMWS content calculated by measuring HMWS (aggregates) of an antibody or a conjugated fragment thereof that binds to human HLA-A2 / NY-ESO at pH 3.5, room temperature, for 1 hour, and then by size exclusion chromatography, is 5% or less, preferably 2% or less, and more preferably 1% or less.

5. A bispecific molecule according to any one of claims 1 to 3, wherein an antibody that binds to human HLA-A2 / NY-ESO or a conjugated fragment thereof is dissolved in a pH 6.0 solution of 25 mM histidine and 5% sorbitol to a concentration of 25 mg / ml, stored at 25°C for 6 days, and then the HMWS (aggregates) content, calculated by measuring HMWS by size exclusion chromatography, is 20% or less.

6. An antibody or its binding fragment that specifically binds to CD3, (CH1) Heavy chain variable region consisting of amino acid sequences 2 to 119 of the amino acid sequence represented by SEQ ID NO: 136, (CH2) Heavy chain variable region consisting of amino acid sequences 2 to 119 of the amino acid sequence represented by SEQ ID NO: 137, (CH3) Heavy chain variable region consisting of amino acid sequences 2 to 119 of the amino acid sequence represented by SEQ ID NO: 147, (CH4) Heavy chain variable region consisting of amino acid sequences 2 to 119 of the amino acid sequence represented by SEQ ID NO: 138, (CH5) Heavy chain variable region consisting of amino acid sequences 2 to 119 of the amino acid sequence represented by SEQ ID NO: 139, (CH6) Heavy chain variable region consisting of amino acid sequences 2 to 119 of the amino acid sequence represented by SEQ ID NO: 140, (CH7) Heavy chain variable region consisting of amino acid sequences 272 to 389 of the amino acid sequence represented by SEQ ID NO: 155, (CH8) A heavy chain variable region consisting of amino acid sequences 277 to 394 of the amino acid sequence represented by SEQ ID NO: 156, or (CH9) A heavy chain variable region consisting of amino acid sequences 277 to 394 of the amino acid sequence represented by SEQ ID NO: 157, and (CL1) Light chain variable region consisting of amino acid sequences 135 to 243 of the amino acid sequence represented by SEQ ID NO: 136, (CL2) Light chain variable region consisting of amino acid sequences 135 to 241 of the amino acid sequence represented by Sequence ID No. 137, (CL3) Light chain variable region consisting of amino acid sequences 135 to 243 of the amino acid sequence represented by Sequence ID No. 147, (CL4) Light chain variable region consisting of amino acid sequences 135 to 241 of the amino acid sequence represented by SEQ ID NO: 138, (CL5) Light chain variable region consisting of amino acid sequences 135 to 243 of the amino acid sequence represented by Sequence ID No. 139, (CL6) Light chain variable region consisting of amino acid sequences 135 to 243 of the amino acid sequence represented by SEQ ID NO: 140, (CL7) Light chain variable region consisting of amino acid sequences 405 to 511 of the amino acid sequence represented by SEQ ID NO: 155, (CL8) The light chain variable region consisting of amino acid sequences 410 to 516 of the amino acid sequence represented by SEQ ID NO: 156, or (CL9) Light chain variable region consisting of amino acid sequences 410 to 516 of the amino acid sequence represented by sequence number 157, A bispecific molecule according to any one of claims 1 to 5, comprising:

7. An antibody or its binding fragment that specifically binds to CD3, (CH1CL1) Heavy chain variable region consisting of amino acid sequences 2 to 119 of the amino acid sequence represented by SEQ ID NO: 136 and light chain variable region consisting of amino acid sequences 135 to 243 of the amino acid sequence represented by SEQ ID NO: 136, (CH2CL2) Heavy chain variable region consisting of amino acid sequences 2 to 119 of the amino acid sequence represented by SEQ ID NO: 137 and light chain variable region consisting of amino acid sequences 135 to 241 of the amino acid sequence represented by SEQ ID NO: 137, (CH3CL3) Heavy chain variable region consisting of amino acid sequences 2 to 119 of the amino acid sequence represented by SEQ ID NO: 147 and light chain variable region consisting of amino acid sequences 135 to 243 of the amino acid sequence represented by SEQ ID NO: 147, (CH4CL4) Heavy chain variable region consisting of amino acid sequences 2 to 119 of the amino acid sequence represented by SEQ ID NO: 138 and light chain variable region consisting of amino acid sequences 135 to 241 of the amino acid sequence represented by SEQ ID NO: 138, (CH5CL5) Heavy chain variable region consisting of amino acid sequences 2 to 119 of the amino acid sequence represented by SEQ ID NO: 139 and light chain variable region consisting of amino acid sequences 135 to 243 of the amino acid sequence represented by SEQ ID NO: 139, (CH6CL6) Heavy chain variable region consisting of amino acid sequences 2 to 119 of the amino acid sequence represented by SEQ ID NO: 140 and light chain variable region consisting of amino acid sequences 135 to 243 of the amino acid sequence represented by SEQ ID NO: 140, (CH7CL7) Heavy chain variable region consisting of amino acid sequences 272 to 389 of the amino acid sequence represented by SEQ ID NO: 155 and light chain variable region consisting of amino acid sequences 405 to 511 of the amino acid sequence represented by SEQ ID NO: 155, (CH8CL8) A heavy chain variable region consisting of amino acid sequences 277 to 394 of the amino acid sequence represented by SEQ ID NO: 156 and a light chain variable region consisting of amino acid sequences 410 to 516 of the amino acid sequence represented by SEQ ID NO: 156, or The bispecific molecule according to claim 6, comprising a heavy chain variable region consisting of amino acid sequences 277 to 394 of the amino acid sequence represented by (CH9CL9)SEQ ID NO: 157, and a light chain variable region consisting of amino acid sequences 410 to 516 of the amino acid sequence represented by SEQ ID NO:

157.

8. The bispecific molecule according to any one of claims 1 to 7, wherein the antibody or its binding fragment that specifically binds to CD3 is scFv.

9. An antibody or its binding fragment, scFv, that specifically binds to CD3, (CS1) scFv consisting of the amino acid sequence from the 2nd to the 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 136, (CS2) scFv consisting of the amino acid sequence from the 2nd to the 241st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 137, (CS3) scFv consisting of the amino acid sequence from the 2nd to the 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 147, (CS4) scFv consisting of the amino acid sequence from the 2nd to the 241st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 138, (CS5) scFv consisting of the amino acid sequence from the 2nd to the 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 139, (CS6) scFv consisting of the amino acid sequence from the 2nd to the 243rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 140, (CS7) scFv consisting of amino acid sequences 272 to 511 of the amino acid sequence represented by sequence number 155, (CS8) scFv consisting of amino acid sequences 277 to 516 of the amino acid sequence represented by SEQ ID NO: 156, or (CS9) scFv consisting of amino acid sequences 277 to 516 of the amino acid sequence represented by SEQ ID NO: 157, A bispecific molecule according to claim 8, comprising:

10. A first polypeptide comprising, in that order 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); and, A bispecific molecule according to claim 8 or 9, comprising a second polypeptide containing an Fc region (ii), preferably associated at the Fc region (i) and the Fc region (ii).

11. The bispecific molecule according to claim 10, wherein the scFv that specifically binds to human HLA / NY-ESO is an antibody or a binding fragment thereof that specifically binds to human HLA / NY-ESO according to claim 1, where scFv is scFv.

12. The bispecific molecule according to claim 10, wherein the scFv that specifically binds to human HLA / NY-ESO is an antibody or a binding fragment thereof that specifically binds to human HLA / NY-ESO according to claim 2, where scFv is scFv.

13. The bispecific molecule according to any one of claims 10 to 12, wherein the scFv that specifically binds to CD3 is an antibody that specifically binds to CD3 according to claim 1, or a binding fragment thereof.

14. The bispecific molecule according to any one of claims 10 to 12, wherein the scFv that specifically binds to CD3 is an antibody that specifically binds to CD3 according to claim 6, or a binding fragment thereof.

15. The bispecific molecule according to any one of claims 10 to 12, wherein the scFv that specifically binds to CD3 is an antibody that specifically binds to CD3 according to claim 7, or a binding fragment thereof.

16. A bispecific molecule according to any one of claims 10 to 15, comprising the amino acid sequence from the 21st to the 511th amino acid sequence of the amino acid sequence represented by Sequence ID No.

96.

17. A bispecific molecule according to any one of claims 10 to 15, comprising an amino acid sequence selected from the group consisting of the amino acid sequences from the 21st to the 516th position of the amino acid sequence represented by SEQ ID NO: 156 and the amino acid sequences from the 21st to the 516th position of the amino acid sequence represented by SEQ ID NO:

157.

18. The bispecific molecule according to any one of claims 10 to 16, wherein the first polypeptide comprises the amino acid sequence from position 529 to 745 of the amino acid sequence represented by SEQ ID NO:

96.

19. A bispecific molecule according to any one of claims 10 to 15 and 17, wherein the first polypeptide comprises the amino acid sequence from position 534 to 750 of the amino acid sequence represented by SEQ ID NO: 156, or the amino acid sequence from position 534 to 750 of the amino acid sequence represented by SEQ ID NO:

157.

20. The bispecific molecule according to claim 16 or 18, wherein the first polypeptide consists of the amino acid sequence from the 20th to the 745th amino acid sequence represented by SEQ ID NO:

96.

21. The bispecific molecule according to claim 17 or 19, wherein the first polypeptide consists of the amino acid sequence from the 20th to the 750th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 156, or the amino acid sequence from the 20th to the 750th amino acid sequence of the amino acid sequence represented by SEQ ID NO:

157.

22. The bispecific molecule according to any one of claims 10 to 21, wherein the second polypeptide comprises the amino acid sequence from the 20th to the 246th amino acid sequence shown in SEQ ID NO:

84.

23. The material comprises a first polypeptide comprising, in that order from the N-terminus to the C-terminus, an scFv that specifically binds to human HLA / NY-ESO, a variable region and a constant region CH1 of the antibody heavy chain that specifically binds to CD3, and an immunoglobulin Fc region (i); a second polypeptide comprising an immunoglobulin hinge region and an Fc region (ii); and a third polypeptide comprising an antibody light chain consisting of a variable region and a constant region. Preferably, the first polypeptide and the second polypeptide are associated at the Fc region (i) and Fc region (ii), and the first polypeptide is associated with the third polypeptide at the variable region and constant region CH1 of the antibody heavy chain. A bispecific molecule according to any one of claims 1 to 7.

24. The bispecific molecule according to claim 23, wherein the third polypeptide comprises an antibody light chain that specifically binds to the CD3, comprising a variable region and a constant region.

25. The bispecific molecule according to claim 23 or 24, wherein the scFv that specifically binds to human HLA / NY-ESO is an antibody or a binding fragment thereof that specifically binds to human HLA / NY-ESO according to claim 1, where scFv is scFv.

26. The bispecific molecule according to claim 23 or 24, wherein the scFv that specifically binds to human HLA / NY-ESO is an antibody or a binding fragment thereof that specifically binds to human HLA / NY-ESO according to claim 2, where scFv is scFv.

27. The bispecific molecule according to any one of claims 23 to 26, wherein the antibody or its binding fragment that specifically binds to the CD3 is Fab.

28. The bispecific molecule according to claim 27, wherein the Fab that specifically binds to the CD3 is the antibody that specifically binds to the CD3 according to claim 1, or a binding fragment thereof.

29. The bispecific molecule according to claim 27, wherein the Fab that specifically binds to the CD3 is the antibody that specifically binds to the CD3 according to claim 6, or a binding fragment thereof.

30. The bispecific molecule according to claim 27, wherein the Fab that specifically binds to the CD3 is the antibody that specifically binds to the CD3 according to claim 7, or a binding fragment thereof.

31. The bispecific molecule according to any one of claims 23 to 30, wherein the first polypeptide comprises the amino acid sequence from the 21st to the 394th amino acid sequence represented by SEQ ID NO:

160.

32. The first polypeptide comprises the amino acid sequence described in (i) or (ii) below, the bispecific molecule according to any one of claims 23 to 31: (i) The amino acid sequence from position 20 to 724 of the amino acid sequence represented by SEQ ID NO: 160; (ii) The amino acid sequence from position 20 to 719 of the amino acid sequence represented by SEQ ID NO:

197.

33. The bispecific molecule according to any one of claims 23 to 32, wherein the second polypeptide comprises the amino acid sequence from the 20th to the 246th amino acid sequence shown in SEQ ID NO:

84.

34. The bispecific molecule according to any one of claims 23 to 33, wherein the third polypeptide comprises the amino acid sequence from the 21st to the 127th amino acid sequence of the amino acid sequence represented by SEQ ID NO:

161.

35. The bispecific molecule according to any one of claims 23 to 34, wherein the third polypeptide comprises the amino acid sequence from the 21st to the 233rd amino acid sequence of the amino acid sequence represented by SEQ ID NO:

161.

36. A bispecific molecule according to any one of claims 1 to 35, wherein the amino acid sequence of one or more polypeptides contained in the bispecific molecule is delimited by the deletion of one or two amino acids from the carboxyl terminus of the sequence.

37. A polynucleotide comprising a nucleotide sequence encoding a bispecific molecule according to any one of claims 1 to 36.

38. A vector comprising the polynucleotide described in claim 37.

39. A host cell comprising the polynucleotide described in claim 37 or the vector described in claim 38.

40. A method for producing a bispecific molecule that specifically binds to human HLA / NY-ESO and human CD3, comprising the steps of (i) culturing the host cells described in claim 39, and (ii) purifying an antibody or a bound fragment thereof from the culture obtained in step (i).

41. A bispecific molecule obtained by the method described in claim 40, which specifically binds to human HLA / NY-ESO and human CD3.

42. A pharmaceutical composition comprising a bispecific molecule according to any one of claims 1 to 36 and 41, a polynucleotide according to claim 37, a vector according to claim 38, or a host cell according to claim 39 as an active ingredient.

43. The pharmaceutical composition according to claim 42, which is an anticancer agent.

44. Cancers include kidney cancer, melanoma, squamous cell carcinoma, basal cell carcinoma, conjunctival cancer, oral cancer, laryngeal cancer, pharyngeal cancer, thyroid cancer, lung cancer (non-small cell lung cancer (adenocarcinoma, squamous cell carcinoma, large cell carcinoma), small cell lung cancer), breast cancer, esophageal cancer, stomach cancer, duodenal cancer, small intestine cancer, colon cancer, rectal cancer, appendiceal cancer, anal cancer, liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, adrenal cancer, bladder cancer, prostate cancer, uterine cancer, vaginal cancer, liposarcoma, angiosarcoma, chondrosarcoma, The pharmaceutical composition according to claim 43, which is one or more selected from the group consisting of rhabdomyosarcoma, Ewing's sarcoma, osteosarcoma, undifferentiated pleomorphic sarcoma, myxoid fibrosarcoma, malignant peripheral schwannoma, retroperitoneal sarcoma, synovial sarcoma, uterine sarcoma, gastrointestinal stromal tumor, leiomyosarcoma, epithelioid sarcoma, B-cell lymphoma, T / NK-cell lymphoma, Hodgkin lymphoma, myeloid leukemia, lymphocytic leukemia, myeloproliferative disorder, myelodysplastic syndrome, multiple myeloma, testicular cancer, and ovarian cancer.

45. A pharmaceutical composition according to any one of claims 42 to 44, which is used in combination with other drugs.

46. A pharmaceutical composition according to any one of claims 42 to 44, comprising other drugs.

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