Antibody-pyrrolobenzodiazepine derivative conjugates

Abstract

The present invention pertains to: a novel antibody-pyrrolobenzodiazepine (PBD) derivative conjugate; a pharmaceutical agent using said antibody-drug conjugate and having a therapeutic effect against tumors; and a therapeutic method for tumors, using said antibody-drug conjugate or pharmaceutical agent.

Description

[Technical field]

[0001] The present invention relates to an antibody-drug conjugate useful as an antitumor drug, which comprises an antibody capable of targeting tumor cells and a pyrrolobenzodiazepine derivative linked via a linker structure moiety. [Background technology]

[0002] Antibody-drug conjugates (ADCs) are used in cancer treatment and the like, and are, for example, antibodies that can bind to antigens expressed on the surface of cancer cells and internalize the antigens into the cells through this binding, to which drugs with cytotoxic activity are bound. ADCs can be expected to efficiently deliver drugs to cancer cells, accumulating the drugs in the cancer cells and killing the cancer cells. One of the useful drugs used in ADCs is pyrrolobenzodiazepine (PBD). PBDs exhibit cytotoxicity by binding to the PuGPu sequence in the DNA minor groove. Anthramycin, a naturally occurring PBD, was first discovered in 1965, and since then, various naturally occurring and analogous PBDs have been discovered (Non-Patent Documents 1 to 4). The general structural formula of PBD is as follows:

[0003] [ka] PBDs differ in the number, type, and position of the substituents in the A and C rings, and are known to have different degrees of unsaturation in the B and C rings. It is known that the cytotoxicity of PBD is dramatically improved by forming a dimer structure (Non-Patent Documents 5 and 6), and various ADCs of dimeric PBDs have been reported (Patent Documents 1 to 15). However, no PBDs or ADCs having a spiro ring at the C2 position are known. Human CLDN6 (Claudin-6, hereinafter referred to as hCLDN6) is a member of the Claudin (CLDN) family of proteins, and is a four-transmembrane protein consisting of 220 amino acid residues. It has been suggested that hCLDN6 is overexpressed in some cancers and is an attractive target for cancer therapy (Non-Patent Documents 7 to 9). In addition, CLDN family proteins are taken up into cells by endocytosis, and some family proteins with short turnover times have been reported (Non-Patent Document 10), making it suitable as a target for antibody-drug conjugates (ADCs). Based on information suggesting such an association with cancer, monoclonal antibodies that specifically recognize hCLDN6 have been discovered (Patent Documents 16 and 17), and ADCs have been reported in which tubulin polymerization inhibitors monomethylauristatin E (MMAE) or maytansinoid (DM1) are linked to a CLDN6-specific monoclonal antibody (Non-Patent Document 11). On the other hand, an ADC has been disclosed in which a pyrrolobenzodiazepine (PBD), which has a strong cytocidal effect, is linked to an antibody that recognizes CLDN6 and CLDN9 (Patent Document 18), as an antibody that recognizes multiple CLDN families may be able to broaden the range of therapeutic applications (Patent Document 19). However, their activity is still not sufficient, and there exists an unmet medical need to target hCLDN6 therapeutically. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2013 / 173496 [Patent Document 2] International Publication No. 2014 / 130879 [Patent Document 3] International Publication No. 2017 / 004330 [Patent Document 4] International Publication No. 2017 / 004025 [Patent Document 5] International Publication No. 2017 / 020972 [Patent Document 6] International Publication No. 2016 / 036804 [Patent Document 7] International Publication No. 2015 / 095124 [Patent Document 8] International Publication No. 2015 / 052322 [Patent Document 9] International Publication No. 2015 / 052534 [Patent Document 10] International Publication No. 2016 / 115191 [Patent Document 11] International Publication No. 2015 / 052321 [Patent Document 12] International Publication No. 2015 / 031693 [Patent Document 13] International Publication No. 2011 / 130613 [Patent Document 14] International Publication No. 2005 / 040170 [Patent Document 15] International Publication No. 2017 / 137556 [Patent Document 16] International Publication No. 2009 / 087978 [Patent Document 17] International Publication No. 2011 / 057788 [Patent Document 18] International Publication No. 2015 / 069794 [Patent Document 19] International Publication No. 2017 / 096163 [Non-patent literature]

[0005] [Non-Patent Document 1] Julia Mantaj,et al.,Angewandte Chemie Internationl Edition 2016,55,2-29 [Non-Patent Document 2] Dyeison Antonow.et al., Chemical Reviews 2010,111, 2815-2864 [Non-Patent Document 3] In Antibiotics III. Springer Verlag, New York, pp.3-11 [Non-Patent Document 4] Accounts of Chemical Research 1986, 19, 230 [Non-Patent Document 5] Journal of the American Chemical Society 1992,114, 4939 [Non-Patent Document 6] Journal of Organic Chemistry 1996, 61, 8141 [Non-Patent Document 7] BMC Cancer, 2006, 6, 186. [Non-Patent Document 8] Histopathology, 2012, 61, 1043-1056. [Non-Patent Document 9] Int J Cancer, 2014, 135, 2206-2214. [Non-Patent Document 10] J Membrane Biol, 2004, 199, 29-38. [Non-Patent Document 11] 14th Annu Meet Cancer Immunother(CIMT)(May 10-12, Mainz) 2016, Abst 185 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention provides novel antibody-pyrrolobenzodiazepine (PBD) derivative conjugates and novel pyrrolobenzodiazepine (PBD) derivatives. The present invention also provides a pharmaceutical composition containing the antibody-PBD derivative conjugate having antitumor activity. Furthermore, the present invention provides a method for treating cancer using the antibody-PBD derivative conjugate. [Means for solving the problem]

[0007] As a result of intensive research, the present inventors have found that a novel antibody-pyrrolobenzodiazepine (PBD) derivative conjugate has strong antitumor activity. They also found that the absolute configuration of the hydroxyl group at the 11'-position of the pyrrolodiazepine derivative in the conjugate is S-configuration. The present invention has been completed based on the above findings. That is, the present invention relates to the following:

[0008] [1] The following formula; [ka]

[0009] (In the formula, m 1 represents an integer of 1 or 2, D is any one selected from the following group:

[0010] [ka]

[0011] In the formula, the asterisk * indicates a bond to L, L is a linker that connects D to the glycan that binds to the glycan that binds to Asn297 of Ab (N297 glycan), The N297 glycan may be remodeled, Ab indicates an antibody or a functional fragment of the antibody. The antibody-drug conjugate shown in FIG.

[0012] [2] L is -Lb-La-Lp-NH-B-CH 2 -O(C=O)-*, In the formula, the asterisk * indicates a bond to D, B is a 1,4-phenyl group, a 2,5-pyridyl group, a 3,6-pyridyl group, a 2,5-pyrimidyl group, or a 2,5-thienyl group; Lp is the following group; -GGVA-, -GG-(D-)VA-, -VA-, -GGFG-, -GGPI-, -GGVCit-, -GGVK-, and -GGPL-, indicates one selected from La is the following group: -C(=O)-CH 2 CH 2 -C(=O)-, -C(=O)-(CH 2 CH 2 ) 2 -C(=O)-, -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 ) 2 -C(=O)-, -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 2 -CH 2 -C(=O)-, -C(=O)-CH 2 CH 2 -NH-C(=O)-(CH 2 CH 2 O) 4 -CH 2 CH 2 -C(=O)-, -CH 2 -OC(=O)- and -OC(=O)-, and Lb is expressed by the following formula:

[0013] [ka]

[0014] [ka] or

[0015] [ka]

[0016] Here, in the structural formula of Lb shown above, the asterisk * indicates binding to La, and the wavy line indicates binding to the N297 glycan or a remodeled N297 glycan, the antibody-drug conjugate described in [1].

[0017] [3] L is one of the following groups: -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGVA-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GG-(D-)VA-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-(CH 2 CH 2 ) 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGPI-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGFG-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGVCit-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGVK-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGPL-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 ) 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 2 -CH 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -NH-C(=O)-(CH 2 CH 2 O) 4 -CH 2 CH 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-, -Z 2 -OC(=O)-GGVA-NH-B-CH 2 -OC(=O)-, and -Z 3 -CH 2 -OC(=O)-GGVA-NH-B-CH 2 -OC(=O)- indicates one selected from where B represents a 1,4-phenyl group; Z 1 has the following structural formula:

[0018] [ka]

[0019] indicates, Z 2 has the following structural formula: [ka] indicates, Z 3 has the following structural formula:

[0020] [ka]

[0021] indicates, Here, Z 1 , Z 2 and Z 3 In the structural formula, an asterisk * indicates the adjacent C(=O), OC(=O) or CH 2 The antibody-drug conjugate according to [1] or [2], wherein the wavy line indicates binding to the N297 glycan or a remodeled N297 glycan.

[0022] [4] L is one of the following groups: -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGVA-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-(CH 2 CH 2 ) 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-, -Z 1-C(=O)-CH 2 CH 2 -C(=O)-GGVCit-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 ) 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 2 -CH 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-, and -Z 1 -C(=O)-CH 2 CH 2 -NH-C(=O)-(CH 2 CH 2 O) 4 -CH 2 CH 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)- indicates one selected from where B is a 1,4-phenyl group; Z 1 has the following structural formula:

[0023] [ka]

[0024] indicates, Here, the above Z 1 In the structural formula, the asterisk * represents Z 1 The antibody-drug conjugate of claim 3, wherein the wavy line indicates binding to the C(=O) adjacent to the N297 glycan or a remodeled N297 glycan.

[0025] [5] The antibody-drug conjugate according to any one of [1] to [4], wherein the N297 glycan is a remodeled glycan.

[0026] [6] The antibody-drug conjugate according to any one of [1] to [5], wherein the N297 sugar chain is any one of N297-(Fuc)MSG1, N297-(Fuc)MSG2 or a mixture thereof, or N297-(Fuc)SG, each having a structure represented by the following formula:

[0027] [ka]

[0028] [ka]

[0029] [ka]

[0030] In the above formula, the wavy line indicates binding to Asn297 of the antibody, L(PEG) in the N297 glycan is *-(CH 2 CH 2 -O)n 5 -CH 2 CH 2 -NH-, where n 5 indicates an integer between 2 and 5, the amino group at the right end is bonded via an amide bond to the 2-position carboxylic acid of the non-reducing terminal sialic acid on the 1-3 side or / and 1-6 side of the branched chain of β-Man in the N297 glycan, and the asterisk * on the left end indicates the Z 1 This indicates that the ring is bonded to the nitrogen atom at the 1st or 3rd position on the triazole ring.

[0031] [7]n 5 The antibody-drug conjugate according to [6], wherein [8] The following formula;

[0032] [ka]

[0033] (In each of the structural formulas shown above, m 1 indicates an integer of 1 or 2, Ab is an antibody or a functional fragment of said antibody, The N297 sugar chain is N297-(Fuc)MSG1, N297-(Fuc)MSG2, or a mixture thereof, or N297-(Fuc)SG, each of which has the structure shown in the following formula:

[0034] [ka]

[0035] [ka]

[0036] [ka]

[0037] In the formula, the wavy line indicates binding to Asn297 of the antibody, L(PEG) in the N297 glycan is *-(CH 2 CH 2 -O) 3 -CH 2 CH 2 -NH-, Here, the amino group on the right end is bonded via an amide bond to the 2-position carboxylic acid of the non-reducing terminal sialic acid on the 1-3 chain side and / or the 1-6 chain side of the branched chain of β-Man of the N297 glycan, and the asterisk * on the left end indicates that it is bonded to the 1- or 3-position nitrogen atom on the triazole ring in the above structural formula.

[0038] [9] The following formula; [ka]

[0039] (In each of the structural formulas shown above, m 1 indicates an integer of 1 or 2, Ab is an antibody or a functional fragment of said antibody, The N297 sugar chain is N297-(Fuc)MSG1, N297-(Fuc)MSG2, or a mixture thereof, or N297-(Fuc)SG, each of which has the structure shown in the following formula:

[0040] [ka]

[0041] [ka]

[0042] [ka]

[0043] In the formula, the wavy line indicates binding to Asn297 of the antibody, L(PEG) in the N297 glycan is *-(CH 2 CH 2 -O) 3 -CH 2 CH 2 -NH-, Here, the amino group on the right end is bonded via an amide bond to the 2-position carboxylic acid of the non-reducing terminal sialic acid on the 1-3 chain side and / or the 1-6 chain side of the branched chain of β-Man of the N297 glycan, and the asterisk * on the left end indicates that it is bonded to the 1- or 3-position nitrogen atom on the triazole ring in the above structural formula.

[0044]

[10] The following formula; [ka]

[0045] (In each of the structural formulas shown above, m 1 indicates an integer of 1 or 2, Ab is an antibody or a functional fragment of said antibody, The N297 sugar chain is N297-(Fuc)MSG1, N297-(Fuc)MSG2, or a mixture thereof, or N297-(Fuc)SG, each of which has the structure shown in the following formula:

[0046] [ka]

[0047] [ka]

[0048] [ka]

[0049] In the formula, the wavy line indicates binding to Asn297 of the antibody, L(PEG) in the N297 glycan is *-(CH 2 CH 2 -O) 3 -CH 2 CH 2 -NH-, Here, the amino group on the right end is bonded via an amide bond to the 2-position carboxylic acid of the non-reducing terminal sialic acid on the 1-3 chain side and / or the 1-6 chain side of the branched chain of β-Man of the N297 glycan, and the asterisk * on the left end indicates that it is bonded to the 1- or 3-position nitrogen atom on the triazole ring in the above structural formula.

[0050]

[11] The following formula; [ka]

[0051] (In each of the structural formulas shown above, m 1 indicates an integer of 1 or 2, Ab is an antibody or a functional fragment of said antibody, The N297 sugar chain is N297-(Fuc)MSG1, N297-(Fuc)MSG2, or a mixture thereof, or N297-(Fuc)SG, each of which has the structure shown in the following formula:

[0052] [ka]

[0053] [ka]

[0054] [ka]

[0055] In the formula, the wavy line indicates binding to Asn297 of the antibody, L(PEG) in the N297 glycan is *-(CH 2 CH 2 -O) 3 -CH 2 CH 2 -NH-, Here, the amino group at the right end is bonded via an amide bond to the 2-position carboxylic acid of the non-reducing terminal sialic acid on the 1-3 chain side and / or the 1-6 chain side of the branched chain of β-Man of the N297 glycan, and the asterisk * on the left end indicates that it is bonded to the 1- or 3-position nitrogen atom on the triazole ring in the above formula.

[0056]

[12] The antibody-drug conjugate according to any one of [1] to

[11] , comprising an antibody having internalization activity.

[0057]

[13] The antibody-drug conjugate according to any one of [1] to

[12] , wherein the antibody has the property of binding to an antigen expressed in tumor cells and being taken up and internalized within the tumor cells.

[0058]

[14] The antibody-drug conjugate according to any one of [1] to

[13] , wherein the antibody has an antitumor effect.

[0059]

[15] Antibodies include anti-CLDN6 antibody, anti-CLDN9 antibody, anti-CLDN6 / CLDN9 antibody, anti-HER2 antibody, anti-HER3 antibody, anti-DLL3 antibody, anti-FAP antibody, anti-CDH11 antibody, anti-A33 antibody, anti- CanAg antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD25 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD37 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD98 antibody, anti-B The antibody-drug conjugate according to any one of [1] to

[14] , which is a 7-H3 antibody, an anti-TROP2 antibody, an anti-CEA antibody, an anti-Cripto antibody, an anti-EphA2 antibody, an anti-FGFR2 antibody, an anti-G250 antibody, an anti-MUC1 antibody, an anti-GPNMB antibody, an anti-integrin antibody, an anti-PSMA antibody, an anti-Tenascin-C antibody, an anti-SLC44A4 antibody, an anti-Mesothelin antibody, an anti-EGFR antibody, an anti-5T4 antibody, an anti-LRRC15 antibody, an anti-DR5 antibody, an anti-CDH3 antibody, an anti-PDPN antibody, or an anti-CD123 antibody.

[0060]

[16] The antibody-drug conjugate according to any one of [1] to

[15] , wherein the antibody specifically binds to CLDN6 and / or CLDN9.

[0061]

[17] The antibody-drug conjugate according to

[16] , wherein the antibody comprises a heavy chain comprising CDRH1, CDRH2 and CDRH3 as set forth in the following (a) or (b), and a light chain comprising CDRL1, CDRL2 and CDRL3: (a) CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO:9, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO:10, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO:11, as well as CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO:5, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO:6, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO:7 or the amino acid sequence in which one or two amino acids have been substituted, or (b) CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 15, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 16, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 17, as well as CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 12, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 13, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 14.

[0062]

[18] The antibody-drug conjugate according to

[17] , wherein the antibody comprises a heavy chain comprising CDRH1, CDRH2 and CDRH3 as set forth in the following (a) or (b), and a light chain comprising CDRL1, CDRL2 and CDRL3: (a) CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO:9, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO:10, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO:11, and CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO:5, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO:6, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO:7 or the amino acid sequence set forth in SEQ ID NO:8, or (b) CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 15, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 16, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 17, and CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 12, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 13, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 14.

[0063]

[19] The antibody-drug conjugate according to any one of

[16] to

[18] , wherein the antibody comprises a heavy chain variable region and a light chain variable region described in the following (a) or (b): (a) the antibody comprises a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 21 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 19; or (b) the antibody comprises a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 25, and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 23.

[0064]

[20] The antibody-drug conjugate according to any one of

[16] to

[19] , comprising a heavy chain variable region consisting of an amino acid sequence selected from the group consisting of the following (a) to (e), and a light chain variable region consisting of an amino acid sequence selected from the group consisting of (f) to (k): (a) the amino acid sequence set forth in SEQ ID NO: 54; (b) the amino acid sequence set forth in SEQ ID NO: 58; (c) the amino acid sequence set forth in SEQ ID NO: 62; (d) an amino acid sequence having at least 95% homology to the sequences of the framework regions other than each CDR sequence in the sequences (a) to (c); (e) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the sequences of framework regions other than each CDR sequence in the sequences (a) to (c); (f) the amino acid sequence set forth in SEQ ID NO: 38; (g) the amino acid sequence set forth in SEQ ID NO: 42; (h) the amino acid sequence set forth in SEQ ID NO: 46; (i) the amino acid sequence set forth in SEQ ID NO: 50; (j) an amino acid sequence having at least 95% homology to the sequences of the framework regions other than each CDR sequence in the sequences (f) to (i); and (k) An amino acid sequence in which one or several amino acids have been deleted, substituted or added in the sequences of the framework regions other than each CDR sequence in the sequences (f) to (i).

[0065]

[21] The antibody-drug conjugate according to

[20] , wherein the antibody comprises a heavy chain variable region and a light chain variable region selected from the group consisting of the following (a) to (e): (a) a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 54 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 38; (b) a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 58 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 42; (c) a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 54 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 46; (d) a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 58 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 50; and (e) a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 62 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 46.

[0066]

[22] The antibody-drug conjugate according to any one of

[16] to

[21] , wherein the antibody is a chimeric antibody.

[0067]

[23] The antibody-drug conjugate according to any one of

[16] to

[21] , wherein the antibody is a humanized antibody.

[0068]

[24] The antibody-drug conjugate according to any one of

[16] to

[23] , wherein the antibody comprises a heavy chain constant region of human IgG1, human IgG2, or human IgG4.

[0069]

[25] The antibody-drug conjugate according to

[23] or

[24] , which comprises a heavy chain and a light chain selected from the group consisting of the following (a) to (e): (a) a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 52 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 36, (b) a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 56, and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 40; (c) a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 52 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 44; (d) a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 56 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 48; and (e) a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 60, and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 44.

[0070]

[26] The antibody-drug conjugate according to

[16] , wherein the antibody competes with the antibody according to any one of

[17] to

[21] and

[25] in binding to CLDN6 and / or CLDN9, or binds to a site on CLDN6 and / or CLDN9 recognized by the antibody according to any one of

[17] to

[21] and

[25] .

[0071]

[27] The antibody-drug conjugate according to any one of [1] to

[15] , wherein the antibody specifically binds to HER2.

[0072]

[28] An antibody-drug conjugate according to any one of

[27] , having antibody-dependent cellular cytotoxicity (ADCC) activity and / or complement-dependent cytotoxicity (CDC) activity.

[0073]

[29] The antibody-drug conjugate according to

[27] , wherein the heavy chain constant region of the antibody is a heavy chain constant region of human IgG1 and contains a mutation that results in reduced ADCC and / or CDC activity.

[0074]

[30] The antibody-drug conjugate according to

[29] , wherein the antibody heavy chain constant region is a heavy chain constant region of human IgG1, and in the heavy chain constant region, leucine at positions 234 and 235 as indicated by the EU Index is substituted with alanine.

[0075]

[31] The antibody-drug conjugate according to

[27] or

[28] , which is a humanized monoclonal antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 65 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 64.

[0076]

[32] The antibody-drug conjugate according to any one of

[27] ,

[29] and

[30] , which is an antibody comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 139 of SEQ ID NO: 75 and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 127 of SEQ ID NO: 73.

[0077]

[33] The antibody-drug conjugate according to any one of

[27] ,

[29] ,

[30] and

[32] , which is a humanized monoclonal antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 469 of SEQ ID NO: 75 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 73.

[0078]

[34] The antibody-drug conjugate according to any one of

[16] to

[33] , wherein the antibody comprises one or more modifications selected from the group consisting of N-linked glycosylation, O-linked glycosylation, N-terminal processing, C-terminal processing, deamidation, isomerization of aspartic acid, oxidation of methionine, addition of a methionine residue at the N-terminus, amidation of a proline residue, and deletion of one or two amino acid residues at the carboxyl terminus of the heavy chain.

[0079]

[35] The antibody-drug conjugate according to

[34] , wherein one or more amino acid residues are deleted at the carboxyl terminus of the antibody heavy chain.

[0080]

[36] The antibody-drug conjugate according to

[34] or

[35] , wherein one amino acid residue is deleted at the carboxyl terminus of both of the two heavy chains of the antibody.

[0081]

[37] The antibody-drug conjugate according to any one of

[34] to

[36] , wherein the proline residue at the carboxyl terminus of the heavy chain is further amidated.

[0082]

[38] The antibody-drug conjugate according to any one of [1] to

[37] , wherein the antibody is a glycan remodeling antibody obtained by the following steps: i) culturing a host cell containing a polynucleotide encoding the antibody according to any one of

[12] to

[37] , and collecting the antibody of interest from the resulting culture; ii) treating the antibody obtained in step i) with a hydrolase to produce a (Fucα1,6)GlcNAc-antibody; and iii) A process in which a PEG linker having an azide group is introduced into the carbonyl group of the carboxylate at the 2-position of the sialic acid of MSG(9) or SG(10), and the reducing end is oxazolinated to obtain a glycan donor molecule, which is then reacted with a (Fucα1,6)GlcNAc-antibody in the presence of a glycosyltransferase.

[0083]

[39] The antibody-drug conjugate according to

[38] , further comprising a step of purifying the (Fucα1,6)GlcNAc-antibody by purifying the reaction solution of step ii) through a hydroxyapatite column.

[0084]

[40] The following formula; [ka]

[0085] Any one of the compounds represented by the following formula (1), its salt, or a hydrate thereof:

[0086]

[41] A method for producing an antibody-drug conjugate according to any one of [1] to

[39] , comprising a step of reacting a glycan remodeling antibody according to

[38] or

[39] with a compound according to

[40] , a salt thereof, or a hydrate thereof.

[0087] An antibody-drug conjugate, characterized in that it is obtained by the production method described in

[42]

[41] .

[0088]

[43] The antibody-drug conjugate according to any one of [1] to

[39] and

[42] , wherein the N297 glycan is N297-(Fuc)MSG1.

[0089]

[44] m 1 The antibody-drug conjugate according to any one of [1] to

[39] ,

[42] and

[43] , wherein is an integer of 1.

[0090]

[45] The antibody-drug conjugate according to any one of [1] to

[39] and

[42] to

[44] , wherein the average number of drugs bound per antibody molecule in the antibody-drug conjugate is 1 to 3 or 3 to 5.

[0091]

[46] A pharmaceutical composition comprising the antibody-drug conjugate according to any one of [1] to

[39] and

[42] to

[45] , a salt thereof, or a hydrate thereof.

[0092]

[47] The pharmaceutical composition according to

[46] , which is an antitumor drug.

[0093]

[48] ​​A method for treating a tumor, comprising administering to an individual an antibody-drug conjugate, a salt thereof, or a hydrate thereof according to any one of [1] to

[39] and

[42] to

[45] .

[0094]

[49] A pharmaceutical composition comprising the antibody-drug conjugate according to any one of [1] to

[39] and

[42] to

[45] , its salt, or a hydrate thereof, and at least one antitumor drug, administered simultaneously, separately or consecutively to an individual. Effect of the Invention

[0095] The novel antibody-pyrrolobenzodiazepine (PBD) derivative conjugate provided by the present invention has excellent antitumor activity and safety, and is therefore useful as an antitumor agent. In addition, the PBD derivative of the present invention has antitumor activity and is useful as a drug for the conjugate. Furthermore, the antibody of the present invention recognizes or binds to an antigen expressed in tumor cells, and is therefore useful as an antibody for the conjugate. [Brief description of the drawings]

[0096] [Figure 1] Schematic representation of the antibody-drug conjugate of the present invention (molecule (I)). (a) represents drug D, (b) represents linker L, (c) represents N3-L(PEG)-, and (d) represents N297 glycan (wherein the white oval represents NeuAc(Sia), the white hexagon represents Man, the filled hexagon represents GlcNAc, the white diamond represents Gal, and the white inverted triangle represents Fuc). (b) and (c) are linked by reaction of the azide group (black teardrop) in (c) with the alkyne structure in the spacer (white semicircle) in (b) to form a triazole ring. The Y-shape represents the antibody Ab. In addition, in this schematic diagram, the N297 sugar chain is conveniently displayed as N297-(Fuc)MSG, and an embodiment is shown in which only one branched chain of each N297 sugar chain has sialic acid bound to a PEG linker having an azide group (N3-L(PEG)-) and the other branched chain has no sialic acid at the non-reducing end, but by adopting N297-(Fuc)SG, an embodiment in which both branched chains have sialic acid bound to a PEG linker having an azide group at the non-reducing end may also be used. Such a display method is applied throughout the present specification unless otherwise specified. [Diagram 2]2A and 2B are schematic diagrams showing the structures of (Fucα1,6)GlcNAc-antibody (molecule (II) in FIG. 2A) and MSG-type glycan remodeling antibody (molecule (III) in FIG. 2B), which are intermediates in the production of the antibody-drug conjugate of the present invention. In both figures, the Y-shape represents the antibody Ab, as in FIG. 1. In FIG. 2A, (e) represents the N297 glycan consisting only of GlcNAc linked by α-glycosidic bond at position 1 and position 6 of Fuc. In FIG. 2B, (d) represents the same N297 glycan as in FIG. 1, and (f) represents the structure of the PEG linker portion having an azide group, which is used for binding to the linker L at the end. The binding mode of the PEG linker having an azide group is the same as that described in FIG. 1. [Diagram 3] This is a schematic diagram of the process of producing an MSG-type glycan remodeling antibody from an antibody produced in an animal cell. Molecules (II) and (III) in the figure represent (Fucα1,6)GlcNAc-antibody and MSG-type glycan remodeling antibody, respectively, as in FIG. 2. Molecule (IV) is an antibody produced in an animal cell, and is a mixture of molecules with heterogeneous N297 glycans. FIG. 3A shows the process of producing a homogeneous (Fucα1,6)GlcNAc-antibody (II) by treating the heterogeneous N297 glycan of (IV) with a hydrolase such as EndoS. FIG. 3B shows the process of producing an MSG-type glycan remodeling antibody (III) by glycosyltransferring the glycan of an MSG-type glycan donor molecule to the GlcNAc of the N297 glycan of antibody (II) using a glycosyltransferase such as EndoS D233Q / Q303L mutant. The MSG-type glycan donor molecule used here is one in which the sialic acid at the non-reducing end of MSG is modified with a PEG linker having an azide group, and the sialic acid at the non-reducing end of the MSG-type N297 glycan remodeling antibody to be produced will also be similarly modified, as explained in Figure 2 B. In Figure 3 B, MSG is shown as the donor molecule for convenience, but by using SG (10) as the glycan donor, the glycan remodeling antibody (III) is synthesized, in which a linker molecule having an azide group is bound to both non-reducing ends of the N297 glycan. [Figure 4]The effects of anti-HER2 antibody-drug conjugates ADC2 and ADC1 on subcutaneously implanted NCI-N87 cells, a human gastric cancer line, are shown. [Diagram 5] The effects of anti-HER2 antibody-drug conjugate ADC7, Trastuzumab, and anti-LPS antibody-drug conjugate ADC13 on subcutaneously implanted human gastric cancer cell line NCI-N87 cells are shown. [Figure 6] 1 shows the effects of anti-HER2 antibody-drug conjugate ADC7, anti-LPS antibody-drug conjugate ADC13, or Trastuzumab-Tesirine (Reference Example 1) on subcutaneously implanted KPL-4 cells, a human breast cancer line. [Figure 7] The figure shows the effects of anti-HER2 antibody-drug conjugate ADC7 or Trastuzumab-Tesirine on subcutaneously implanted JIMT-1 cells, a human breast cancer line. [Figure 8] 1 shows the effect of anti-CLDN6 antibody-drug conjugate ADC8, or anti-CLDN6 antibody (H1L1)-Tesirine on subcutaneously implanted OV-90 cells, a human ovarian cancer line. [Figure 9] 1 shows the effect of anti-CLDN6 antibody-drug conjugate ADC8 or anti-CLDN6 antibody (H1L1)-Tesirine on subcutaneously implanted human ovarian cancer line, NIH:OVCAR-3 cells. [Figure 10] 1 shows the effect of antibody-drug conjugates, anti-TROP2 antibody-drug conjugate ADC11, or anti-LPS antibody-drug conjugate ADC13, on subcutaneously implanted FaDu cells, a human head and neck cancer cell line. [Figure 11] The full-length amino acid sequence of human CLDN6 (SEQ ID NO: 1) and the full-length cDNA nucleotide sequence (SEQ ID NO: 2) are shown. [Figure 12] The full-length amino acid sequence of human CLDN9 (sequence number 3) and the full-length cDNA nucleotide sequence (sequence number 4) are shown. [Figure 13] The amino acid sequences of CDRL1 to 3 of the B1 antibody light chain (SEQ ID NOs: 5 to 7) are shown. [Figure 14]The amino acid sequence of CDRL3 of humanized B1 antibody light chain L4 (sequence number 8) is shown. [Figure 15] The amino acid sequences of CDRH1 to 3 of the B1 antibody heavy chain (SEQ ID NOs: 9 to 11) are shown. [Figure 16] The amino acid sequences of CDRL1 to 3 of the C7 antibody light chain (SEQ ID NOs: 12 to 14) are shown. [Figure 17] The amino acid sequences of CDRH1 to 3 of the C7 antibody heavy chain (SEQ ID NOs: 15 to 17) are shown. [Figure 18] The nucleotide sequence of cDNA encoding the variable region of the B1 antibody light chain (SEQ ID NO: 18) and the amino acid sequence of the variable region of the B1 antibody light chain (SEQ ID NO: 19) are shown. The underlines in the amino acid sequence indicate the CDR sequences. [Figure 19] The nucleotide sequence of cDNA encoding the variable region of the heavy chain of B1 antibody (SEQ ID NO: 20) and the amino acid sequence of the variable region of the heavy chain of B1 antibody (SEQ ID NO: 21) are shown. The underlines in the amino acid sequence indicate the CDR sequences. [Figure 20] The nucleotide sequence of cDNA encoding the variable region of the C7 antibody light chain (SEQ ID NO: 22) and the amino acid sequence of the variable region of the C7 antibody light chain (SEQ ID NO: 23) are shown. The underlines in the amino acid sequence indicate the CDR sequences. [Figure 21] The nucleotide sequence of cDNA encoding the variable region of the heavy chain of the C7 antibody (SEQ ID NO: 24) and the amino acid sequence of the variable region of the heavy chain of the C7 antibody (SEQ ID NO: 25) are shown. The underlines in the amino acid sequence indicate the CDR sequences. [Figure 22] The amino acid sequence of the chB1 light chain (SEQ ID NO: 28) and a DNA fragment containing a DNA sequence encoding the amino acid sequence of the chB1 light chain (SEQ ID NO: 29) are shown. The underlines in the amino acid sequence indicate CDR sequences. [Figure 23] The amino acid sequence of the variable region of the chB1 light chain (SEQ ID NO: 30) and the nucleotide sequence encoding the chB1 light chain variable region (SEQ ID NO: 31) are shown. The underlines in the amino acid sequence indicate the CDR sequences. [Figure 24]The amino acid sequence of the chB1 heavy chain (SEQ ID NO: 32) and the nucleotide sequence encoding the chB1 heavy chain (SEQ ID NO: 33) are shown. The underlines in the amino acid sequence indicate the CDR sequences. [Diagram 25] The amino acid sequence of the variable region of the chB1 heavy chain (SEQ ID NO: 34) and the nucleotide sequence encoding the variable region of the chB1 heavy chain (SEQ ID NO: 35) are shown. The underlines in the amino acid sequence indicate the CDR sequences. [Figure 26] The amino acid sequence of the humanized antibody light chain hL1 (SEQ ID NO: 36) and the nucleotide sequence encoding the humanized antibody light chain hL1 (SEQ ID NO: 37) are shown. The underlines in the amino acid sequence indicate CDR sequences. [Figure 27] The amino acid sequence of the variable region of the humanized antibody light chain hL1 (SEQ ID NO: 38) and the nucleotide sequence encoding the variable region of the humanized antibody light chain hL1 (SEQ ID NO: 39) are shown. The underlines in the amino acid sequence indicate CDR sequences. [Figure 28] The amino acid sequence of the humanized antibody light chain hL2 (SEQ ID NO: 40) and the nucleotide sequence encoding the humanized antibody light chain hL2 (SEQ ID NO: 41) are shown. The underlines in the amino acid sequence indicate the CDR sequences. [Figure 29] The amino acid sequence of the variable region of the humanized antibody light chain hL2 (sequence number 42) and the nucleotide sequence encoding the variable region of the humanized antibody light chain hL2 (sequence number 43) are shown. [Diagram 30] The amino acid sequence of the humanized antibody light chain hL3 (SEQ ID NO: 44) and the nucleotide sequence encoding the humanized antibody light chain hL3 (SEQ ID NO: 45) are shown. The underlines in the amino acid sequence indicate CDR sequences. [Diagram 31] The amino acid sequence (SEQ ID NO: 46) of the variable region of the humanized antibody light chain hL3 and the nucleotide sequence (SEQ ID NO: 47) encoding the variable region of the humanized antibody light chain hL3 are shown. The underlines in the amino acid sequence indicate CDR sequences. [Diagram 32] The amino acid sequence of the humanized antibody light chain hL4 (SEQ ID NO: 48) and the nucleotide sequence encoding the humanized antibody light chain hL4 (SEQ ID NO: 49) are shown. The underlines in the amino acid sequence indicate CDR sequences. [Diagram 33]The amino acid sequence of the variable region of the humanized antibody light chain hL4 (SEQ ID NO: 50) and the nucleotide sequence encoding the variable region of the humanized antibody light chain hL4 (SEQ ID NO: 51) are shown. The underlines in the amino acid sequence indicate CDR sequences. [Diagram 34] The amino acid sequence of the humanized antibody heavy chain hH1 (SEQ ID NO: 52) and the nucleotide sequence encoding the humanized antibody heavy chain hH1 (SEQ ID NO: 53) are shown. The underlines in the amino acid sequence indicate CDR sequences. [Diagram 35] The amino acid sequence of the variable region of the humanized antibody heavy chain hH1 (SEQ ID NO: 54) and the nucleotide sequence encoding the variable region of the humanized antibody heavy chain hH1 (SEQ ID NO: 55) are shown. The underlines in the amino acid sequence indicate the CDR sequences. [Diagram 36] The amino acid sequence of the humanized antibody heavy chain hH2 (SEQ ID NO: 56) and the nucleotide sequence encoding the humanized antibody heavy chain hH2 (SEQ ID NO: 57) are shown. The underlines in the amino acid sequence indicate the CDR sequences. [Figure 37] The amino acid sequence of the variable region of the humanized antibody heavy chain hH2 (sequence number 58) and the nucleotide sequence encoding the variable region of the humanized antibody heavy chain hH2 (sequence number 59) are shown. [Figure 38] The amino acid sequence of the humanized antibody heavy chain hH3 (SEQ ID NO: 60) and the nucleotide sequence encoding the humanized antibody heavy chain hH3 (SEQ ID NO: 61) are shown. The underlines in the amino acid sequence indicate the CDR sequences. [Figure 39] The amino acid sequence (SEQ ID NO: 62) of the variable region of the humanized antibody heavy chain hH3 and the nucleotide sequence (SEQ ID NO: 63) encoding the variable region of the humanized antibody heavy chain hH3 are shown. The underlines in the amino acid sequence indicate CDR sequences. [Diagram 40] 1 shows the binding ability of B1 antibody and C7 antibody to human CLDN6 and family molecules CLDN3, CLDN4, and CLDN9 by flow cytometry. [Diagram 41] 1 shows the antibody internalization activity of B1 antibody and C7 antibody by Mab-ZAP. [Diagram 42]1 shows the binding ability of humanized anti-CLDN6 antibodies H1L1, H2L2, H1L3, H2L4, and H3L3 to CLDN6 and family molecules by flow cytometry. [Diagram 43] The amino acid sequence of the Trastuzumab light chain (SEQ ID NO:64) and heavy chain (SEQ ID NO:65) are shown. [Diagram 44] The amino acid sequence of the light chain (SEQ ID NO: 73) and the heavy chain (SEQ ID NO: 75) of a Trastuzumab variant are shown. [Diagram 45] A diagram showing a comparison of the amino acid sequences of chB1_H, which is the heavy chain of human chimeric anti-CLDN6 antibody chB1, and the humanized antibody heavy chains hH1, hH2, and hH3. "·" indicates an amino acid residue that is identical to that in chB1_H, and the positions where amino acid residues are described indicate substituted amino acid residues. [Diagram 46] A diagram showing a comparison of the amino acid sequences of chB1_L, which is the light chain of human chimeric anti-CLDN6 antibody chB1, and hL1, hL2, hL3, and hL4, which are humanized antibody light chains. "·" indicates an amino acid residue that is identical to that in chB1_L, and the positions where amino acid residues are described indicate substituted amino acid residues. [Figure 47] 1 shows the effects of anti-HER2 antibody-drug conjugates ADC7 and ADC14 on subcutaneously implanted KPL-4 cells, a human breast cancer line. [Figure 48] 1 shows the effect of anti-HER2 antibody-drug conjugate ADC14 on subcutaneously implanted JIMT-1 cells, a human breast cancer line. [Figure 49] 1 shows the effects of anti-HER2 antibody-drug conjugates ADC7 and ADC14, and anti-LPS antibody-drug conjugate ADC13 on subcutaneously implanted CFPAC-1 cells, a human pancreatic cancer line. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0097] [Antibody-drug conjugates] The antibody-drug conjugate of the present invention is an antitumor drug in which an antitumor compound is bound via a linker structure to an antibody capable of recognizing or binding to an antigen expressed in tumor cells.

[0098] The antibody-drug conjugate of the present invention has the following formula: [ka]

[0099] is shown in m 1 is an integer of 1 or 2 (preferably 1), D is a drug, L is a linker connecting the N297 sugar chain and D, Ab is an antibody or a functional fragment of the antibody, and the N297 sugar chain is a sugar chain that binds to the side chain of Asn297 of the antibody. The N297 sugar chain may be a remodeled sugar chain.

[0100] <Drugs> Drug D of the present invention is preferably an antitumor compound. This antitumor compound exerts an antitumor effect when a part or all of the linker of the antibody-drug conjugate of the present invention is cleaved in tumor cells, liberating the antitumor compound moiety. The drug, i.e., the PBD derivative, in the antibody-drug conjugate of the present invention is, for example, any one selected from the following group.

[0101] [ka]

[0102] In the formula, the asterisk * indicates a bond to L. In the PBD derivative of the present invention, the absolute configuration of the hydroxyl group at the 11'-position is the S configuration.

[0103] <Linker structure> The linker L of the present invention is a linker that connects the N297 sugar chain and D. The linker L is represented by the following formula: -Lb-La-Lp-NH-B-CH2 -O(C=O)-* The asterisk * indicates binding to the nitrogen atom at the N10' position of drug D, and Lb indicates a spacer that connects La to the N297 glycan or the remodeled N297 glycan.

[0104] B represents a phenyl group or a heteroaryl group, and is preferably a 1,4-phenyl group, a 2,5-pyridyl group, a 3,6-pyridyl group, a 2,5-pyrimidyl group, or a 2,5-thienyl group, and more preferably a 1,4-phenyl group.

[0105] Lp represents a linker consisting of an amino acid sequence that is cleavable in vivo or in target cells, for example, by the action of an enzyme such as esterase or peptidase. Lp is a peptide residue composed of 2 to 7 (preferably 2 to 4) amino acids, i.e., composed of a residue of an oligopeptide in which 2 to 7 amino acids are peptide-bonded. Lp binds to the carbonyl group of La in Lb-La- at the N-terminus and to the linker -NH-B-CH at the C-terminus. 2 An amide bond is formed with the amino group (-NH-) of the -O(C=O)- portion. The bond between the C-terminus of Lp and -NH- is cleaved by an enzyme such as the esterase.

[0106] The amino acids constituting Lp are not particularly limited, but may be, for example, L- or D-amino acids, preferably L-amino acids, and may be, in addition to α-amino acids, amino acids having structures such as β-alanine, ε-aminocaproic acid, and γ-aminobutyric acid, or may be non-natural amino acids such as N-methylated amino acids.

[0107] The amino acid sequence of Lp is not particularly limited, but the constituent amino acids include glycine (Gly; G), valine (Val; V), alanine (Ala; A), phenylalanine (Phe; F), glutamic acid (Glu; E), isoleucine (Ile; I), proline (Pro; P), citrulline (Cit), leucine (Leu; L), serine (Ser; S), lysine (Lys; K), and aspartic acid (Asp; D). Among these, glycine (Gly; G), valine (Val; V), alanine (Ala; A), and citrulline (Cit) are preferred. These amino acids may overlap, and the amino acid sequence may include any amino acid selected from the group consisting of 1 to 3. Furthermore, the drug release pattern can be controlled by the type of amino acid.

[0108] Specific examples of the linker Lp include: -GGVA-, -GG-(D-)VA-, -VA-, -GGFG-, -GGPI-, -GGVCit-, -GGVK-, -GG(D-)PI-, -GGPL-, -EGGVA, -PI-, -G GF-, -DGGF-, (D-)D-GGF-, -EGGF-, -SGGF-, -KGGF-, -DGGFG-, -GGGFG-, -DDGGFG-, -KDGGFG-, -GGGFGGF- Examples include: Here, the above "(D-)V" means D-valine, "(D-)P" means D-proline, and "(D-)D" means D-aspartic acid.

[0109] The linker Lp is preferably: -GGVA-, -GG-(D-)VA-, -VA-, -GGFG-, -GGPI-, -GGVCit-, -GGVK-, -GG(D-)PI-, -GGPL-

[0110] The linker Lp is more preferably: -GGVA-, -GGVCit-, -VA-

[0111] La represents any one selected from the following group: -C(=O)-(CH 2 CH 2 )n 2 -C(=O)-, -C(=O)-(CH 2 CH 2 )n 2 -C(=O)-NH-(CH 2 CH 2 )n 3 -C(=O)-, -C(=O)-(CH 2 CH 2 )n 2 -C(=O)-NH-(CH 2 CH 2 O)n 3 -CH 2 -C(=O)-, -C(=O)-(CH 2 CH 2 )n 2 -NH-C(=O)-(CH 2 CH 2 O)n 3 -CH 2 CH 2 -C(=O)-, -(CH 2 )n 4 -OC(=O)- Wherein, n 2 is an integer of 1 to 3 (preferably 1 or 2), n 3 is an integer of 1 to 5 (preferably an integer of 2 to 4, more preferably 2 or 4), n 4 represents an integer of 0 to 2 (preferably 0 or 1).

[0112] La preferably represents any one selected from the following group: -C(=O)-CH 2 CH 2 -C(=O)-, -C(=O)-(CH 2 CH 2 ) 2 -C(=O)-, -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 ) 2 -C(=O)- -C(=O)-CH 2CH 2 -C(=O)-NH-(CH 2 CH 2 O) 2 -CH 2 -C(=O)-, -C(=O)-CH 2 CH 2 -NH-C(=O)-(CH 2 CH 2 O) 4 -CH 2 CH 2 -C(=O)-, -CH 2 -OC(=O)- and -OC(=O)- La is more preferably -C(=O)-CH 2 CH 2 -C(=O)- or -C(=O)-(CH 2 CH 2 ) 2 -C(=O)-.

[0113] The spacer of Lb is not particularly limited, but examples thereof include spacers represented by the following formula:

[0114] [ka]

[0115] [ka]

[0116] [ka]

[0117] In each of the structural formulas of Lb shown above, the asterisk represents the -(C=O) or -(CH 2 )n 4 The wavy line indicates binding to the N297 glycan of Ab or the remodeled N297 glycan. In each of the structural formulas of Lb (Lb-1, Lb-2, or Lb-3) shown above, the triazole ring moiety formed by the click reaction of the azide group with DBCO has a geometric isomeric structure, and one Lb exists as one of these two types of structures or a mixture thereof. One molecule of the antibody-drug conjugate of the present invention contains 2 or 4 (m 1 There are 1 or 2) ``-LD''s, and each Lb (Lb-1, Lb-2, or Lb-3) in L in each of the 2 or 4 ``-LD''s has either one of these two types of structures or a mixture of both.

[0118] L is preferably -Lb-La-Lp-NH-B-CH 2 -O(C=O)-*, B is a 1,4-phenyl group; Lp represents any one selected from the following group: -GGVA-, -GG-(D-)VA-, -VA-, -GGFG-, -GGPI-, -GGVCit-, -GGVK-, -GG(D-)PI-, -GGPL- La represents one selected from the following group: -C(=O)-CH 2 CH 2 -C(=O)-, -C(=O)-(CH 2 CH 2 ) 2 -C(=O)-, -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 ) 2 -C(=O)-, -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 2 -CH 2 -C(=O)-, -C(=O)-CH 2 CH 2 -NH-C(=O)-(CH 2 CH2 O) 4 -CH 2 CH 2 -C(=O)-, -CH 2 -OC(=O)-, -OC(=O)- Lb represents any one of the structural formulas of Lb shown above.

[0119] L is more preferably any one selected from the following group: -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGVA-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GG-(D-)VA-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-(CH 2 CH 2 ) 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGPI-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGFG-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGVCit-NH-B-CH 2 -OC(=O)-, -Z 1-C(=O)-CH 2 CH 2 -C(=O)-GGVK-NH-B-CH 2 -OC(=O)-、 -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGPL-NH-B-CH 2 -OC(=O)-、 -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 ) 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-、 -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 2 -CH 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-、 -Z 1 -C(=O)-CH 2 CH 2 -NH-C(=O)-(CH 2 CH 2 O) 4 -CH 2 CH 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-、 -Z 2 -OC(=O)-GGVA-NH-B-CH 2 -OC(=O)-、-Z 3 -CH 2 -OC(=O)-GGVA-NH-B-CH 2 -OC(=O)- Here, Z 1 is the structural formula shown below for the above Lb:

[0120]

Chemical formula

[0121] indicates Z 2 is the structural formula of Lb shown below:

[0122] [ka]

[0123] indicates Z 3 is the structural formula of Lb shown below:

[0124] [ka]

[0125] where B is a 1,4-phenyl group.

[0126] L is most preferably any of the following: -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGVA-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-(CH 2 CH 2 ) 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGVCit-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 ) 2-C(=O)-VA-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 2 -CH 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-, and -Z 1 -C(=O)-CH 2 CH 2 -NH-C(=O)-(CH 2 CH 2 O) 4 -CH 2 CH 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)- where B is a 1,4-phenyl group; Z 1 is the structural formula of Lb shown below:

[0127] [ka]

[0128] It is.

[0129] <Free drug> The free drug of the antibody-drug conjugate of the present invention is one selected from the following group:

[0130] [ka]

[0131] The free drug of the present invention is generated by cleavage of the linker L moiety after the antibody-drug conjugate of the present invention is transferred into tumor cells, and the free drug has been confirmed to have an antitumor cell effect.

[0132] <Antibody> In the present invention, the terms "cancer" and "tumor" are used interchangeably. In the present invention, the term "gene" refers to a nucleotide or nucleotide sequence containing a nucleotide sequence encoding the amino acids of a protein, or a complementary strand thereof, and for example, a polynucleotide, oligonucleotide, DNA, mRNA, cDNA, RNA, etc., which is a nucleotide sequence containing a nucleotide sequence encoding the amino acids of a protein, or a complementary strand thereof, is included in the meaning of "gene". Examples of the "CLDN6 gene" of the present invention include DNA, mRNA, cDNA, cRNA, etc., which contain a nucleotide sequence encoding the amino acid sequence of the CLDN6 protein. In the present invention, "nucleotide", "polynucleotide", or "nucleotide sequence" is synonymous with "nucleic acid", and for example, DNA, RNA, probes, oligonucleotides, polynucleotides, primers, etc. are also included in the meaning of "nucleotide" or "nucleotide sequence". In the present invention, the terms "polypeptide", "peptide" and "protein" are used interchangeably. In the present invention, "CLDN6" is used synonymously with CLDN6 protein.

[0133] In the present invention, the term "cell" includes cells in an animal individual and cultured cells. In the present invention, "cytotoxic activity" refers to causing pathological changes in cells in some form, including not only direct trauma but also any damage to the structure or function of cells, such as DNA breakage, formation of base dimers, chromosome breakage, damage to the cell division apparatus, and reduced activity of various enzymes.

[0134] In the present invention, the term "functional antibody fragment", also called "antigen-binding fragment of antibody", refers to a partial fragment of an antibody that has binding activity to an antigen, and includes Fab, F(ab')2, Fv, scFv, diabody, linear antibody, and multispecific antibody formed from antibody fragments. In addition, Fab', which is a monovalent fragment of the variable region of an antibody obtained by treating F(ab')2 under reducing conditions, is also included in the antigen-binding fragment of an antibody. However, it is not limited to these molecules as long as it has the ability to bind to an antigen. In addition, these antigen-binding fragments include not only those obtained by treating the full-length molecule of an antibody protein with an appropriate enzyme, but also proteins produced in an appropriate host cell using an antibody gene modified by genetic engineering. The functional fragments of the present invention include functional fragments that retain an asparagine (Asn297) that is modified by N-linked glycans that is well conserved in the Fc region of an IgG heavy chain and the amino acids surrounding it, and have the ability to bind to an antigen.

[0135] In the present invention, the term "epitope" refers to a partial peptide or partial three-dimensional structure of an antigen (e.g., a partial peptide or partial three-dimensional structure of CLDN6) to which a specific antibody (e.g., an anti-CLDN6 antibody) binds. The epitope, which is the partial peptide (e.g., a partial peptide of CLDN6), can be determined by a method well known to those skilled in the art, such as an immunoassay method.

[0136] In the present invention, "CDR" means a complementarity determining region (CDR). It is known that the heavy and light chains of an antibody molecule each have three CDRs. CDRs are also called hypervariable regions, and are located in the variable regions of the heavy and light chains of an antibody, and are sites with particularly high variability in the primary structure, and are separated into three sites on the primary structure of the heavy and light chain polypeptide chains. In this specification, the CDRs of the heavy chain are represented as CDRH1, CDRH2, and CDRH3 from the amino terminal side of the heavy chain amino acid sequence, and the CDRs of the light chain are represented as CDRL1, CDRL2, and CDRL3 from the amino terminal side of the light chain amino acid sequence. These sites are close to each other in the three-dimensional structure, and determine the specificity for the antigen to which they bind.

[0137] In the present invention, "hybridizing under stringent conditions" means hybridizing at 68°C in a commercially available hybridization solution, ExpressHyb Hybridization Solution (Clontech), or hybridizing using a DNA-fixed filter at 68°C in the presence of 0.7-1.0 M NaCl, followed by washing at 68°C using a 0.1-2x SSC solution (1x SSC consists of 150 mM NaCl and 15 mM sodium citrate), which can be identified by hybridization under equivalent conditions. In the present invention, "one to several" means 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2.

[0138] In the present invention, antibodies that recognize or bind to CLDN6 or CLDN6 and CLDN9 may be referred to as "anti-CLDN6 antibodies" and "anti-CLDN6 / CLDN9 antibodies", respectively. Such antibodies include chimeric antibodies, humanized antibodies, human antibodies, etc. Antibodies that recognize or bind to CLDN6 and CLDN9 may be referred to as "anti-CLDN6 antibodies".

[0139] The antibody used in the antibody-drug conjugate of the present invention means an immunoglobulin, and is a molecule containing an antigen-binding site that immunospecifically binds to an antigen. The antibody of the present invention may be of any class, IgG, IgE, IgM, IgD, IgA, or IgY, but is preferably IgG. The subclass may be any of IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2, but is preferably IgG1, IgG2, or IgG4. When IgG1 or IgG4 is used, the effector function can be adjusted by substituting a portion of the amino acid residues in the constant region (see WO88 / 07089, WO94 / 28027, and WO94 / 29351).

[0140] In addition, when IgG1 is used as the isotype of the antibody of the present invention, it is possible to adjust the effector function by substituting a part of the amino acid residues in the constant region. Examples of IgG1 mutants with reduced or weakened effector function include IgG1 LALA (IgG1-L234A, L235A) and IgG1 LAGA (IgG1-L235A, G237A), and preferably IgG1 LALA. The L234A and L235A indicate the substitution of leucine at positions 234 and 235 with alanine as specified by the EU index (Proc. Natl. Acad. Sci. USA, Vol. 63, No. 1 (May 15, 1969), pp. 78-85), and G237A indicates the substitution of glycine at position 237 with alanine as specified by the EU index.

[0141] The antibody of the present invention may be derived from any species, but preferably includes human, rat, mouse, and rabbit. When derived from a species other than human, it is preferable to chimerize or humanize it using a well-known technique. The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody. Monoclonal antibodies include monoclonal antibodies derived from non-human animals such as rat antibodies, mouse antibodies, and rabbit antibodies, chimeric antibodies, humanized antibodies, human antibodies, functional fragments thereof, or modified forms thereof.

[0142] The antibodies of the present invention are preferably antibodies capable of targeting tumor cells, ie, antibodies that bind to antigens expressed on the surface of tumor cells. Since the antibody-drug conjugate of the present invention is bound to a compound that exerts an antitumor effect, it is preferable, but not essential, that the antibody itself has an antitumor effect. For the purpose of specifically and selectively exerting the cytotoxicity of an antitumor compound in tumor cells, it is important and preferable that the antibody has the property of being internalized and transferred into tumor cells. When the antibody is internalized, the antibody-drug conjugate can also be transferred into the cell. From the viewpoint of antitumor effect, it is important and preferable that the antibody or antibody-drug conjugate has the property of being internalized and transferred into tumor cells in order to specifically and selectively damage tumor cells with a drug. The antitumor activity of an antibody refers to the cytotoxic activity and anticellular effect on tumor cells. The antitumor activity can be confirmed using a known in vitro or in vivo evaluation system. The internalization ability of an antibody can be measured using a known evaluation system. Such antibodies include antibodies against tumor-associated antigens, such as anti-CLDN6 antibody, anti-CLDN9 antibody, anti-CLDN6 / CLDN9 antibody, anti-HER2 antibody, anti-HER3 antibody, anti-DLL3 (Delta like protein 3) antibody, anti-A33 antibody, anti-CanAg antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD25 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD37 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD98 antibody, anti-B7-H3 (CD276) antibody, anti-TROP2 antibody, and anti-CEA antibody. Antibodies, anti-Cripto antibodies, anti-EphA2 antibodies, anti-FGFR2 antibodies (WO201315206, etc.), anti-G250 antibodies, anti-MUC1 antibodies (WO2011012309, etc.), anti-GPNMB antibodies, anti-Integrin antibodies, anti-PSMA antibodies, anti-Tenascin-C antibodies, anti-SLC44A4 antibodies, anti-Mesothelin antibodies, anti-EGFR antibodies, anti-5T4 Examples include, but are not limited to, oncofetal antigen 5T4 (also TPBG and trophoblast glycoprotein) antibody, anti-LRRC15 (Leucine-rich repeat-containing protein 15) antibody, anti-DR5 antibody, anti-CDH3 (cadherin 3) antibody, anti-PDPN (podoplanin) antibody, and anti-CD123 antibody. The antibody of the present invention is preferably an anti-CLDN6 antibody, an anti-CLDN6 / CLDN9 antibody, an anti-HER2 antibody, an anti-CD98 antibody, or an anti-TROP2 antibody, and more preferably an anti-CLDN6 antibody or an anti-HER2 antibody (eg, Trastuzumab, a Trastuzumab mutant).

[0143] The anti-CLDN6 antibody used in the present invention is described below.

[0144] 1. CLDN6 and CLDN9 CLDN6 is a four-transmembrane protein consisting of 220 amino acids and belonging to the Claudin family, with both N- and C-termini inside the cell. The amino acid sequence and DNA sequence of human CLDN6 are publicly available in databases and can be referenced by accession numbers such as NP_067018 (SEQ ID NO: 1) and NM_021195 (SEQ ID NO: 2 (both from NCBI)). Regarding the amino acid sequence of human CLDN6 protein (hereinafter referred to as "CLDN6 amino acid sequence"), the extracellular region is composed of an extracellular domain (EC1) consisting of amino acid numbers 29 to 81 of SEQ ID NO: 1 in the sequence listing, and an extracellular domain (EC2) consisting of amino acid numbers 138 to 160. CLDN9 is a 4-transmembrane protein consisting of 217 amino acids, belonging to the Claudin family, and has intracellular N- and C-termini. CLDN9 has high homology to CLDN6. The amino acid sequence and DNA sequence of human CLDN9 are published on public databases and can be referenced by accession numbers such as NP_066192 (sequence number 3) and NM_020982 (sequence number 4) (both NCBI).

[0145] 2. Anti-CLDN6 antibody An example of an anti-CLDN6 antibody of the present invention is an anti-CLDN6 antibody that recognizes a higher-order structure consisting of two extracellular domains, the amino acid sequence from 29 to 81 and the amino acid sequence from 138 to 160 from the N-terminus of CLDN6 shown in sequence number 1 in the sequence listing, and has internalization activity. The anti-CLDN6 antibody of the present invention is an antibody capable of targeting tumor cells, i.e., it has the properties of recognizing tumor cells, binding to tumor cells, and being taken up and internalized in tumor cells, etc. Therefore, the anti-CLDN6 antibody of the present invention can be bound to a compound having antitumor activity via a linker to form an antibody-drug conjugate. The anti-CLDN6 antibody of the present invention may have anti-tumor activity.

[0146] (1) The anti-CLDN6 antibody of the present invention has the following characteristics (a) and (b): (a) Recognizes or binds to the CLDN family. The antibody of the present invention recognizes the CLDN family. In other words, the antibody of the present invention binds to the CLDN family. The antibody of the present invention preferably binds to CLDN6, and more preferably specifically binds to CLDN6. Furthermore, the antibody of the present invention may recognize or bind to CLDN9. In the present invention, "specific recognition", i.e., "specific binding" means binding that is not nonspecific adsorption. The criterion for determining whether or not binding is specific can be, for example, the dissociation constant (hereinafter referred to as "KD"). The KD value of a suitable antibody of the present invention for CLDN6 and / or CLDN9 is 1×10 -5 M or less, 5×10 -6 M or less, 2×10 -6 M or less or 1×10 -6 M or less, more preferably 5×10 -7 M or less, 2×10 -7 M or less or 1×10 -7 It is below M. In the present invention, the binding between an antigen and an antibody can be measured or determined by ELISA, RIA, Surface Plasmon Resonance (hereinafter referred to as "SPR") analysis, etc. The binding between an antigen expressed on a cell surface and an antibody can be measured by flow cytometry, etc. (b) Having an activity of being internalized into CLDN6 and / or CLDN9 expressing cells by binding to CLDN6 and / or CLDN9. (2) The antibody according to (1) above, wherein CLDN6 and / or CLDN9 is human CLDN6 and / or human CLDN9.

[0147] The anti-CLDN6 monoclonal antibody of the present invention can be obtained by a method using a hybridoma or the like. Examples of the anti-CLDN6 monoclonal antibody include mouse anti-CLDN6 antibodies B1 and C7. In the present invention, the "B1" may be referred to as the "B1 antibody" and the "C7" may be referred to as the "C7 antibody". The nucleotide sequence of the heavy chain variable region of the B1 antibody is shown in SEQ ID NO: 20 of the Sequence Listing, and the amino acid sequence is shown in SEQ ID NO: 21. The nucleotide sequence of the light chain variable region of the B1 antibody is shown in SEQ ID NO: 18 of the Sequence Listing, and the amino acid sequence is shown in SEQ ID NO: 19. The amino acid sequence of CDRH1 of the B1 antibody is set forth in SEQ ID NO:9, the amino acid sequence of CDRH2 in SEQ ID NO:10, the amino acid sequence of CDRH3 in SEQ ID NO:11, the amino acid sequence of CDRL1 in SEQ ID NO:5, the amino acid sequence of CDRL2 in SEQ ID NO:6, and the amino acid sequence of CDRL3 in SEQ ID NO:7. The nucleotide sequence of the heavy chain variable region of the C7 antibody is shown in SEQ ID NO: 24 in the sequence listing, and the amino acid sequence is shown in SEQ ID NO: 25. The nucleotide sequence of the light chain variable region of the C7 antibody is shown in SEQ ID NO: 22 in the sequence listing, and the amino acid sequence is shown in SEQ ID NO: 23. The amino acid sequence of CDRH1 of the C7 antibody is set forth in SEQ ID NO: 15, the amino acid sequence of CDRH2 in SEQ ID NO: 16, the amino acid sequence of CDRH3 in SEQ ID NO: 17, the amino acid sequence of CDRL1 in SEQ ID NO: 12, the amino acid sequence of CDRL2 in SEQ ID NO: 13, and the amino acid sequence of CDRL3 in SEQ ID NO: 14.

[0148] The anti-CLDN6 antibody of the present invention may be an antibody that binds to the same epitope as the B1 or C7 antibody. If the antibody binds to a partial peptide or partial three-dimensional structure to which the B1 or C7 antibody binds, it can be determined that the antibody binds to the same epitope as the B1 or C7 antibody. In addition, by confirming that the antibody competes with the binding of the B1 or C7 antibody to CLDN6 (i.e., the antibody inhibits the binding of the B1 or C7 antibody to CLDN6), it can be determined that the antibody binds to the same epitope as the anti-CLDN6 antibody, even if the sequence or structure of the specific epitope has not been determined. If it is confirmed that the epitope is the same, it is strongly expected that the antibody has the same antigen-binding ability, biological activity, and / or internalization activity as the B1 or C7 antibody.

[0149] The antibodies of the present invention include not only the above-mentioned monoclonal antibodies against CLDN6, but also genetically engineered antibodies that have been artificially modified for the purpose of reducing heterologous antigenicity against humans, such as chimeric antibodies, humanized antibodies, and human antibodies. These antibodies can be produced using known methods.

[0150] (1) Chimeric antibody Chimeric antibodies include antibodies in which the variable and constant regions are of different species, for example, chimeric antibodies in which the variable regions of an antibody derived from a mouse or rat are joined to constant regions derived from a human. A chimeric antibody derived from a mouse anti-human CLDN6 antibody B1 antibody, exemplified as a chimeric antibody of the present invention, is an antibody comprising a heavy chain containing a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 21 and a light chain containing a light chain variable region shown in SEQ ID NO: 19, and may have a constant region derived from any human. A specific example of a chimeric antibody derived from mouse anti-human CLDN6 antibody B1 antibody is the chimeric antibody chB1 antibody (hereinafter also referred to as "chB1") derived from mouse anti-human CLDN6 antibody B1 antibody. The amino acid sequence of the chB1 antibody includes an antibody having a heavy chain having an amino acid sequence consisting of amino acid residues at positions 20 to 471 of SEQ ID NO: 32 in the sequence listing, and a light chain having an amino acid sequence consisting of amino acid residues at positions 21 to 234 of SEQ ID NO: 28 in the sequence listing. In the heavy chain sequence shown in SEQ ID NO: 32 in the sequence listing, the amino acid sequence consisting of amino acid residues at positions 1 to 19 is a signal sequence, the amino acid sequence consisting of amino acid residues at positions 20 to 141 is a heavy chain variable region, and the amino acid sequence consisting of amino acid residues at positions 142 to 471 is a heavy chain constant region. In the light chain sequence shown in SEQ ID NO: 28 in the sequence listing, the amino acid sequence consisting of amino acid residues at positions 1 to 20 is a signal sequence, the amino acid sequence consisting of amino acid residues at positions 21 to 127 is a light chain variable region, and the amino acid sequence consisting of amino acid residues at positions 128 to 234 is a light chain constant region. The amino acid sequences of the heavy and light chain variable regions of the chB1 antibody are set forth in SEQ ID NO:34 and SEQ ID NO:30 in the sequence listing. The heavy chain amino acid sequence of the chB1 antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 33 in the Sequence Listing. The nucleotide sequence consisting of nucleotides 1 to 57 of the nucleotide sequence shown in SEQ ID NO: 33 in the Sequence Listing encodes the signal sequence of the chB1 antibody heavy chain, the nucleotide sequence consisting of nucleotides 58 to 423 of the nucleotide sequence shown in SEQ ID NO: 33 in the Sequence Listing encodes the heavy chain variable region of the chB1 antibody, and the nucleotide sequence consisting of nucleotides 424 to 1413 of the nucleotide sequence shown in SEQ ID NO: 33 in the Sequence Listing encodes the heavy chain constant region of the chB1 antibody. The base sequence of the heavy chain variable region of the chB1 antibody is set forth in SEQ ID NO: 35 of the sequence listing. The light chain amino acid sequence of the chB1 antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 29 of the Sequence Listing. The nucleotide sequence consisting of nucleotides 26 to 85 of the nucleotide sequence shown in SEQ ID NO: 29 of the Sequence Listing encodes the signal sequence of the chB1 antibody light chain, the nucleotide sequence consisting of nucleotides 86 to 406 of the nucleotide sequence shown in SEQ ID NO: 29 of the Sequence Listing encodes the light chain variable region of the chB1 antibody, and the nucleotide sequence consisting of nucleotides 407 to 727 of the nucleotide sequence shown in SEQ ID NO: 29 of the Sequence Listing encodes the light chain constant region of the chB1 antibody. The nucleotide sequence of the light chain variable region of the chB1 antibody is set forth in SEQ ID NO: 31 of the sequence listing.

[0151] (2) Humanized antibodies Examples of humanized antibodies include antibodies in which only the complementarity determining region (CDR) has been incorporated into a human-derived antibody (see Nature (1986) 321, p. 522-525), antibodies in which some framework amino acid residues have been grafted to a human antibody in addition to the CDR sequence by a CDR grafting method (WO90 / 07861), and antibodies in which some CDR amino acid sequences have been modified while maintaining the antigen-binding ability (WO2012 / 075581, WO2011 / 084496, US2018 / 0501692). The amino acid sequence of the CDR can be determined by known methods such as the Kabat definition, Chothia definition, Abm definition, and IMGT, but the CDR in the present invention may be defined by any method. However, the humanized antibody derived from the B1 antibody or the C1 antibody is not limited to a specific humanized antibody as long as it retains all six CDR sequences of the B1 antibody or the C1 antibody and has CLDN6 binding activity, and furthermore, a humanized antibody mutant in which the amino acid sequence of one to several (preferably one to two, more preferably one) CDRs has been modified is also not limited to a specific humanized antibody as long as it recognizes the CLDN6 protein or has the CLDN6 protein binding activity of the antibody. Examples of the anti-CLDN6 humanized antibody or a functional fragment thereof of the present invention include: CDRH1 consisting of the amino acid sequence shown in SEQ ID NO:9 in the sequence listing or an amino acid sequence in which one or several (preferably one or two) amino acids in said amino acid sequence have been substituted; CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 10 in the sequence listing or an amino acid sequence in which one or several (preferably one or two) amino acids in the amino acid sequence have been substituted; and A heavy chain having a variable region including CDRH3 consisting of the amino acid sequence shown in SEQ ID NO:11 in the sequence listing or an amino acid sequence in which one or several (preferably one or two) amino acids in said amino acid sequence have been substituted, and CDRL1 consisting of the amino acid sequence shown in SEQ ID NO:5 in the sequence listing or an amino acid sequence in which one or several (preferably one or two) amino acids in said amino acid sequence have been substituted; CDRL2 consisting of the amino acid sequence shown in SEQ ID NO:6 in the sequence listing or an amino acid sequence in which one or several (preferably one or two) amino acids in the amino acid sequence have been substituted, and a light chain having a variable region including CDRL3 consisting of the amino acid sequence shown in SEQ ID NO:7 in the sequence listing or an amino acid sequence in which one or several (preferably one or two) amino acids in said amino acid sequence have been substituted, Examples of such antibodies include those that recognize the CLDN6 protein of the present invention or that retain the CLDN6 protein-binding activity of the antibody, or functional fragments of the antibody. An example of an amino acid substitution in the CDR in the above-mentioned anti-CLDN6 humanized antibody or a functional fragment thereof preferably includes one to several (preferably 1 to 2) amino acid substitutions in the above-mentioned CDRL3, such as CDRL3 shown in SEQ ID NO: 8 in the sequence listing in which amino acids 4 and 5 of SEQ ID NO: 7 in the sequence listing are substituted.

[0152] Examples of the heavy chain variable region of the humanized antibody having the above-mentioned CDRH include the amino acid sequence shown in SEQ ID NO: 54, the amino acid sequence shown in SEQ ID NO: 58, and the amino acid sequence shown in SEQ ID NO: 62 in the Sequence Listing, and examples of the light chain variable region of the humanized antibody having the above-mentioned CDRL include the amino acid sequence shown in SEQ ID NO: 38, the amino acid sequence shown in SEQ ID NO: 42, the amino acid sequence shown in SEQ ID NO: 46, and the amino acid sequence shown in SEQ ID NO: 50 in the Sequence Listing.

[0153] As the humanized antibody comprising the above-mentioned combination of a heavy chain variable region and a light chain variable region, A humanized antibody comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO:54 in the sequence listing, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO:38 in the sequence listing. A humanized antibody comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO:58 of the sequence listing, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO:42 of the sequence listing; A humanized antibody comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO:54 in the sequence listing, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO:46 in the sequence listing. A humanized antibody comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO:58 of the sequence listing, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO:50 of the sequence listing. A suitable example is a humanized antibody comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 62 in the sequence listing, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 46 in the sequence listing.

[0154] The full-length sequence of a humanized antibody comprising the above-mentioned combination of heavy chain variable region and light chain variable region is: A humanized antibody (H1L1) comprising a heavy chain consisting of the amino acid sequence shown in amino acid numbers 20 to 471 of SEQ ID NO:52 in the sequence listing, and a light chain consisting of the amino acid sequence shown in amino acid numbers 21 to 234 of SEQ ID NO:36 in the sequence listing; A humanized antibody (H2L2) comprising a heavy chain consisting of the amino acid sequence shown in amino acid numbers 20 to 471 of SEQ ID NO: 56 in the sequence listing, and a light chain consisting of the amino acid sequence shown in amino acid numbers 21 to 234 of SEQ ID NO: 40 in the sequence listing; A humanized antibody (H1L3) comprising a heavy chain consisting of the amino acid sequence shown in amino acid numbers 20 to 471 of SEQ ID NO: 52 in the sequence listing, and a light chain consisting of the amino acid sequence shown in amino acid numbers 21 to 234 of SEQ ID NO: 44 in the sequence listing; A humanized antibody (H2L4) comprising a heavy chain consisting of the amino acid sequence shown in amino acid numbers 20 to 471 of SEQ ID NO: 56 in the sequence listing, and a light chain consisting of the amino acid sequence shown in amino acid numbers 21 to 234 of SEQ ID NO: 48 in the sequence listing, or An example is a humanized antibody (H3L3) comprising a heavy chain consisting of the amino acid sequence shown by amino acid numbers 20 to 471 of SEQ ID NO: 60 in the sequence listing, and a light chain consisting of the amino acid sequence shown by amino acid numbers 21 to 234 of SEQ ID NO: 44 in the sequence listing. In the heavy chain amino acid sequence shown in SEQ ID NO: 52, 56, or 60 in the sequence listing, the amino acid sequence consisting of amino acid residues 1 to 19 is a signal sequence, the amino acid sequence consisting of amino acid residues 20 to 141 is a heavy chain variable region, and the amino acid sequence consisting of amino acid residues 142 to 471 is a heavy chain constant region. In addition, in the light chain amino acid sequence shown in SEQ ID NO: 36, 40, 44 or 48 in the sequence listing, the amino acid sequence consisting of the 1st to 20th amino acid residues is a signal sequence, the amino acid sequence consisting of the 21st to 127th amino acid residues is a light chain variable region, and the amino acid sequence consisting of the 128th to 234th amino acid residues is a light chain constant region.

[0155] The carboxyl terminus of the heavy chain of the above-mentioned humanized antibodies H1L1, H2L2, H1L3, H2L4 and H3L3 may be deleted by one or two amino acids, as described below, and such deleted forms are also included in the present invention. Examples of the heavy chain of the deletion variant include a heavy chain comprising the amino acid sequence set forth in amino acid numbers 20 to 470 of SEQ ID NOs: 52, 56 and 60 in the sequence listing. The deletion is A humanized antibody (H1L1) comprising a heavy chain consisting of the amino acid sequence shown in amino acid numbers 20 to 470 of SEQ ID NO:52 in the sequence listing, and a light chain consisting of the amino acid sequence shown in amino acid numbers 21 to 234 of SEQ ID NO:36 in the sequence listing; A humanized antibody (H2L2) comprising a heavy chain consisting of the amino acid sequence shown in amino acid numbers 20 to 470 of SEQ ID NO:56 in the sequence listing, and a light chain consisting of the amino acid sequence shown in amino acid numbers 21 to 234 of SEQ ID NO:40 in the sequence listing; A humanized antibody (H1L3) comprising a heavy chain consisting of the amino acid sequence shown in amino acid numbers 20 to 470 of SEQ ID NO: 52 in the sequence listing, and a light chain consisting of the amino acid sequence shown in amino acid numbers 21 to 234 of SEQ ID NO: 44 in the sequence listing; A humanized antibody (H2L4) comprising a heavy chain consisting of the amino acid sequence shown in amino acid numbers 20 to 470 of SEQ ID NO: 56 in the sequence listing, and a light chain consisting of the amino acid sequence shown in amino acid numbers 21 to 234 of SEQ ID NO: 48 in the sequence listing, or An example is a humanized antibody (H3L3) comprising a heavy chain consisting of the amino acid sequence shown by amino acid numbers 20 to 470 of SEQ ID NO: 60 in the sequence listing, and a light chain consisting of the amino acid sequence shown by amino acid numbers 21 to 234 of SEQ ID NO: 44 in the sequence listing.

[0156] The base sequence encoding the heavy chain amino acid sequence of the humanized antibody H1L1 is SEQ ID NO: 53, the base sequence encoding the light chain amino acid sequence is SEQ ID NO: 37, The base sequence encoding the heavy chain amino acid sequence of the humanized antibody H2L2 is SEQ ID NO: 57, the base sequence encoding the light chain amino acid sequence is SEQ ID NO: 41, The base sequence encoding the heavy chain amino acid sequence of the humanized antibody H1L3 is SEQ ID NO: 53, the base sequence encoding the light chain amino acid sequence is SEQ ID NO: 45, The base sequence encoding the heavy chain amino acid sequence of the humanized antibody H2L4 is SEQ ID NO: 57, the base sequence encoding the light chain amino acid sequence is SEQ ID NO: 49, The nucleotide sequence encoding the heavy chain amino acid sequence of the humanized antibody H3L3 is a polynucleotide shown in SEQ ID NO:61, and the nucleotide sequence encoding the light chain amino acid sequence is a polynucleotide shown in SEQ ID NO:45. The base sequence encoding the amino acid sequence of the heavy chain variable region of the humanized antibody H1L1 is SEQ ID NO: 55, the base sequence encoding the amino acid sequence of the light chain variable region is SEQ ID NO: 39, The base sequence encoding the amino acid sequence of the heavy chain variable region of the humanized antibody H2L2 is SEQ ID NO: 59, the base sequence encoding the amino acid sequence of the light chain variable region is SEQ ID NO: 43, The base sequence encoding the amino acid sequence of the heavy chain variable region of the humanized antibody H1L3 is SEQ ID NO: 55, the base sequence encoding the amino acid sequence of the light chain variable region is SEQ ID NO: 47, The base sequence encoding the amino acid sequence of the heavy chain variable region of the humanized antibody H2L4 is SEQ ID NO: 59, the base sequence encoding the amino acid sequence of the light chain variable region is SEQ ID NO: 51, The nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of humanized antibody H3L3 is a polynucleotide shown in SEQ ID NO:63, and the nucleotide sequence encoding the amino acid sequence of the light chain variable region is a polynucleotide shown in SEQ ID NO:47. In addition, the nucleotide sequence consisting of nucleotides 1 to 57 of each of the nucleotide sequences shown in SEQ ID NOs: 53, 57, and 61 in the sequence listing encodes the signal sequence of the humanized antibody heavy chain, the nucleotide sequence consisting of nucleotides 58 to 423 encodes the amino acid sequence of the humanized antibody heavy chain variable region, and the nucleotide sequence consisting of nucleotides 424 to 1413 encodes the antibody heavy chain constant region. In addition, the nucleotide sequence consisting of nucleotides 1 to 60 of each of the nucleotide sequences shown in SEQ ID NOs: 37, 41, 45, and 49 in the Sequence Listing encodes the signal sequence of the humanized antibody light chain, the nucleotide sequence consisting of nucleotides 61 to 381 encodes the amino acid sequence of the humanized antibody light chain variable region, and the nucleotide sequence consisting of nucleotides 382 to 702 encodes the antibody light chain constant region.

[0157] The antibodies of the present invention also include antibodies comprising the combination of the heavy chain variable region and light chain variable region described above, or antibodies having an identity or homology of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and most preferably 99% or more with the amino acid sequence of an antibody comprising the combination of the heavy chain and light chain described above, as long as they have binding activity to CLDN6. Furthermore, antibodies comprising a combination of the above-mentioned heavy chain variable region and light chain variable region, or antibodies having a CDR consisting of the same amino acid sequence as the CDR of an antibody comprising the above-mentioned combination of a heavy chain and a light chain, and having an identity or homology of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and most preferably 99% or more, with respect to the amino acid sequence excluding the amino acid sequence of the CDR of said antibody, are also included in the antibodies of the present invention, as long as they have binding activity to CLDN6. Furthermore, it is possible to select antibodies having biological activity equivalent to each of the above antibodies by combining amino acid sequences in which one to several amino acid residues are substituted, deleted, or added to the amino acid sequence of the heavy chain or light chain. Furthermore, conservative amino acid substitutions are preferred for the amino acid substitutions herein (WO2013154206). Conservative amino acid substitutions are substitutions that occur within a group of amino acids that are related in their side chains. It is preferable that such amino acid substitutions are made within a range that does not impair the properties of the substance having the original amino acid sequence. The homology between two amino acid sequences can be determined using the default parameters of the Blast algorithm version 2.2.2 (Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402). The Blast algorithm can also be accessed on the Internet at www.ncbi.nlm.nih.gov / blast. (3) Human antibodies The antibody of the present invention further includes a human antibody that binds to CLDN6 and / or CLDN9. An anti-CLDN6 and / or CLDN9 human antibody means a human antibody having only the gene sequence of the antibody derived from a human chromosome. The anti-CLDN6 human antibody can be obtained by known methods (Nature Genetics (1997) 16, p. 133-143; Nucl. Acids Res. (1998) 26, p. 3447-3448; Animal Cell Technology: Basic and Applied Aspects, vol. 10, p. 69-73, Kluwer Academic Publishers, 1999; Proc. Natl. Acad. Sci. USA (2000) 97, p. 722-727; Investigative Ophthalmology & Visual Science. (2002) 43(7), p. 2301-2308; Briefings in Functional Genomics and Proteomics (2002), 1(2), p.189-203, Ophthalmology (2002) 109(3), p.427-431, 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) are also known.

[0158] The anti-HER2 antibody used in the present invention will be described below. The anti-HER2 antibodies of the present invention have the following properties: (1) An anti-HER2 antibody characterized by the following properties: (a) Specifically binds to HER2. (b) It has the activity of being internalized into HER2-expressing cells by binding to HER2. (2) The antibody described in (1) above, which binds to the extracellular domain of HER2. (3) The antibody according to (1) or (2) above, wherein the antibody is a monoclonal antibody. (4) The antibody according to any one of (1) to (3) above, which has antibody-dependent cellular cytotoxicity (ADCC) activity and / or complement-dependent cytotoxicity (CDC) activity. (5) The antibody according to any one of (1) to (4) above, which is a mouse monoclonal antibody, a chimeric monoclonal antibody, or a humanized monoclonal antibody. (6) The antibody according to any one of (1) to (3) and (5) above, wherein the heavy chain constant region is a heavy chain constant region of human IgG1 and contains a mutation that results in reduced ADCC and / or CDC activity. (7) The antibody according to (6) above, wherein the heavy chain constant region is a heavy chain constant region of human IgG1, and leucine at positions 234 and 235 as determined by the EU index is substituted with alanine. (8) The antibody according to any one of (1) to (5) above, which comprises a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 65 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 64. (9) The antibody according to any one of (1) to (3) and (5) to (7) above, which comprises a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 139 of SEQ ID NO: 75 and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 127 of SEQ ID NO: 73. (10) The antibody according to any one of (1) to (3), (5) to (7), and (9), which comprises a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 469 of SEQ ID NO: 75 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 73. (11) The antibody according to any one of (1) to (10) above, wherein one or two amino acids are deleted at the carboxyl terminus of the heavy chain. (12) The antibody according to any one of (1) to (5), (8) and (11) above, comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 of SEQ ID NO: 65 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 of SEQ ID NO: 64. (13) The antibody according to any one of (1) to (3), (5) to (7), and (9) to (11) above, comprising a heavy chain consisting of an amino acid sequence set forth in amino acid numbers 20 to 468 of SEQ ID NO: 75 and a light chain consisting of an amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 73. (14) An antibody obtained by a method for producing the antibody, the method comprising the steps of culturing a host cell transformed with an expression vector containing a polynucleotide encoding the antibody according to any one of (1) to (13) above, and collecting the antibody of interest from the culture obtained in the above step. In the present application, an antibody in which the leucine at positions 234 and 235, as determined by the EU index, of the heavy chain constant region of trastuzumab (SEQ ID NO: 65) is substituted with alanine is referred to as a trastuzumab variant. Further, the anti-HER2 antibody of the present invention includes Trastuzumab variant 2. The heavy chain amino acid sequence of Trastuzumab variant 2 is set forth in SEQ ID NO:77, and the light chain amino acid sequence is set forth in SEQ ID NO:76.

[0159] The antibody of the present invention also includes modified antibodies. The modified antibody means an antibody of the present invention that has been chemically or biologically modified. Chemical modifications include those in which a chemical moiety is bonded to the amino acid backbone, and those in which an N- or O-linked carbohydrate chain is chemically modified. Biological modifications include those that have been post-translationally modified (e.g., N- or O-linked glycosylation, N- or C-terminal processing, deamidation, isomerization of aspartic acid, and oxidation of methionine), and those in which a methionine residue has been added to the N-terminus by expression using a prokaryotic host cell. In addition, those labeled to enable detection or isolation of the antibody or antigen of the present invention, such as enzyme-labeled, fluorescent-labeled, and affinity-labeled, are also included in the meaning of such modified antibodies. Such modified antibodies of the present invention are useful for improving the stability and blood retention of the antibody, reducing antigenicity, and detecting or isolating the antibody or antigen, and the like. In addition, antibody-dependent cellular cytotoxicity can be enhanced by regulating the sugar chain modification bound to the antibody of the present invention (glycosylation, defucosylation, etc.). Techniques for regulating the sugar chain modification of antibodies are known, including, but not limited to, WO1999 / 54342, WO2000 / 61739, WO2002 / 31140, WO2007133855, and WO2013120066. The antibody of the present invention also includes an antibody in which the sugar chain modification has been regulated. Such modifications may be made at any or desired position in the antibody or functional fragment thereof, and the same or two or more different modifications may be made at one or more positions. In the present invention, the term "modified antibody fragment" also includes within its meaning "a fragment of a modified antibody".

[0160] When an antibody gene is isolated and then introduced into a suitable host to produce an antibody, a suitable combination of a host and an expression vector can be used. Specific examples of antibody genes include a combination of a gene encoding the heavy chain sequence, etc., of an antibody described herein and a gene encoding the light chain sequence, etc. When transforming a host cell, the heavy chain sequence gene, etc. and the light chain sequence gene, etc. can be inserted into the same expression vector, or they can be inserted into separate expression vectors. When eukaryotic cells are used as hosts, animal cells, plant cells, and eukaryotic microorganisms can be used. In particular, examples of animal cells include mammalian cells, such as monkey cells, COS cells (Cell (1981) 23, p. 175-182, ATCC CRL-1650), mouse fibroblasts NIH3T3 (ATCC No. CRL-1658), dihydrofolate reductase-deficient strains of Chinese hamster ovary cells (CHO cells, ATCC CCL-61) (Proc. Natl. Acad. Sci. USA (1980) 77, p. 4126-4220), and FreeStyle 293F cells (Invitrogen). When prokaryotic cells are used, examples include Escherichia coli and Bacillus subtilis. The antibody gene of interest is introduced into these cells by transformation, and the transformed cells are cultured in vitro to obtain the antibody. In this culture, the yield may vary depending on the antibody sequence, and it is possible to select antibodies that are easy to produce as a pharmaceutical using the yield as an indicator from among antibodies with equivalent binding activity. Thus, the antibody of the present invention also includes an antibody obtained by a production method for the antibody, which is characterized by comprising the steps of culturing the transformed host cells and recovering the antibody of interest or a functional fragment of the antibody from the culture obtained in this step.

[0161] The above-mentioned antibody gene is preferably a polynucleotide comprising any one of the polynucleotides (a) to (e) below: (a) a combination of a polynucleotide encoding a heavy chain amino acid sequence and a polynucleotide encoding a light chain amino acid sequence of any one of the antibodies B1 or C7 antibody, chB1 antibody, humanized antibody H1L1, H2L2, H1L3, H2L4, H3L3, Trastuzumab, and variants thereof; (b) A combination of a polynucleotide encoding a heavy chain amino acid sequence including CDRH1 to CDRH3 and a polynucleotide encoding a light chain amino acid sequence including CDRL1 to CDRL3 of any one of the antibodies B1 or C7 antibody, chB1 antibody, and humanized antibodies H1L1, H2L2, H1L3, H2L4, H3L3, Trastuzumab, and variants thereof. (c) a combination of a polynucleotide encoding a heavy chain amino acid sequence including the amino acid sequence of the heavy chain variable region of any one of the antibodies B1 or C7 antibody, chB1 antibody, and humanized antibodies H1L1, H2L2, H1L3, H2L4, H3L3, Trastuzumab, and variants thereof, and a polynucleotide encoding a light chain amino acid sequence including the amino acid sequence of the light chain variable region. (d) a polynucleotide that hybridizes under stringent conditions with a nucleotide consisting of a polynucleotide complementary to any one of the polynucleotides (a) to (c) and encodes an amino acid sequence of an antibody that binds to CDLN6 or HER2; and (e) A polynucleotide encoding the amino acid sequence of a polypeptide in which 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1 amino acid has been substituted, deleted, added, or inserted in the polynucleotide according to any one of (a) to (c), and encoding the amino acid sequence of an antibody that binds to CLDN6 or HER2. The present invention includes nucleotides encoding the antibodies of the present invention or functional fragments thereof, or modified forms thereof, recombinant vectors into which the genes have been inserted, and cells into which the genes or the vectors have been introduced. The present invention also includes a method for producing an antibody, a functional fragment thereof, or a modified antibody, comprising the steps of culturing the cells and recovering the antibody, a functional fragment thereof, or a modified antibody from the culture.

[0162] It is known that the lysine residue at the carboxyl terminus of the heavy chain of an antibody produced in cultured mammalian cells is deleted (Journal of Chromatography A, 705:129-134(1995)), and that two amino acid residues, glycine and lysine, are deleted at the carboxyl terminus of the heavy chain, and a proline residue located at the carboxyl terminus is newly amidated (Analytical Biochemistry, 360:75-83(2007)). However, these deletions and modifications of the heavy chain sequence do not affect the antigen-binding ability and effector functions (complement activation, antibody-dependent cellular cytotoxicity, etc.) of the antibody. Therefore, the antibody according to the present invention also includes antibodies and functional fragments of the antibodies that have been modified in this way, and also includes deletion products in which one or two amino acids are deleted at the carboxyl terminus of the heavy chain, and deletion products that have been amidated (for example, heavy chains in which the proline residue at the carboxyl terminus is amidated). However, as long as the antigen-binding ability and effector function are maintained, the deletion of the carboxyl terminus of the heavy chain of the antibody according to the present invention is not limited to the above types. The two heavy chains constituting the antibody according to the present invention may be any one type of heavy chain selected from the group consisting of full-length and the above deletions, or a combination of any two types. The quantitative ratio of each deletion may be influenced by the type and culture conditions of the cultured mammalian cells producing the antibody according to the present invention, but the main component of the antibody according to the present invention may be one in which one amino acid residue is deleted at the carboxyl terminus in both of the two heavy chains.

[0163] The obtained antibody can be purified to homogeneity. For the separation and purification of the antibody, the separation and purification methods commonly used for ordinary proteins can be employed. For example, column chromatography, filter filtration, ultrafiltration, salting out, dialysis, preparative polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, etc. can be appropriately selected and combined to separate and purify the antibody (Strategies for Protein Purification and Characterization: A Laboratory Course Manual, Daniel R. Marshak et al. eds., Cold Spring Harbor Laboratory Press (1996); Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)), but it is not limited thereto.

[0164] <N297 sugar chain> In recent years, methods have been reported for remodeling heterogeneous antibody glycoproteins by enzymatic reactions, etc. to uniformly introduce sugar chains having functional groups (ACS Chemical Biology 2012, 7, 110, ACS Medicinal Chemistry Letters 2016, 7, 1005, Bioconjugate Chemistry 2015, 26, 2233, Angew. Chem. Int. Ed. 2016, 55, 2361 - 2367, US2016361436).

[0165] For the remodeling of the sugar chain of the present invention, first, a hydrolase is used to excise the heterogeneous sugar chains attached to a protein (such as an antibody) leaving only the terminal GlcNAc, and a homogeneous protein moiety with GlcNAc added is prepared (hereinafter referred to as the "acceptor"). Next, an arbitrarily prepared sugar chain (hereinafter referred to as the "donor") is prepared, and this acceptor and donor are linked using a glycosyltransferase. Thereby, a homogeneous glycoprotein having an arbitrary sugar chain structure can be synthesized.

[0166] In the present invention, the term "glycan" refers to a structural unit in which two or more monosaccharides are linked by glycosidic bonds. Specific monosaccharides and glycans may be represented by abbreviations, such as "GlcNAc-" or "MSG-". When these abbreviations are used in structural formulae, the oxygen atom or nitrogen atom that belongs to the glycosidic bond with another structural unit at the reducing end is not included in the abbreviation representing the glycan, unless otherwise defined.

[0167] In the present invention, unless otherwise specified, monosaccharides that are the basic units of sugar chains are described by assuming that the carbon atom that is bonded to the oxygen atom constituting the ring and that is directly bonded to a hydroxyl group (or an oxygen atom belonging to a glycosidic bond) in the ring structure is in the 1st position (only in the case of sialic acid, the 2nd position). The names of the example compounds are given based on the entire chemical structure, and this rule does not necessarily apply.

[0168] In the present invention, when a glycan is described as a symbol (e.g., GLY, SG, MSG, GlcNAc, etc.), unless otherwise defined, the symbol includes the carbon atom at the reducing end, and does not include the N or O belonging to the N- or O-glycosidic bond.

[0169] In the present invention, unless otherwise specified, a partial structure in which an amino acid is linked to a glycan at its side chain will be represented by displaying the side chain portion in parentheses, for example, as "(SG-)Asn."

[0170] The antibody-drug conjugate of the present invention has the following formula: [ka]

[0171] and the antibody Ab or a functional fragment thereof is bound to L from the N297 glycan or a remodeled N297 glycan, preferably to L from the remodeled N297 glycan of the Ab.

[0172] The sugar chain of Ab in the present invention is an N-linked sugar chain or an O-linked sugar chain, preferably an N-linked sugar chain. N-linked glycans are bound to amino acid side chains of antibodies via N-glycosidic bonds, and O-linked glycans are bound to amino acid side chains of antibodies via O-glycosidic bonds.

[0173] IgG has a well-conserved N-linked glycan at the 297th asparagine residue (hereinafter referred to as "Asn297 or N297") in the Fc region of its heavy chain, which is known to contribute to the activity and dynamics of antibody molecules (Biotechnol. Prog., 2012, 28, 608-622, Anal. Chem., 2013, 85, 715-736).

[0174] The amino acid sequence in the constant region of IgG is well conserved, and each amino acid is identified by Eu number (Eu INDEX) in Edelman et al. (Proc. Natl. Acad. Sci. USA, Vol. 63, No. 1 (May 15, 1969), p. 78-85). For example, Asn297, to which N-linked glycan is added in the Fc region, corresponds to position 297 in the Eu numbering, and even if the actual amino acid position changes due to molecular fragmentation or region deletion, the amino acid is uniquely identified by displaying it by Eu numbering.

[0175] In the antibody-drug conjugate of the present invention, preferably, the antibody or a functional fragment thereof is bound to L from a glycan attached to the side chain of Asn297 (hereinafter referred to as "N297 glycan"), and more preferably, the antibody or a functional fragment thereof is bound to L from the N297 glycan, and the N297 glycan is a remodeled glycan.

[0176] SGP is an abbreviation for sialylated glycopeptide, and is a representative N-linked complex glycan. SGP can be isolated and purified from egg yolk of chicken eggs, for example, according to the method described in WO2011 / 0278681. In addition, purified products of SGP are commercially available (Tokyo Kasei Co., Ltd., Fushimi Pharmaceutical Co., Ltd.) and can be purchased. Disialooctasaccharide (Tokyo Kasei Co., Ltd.), which consists only of a glycan lacking one GlcNAc at the reducing end in the glycan portion of SG (hereinafter referred to as "SG(10)"), is commercially available.

[0177] In the present invention, a glycan structure in which sialic acid is deleted from the non-reducing end of only one of the branched chains of β-Man of SG(10) is referred to as MSG(9), and one having sialic acid only in the 1-3 glycan of the branched chain is referred to as MSG1, and one having sialic acid only in the 1-6 glycan of the branched chain is referred to as MSG2.

[0178] The remodeled N297 glycan of the present invention is N297-(Fuc)MSG1, N297-(Fuc)MSG2, a mixture of N297-(Fuc)MSG1 and N297-(Fuc)MSG2, or N297-(Fuc)SG, preferably N297-(Fuc)MSG1, N297-(Fuc)MSG2 or N297-(Fuc)SG, more preferably N297-(Fuc)MSG1 or N297-(Fuc)MSG2.

[0179] N297-(Fuc)MSG1 is represented by the following structural formula or sequence formula: [ka]

[0180] [ka]

[0181] In the above formula, the wavy line indicates binding to Asn297 of the antibody, L(PEG) is *-(CH 2 CH 2-O)n 5 -CH 2 CH 2 -NH-, the amino group at the right end is bonded via an amide bond to the 2-position carboxylic acid of the non-reducing terminal sialic acid on the 1-3 side of the branched chain of β-Man in the N297 sugar chain, and the asterisk * at the left end indicates that it is bonded to the 1- or 3-position nitrogen atom on the 1,2,3-triazole ring of Lb in the linker L, 5 is an integer of 2 to 10, and preferably an integer of 2 to 5.

[0182] N297-(Fuc)MSG2 is represented by the following structural formula or sequence formula: [ka]

[0183] [ka]

[0184] In the above formula, the wavy line indicates binding to Asn297 of the antibody, L(PEG) is *-(CH 2 CH 2 -O)n 5 -CH 2 CH 2 -NH-, the amino group at the right end is bonded via an amide bond to the 2-position carboxylic acid of the non-reducing terminal sialic acid on the 1-6 chain side of the branched chain of β-Man in the N297 sugar chain, and the asterisk * at the left end indicates bonding to the 1- or 3-position nitrogen atom on the 1,2,3-triazole ring of Lb in the linker L, 5 is an integer of 2 to 10, and preferably an integer of 2 to 5.

[0185] N297-(Fuc)SG is represented by the following structural formula or sequence formula: [ka]

[0186] [ka]

[0187] In the above formula, the wavy line indicates binding to Asn297 of the antibody, L(PEG) is *-(CH 2 CH 2 -O)n 5 -CH 2 CH 2 -NH-, the amino group at the right end is bonded via an amide bond to the 2-position carboxylic acid of the non-reducing terminal sialic acid on both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man in the N297 sugar chain, and the asterisk * at the left end indicates that it is bonded to the 1-position or 3-position nitrogen atom on the 1,2,3-triazole ring of Lb in the linker L, 5 is an integer of 2 to 10, and preferably an integer of 2 to 5.

[0188] When the N297 sugar chain of the antibody in the antibody-drug conjugate of the present invention is N297-(Fuc)MSG1 or N297-(Fuc)MSG2 or a mixture thereof, the antibody is a dimer, and therefore the antibody-drug conjugate is a molecule in which two drug linkers (-LD) are bound (the above m 1 =1) (see Figure 1). For example, Example 27: ADC5 is a case where the N297 sugar chain is N297-(Fuc)MSG1.

[0189] When the N297 sugar chain of the antibody in the antibody-drug conjugate of the present invention is N297-(Fuc)SG, the antibody is a dimer, and therefore the antibody-drug conjugate is a molecule to which four drug linkers (-LD) are bound (the above m 1 =2).

[0190] The N297 sugar chain is preferably N297-(Fuc)MSG1 or N297-(Fuc)MSG2 or N297-(Fuc)SG, more preferably N297-(Fuc)MSG1 or N297-(Fuc)MSG2, and most preferably N297-(Fuc)MSG1. When the N297 sugar chain of the antibody in the antibody-drug conjugate of the present invention is N297-(Fuc)MSG1, N297-(Fuc)MSG2, or N297-(Fuc)SG, an ADC of uniform quality can be obtained.

[0191] The present invention provides a method for producing a sugar chain remodeling antibody or a functional fragment of the antibody, which comprises the following steps i) to iii): i) culturing the host cells (e.g., animal cells (CHO cells, etc.)) and collecting the antibody of interest from the resulting culture; ii) treating the antibody obtained in step i) with a hydrolase to produce an antibody in which the N297 sugar chain is (Fucα1,6)GlcNAc ((Fucα1,6)GlcNAc-antibody) ( FIG. 3A ); Preferably, the method further comprises the step of purifying the (Fucα1,6)GlcNAc-antibody from the reaction solution by a step including purification using a hydroxyapatite column; and iii) A PEG linker-(N) having an azide group at the carbonyl group of the 2-carboxylate of the sialic acid of MSG(9) or SG(10) 3 A process for synthesizing a glycan remodeling antibody in which an azide group has been introduced into sialic acid by reacting a glycan donor molecule having an oxazolinyl group at its reducing end with a (Fucα1,6)GlcNAc-antibody in the presence of a glycosyltransferase. Furthermore, the present invention also includes the sugar chain remodeling antibody or a functional fragment thereof obtained by such a production method, or modified versions thereof.

[0192] The intermediate in the production of the antibody-drug conjugate has an alkyne structure that reacts with an azide group such as DBCO (dibenzocyclooctyne) (see Example 2-1, compound 3-14). Therefore, the antibody-drug conjugate of the present invention can be produced by reacting the intermediate in the production with an MSG1-, MSG2- or SG-type sugar chain remodeling antibody or a functional fragment of the antibody, in which a PEG linker having an azide group has been introduced into the sialic acid of the sugar chain obtained in the steps i) to iii).

[0193] In the N297 sugar chain of the present invention, the fucose-added GlcNAc-(Fucα1,6)GlcNAc) at the reducing end is derived from an antibody produced in animal cells, and the sugar chain on the non-reducing end side is preferably one remodeled to the same sugar chain structure as the above-mentioned MSG (MSG1, MSG2) or SG. In either case, L(PEG) is bound to the carboxylic acid bound to the 2-position of the sialic acid at the non-reducing end. Such glycan remodeling antibodies having MSG (MSG1, MSG2) or SG-type N297 glycans can be produced, for example, according to the method described in WO2013 / 120066, as shown in Figure 3. When an antibody is produced as a recombinant protein using an animal cell as a host according to a known method (step i above), the N297 glycan has a fucose-added N-linked glycan structure as a basic structure, but is obtained as a mixture of antibodies or fragments thereof having glycans of various structures with various modifications to the non-reducing terminal structure or the constituent sugars (IV in Figure 3A). When the antibody produced in animal cells in this manner is treated with a hydrolase such as EndoS, the glycosidic bond between GlcNAcβ1-4GlcNAc in the reducing end chitobiose structure is hydrolyzed to obtain an antibody molecule having a single glycan structure containing only (Fucα1,6)GlcNAc as the N297 glycan (referred to as "(Fucα1,6)GlcNAc-antibody" (see A in Figure 2) (Figure 3A) (step ii) above).

[0194] The enzyme used in the hydrolysis reaction of the N297 sugar chain may be Endo S or a mutant enzyme that retains its hydrolysis activity.

[0195] The (Fucα1,6)GlcNAc-antibody obtained by the above hydrolysis reaction can be reacted as a glycan acceptor molecule with an MSG (MSG1, MSG2) or SG-type glycan donor molecule using a glycosyltransferase such as EndoS D233Q or EndoS D233Q / Q303L mutant (WO2017010559, etc.) to obtain an antibody having an MSG (MSG1, MSG2) or SG-type N297 glycan having the above structure (see B in Figure 2) (Figure 3B) (steps iii)-1 and iii)-2 above).

[0196] The number of drugs bound per antibody molecule in an antibody-drug conjugate, m 1 When is 1, a glycan donor molecule having MSG (MSG1, MSG2) as the glycan is adopted. Such glycans can be adopted by separating (MSG-)Asn1 or (MSG2-)Asn from commercially available monosialo-Asn free (1S2G / 1G2S-10NC-Asn, Glycotechnology Institute, hereinafter referred to as "(MSG-)Asn") as a raw material in accordance with the method described in Example 3, or can be adopted as a mixture without separation.

[0197] The number of drugs bound per antibody molecule in an antibody-drug conjugate, m 1 In the case of 2, a glycan donor molecule having SG(10) as a glycan is used in this glycosyltransfer reaction. Such an SG(10) glycan may be obtained, for example, from SGP by hydrolysis or the like, or a commercially available SG(10) glycan such as disialooctasaccharide (Tokyo Chemical Industry Co., Ltd.) may be used.

[0198] The MSG (MSG1, MSG2) or SG-type glycan contained in the donor molecule is linked to a PEG linker (N 3 -L(PEG)).

[0199] The GlcNAc at the reducing end of the MSG (MSG1, MSG2) or SG-type glycan contained in the donor molecule is preferably activated, for example, by oxazolination using 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride treatment (J. Org. Chem., 2009, 74(5), 2210-2212).

[0200] As the enzyme (glycosyltransferase) used in the glycosyltransferase reaction, various enzymes can be used as long as they have the activity of transferring a complex-type glycan to the N297 glycan, but a preferred one is EndoS D233Q, a modified form in which the 233rd Asp of EndoS is replaced with Gln to suppress hydrolysis. The glycosyltransferase reaction using EndoS D233Q is described in WO2013 / 120066 and the like. In addition, a modified enzyme such as EndoS D233Q / Q303L, which is further mutated in EndoS D233Q (WO2017010559), may be used.

[0201] The antibody purification procedure after the glycoremodeling (glycolysis and glycosyltransferase reaction) of the antibody is aimed at separating the antibody from the low molecular weight compounds and enzymes used in the reaction. For such purification, gel filtration chromatography, ion exchange chromatography, affinity chromatography, etc. are usually used, but additional purification using a hydroxyapatite column may be performed. That is, the present invention provides a method for producing an antibody-drug conjugate, which further includes a purification step using a hydroxyapatite column in the purification step of an intermediate from the reaction solution after glycolysis of the antibody. According to the glycoremodeling report (JACS. 2012, 134,12308-12318., Angew. Chem. Int. Ed. 2016, 55, 2361-2367), the reaction solution in which the antibody was treated with a hydrolase is simply purified using a protein A column (affinity chromatography column), but it was found that this purification method does not completely remove the hydrolase (EndoS, etc.), and the residual enzyme has an effect on the subsequent glycosyltransferase reaction. Here, we investigated purification methods and found that by purifying the reaction solution in which the antibody was treated with hydrolases using a protein A column and then a hydroxyapatite column (CHT column, Bio-Rad Laboratories, Inc.), the reaction efficiency of the subsequent glycosyltransferase reaction was improved without being affected by residual enzymes.

[0202] The antibody-drug conjugate of the present invention is most preferably an antibody-drug conjugate selected from the following group:

[0203] [ka]

[0204] [ka]

[0205] [ka]

[0206] [ka]

[0207] In each of the structural formulas shown above, m 1 is an integer of 1 or 2 (preferably, m 1 is an integer equal to 1), Antibody Ab is anti-CLDN6 antibody, anti-CLDN9 antibody, anti-CLDN6 / CLDN9 antibody, anti-HER2 antibody, anti-HER3 antibody, anti-DLL3 antibody, anti-A33 antibody, anti-CanAg antibody, anti-CD19 antibody, anti-CD20 antibody. Antibodies, anti-CD22 antibodies, anti-CD25 antibodies, anti-CD30 antibodies, anti-CD33 antibodies, anti-CD37 antibodies, anti-CD56 antibodies, anti-CD70 antibodies, anti-CD98 antibodies, anti-B7-H3 antibodies, anti-TROP2 antibodies, anti-CEA antibodies, anti- Cripto antibody, anti-EphA2 antibody, anti-FGFR2 antibody (WO201315206, etc.), anti-G250 antibody, anti-MUC1 antibody (WO2011012309, etc.), anti-GPNMB antibody, anti-Integrin antibody , anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody, anti-Mesothelin antibody, anti-EGFR antibody, anti-5T4 antibody, anti-LRRC15 antibody, anti-DR5 antibody, anti-CDH3 antibody, anti-PDPN antibody, or anti-CD123 antibody (preferably the anti-CLDN6 antibody or anti-HER2 antibody, The N297 sugar chain is any one of N297-(Fuc)MSG1, N297-(Fuc)MSG2, a mixture thereof, and N297-(Fuc)SG (preferably, N297-(Fuc)MSG1), L(PEG) is *-(CH 2 CH 2 -O) 3 -CH 2 CH 2 -NH-, the amino group at the right end is bonded via an amide bond to the 2-position carboxylic acid of the non-reducing terminal sialic acid on the 1-3 chain side and / or the 1-6 chain side (preferably the 1-3 chain side) of the branched β-Man chain of the N297 sugar chain, and the asterisk at the left end indicates that it is bonded to the 1- or 3-position nitrogen atom on the triazole ring in the above structural formula. For convenience, the most preferred antibody-drug conjugate is one in which one molecule of the conjugate contains two or four (m 2 = 1 or 2), or have two or four "-(N297 sugar chain)-LD" ("(N297 sugar chain)-(N3Lb)LD") bound to the nitrogen atom at position 3 (m 2 = 1 or 2), but one molecule of the conjugate contains "(N297 sugar chain)-(N1Lb)LD" (m 2 If =1, 1 piece, m 2 In the case of =2, 1, 2, 3) and "(N297 sugar chain)-(N3Lb)LD" (m 2 If =1, 1 piece, m 2 In the case of = 2, it also includes antibody-drug conjugates having both (3, 2, 1). In other words, one molecule of the conjugate contains either "(N297 glycan)-(N1Lb)LD" or "(N297 glycan)-(N3Lb)LD", or both are present.

[0208] The antibody-drug conjugate of the present invention, its free drug, or its production intermediate may exist as a stereoisomer or an optical isomer derived from an asymmetric carbon atom, a geometric isomer, a tautomer, or an optical isomer such as a d-form, an l-form, or an atropisomer, and all of these isomers, optical isomers, and mixtures thereof are included in the present invention.

[0209] The antibody-drug conjugate of the present invention exhibits strong antitumor activity (in vivo antitumor activity, in vitro anticellular activity), favorable pharmacokinetics and physical properties, and is highly safe, and therefore is useful as a pharmaceutical.

[0210] In the antibody-drug conjugate of the present invention, the number of drugs bound to one antibody molecule is an important factor that affects its efficacy and safety. The production of antibody-drug conjugates is carried out by specifying reaction conditions such as the amounts of raw materials and reagents used to react so that the number of drugs bound is a certain number, but unlike chemical reactions of low molecular weight compounds, it is usually obtained as a mixture in which different numbers of drugs are bound. The number of drugs bound to one antibody molecule can be specified as an average value, that is, the average drug binding number (DAR: Drug to Antibody Ratio). The number of pyrrolobenzodiazepine derivatives bound to an antibody molecule can be controlled, and the average drug binding number (DAR) per antibody can be in the range of 1 to 10 pyrrolobenzodiazepine derivatives, but is preferably 1 to 8, and more preferably 1 to 5. In the antibody-drug conjugate of the present invention, when the antibody is bound to L via a remodeled sugar chain of the antibody, the number of drugs bound per antibody molecule in the antibody-drug conjugate, m 2 is an integer of 1 or 2. When the glycan is an N297 glycan and the glycan is N297-(Fuc)MSG1, N297-(Fuc)MSG2, or a mixture of N297-(Fuc)MSG1 and N297-(Fuc)MSG2, m 2 is 1, and DAR is in the range of 1 to 3 (preferably in the range of 1.0 to 2.5, more preferably in the range of 1.2 to 2.2 or 1.6 to 2.2). When the N297 sugar chain is N297-(Fuc)SG, m 2 is 2, and DAR is in the range of 3 to 5 (preferably in the range of 3.2 to 4.8, and more preferably in the range of 3.5 to 4.2). Furthermore, a person skilled in the art would be able to design a reaction for binding a required number of drugs to an antibody from the description of the examples of the present application, and would be able to obtain an antibody in which the number of pyrrolobenzodiazepine derivatives bound is controlled.

[0211] Furthermore, the antibody-drug conjugate, free drug, or production intermediate of the present invention may absorb moisture and become adsorbed water or become a hydrate when left in the air or when recrystallized, and such compounds and salts containing water are also encompassed by the present invention.

[0212] When the antibody-drug conjugate, free drug, or production intermediate of the present invention has a basic group such as an amino group, it can be converted into a pharma- ceutically acceptable salt, if desired. Examples of such salts include hydrohalide salts such as hydrochloride and hydroiodide salts; inorganic acid salts such as nitrate, perchlorate, sulfate, and phosphate salts; lower alkanesulfonate salts such as methanesulfonate, trifluoromethanesulfonate, and ethanesulfonate salts; arylsulfonate salts such as benzenesulfonate and p-toluenesulfonate salts; organic acid salts such as formate, acetic acid, malic acid, fumarate, succinate, citrate, tartrate, oxalate, and maleate salts; and amino acid salts such as ornithine, glutamate, and aspartate salts.

[0213] When the antibody-drug conjugate, free drug, or production intermediate of the present invention has an acidic group such as a carboxy group, it is generally possible to form a base addition salt. Examples of pharma-ceutically acceptable salts include alkali metal salts such as sodium salt, potassium salt, and lithium salt; alkaline earth metal salts such as calcium salt and magnesium salt; inorganic salts such as ammonium salt; dibenzylamine salt, morpholine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, diethylamine salt, triethylamine salt, cyclohexylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, diethanolamine salt, N-benzyl-N-(2-phenylethoxy)amine salt, piperazine salt, tetramethylammonium salt, and tris(hydroxymethyl)aminomethane salt, and other organic amine salts.

[0214] The antibody-drug conjugate, free drug, or production intermediate of the present invention may exist as a hydrate by absorbing moisture in the air, etc. The solvate of the present invention is not particularly limited as long as it is medicamentously acceptable, and specifically, hydrates, ethanol solvates, 2-propanol solvates, etc. are preferred. In addition, when a nitrogen atom is present in the antibody-drug conjugate, free drug, or production intermediate of the present invention, it may be in the form of an N-oxide, and these solvates and N-oxides are also included in the scope of the present invention.

[0215] The present invention also encompasses compounds labeled with various radioactive or non-radioactive isotopes. One or more atoms constituting the antibody-drug conjugate, free drug, or production intermediate of the present invention may contain unnatural proportions of atomic isotopes. Examples of atomic isotopes include deuterium ( 2 H), tritium ( 3 H), iodine-125( 125 I) or carbon-14( 14 C) and the like. In addition, the compound of the present invention can be used, for example, with tritium ( 3 H), iodine-125( 125 I) or carbon-14( 14 The antibody-drug conjugate of the present invention may be radiolabeled with a radioisotope such as C). Radiolabeled compounds are useful as therapeutic or prophylactic agents, research reagents, e.g., assay reagents, and diagnostic agents, e.g., in vivo imaging diagnostic agents. All isotopic variants of the antibody-drug conjugate of the present invention, whether radioactive or not, are within the scope of the present invention. [Manufacturing method]

[0216] R method: Preparation of antibodies Glycochain remodeling antibodies can be produced, for example, by a method similar to that described in WO2013 / 120066, as shown in FIG. 3.

[0217] [ka]

[0218] R-1 process: Hydrolysis of the glycosidic bond between GlcNAcβ1-4GlcNAc in the reducing end chitobiose structure This step involves cleaving the N-linked glycan (N297-linked glycan) bound to asparagine at position 297 in the amino acid sequence of the antibody of interest using a known enzymatic reaction to prepare a glycan-cleaved antibody. The antibody of interest (20 mg / ml) is placed in a buffer solution (such as 50 mM phosphate buffer solution) at 0°C to 40°C to carry out hydrolysis of the glycosidic bond between GlcNAcβ1 and 4GlcNAc in the chitobiose structure at the reducing end using a hydrolase such as EndoS enzyme. The reaction time is 10 minutes to 72 hours, preferably 1 hour to 6 hours. 0.1 to 10 mg, preferably 0.1 to 3 mg of wild-type EndoS enzyme is used per 100 mg of antibody. After completion of the reaction, affinity chromatography purification and / or hydroxyapatite column purification described below can be carried out to produce a (Fucα1,6)GlcNAc antibody in which the sugar chain between GlcNAcβ1 and 4GlcNAc has been hydrolyzed.

[0219] R-2 process: Glycosylation reaction This process involves binding MSG (MSG1, MSG2) or SG-type glycan oxazoline (hereinafter referred to as "azido glycan oxazoline") having an azide group-containing PEG linker to the above-mentioned (Fucα1,6)GlcNAc antibody using an enzymatic reaction to produce a glycan remodeling antibody. The glycosylation reaction is carried out by reacting the above-mentioned glycosylated antibody with an azido-glycosylated oxazoline in the presence of a catalytic amount of a glycosyltransferase such as EndoS (D233Q / Q303L) in a buffer solution (such as a phosphate buffer solution) at 0°C to 40°C. The reaction time is 10 minutes to 72 hours, preferably 1 hour to 6 hours. EndoS enzyme (D233Q / Q303L) is used in an amount of 1 to 10 mg, preferably 1 to 3 mg, per 100 mg of antibody, and the azido-glycosylated oxazoline is used in an amount of 2 to an excess equivalent, preferably 2 to 20 equivalents. After completion of the reaction, a purified glycan remodeling antibody can be obtained by carrying out affinity chromatography purification and hydroxyapatite column purification. Azide sugar chain oxazoline compounds can be prepared according to the methods described in Examples 3 to 5. A PEG linker (N) containing an azide group is added to MSG (MSG1, MSG2) or disialooctasaccharide (Tokyo Chemical Industry Co., Ltd.) using a reaction (condensation reaction, etc.) known in the field of organic synthesis science. 3 -L(PEG) 3 -(CH 2 CH 2 -O)n 5 -CH 2 CH 2 -NH 2 That is, a carboxyl group and N-acetylglucosyl group can be introduced into the 2-position of sialic acid. 3 -(CH 2 CH 2 -O)n 5 -CH 2 CH 2 -NH 2 The amino group at the right end of the formula (I) forms an amide bond by a known condensation reaction. MSG, MSG1 or MSG2 can be obtained by hydrolyzing the above-mentioned (MSG-)Asn or isolated and purified (MSG1-)Asn or (MSG2-)Asn (Example 56) with a hydrolase such as EndoM.

[0220] In the preparation of the above-mentioned sugar chain remodeling antibody, concentration of the aqueous antibody solution, concentration measurement, and buffer exchange can be carried out according to the following common procedures A to C. (Common Procedure A: Concentration of antibody aqueous solution) The antibody or antibody-drug conjugate solution was placed in an Amicon Ultra (30,000 to 50,000 MWCO, Millipore Co.) container, and the antibody and the antibody-drug conjugate solution described below were concentrated by centrifugation (at 2000 G to 4000 G for 5 to 20 minutes) using a centrifuge (Allegra X-15R, Beckman Coulter, Inc.). (Common Procedure B: Antibody concentration measurement) The antibody concentration was measured using a UV meter (Nanodrop 1000, Thermo Fisher Scientific Inc.) according to the method specified by the manufacturer. -1 cm -1 ~1.8mLmg -1 cm -1 ) was used. (Common Procedure C: Buffer Exchange of Antibody) The aqueous antibody solution was concentrated by adding a buffer solution (phosphate buffered saline (pH 6.0), phosphate buffer (pH 6.0), etc.) using common procedure A. After repeating this procedure several times, the antibody concentration was measured using common procedure B, and the antibody concentration was adjusted to 10 mg / mL using a buffer solution (phosphate buffered saline (pH 6.0), phosphate buffer (pH 6.0), etc.).

[0221] S Method: Conjugation This production method is a method for producing an antibody-drug conjugate by binding the above-mentioned glycan remodeling antibody with production intermediate (2) by the SPAAC reaction (strain-promoted alkyne azide cycloaddition: JACS. 2004, 126, 15046-15047).

[0222] [ka]

[0223] In the formula, Ab represents a glycan remodeling antibody, La', Lp', and B' are synonymous with La, Lp, and B. J represents one of the following structural formulas: In the formula, the asterisk indicates a bond to La'.

[0224] [ka]

[0225] J-La'-Lp'-NH-B'-CH2 -O(C=O)-PBD can be synthesized by the methods described in Examples 2-1 to 2-6.

[0226] The SPAAC reaction proceeds by mixing a buffer solution of the antibody Ab (sodium acetate solution, sodium phosphate solution, sodium borate solution, etc., or a mixture thereof) with a solution of compound (2) dissolved in an appropriate solvent (dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), N-methyl-2-pyridone (NMP), propylene glycol (PG), etc., or a mixture thereof). The amount of compound (2) is 2 moles to an excess molar amount, preferably 1 mole to 30 moles, per mole of antibody, and the ratio of the organic solvent to the antibody buffer is preferably 1 to 200% v / v. The reaction temperature is 0° C. to 37° C., preferably 10° C. to 25° C., and the reaction time is 1 to 150 hours, preferably 6 hours to 100 hours. The pH during the reaction is preferably 5 to 9.

[0227] The antibody-drug conjugate can be subjected to buffer exchange, purification, antibody concentration, and measurement of the average number of drugs bound per antibody molecule by common procedures A to C described above and common procedures D to F described below, and the antibody-drug conjugate compound (ADC) can be identified.

[0228] Common Procedure D: Purification of Antibody-Drug Conjugates A NAP-25 column was equilibrated with a commercially available acetate buffer (10 mM, pH 5.5; referred to as ABS in this specification) containing sorbitol (5%). The antibody-drug conjugate reaction solution (about 1.5 to 2.5 mL) was loaded onto this NAP-25 column, and the antibody fraction was separated by eluting with the amount of buffer specified by the manufacturer. The separated fraction was again loaded onto the NAP-25 column, and the gel filtration purification procedure of eluting with buffer was repeated 2 to 3 times in total to obtain an antibody-drug conjugate from which unbound drug linkers, dimethyl sulfoxide, and propylene glycol had been removed. If necessary, the concentration of the antibody-drug conjugate solution was adjusted by common procedures A and C. Common Procedure E: Measurement of antibody concentration in antibody-drug conjugates The bound drug concentration in an antibody-drug conjugate can be calculated using the Beer-Lambert law as shown below. Equation (I) using the Beer-Lambert law is shown below.

[0229]

number

[0230] where A280 is the absorbance at 280 nm of the antibody-drug conjugate aqueous solution, ε280 is the molar absorption coefficient at 280 nm of the antibody-drug conjugate, and C (mol L -1 ) indicates the molar concentration of the antibody-drug conjugate. From the above formula (I), the molar concentration of the antibody-drug conjugate, C (mol L -1 ) is calculated by the following formula (II).

[0231]

number

[0232] In addition, on both sides, the molar mass MW (g mol -1 ) to obtain the weight concentration of the antibody-drug conjugate, C' (mg mL -1 ) can be obtained (formula (III)).

[0233]

number

[0234] The values ​​used in the above formula and applied to this embodiment are described below. The absorbance A280 was measured using the UV absorbance at 280 nm of the antibody-drug conjugate aqueous solution. Molar mass MW (g mol -1) uses the calculated estimated molecular weight of the antibody obtained from the amino acid sequence of the antibody as an approximation of the molar mass of the antibody-drug conjugate. The optical path length l (cm) was 1 cm. The molar extinction coefficient ε280 of an antibody-drug conjugate can be determined by the following formula (IV):

[0235]

number

[0236] Here, ε Ab,280 denotes the molar extinction coefficient of the antibody at 280 nm, and ε DL,280 indicates the molar extinction coefficient of the drug at 280 nm. ε Ab,280 can be estimated from the amino acid sequence of the antibody by a known calculation method (Protein Science, 1995, vol. 4, 2411-2423). In the examples, the molar extinction coefficient of Trastuzumab is Ab,280 = 215400 (calculated estimated value). The molar extinction coefficient of the CLDN6 antibody is ε Ab,280 = 221340 (calculated estimated value), the molar extinction coefficient of TROP2 antibody is ε Ab,280 = 226400 (calculated estimated value), and the molar extinction coefficient of the CD98 antibody is ε Ab,280 = 240400 (calculated estimated value), the molar extinction coefficient of LPS antibody is ε Ab,280 = 230300 (calculated estimated value), and the molar extinction coefficient of the Trastuzumab variant is ε Ab,280 = 215057 (calculated estimate) was used. ε DL,280 was calculated from the actual value obtained by UV measurement each time. That is, the absorbance of a solution in which the conjugate precursor (drug) was dissolved at a certain molar concentration was measured, and the value obtained by applying the Beer-Lambert law, formula (I), was used.

[0237] (Common Procedure F: Measurement of the average number of drugs bound per antibody molecule in antibody-drug conjugates) The average number of drugs bound per antibody molecule in an antibody-drug conjugate can be determined by high performance liquid chromatography (HPLC) analysis using the following method. [F-1. Preparation of samples for HPLC analysis (reduction of antibody-drug conjugates)] The antibody-drug conjugate solution (approximately 1 mg / mL, 60 μL) is mixed with an aqueous solution of dithiothreitol (DTT) (100 mM, 15 μL). The mixture is incubated at 37° C. for 30 minutes to cleave the disulfide bond between the L chain and the H chain of the antibody-drug conjugate, and the resulting sample is used for HPLC analysis.

[0238] [F-2.HPLC analysis] The HPLC analysis is carried out under the following measurement conditions. HPLC system: Agilent 1290 HPLC system (Agilent Technologies) Detector: Ultraviolet spectrophotometer (measurement wavelength: 280 nm, 329 nm) Column: BEH Phenyl (2.1 x 50 mm, 1.7 μm, Waters Acquity) Column temperature: 75℃ Mobile phase A: 0.1% trifluoroacetic acid (TFA), 15% isopropyl alcohol aqueous solution Mobile phase B: 0.075% TFA, 15% isopropyl alcohol in acetonitrile Gradient program: 14%-36% (0 min-15 min), 36%-80% (15 min-17 min), 80%-14% (17 min-17.1 min), 14%-14% (17.1 min-23 min) Sample injection volume: 5μL

[0239] [F-3. Data Analysis] [F-3-1] Antibody H chain (H 0 ) to the H chain with the drug bound (H chain with one drug bound: H 1 , H chain with two drugs bound: H 2 ) increases in hydrophobicity in proportion to the number of bound drugs, and the retention time increases. 0 , H0 , H 1 , H 2 , and are eluted in that order. 0 and H 0 The detected peaks were identified by comparing the retention times with L 0 , H 0 , H 1 , H 2 Drug binding can also be confirmed by the drug's characteristic absorption wavelength of 329 nm. [F-3-2] Since the drug linker has UV absorption, the peak area value is corrected according to the following formula using the molar absorption coefficients of the L chain, H chain, and drug linker depending on the number of drug linkers bonded.

[0240]

number

[0241] Here, the molar extinction coefficient (280 nm) of the L chain and H chain of each antibody can be a value estimated from the amino acid sequence of the L chain and H chain of each antibody by a known calculation method (Protein Science, 1995, vol. 4, 2411-2423). In the case of Trastuzumab, the molar extinction coefficient of the H chain was estimated to be 81290 according to the amino acid sequence. Similarly, for the CLDN6 antibody, the molar absorption coefficient of the H chain was 77280, for the TROP2 antibody, the molar absorption coefficient of the H chain was 68990, for the CD98 antibody, the molar absorption coefficient of the H chain was 78500, for the LPS antibody, the molar absorption coefficient of the H chain was 77470, and for the Trastuzumab mutant, the molar absorption coefficient of the H chain was 81488. The molar absorption coefficient (280 nm) of the drug linker was the measured molar absorption coefficient (280 nm) of the conjugate precursor, compound (1). [F-3-3] Calculate the peak area ratio (%) of each chain to the total corrected peak area value according to the following formula.

[0242]

number

[0243] [F-3-4] Calculate the average number of drugs bound per antibody molecule in the antibody-drug conjugate according to the following formula.

[0244]

number

[0245] <Medicine> The antibody-drug conjugate of the present invention exhibits cytotoxic activity against cancer cells and can therefore be used as a pharmaceutical, particularly as a therapeutic and / or preventive agent for cancer.

[0246] Cancer types to which the anti-CLDN6 antibody-drug conjugate of the present invention can be applied include lung cancer (non-small cell lung cancer, small cell lung cancer, etc.), renal cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer (superficial epithelial tumor, stromal tumor, germ cell tumor, etc.), pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, esophageal cancer, uterine cancer, testicular cancer (seminoma, non-seminoma), cervical cancer, placental choriocarcinoma, brain tumor, head and neck cancer, and metastatic forms thereof, etc. Cancer types to which the anti-HER2 antibody-drug conjugate can be applied include lung cancer (non-small cell lung cancer, small cell lung cancer, etc.), renal cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer (superficial epithelial tumor, stromal tumor, germ cell tumor, etc.), pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, esophageal cancer, uterine cancer, testicular cancer (seminoma, non-seminoma), cervical cancer, placental choriocarcinoma, brain tumor, head and neck cancer, and metastatic forms thereof. Examples of cancers that can be treated include lung cancer, urothelial cancer, colon cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, and penile cancer, as well as metastatic forms thereof, but are not limited to these, so long as the cancer cells to be treated express a protein that can be recognized by the antibody in the antibody-drug conjugate.

[0247] The antibody-drug conjugate of the present invention can be suitably administered to mammals, more preferably humans.

[0248] Substances used in pharmaceutical compositions containing the antibody-drug conjugates of the present invention can be appropriately selected and applied in terms of dosage amount and administration concentration from formulation additives and others commonly used in this field.

[0249] The antibody-drug conjugate of the present invention can be administered as a pharmaceutical composition containing one or more pharma- ceutical compatible components. For example, the pharmaceutical composition typically contains one or more pharmaceutical carriers, such as sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is the more typical carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients are known in the art. The composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin. The formulation corresponds to the mode of administration.

[0250] A variety of delivery systems are known and can be used to administer the antibody-drug conjugate of the present invention.Introduction methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes.Administration can be, for example, by infusion or bolus injection.In certain preferred embodiments, administration of the ligand-drug conjugate is by infusion.Parenteral administration is the preferred administration route.

[0251] In a representative embodiment, the pharmaceutical composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the medicament may also include a solubilizing agent and a local anesthetic to ease pain at the site of injection (e.g., lignocaine). Generally, the ingredients are supplied either separately or mixed together in a unit dosage form (e.g., as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent). If the medicament is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. If the medicament is administered by injection, an ampoule of sterile water for injection or saline can be provided, for example, so that the ingredients can be mixed prior to administration.

[0252] The pharmaceutical composition of the present invention may be a pharmaceutical composition containing only the antibody-drug conjugate of the present invention, or may be a pharmaceutical composition containing the antibody-drug conjugate and at least one other cancer therapeutic agent. The antibody-drug conjugate of the present invention may be administered together with another cancer therapeutic agent, thereby enhancing the anti-cancer effect. The other anti-cancer agent used for such a purpose may be administered to an individual simultaneously with the antibody-drug conjugate, separately, or consecutively, or may be administered at different administration intervals. Examples of such cancer therapeutic agents include abraxane, carboplatin, cisplatin, gemcitabine, irinotecan (CPT-11), paclitaxel, pemetrexed, sorafenib, vinblastin, or agents described in WO2003 / 038043, as well as LH-RH analogues (leuprorelin, goserelin, etc.), estramustine phosphate, estrogen antagonists (tamoxifen, raloxifene, etc.), aromatase inhibitors (anastrozole, letrozole, exemestane, etc.), and the like, but are not limited thereto as long as they have antitumor activity.

[0253] Such pharmaceutical compositions may be formulated as lyophilized or liquid preparations with the selected composition and required purity. When formulated as a lyophilized preparation, the preparation may contain appropriate formulation additives used in this field. Similarly, liquid preparations may be formulated as liquid preparations containing various formulation additives used in this field.

[0254] Although the composition and concentration of the pharmaceutical composition vary depending on the administration method, the antibody-drug conjugate contained in the pharmaceutical composition of the present invention can exert its medicinal effect even at a small dose, as the affinity of the antibody-drug conjugate to the antigen, i.e., the dissociation constant (Kd value) to the antigen, of the antibody-drug conjugate is higher (the Kd value is lower). Therefore, when determining the dose of the antibody-drug conjugate, the dose can be set based on the affinity between the antibody-drug conjugate and the antigen. When the antibody-drug conjugate of the present invention is administered to a human, for example, about 0.001 to 100 mg / kg may be administered once or multiple times at intervals of once every 1 to 180 days.

[0255] The antibody of the present invention or a functional fragment of the antibody can also be used as a medicine. In this case, the description of the "antibody-drug conjugate" in the above <medicine> can be appropriately read as the description of the "antibody or a functional fragment of the antibody".

[0256] Furthermore, the free drug (novel PBD derivative compound), its salt, or their hydrates of the present invention can also be used as a medicine. In this case, the description of the "antibody-drug conjugate" in the above <medicine> can be appropriately read as a description of the "free drug (novel PBD derivative compound), its salt, or their hydrates." EXAMPLES

[0257] The present invention will be specifically described with reference to the following examples, but the present invention is not limited thereto. Furthermore, the examples should not be construed as being limiting in any sense. Furthermore, reagents, solvents and starting materials not specifically described herein are readily available from commercial sources.

[0258] Reference Example 1: Trastuzumab Tesirine Step 1: Conjugation of antibody and drug linker A 5 mM tetraacetate ethylenediamine-phosphate buffered saline (pH 6.5) solution (9.91 mg / mL, 0.70 mL) of Trastuzumab (Reference Example 3) was added with a dipotassium phosphate aqueous solution (1.0 M, 0.0112 mL) and a tris(2-carboxyethyl)phosphine hydrochloride aqueous solution (10 mM, 0.0086 mL) at 20° C., and reacted for 60 minutes at 20° C. and 30 minutes at room temperature. A dimethylacetamide solution (0.0415 mL) of tesirine (0.36 mg) synthesized with reference to a literature (Med. Chem. Lett. 2016, 7, 983-987) was added to the reaction solution, and the reaction was allowed to proceed for 1 hour at room temperature. An N-acetylcysteine ​​aqueous solution (100 mM, 0.0024 mL) was added to the reaction solution, and the reaction was stopped by reacting for 30 minutes. Purification procedure: The above solution was purified using common procedure D to obtain 3.5 mL of a solution containing the target compound. Characterization: The following characteristic values ​​were obtained using common procedures E and F. Antibody concentration: 1.40mg / mL, antibody yield: 4.90mg (71%), average number of drugs bound per antibody molecule (n): 2.0

[0259] Reference example 2: Anti-CLDN6 (H1L1) antibody Tesirine Step 1: Conjugation of antibody and drug linker A 5 mM tetraacetate ethylenediamine-phosphate buffered saline (pH 6.5) solution (9.87 mg / mL, 0.45 mL) of anti-CLDN6 (H1L1) antibody was added to a dipotassium phosphate aqueous solution (1.0 M, 0.0072 mL) and tris(2-carboxyethyl)phosphine hydrochloride aqueous solution (10 mM, 0.0041 mL) at 20 ° C., and the reaction was allowed to proceed for 90 minutes at 20 ° C. Tesirine (0.15 mg) synthesized with reference to the literature (Med. Chem. Lett. 2016, 7, 983-987) was added to the reaction solution in N, N-dimethylacetamide solution (0.0277 mL), and the reaction was allowed to proceed for 1 hour at 20 ° C. An N-acetylcysteine ​​aqueous solution (100 mM, 0.001 mL) was added to the reaction solution, and the reaction was stopped by reacting for 30 minutes. Purification procedure: The above solution was purified using common procedure D to obtain 3.5 mL of a solution containing the target compound. Characterization: The following characteristic values ​​were obtained using common procedures E and F. Antibody concentration: 1.56 mg / mL, antibody yield: 3.90 mg (88%), average number of drugs bound per antibody molecule (n): 2.1

[0260] Reference example 3: Anti-HER2 antibody Trastuzumab The anti-HER2 antibody was prepared with reference to US 5821337. The amino acid sequences of the light chain and heavy chain of Trastuzumab are shown in SEQ ID NO:64 and SEQ ID NO:65.

[0261] Reference example 4: Anti-LPS antibody h#1G5-H1L1 The anti-LPS antibody was produced with reference to WO2015 / 046505. The amino acid sequences of the light chain and heavy chain of h#1G5-H1L1 are shown in SEQ ID NO:66 and SEQ ID NO:67.

[0262] Reference example 5: Anti-TROP2 antibody hRS7 The anti-TROP2 antibody was produced with reference to WO2003 / 074566 and WO2015 / 098099 (Reference Example 1). The amino acid sequences of the light chain and heavy chain of hRS7 are shown in SEQ ID NO:68 and SEQ ID NO:69.

[0263] Reference example 6: Anti-CD98 antibody hM23-H1L1 The anti-CD98 antibody was produced with reference to WO2015 / 146132. The amino acid sequences of the light chain and heavy chain of hM23-H1L1 are shown in SEQ ID NO:70 and SEQ ID NO:71.

[0264] [Synthesis of manufacturing intermediates (drug linkers)] Example 1 [Example 1-1: Intermediate 1]

[0265] [ka]

[0266] Step 1: Benzyl (6S)-6-(hydroxymethyl)-5-azaspiro[2.4]heptane-5-carboxylate (1-2) Lithium borohydride (4.30 g, 178 mmol) was added in small portions to a solution of 5-benzyl 6-methyl(6S)-5-azaspiro[2.4]heptane-5,6-dicarboxylate (1-1) (104 mmol, WO2012087596) in tetrahydrofuran (500 mL) at 0° C. After stirring for 30 minutes at 0° C., the mixture was stirred at room temperature for 2 hours. Water (180 mL) and 2N hydrochloric acid (186 mL) were added at 0° C., and the mixture was distilled under reduced pressure. The resulting residue was extracted four times with ethyl acetate, and the organic layer was washed with saturated saline and then dried over anhydrous sodium sulfate. The residue (1-2) (27.9 g, 90%) obtained by distillation under reduced pressure was used as it was in the next reaction.

[0267] Step 2: Benzyl (6S)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-5-azaspiro[2.4]heptane-5-carboxylate (1-3) To a solution of compound (1-2) (27.9 g, 107 mmol) obtained in the above step 1 and imidazole (14.5 g, 214 mmol) in dichloromethane (300 mL), tert-butyldimethylsilyl chloride (24.2 g, 160 mmol) was added at room temperature, and the mixture was stirred at room temperature for 18 hours. The reaction solution was washed with a saturated aqueous solution of citric acid, a saturated aqueous solution of sodium bicarbonate, and a saturated saline solution, dried over anhydrous sodium sulfate, and then distilled under reduced pressure. The resulting residue was purified by silica gel column chromatography [hexane:ethyl acetate=100:0 (v / v) to 50:50 (v / v)] to obtain the target compound (1-3) (32.5 g, 81%). 1H-NMR(CDCl3)δ:7.39-7.34(5H,m),5.23-5.11(2H,m),4.10-3.48(4H,m), 3.16-3.14(1H,m),2.15-2.04(1H,m),1.81-1.77(1H,m),0.91-0.88(9H,m),0.65-0.55(4H,m),0.08-0.01(6H,m). MS(APCI)m / z:376(M+H)+

[0268] Step 3: (6S)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-5-azaspiro[2.4]heptane (1-4) To a solution of the compound (1-3) (32.5 g, 86.5 mmol) obtained in the above step 2 in ethanol (400 mL), 7.5% palladium carbon catalyst (54% water, 5.00 g) was added at room temperature, and the mixture was stirred at room temperature under a hydrogen atmosphere for 6 hours. The reaction solution was filtered through Celite, and the filtrate was evaporated under reduced pressure to obtain the target product (1-4) (21.3 g, quantitative). 1H-NMR (CDCl 3 )δ:3.79-3.77(1H,m),3.71-3.69(1H,m),3.65-3.60(1H,m),3.01-2.98(2H,m) ,1.81-1.71(2H,m),0.90(9H,s),0.65-0.57(4H,m),0.08(3H,s),0.07(3H,s). MS (APCI, ESI) m / z: 242 (M+H)+

[0269] Step 4: [(6S)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-5-azaspiro[2.4]hept-5-yl](5-methoxy-2-nitro-4-{[tri(propan-2-yl)silyl]oxy}phenyl)methanone (1-5) To a solution of 5-methoxy-2-nitro-4-{tri(propan-2-yl)silyl]oxy}benzoic acid (52.2 g, 141 mmol, US20150283262) and 1-hydroxybenzotriazole monohydrate (23.8 g, 155 mmol) in dichloromethane (500 mL), N,N'-dicyclohexylcarbodiimide (35.0 g, 170 mmol) was added under ice cooling. The reaction mixture was stirred at room temperature. After the carboxylic acid disappeared, a dichloromethane (100 mL) solution of compound (1-4) (34.1 g, 141 mmol) obtained in the above step 3 and triethylamine (29.4 mL, 212 mmol) was slowly added dropwise at -60 ° C. The reaction solution was stirred overnight at room temperature, and then a saturated aqueous solution of sodium bicarbonate was added to the reaction mixture, and the reaction mixture was extracted with chloroform. The organic layer was washed with water and saturated saline, and dried over anhydrous magnesium sulfate. Ethyl acetate and diethyl ether were added to the residue obtained by distillation under reduced pressure, the solid components were removed by filtration, the filtrate was distilled under reduced pressure, and the resulting residue was purified by silica gel column chromatography [hexane:ethyl acetate=100:0 (v / v) to 25:75 (v / v)] to obtain the target compound (1-5) (55.0 g, 66%). 1H-NMR (CDCl 3)δ:7.72-7.66(1H,m),6.80-6.73(1H,m),4.53-4.49(1H,m),4.04-3.95(1H,m),3.91-3.88(3H,m),3 .59-3.54(1H,m),3.36-3.25(0.5H,m),3.01-2.96(1.5H,m),2.24-2.20(0.3H,m),2.09-2.05(0.7H, m),2.00-1.97(0.7H,m),1.69-1.67(0.3H,m),1.32-1.24(3H,m),1.12-1.05(18H,m),0.93-0.91(6H ,m),0.79-0.77(3H,m),0.71-0.62(2H,m),0.57-0.40(2H,m),0.12-0.10(4H,m),0.11-0.15(2H,m). MS (APCI, ESI) m / z: 593 (M+H)+

[0270] Step 5: (2-amino-5-methoxy-4-{[tri(propan-2-yl)silyl]oxy}phenyl)[(6S)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-5-azaspiro[2.4]hept-5-yl]methanone (1-6) To a solution of the compound (1-5) (55.0 g, 92.8 mmol) obtained in the above step 4 in ethanol (300 mL), 7.5% palladium carbon (10.0 g) was added under a nitrogen atmosphere. The nitrogen balloon was immediately replaced with a hydrogen balloon, and the reaction mixture was vigorously stirred at room temperature under a hydrogen atmosphere. After the raw materials had disappeared, the reaction mixture was filtered, and the filtrate was distilled under reduced pressure to obtain the target product (1-6) (52.2 g, 100%), which was used as it was in the next reaction. 1H-NMR (CDCl 3 )δ:6.71(1H,s),6.25(1H,s),4.55-4.28(2H,m),3.97(1H,m),3.75-3.62(3H,m),3.70(3H,s),3.09-3.07(1H,m),2.24-2 .19(1H,m),1.81-1.68(1H,m),1.27-1.22(3H,m),1.09-1.05(18H,m),0.90(9H,s),0.65-0.46(4H,m),0.07-0.03(6H,m). MS (APCI, ESI) m / z: 563 (M+H)+

[0271] Step 6: N-[(prop-2-en-1-yloxy)carbonyl]-L-valyl-N-[4-({[(2-{[(6S)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-5-azaspiro[2.4]hept-5-yl]carbonyl}-4-methoxy-5-{[tri(propan-2-yl)silyl]oxy}phenyl)carbamoyl]oxy}methyl)phenyl]-L-alaninamide (1-7) To a solution of the compound (1-6) (18.6 g, 33.0 mmol) obtained in the above step 5 and triethylamine (6.26 mL, 45.2 mmol) in THF (300 mL), triphosgene (4.22 g, 14.2 mmol) was slowly added on an ethanol-ice bath. After the addition, a mixed solution of N-[(prop-2-en-1-yloxy)carbonyl]-L-valyl-N-[4-(hydroxymethyl)phenyl]-L-alaninamide (11.4 g, 30.2 mmol, WO2011130598) and triethylamine (6.26 mL, 45.2 mmol) in tetrahydrofuran (100 mL) and N,N-dimethylformamide (30 mL) was slowly added dropwise to the ice-cooled reaction mixture. After the dropwise addition, the ice bath was removed, and the reaction mixture was stirred at 40° C. under a nitrogen atmosphere. After the raw materials disappeared, water was added to the reaction mixture, and the reaction mixture was extracted with ethyl acetate. The organic layer was washed with saturated saline and dried over anhydrous sodium sulfate. After filtration, the residue obtained by distillation under reduced pressure was purified by silica gel column chromatography [hexane:ethyl acetate=100:0(v / v) to 40:60(v / v)] to obtain the target product (1-7) (23.5 g, 74%). 1H-NMR (CDCl 3)δ:8.99(1H,m),8.58(1H,s),7.80(1H,s),7.55-7.53(2H,m),7.34-7.32(2H,m),6.77-6.75(2H,m),5.94-5.87(1 H,m),5.40-5.38(1H,m),5.33-5.29(1H,m),5.23-5.21(1H,m),5.13(1H,m),5.10(2H,m),4.69-4.64(1H,m),4.62 -4.52(2H,m),4.06-4.03(1H,m),3.98(1H,m),3.76-3.65(6H,m),3.04(1H,m),2.28-2.26(1H,m),2.18-2.13(1H, m),1.46(3H,m),1.32-1.25(3H,m),1.11-1.09(18H,m),0.99-0.84(15H,m),0.65-0.40(4H,m),0.08-0.00(6H,m). MS (APCI, ESI) m / z: 966 (M+H)+

[0272] Step 7: N-[(prop-2-en-1-yloxy)carbonyl]-L-valyl-N-[4-({[(2-{[(6S)-6-(hydroxymethyl)-5-azaspiro[2.4]hept-5-yl]carbonyl}-4-methoxy-5-{[tri(propan-2-yl)silyl]oxy}phenyl)carbamoyl]oxy}methyl)phenyl]-L-alaninamide (1-8) Acetic acid (200 mL) was added to a solution of the compound (1-7) (23.5 g, 24.3 mmol) obtained in the above step 6 in tetrahydrofuran (50 mL), methanol (50 mL), and water (44 mL) at room temperature. The reaction mixture was stirred at room temperature. After the raw materials disappeared, the reaction mixture was extracted with ethyl acetate. The organic layer was washed with water and saturated saline, and dried over anhydrous sodium sulfate. After filtration, the residue obtained by distillation under reduced pressure was purified by silica gel column chromatography [hexane:ethyl acetate = 100:0 (v / v) to 0:100 (v / v)] to obtain the target product (1-8) (18.0 g, 87%). 1H-NMR (CDCl 3)δ:8.64-8.62(1H,m),8.50(1H,m),7.69(1H,m),7.55-7.53(2H,m),7.34-7.32(2H,m),6.79-6.75(3H,m),5 .91-5.89(1H,m),5.39(1H,m),5.32-5.29(1H,m),5.23-5.21(1H,m),4.68-4.54(4H,m),4.31(1H,m),4.06-4 .04(1H,m),3.81-3.79(3H,m),3.76(3H,s),3.63-3.61(1H,m),3.13-3.11(1H,m),2.16-2.13(1H,m),1.87-1 .81(2H,m),1.46-1.43(3H,m),1.30-1.24(3H,m),1.12-1.08(18H,m),0.98-0.91(6H,m),0.63-0.45(4H,m). MS (APCI, ESI) m / z: 852 (M+H)+

[0273] Step 8: N-[(prop-2-en-1-yloxy)carbonyl]-L-valyl-N-{4-[({[(11'S,11a'S)-11'-hydroxy-7'-methoxy-5'-oxo-8'-{[tri(propan-2-yl)silyl]oxy}-11',11a'-dihydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide (1-9) Oxalyl chloride (2.17 mL, 25.3 mmol) was slowly added dropwise to a solution of dimethyl sulfoxide (3.75 mL, 52.8 mmol) in dichloromethane (300 mL) at -78°C under a nitrogen atmosphere. After the dropwise addition, the reaction mixture was stirred at -78°C. A solution of compound (1-8) (18.0 g, 21.1 mmol) obtained in step 7 above in dichloromethane (50.0 mL) was slowly added dropwise to the reaction mixture. Triethylamine (14.6 mL, 105 mmol) was added to the reaction solution at -78°C. After the addition, the cooling bath was removed and the temperature was slowly raised to room temperature. After the raw materials disappeared, water was added to the reaction mixture, and the reaction mixture was extracted with chloroform (200 mL). The organic layer was washed with water and saturated saline, and dried over anhydrous magnesium sulfate. After filtration, the residue obtained by evaporation under reduced pressure was purified by silica gel column chromatography [hexane:ethyl acetate=100:0 (v / v) to 0:60 (v / v)] to obtain the target compound (1-9) (16.5 g, 92%). 1H-NMR (CDCl 3 )δ:8.51-8.36(1H,m),7.54-7.38(2H,m),7.22-7.07(3H,m),6.73-6.64(1H,m),5.94-5.87(2H,m),5 .33-5.22(3H,m),5.09(1H,m),4.97(1H,m),4.64-4.58(4H,m),4.02-4.00(1H,m),3.86-3.83(3H,m) ,3.75-3.70(1H,m),3.61-3.54(2H,m),3.38-3.29(1H,m),2.40(1H,m),2.16-2.14(1H,m),1.74-1.7 1(1H,m),1.44(3H,m),1.18-1.16(3H,m),1.05-1.00(18H,m),0.97-0.92(6H,m),0.72-0.60(4H,m). MS (APCI, ESI) m / z: 850 (M+H)+

[0274] Step 9: N-[(prop-2-en-1-yloxy)carbonyl]-L-valyl-N-{4-[({[(11'S,11a'S)-11'-{[tert-butyl(dimethyl)silyl]oxy}-7'-methoxy-5'-oxo-8'-{[tri(propan-2-yl)silyl]oxy}-11',11a'-dihydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide (1-10) To a solution of the compound (1-9) (12.0 g, 14.1 mmol) obtained in the above step 8 and 2,6-lutidine (6.58 mL, 56.5 mmol) in dichloromethane (200 mL), tert-butyldimethylsilyl trifluoromethylsulfonate (9.73 mL, 42.3 mmol) was slowly added dropwise at 0°C under a nitrogen atmosphere. After stirring for 10 minutes under ice cooling, the ice bath was removed and the mixture was stirred at room temperature. After the disappearance of the raw materials, water was added to the reaction mixture, and the reaction mixture was washed with water extracted with chloroform and saturated saline, and dried over anhydrous sodium sulfate. After filtration, the residue obtained by distillation under reduced pressure was purified by silica gel column chromatography [hexane:ethyl acetate = 100:0 (v / v) to 25:75 (v / v)] to obtain the target product (1-10) (8.12 g, 60%). 1H-NMR (CDCl 3 )δ:8.67-8.45(1H,m),7.50-7.44(2H,m),7.19(1H,s),7.13(2H,m),6.95(2H,m),6.62-6.57(2H,m),6.01(1H, m),5.95-5.86(1H,m),5.33-5.13(3H,m),4.82(1H,m),4.65-4.54(3H,m),4.03-4.01(1H,m),3.84-3.82(3H,m) ),3.73-3.66(1H,m),3.50-3.48(1H,m),3.27(1H,m),2.37-2.33(1H,m),2.19-2.13(1H,m),1.54-1.43(3H,m) ,1.22-1.13(3H,m),1.10-1.00(18H,m),0.97-0.91(6H,m),0.81(9H,s),0.76-0.59(4H,m),0.19-0.09(6H,m). MS (APCI, ESI) m / z: 964 (M+H)+

[0275] Step 10: N-[(prop-2-en-1-yloxy)carbonyl]-L-valyl-N-{4-[({[(11'S,11a'S)-11'-{[tert-butyl(dimethyl)silyl]oxy}-8'-hydroxy-7'-methoxy-5'-oxo-11',11a'-dihydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide (1-11) Lithium acetate (0.611 g, 9.26 mmol) was added to a solution of the compound (1-10) (8.12 g, 8.42 mmol) obtained in the above step 9 in N,N-dimethylformamide (90 mL) and water (2 mL), and the mixture was stirred at room temperature. After the raw materials disappeared, water was added to the reaction mixture, and the reaction mixture was extracted with ethyl acetate. The organic layer was washed with water and saturated saline, and dried over anhydrous sodium sulfate. After filtration, the residue obtained by distillation under reduced pressure was purified by silica gel column chromatography [hexane:ethyl acetate = 100:0 (v / v) to 0:100 (v / v)] to obtain the target product (1-11) (5.48 g, 81%). 1 H-NMR (400MHz, CDCl 3, 20.9℃)δ:8.76-8.60(1H,m),7.45-7.44(2H,m),7.21(1H,s),7.10-7.09(2H,m),6.81-6.74(1H,m),6.65(1 H,s),6.23(1H,s),6.01-5.99(1H,m),5.95-5.84(1H,m),5.41-5.20(2H,m),5.16(1H,m),4.84(1H,m),4.6 7-4.54(4H,m),4.05-4.03(1H,m),3.87(3H,s),3.71(1H,m),3.55-3.51(1H,m),3.26(1H,m),2.35(1H,m), 2.18-2.12(1H,m),1.55-1.42(4H,m),0.97-0.92(6H,m),0.81(9H,s),0.76-0.61(4H,m),0.20-0.06(6H,m) MS (APCI, ESI) m / z: 808 (M+H)+

[0276] 1 H-NMR (500MHz, CDCl 3, 27℃)δ:8.76(1H,s),7.43(2H,brd),7.20(1H,s),7.08(2H,d,J=8.3Hz),7.00(1H,br),6.66(1H,s),6.44(1H,s),6.00(1H,H-11', d,J11',11'a=9.2Hz),5.89(1H,m),5.53(1H,brd),5.30(1H,d,J=17.2Hz),5.20(1H,d,J=10.3Hz),5.15(1H,d,JABq=12.5Hz),4. 85(1H,d,JABq=12.5Hz),4.66(1H,m),4.60-4.52(2H,m),4.07(1H,m),3.84(3H,s),3.71(1H,H-3'β,d,Jgem=11.7Hz),3.53(1H,H -11'a,m),3.26(1H,H-3'α,d,Jgem=11.7Hz),2.35(1H,H-1'β,dd,J1'β,11'a=8.30Hz,Jgem=13.1Hz),2.14(1H,m),1.54(1H,H-1' α,d,Jgem=13.1Hz),1.41(3H,d,J=6.90Hz),0.95(3H,d,J=6.80Hz),0.92(3H,d, J=6.80Hz),0.81(9H,s),0.80-0.70(1H,m),0.70-0.59(3H,m),0.2-0.06(6H,m)

[0277] The absolute configuration of the 11' position of compound (1-11) was analyzed from the correlation (see figure below) obtained from the selective 1D ROESY spectrum. Correlations were observed between 1'α-H and 11'-H, 3'α-H and 11'-H, and 1'β-H and 3' β-H, indicating that the absolute configuration of the 11' position is S-configuration.

[0278] [ka] Significant correlations obtained with selective 1D ROESY spectra

[0279] Therefore, it was found that the absolute configuration at the 11' position in compound (1-11), compound (1-9) and compound (1-10) having the same absolute configuration as compound (1-11), compound (3-11), compound (3-12), compound (3-13) and drug linker 1 (compound (3-14)), compound (4-9), compound (4-10), compound (4-11) and drug linker 2 (compound (4-12)), as well as compound (6-10), compound (6-11), compound (6-12) and drug linker 4 (compound (6-13)) is S-configuration. In addition, the absolute configuration at the 11' position in compound (5-9), compound (5-10) and drug linker 3 (compound (5-11)) obtained by the same synthesis method was determined to be S-configuration.

[0280] [Example 1-2: Intermediate 2] [ka]

[0281] Step 1: N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycine (2-2) Glycylglycine (0.328g, 2.49mmol), N,N-diisopropylethylamine (0.433mL, 2.49mmol) in N,N-dimethylformamide (20mL) solution, 1-{[4-(11,12-didehydrodibenzo[b,f]azocine-5(6H)-yl)-4-oxobutanoyl]oxy}pyrrolidine-2,5-dione (2-1) (1.00g, 2.49mmol, Click Chemistry Tools), water (10mL) were added at room temperature and stirred at the same temperature overnight. The residue obtained by distillation under reduced pressure was purified by silica gel column chromatography [chloroform to chloroform:methanol:water = 7:3:1 (v / v / v) partition organic layer] to obtain the target product (0.930g, 89%). 1H-NMR(DMSO-D6)δ:12.58(1H,s),8.14-8.12(1H,m),8.08-8.07(1H,m),7.69-7.68(1H,m),7.62-7.61(1H,m),7.53-7.45(3H,m),7.40-7.29 (3H,m),5.05-5.01(1H,m),3.73-3.72(2H,m),3.66-3.60(3H,m),2.66 -2.60(1H,m),2.33-2.24(1H,m),2.08-2.04(1H,m),1.81-1.77(1H,m). MS(APCI, ESI)m / z:420[(M+H)+].

[0282] Example 2 [Example 2-1: Drug Linker 1] [ka]

[0283] Step 1: (2R,11aS)-2-{[tert-butyl(dimethyl)silyl]oxy}-8-hydroxy-7-methoxy-10-{[2-(trimethylsilyl)ethoxy]methyl}-2,3-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (3-2) (2R,11aS)-8-(benzyloxy)-2-{[tert-butyl(dimethyl)silyl]oxy}-7-methoxy-10-{[2-(trimethylsilyl)ethoxy]methyl}-2,3-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (3-1) (25.5g, 41.6mmol, WO2016149546) in tetrahydrofuran (150mL) and ethanol (150mL) were added under a nitrogen atmosphere, and the reaction solution was stirred at room temperature under a hydrogen atmosphere for three days. Chloroform was added to the reaction solution, and the solution was filtered through Celite, and the filtrate was distilled off under reduced pressure. The resulting residue was purified by silica gel column chromatography [hexane:ethyl acetate=100:0 (v / v) to 50:50 (v / v)] to obtain the target product (3-2) (19.4 g, 89%). 1H-NMR (CDCl 3 )δ:7.36(1H,s),7.25(1H,s),6.01(1H,s),5.45-5.43(1H,m),4.69-4.6 7(1H,m),4.60-4.55(1H,m),4.23-4.21(1H,m),3.96(3H,s),3.76-3.68 (2H,m),3.63-3.61(1H,m),3.56-3.53(1H,m),2.88-2.83(1H,m),2.03- 2.00(1H,m),1.00-0.98(2H,m),0.87(9H,s),0.10(6H,s),0.02(9H,s). MS (APCI, ESI) m / z: 523 (M+H)+

[0284] Step 2: (2R,11aS)-8-[(5-bromopentyl)oxy]-2-{[tert-butyl(dimethyl)silyl]oxy}-7-methoxy-10-{[2-(trimethylsilyl)ethoxy]methyl}-2,3-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (3-3) 1,5-Dibromopentane (23.8g, 103mmol) and potassium carbonate (3.43g, 24.8mmol) were added to a solution of the compound (3-2) (10.8g, 20.7mmol) obtained in the above step 1 in N,N-dimethylformamide (30mL) at room temperature. After stirring at room temperature for 3 hours, water was added to the reaction solution and extracted with ethyl acetate. The obtained organic layer was washed with saturated saline, dried over sodium sulfate, and then evaporated under reduced pressure. The obtained residue was purified by silica gel column chromatography [hexane:ethyl acetate = 90:10 (v / v) to 50:50 (v / v)] to obtain the target product (3-3) (14.5g, quantitative). 1H-NMR (CDCl 3)δ:7.34(1H,s),7.21(1H,s),5.52-5.49(1H,m),4.63-4.62(1H,m),4.58-4.55(1H ,m),4.24-4.22(1H,m),4.07-4.04(2H,m),3.92(3H,s),3.82-3.64(3H,m),3.56-3 .53(1H,m),3.45-3.43(2H,m),2.86-2.84(1H,m),2.04-2.00(1H,m),1.97-1.87(4 H,m),1.66-1.62(2H,m),1.01-0.98(2H,m),0.87(9H,s),0.10(6H,s),0.04(9H,s) MS(APCI, ESI)m / z:673[81Br,(M+H)+],671[79Br,(M+H)+].

[0285] Step 3: (2R,11aS)-8-[(5-bromopentyl)oxy]-2-hydroxy-7-methoxy-10-{[2-(trimethylsilyl)ethoxy]methyl}-2,3-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (3-4) To a solution of the compound (3-3) (21.5 mmol) obtained in the above step 2 in tetrahydrofuran (40 mL), 1 mol / L tetrabutylammonium fluoride tetrahydrofuran solution (28.0 mL, 28.0 mmol) was added at 0°C. After stirring at room temperature for 30 minutes, water was added to the reaction solution, extracted with ethyl acetate, and the obtained organic layer was washed with saturated saline. After drying with sodium sulfate, it was distilled under reduced pressure. The obtained residue was purified by silica gel column chromatography [chloroform:methanol = 97.5:2.5 (v / v) to 92.5:7.5 (v / v)] to obtain the target product (3-4) (11.3 g, 94%). 1H-NMR (CDCl 3)δ:7.34(1H,s),7.21(1H,s),5.53-5.50(1H,m),4.69-4.64(2H,m),4. 32-4.30(1H,m),4.10-4.00(2H,m),3.91(3H,s),3.88-3.75(2H,m),3.7 3-3.64(2H,m),3.45-3.44(2H,m),2.99-2.96(1H,m),2.15-2.09(1H,m) ,1.99-1.85(5H,m),1.68-1.62(2H,m),1.01-0.95(2H,m),0.04(9H,s). MS(APCI, ESI)m / z:559[81Br,(M+H)+],557[79Br,(M+H)+].

[0286] Step 4: (11aS)-8-[(5-bromopentyl)oxy]-7-methoxy-10-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,1-c][1,4]benzodiazepine-2,5,11(3H,10H,11aH)-trione (3-5) The compound (3-4) (11.3 g, 20.2 mmol) obtained in the above step 3, tetrabutylammonium bromide (0.325 g, 1.01 mmol), potassium bromide (0.240 g, 2.02 mmol) were dissolved in saturated sodium bicarbonate aqueous solution (60 mL) and dichloromethane (60 mL), and nor-AZADO (0.0279 g, 0.202 mmol) and sodium hypochlorite pentahydrate (2.03 g, 27.2 mmol) were added at 0 ° C. and stirred for 30 minutes at 0 ° C. Since the raw material remained, sodium hypochlorite pentahydrate (1.00 g, 13.4 mmol) was added at 0 ° C. and stirred for 15 minutes at 0 ° C. Sodium hypochlorite pentahydrate (0.300 g, 4.03 mmol) was further added at 0°C, and the mixture was stirred at 0°C for 15 minutes, and the disappearance of the raw materials was confirmed by TLC. Aqueous sodium thiosulfate solution was added to the reaction solution, and the mixture was extracted with chloroform, and the obtained organic layer was dried over sodium sulfate. The mixture was evaporated under reduced pressure, and the obtained residue was purified by silica gel column chromatography [hexane:ethyl acetate=75:25 (v / v) to 40:60 (v / v)] to obtain the target product (3-5) (9.74 g, 87%). 1H-NMR (CDCl 3)δ:7.33(1H,s),7.24(1H,s),5.56-5.53(1H,m),4.71-4.69(1H,m),4.66-4.6 3(1H,m),4.27-4.22(1H,m),4.12-4.02(2H,m),3.93-3.88(4H,m),3.82-3.75 (1H,m),3.69-3.67(1H,m),3.61-3.56(1H,m),3.46-3.44(2H,m),2.82-2.77( 1H,m),1.97-1.89(4H,m),1.68-1.64(2H,m),1.05-0.93(2H,m),0.04(9H,s). MS(APCI, ESI)m / z:557[81Br,(M+H)+],555[79Br,(M+H)+].

[0287] Step 5: (11aS)-8-[(5-bromopentyl)oxy]-7-methoxy-5,11-dioxo-10-{[2-(trimethylsilyl)ethoxy]methyl}-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl trifluoromethanesulfonate (3-6) 2,6-lutidine (8.17mL, 70.1mmol) was added to a dichloromethane (160mL) solution of the compound (3-5) (9.74g, 17.5mmol) obtained in the above step 4 at -40°C, and the mixture was stirred at -40°C for 10 minutes. Trifluoromethanesulfonic anhydride (8.85mL, 52.6mmol) was added to the reaction solution at -40°C, and the mixture was stirred at -40°C for 30 minutes. 10% citric acid aqueous solution was added to the reaction solution, extracted with chloroform, and the obtained organic layer was dried over sodium sulfate. The residue obtained by distillation under reduced pressure was purified by silica gel column chromatography [hexane:ethyl acetate = 95:5 → 70:35], and then purified by NH2 silica gel chromatography [hexane:ethyl acetate = 95:5 (v / v) to 65:35 (v / v)] to obtain the target product (3-6) (7.10g, 59%). 1H-NMR (CDCl 3)δ:7.32(1H,s),7.24(1H,s),7.15-7.14(1H,m),5.56-5.53(1H,m),4.70 -4.68(1H,m),4.66-4.63(1H,m),4.11-4.01(2H,m),3.94-3.90(4H,m),3 .84-3.75(1H,m),3.73-3.68(1H,m),3.46-3.44(2H,m),3.18-3.14(1H,m ),1.96-1.88(4H,m),1.69-1.61(2H,m),1.02-0.92(2H,m),0.04(9H,s). MS(APCI, ESI)m / z:689[81Br,(M+H)+],687[79Br,(M+H)+].

[0288] Step 6: (11aS)-8-[(5-bromopentyl)oxy]-7-methoxy-2-(4-methoxyphenyl)-10-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (3-7) Toluene (20 mL), ethanol (10 mL), and water (10 mL) were added to a mixture of the compound (3-6) (2.00 g, 2.91 mmol) obtained in the above step 5, 4-methoxyphenylboronic acid (0.884 g, 5.82 mmol), tetrakis(triphenylphosphine)palladium(0) (0.336 g, 0.291 mmol), and sodium carbonate (1.23 g, 11.6 mmol) at room temperature. After stirring the reaction solution at room temperature for 30 minutes, the reaction solution was extracted with ethyl acetate and washed with water and saturated saline. The organic layer was dried over sodium sulfate and then evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography [hexane:ethyl acetate = 90:10 (v / v) to 50:50 (v / v)] to obtain the target product (3-7) (1.71 g, 91%). 1H-NMR (CDCl 3)δ:7.38-7.37(3H,m),7.33(1H,s),7.25(1H,s),6.89-6.88(2H,m),5.56 -5.54(1H,m),4.71-4.68(1H,m),4.65-4.62(1H,m),4.09-4.04(2H,m),3 .96-3.91(4H,m),3.85-3.66(5H,m),3.46-3.45(2H,m),3.16-3.12(1H,m ),1.99-1.94(4H,m),1.69-1.64(2H,m),1.00-0.98(2H,m),0.04(9H,s). MS(APCI, ESI)m / z:647[81Br,(M+H)+],645[79Br,(M+H)+].

[0289] Step 7: (11aS)-8-[(5-bromopentyl)oxy]-7-methoxy-2-(4-methoxyphenyl)-1,11a-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (3-8) The compound (3-7) (0.789 g, 1.22 mmol) obtained in the above step 6 was dissolved in ethanol (10 mL) and tetrahydrofuran (10 mL), and 2.0 M lithium borohydride tetrahydrofuran solution (6.11 mL, 12.2 mmol) was added at 0°C, and the mixture was stirred at 0°C for 3 hours. Water was added to the reaction solution, and the mixture was extracted with chloroform, and the resulting organic layer was dried over sodium sulfate. The mixture was evaporated under reduced pressure, and the resulting residue was dissolved in dichloromethane (10 mL), ethanol (20 mL), and water (10 mL), and silica gel (4 g) was added at room temperature, and the mixture was stirred at room temperature for 4 days. The silica gel was removed by filtration, water was added, and the mixture was extracted with chloroform, and the resulting organic layer was dried over sodium sulfate. The mixture was evaporated under reduced pressure, and the resulting residue was purified by silica gel column chromatography [hexane:ethyl acetate = 60:40 (v / v) to 25:75 (v / v)] to obtain the target compound (3-8) (0.496 g, 81%). 1H-NMR (CDCl 3)δ:7.90-7.89(1H,m),7.53(1H,s),7.40-7.40(1H,m),7.35-7.34(2H,m),6.92-6.90(2H,m),6.83-6.81(1H,m),4.43-4.40(1H ,m),4.13-4.06(2H,m),3.96(3H,s),3.84(3H,s),3.61-3.57(1H,m),3.47-3.36(3H,m),2.00-1.92(4H,m),1.67-1.63(2H,m). MS(APCI, ESI)m / z:501[81Br,(M+H)+],499[79Br,(M+H)+].

[0290] Step 8: (11aS)-8-[(5-bromopentyl)oxy]-7-methoxy-2-(4-methoxyphenyl)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (3-9) Sodium triacetoxyborohydride (0.421 g, 1.99 mmol) was added to a dichloromethane (20 mL) solution of compound (3-8) (0.496 g, 0.992 mmol) obtained in the above step 7 at 0°C. After stirring at room temperature for 2 hours, a saturated aqueous solution of sodium bicarbonate was added and the mixture was extracted with chloroform. The organic layer was dried over sodium sulfate and evaporated under reduced pressure, and the resulting residue was purified by silica gel column chromatography [hexane:ethyl acetate = 60:40 (v / v) to 25:75 (v / v)] to obtain the target compound (3-9) (0.426 g, 86%). 1H-NMR (CDCl 3 )δ:7.53-7.53(2H,m),7.32-7.30(2H,m),6.89-6.87(2H,m),6.05(1H,s),4.33-4.27(2H,m),4.00-3.98(2H,m),3.86 (3H,s),3.82(3H,s),3.57-3.55(2H,m),3.42-3.38(3H,m),2.76-2.72(1H,m),1.96-1.88(4H,m),1.65-1.62(2H,m). MS(APCI, ESI)m / z:503[81Br,(M+H)+],501[79Br,(M+H)+].

[0291] Step 9: Prop-2-en-1-yl (11aS)-8-[(5-bromopentyl)oxy]-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate (3-10) To a dichloromethane (30 mL) solution of compound (3-9) (0.426 g, 0.849 mmol) obtained in the above step 8, pyridine (0.102 mL, 1.27 mmol) and allyl chloroformate (0.374 mL, 3.54 mmol) were added at 0°C, and the mixture was stirred at 0°C for 15 minutes. A 10% aqueous citric acid solution was added to the reaction solution, extracted with chloroform, and the obtained organic layer was washed with a saturated aqueous sodium bicarbonate solution and then dried over sodium sulfate. The residue obtained by distillation under reduced pressure was purified by silica gel column chromatography [hexane:ethyl acetate=90:10 (v / v) to 50:50 (v / v)] to obtain the target compound (3-10) (0.465 g, 94%). 1H-NMR (CDCl 3 )δ:7.38(1H,s),7.31-7.29(2H,m),7.26-7.25(1H,m),6.89-6.87(2H,m),6.71( 1H,s),5.80-5.78(1H,m),5.14-5.11(2H,m),4.65-4.62(1H,m),4.39-4.26(3H,m ),4.03-4.01(2H,m),3.92(3H,s),3.82(3H,s),3.66-3.64(1H,m),3.46-3.44(2H ,m),3.30-3.27(1H,m),2.72-2.68(1H,m),1.96-1.88(4H,m),1.68-1.60(2H,m). MS(APCI, ESI)m / z:587[81Br,(M+H)+],585[79Br,(M+H)+].

[0292] Step 10: N-[(prop-2-en-1-yloxy)carbonyl]-L-valyl-N-{4-[({[(11'S,11a'S)-11'-{[tert-butyl(dimethyl)silyl]oxy}-7'-methoxy-8'-{[5-({(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-10-[(prop-2-en-1-yloxy)carbonyl]-5,10 ,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yl}oxy)pentyl]oxy}-5'-oxo-11',11a'-dihydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide (3-11) Potassium carbonate (0.0266 g, 0.193 mmol) was added to a solution of compound (1-11) (0.130 g, 0.161 mmol) obtained in step 10 of Example 1-1 and compound (3-10) (0.104 g, 0.177 mmol) obtained in step 9 in N,N-dimethylformamide (3 mL) at room temperature, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with ethyl acetate, washed with water and saturated saline, and then dried over sodium sulfate. After distillation under reduced pressure, the resulting residue was dissolved in NH 2 The mixture was purified by silica gel column chromatography [hexane:ethyl acetate = 70:30 (v / v) to 0:100 (v / v)] to obtain the target compound (3-11) (0.184 g, 87%). 1H-NMR (CDCl 3)δ:8.76(1H,s),7.58-7.56(2H,m),7.39(1H,s),7.32-7.30(2H,m),7.26-7.24(2H,m),7.19-7.17(3H,m),6.90-6. 88(2H,m),6.78(1H,s),6.68-6.66(1H,m),6.37(1H,s),5.99-5.93(3H,m),5.34-5.20(6H,m),4.66-4.01(11H,m), 3.90(3H,s),3.89(3H,s),3.78-3.54(9H,m),3.31-3.28(2H,m),2.73-2.69(1H,m),2.38-2.35(1H,m),2.19-2.13( 1H,m),1.82-1.80(2H,m),1.46-1.29(6H,m),0.98-0.90(6H,m),0.83(9H,s),0.69-0.63(4H,m),0.19-0.16(6H,m). MS (APCI, ESI) m / z: 1312 (M+H)+

[0293] Step 11: N-[(prop-2-en-1-yloxy)carbonyl]-L-valyl-N-{4-[({[(11'S,11a'S)-11'-hydroxy-7'-methoxy-8'-{[5-({(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-10-[(prop-2-en-1-yloxy)carbonyl]-5,10,11,11a-tetramethylphenyl)-N ... tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yl}oxy)pentyl]oxy}-5'-oxo-11',11a'-dihydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide (3-12) A 1 mol / L tetrabutylammonium fluoride tetrahydrofuran solution (0.700 mL, 0.700 mmol) was added to a tetrahydrofuran (5.00 mL) solution of the compound (3-11) (0.1837 g, 0.140 mmol) obtained in the above step 10 and acetic acid (0.048 mL, 0.840 mmol) at room temperature, and the mixture was stirred at room temperature for 3 hours. The reaction solution was diluted with ethyl acetate, and the organic layer was washed with a saturated aqueous sodium bicarbonate solution and saturated saline, and then dried over sodium sulfate. After distillation under reduced pressure, the resulting residue was purified by silica gel chromatography [chloroform:methanol = 99.5:0.5 (v / v) to 95:5 (v / v)] to obtain the target product (3-12) (0.178 g, quantitative). 1H-NMR (CDCl 3 )δ:8.86(1H,s),7.60-7.59(2H,m),7.39(1H,s),7.32-7.20(7H,m),6.90-6.88(2H,m),6.78(1H,s) ,6.68(1H,s),6.38(1H,s),5.90-5.87(3H,m),5.39-5.22(6H,m),4.72-4.02(11H,m),3.90(3H,s), 3.88(3H,s),3.83(3H,s),3.70-3.63(6H,m),3.32-3.29(3H,m),2.73-2.69(1H,m),2.43-2.40(1H, m),2.12-2.06(1H,m),1.77-1.74(2H,m),1.39-1.25(6H,m),0.96-0.89(6H,m),0.73-0.66(4H,m). MS (APCI, ESI) m / z: 1198 (M+H)+

[0294] Step 12: L-valyl-N-{4-[({[(11'S,11a'S)-11'-hydroxy-7'-methoxy-8'-[(5-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yl]oxy}pentyl)oxy]-5'-oxo-11',11a'-dihydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide (3-13) To a dichloromethane (2 mL) solution of the compound (3-12) (0.140 mmol) obtained in the above step 11, pyrrolidine (0.0579 mL, 0.700 mmol) and tetrakis(triphenylphosphine)palladium(0) (0.0162 g, 0.0140 mmol) were added at room temperature, and the mixture was stirred at room temperature for 15 minutes. After distillation under reduced pressure, the resulting residue was purified by silica gel chromatography [chloroform:methanol = 99.5:0.5 (v / v) to 92.5:7.5 (v / v)] to obtain the target compound (3-13) (0.143 g, 99%). 1H-NMR (CDCl 3 )δ:9.12(1H,s),7.94-7.92(1H,m),7.57-7.53(4H,m),7.33-7.31(2H,m),7.20-7.18(3H,m),6.90-6.88(2H ,m),6.36(1H,s),6.07(1H,s),5.91-5.88(1H,m),5.47-5.44(1H,m),5.21-5.13(1H,m),4.66-4.58(3H,m),4 .32(1H,s),4.03-3.49(17H,m),3.38-3.29(4H,m),3.15-3.14(1H,m),2.77-2.73(1H,m),2.57(2H,s),2.43- 2.40(1H,m),2.32-2.27(1H,m),1.81-1.39(8H,m),0.98-0.96(3H,m),0.85-0.83(3H,m),0.75-0.62(4H,m). MS (APCI, ESI) m / z: 1030 (M+H)+

[0295] Step 13: N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11'S,11a'S)-11'-hydroxy-7'-methoxy-8'-[(5-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a -Tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yl]oxy}pentyl)oxy]-5'-oxo-11',11a'-dihydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide (3-14) To a mixture of compound (2-2) (0.0640g, 0.153mmol) obtained in Example 1-2, step 1, and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (0.0446g, 0.180mmol), dichloromethane (2mL) was added at room temperature, and the mixture was stirred at room temperature for 15 minutes. To the reaction solution, a solution of compound (3-13) (0.143g, 0.139mmol) obtained in the above step 12 in dichloromethane (2mL) was added, and the mixture was stirred at room temperature for five hours, and then distilled under reduced pressure. The obtained residue was purified by silica gel chromatography [chloroform:methanol = 99.5:0.5 (v / v) to 92.5:7.5 (v / v)] to obtain the target compound (3-14) (0.103g, 52%). 1H-NMR(DMSO-D6)δ:9.93(1H,s),8.21-8.16(2H,m),8.07-8.04(1H,m),7.83-7.64(2H,m),7.60-7.55(3H,m),7.51-7.28(10H,m),7 .19-7.16(2H,m),7.10-7.04(1H,m),6.92-6.90(2H,m),6.76-6.70(1H,m),6.39(1H,s),5.77-5.75(1H,m),5.21-5.18(1H,m),5.03 -4.99(1H,m),4.82-4.79(1H,m),4.37-4.35(1H,m),4.21-4.20(2H,m),4.02-3.24(26H,m),3.16-3.13(1H,m),2.79-2.59(2H,m),2 .39-2.28(2H,m),2.05-1.97(2H,m),1.91-1.77(4H,m),1.57-1.54(3H,m),1.28-1.23(3H,m),0.85-0.80(6H,m),0.67-0.61(4H,m). MS (APCI, ESI) m / z: 1431 (M+H)+

[0296] [Example 2-2: Drug Linker 2] [ka]

[0297] Step 1: (2R,11aS)-8-(3-bromopropoxy)-2-{[tert-butyl(dimethyl)silyl]oxy}-7-methoxy-10-{[2-(trimethylsilyl)ethoxy]methyl}-2,3-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (4-1) The compound (3-2) (5.06 g, 9.67 mmol) obtained in step 1 of Example 2-1 was reacted with 1,3-dibromopropane (4.93 mL, 48.4 mmol) in the same manner as in step 2 of Example 2-1 to obtain the target compound (4-1) (4.85 g, 78%). MS(APCI, ESI)m / z:645[81Br,(M+H)+],643[79Br,(M+H)+].

[0298] Step 2: (2R,11aS)-8-(3-bromopropoxy)-2-hydroxy-7-methoxy-10-{[2-(trimethylsilyl)ethoxy]methyl}-2,3-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (4-2) The compound (4-1) (4.85 g, 7.54 mmol) obtained in the above step 1 was reacted in the same manner as in step 3 of Example 2-1 to obtain the target compound (4-2) (4.05 g, quantitative). MS(APCI, ESI)m / z:531[81Br,(M+H)+],529[79Br,(M+H)+].

[0299] Step 3: (11aS)-8-(3-bromopropoxy)-7-methoxy-10-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,1-c][1,4]benzodiazepine-2,5,11(3H,10H,11aH)-trione (4-3) The compound (4-2) (7.54 mmol) obtained in the above step 2 was reacted in the same manner as in step 4 of Example 2-1 to obtain the target compound (4-3) (3.73 g, 93%). 1H-NMR(CDCl3)δ:7.34(1H,s),7.29(1H,s),5.56-5.53(1H,m),4.72-4.69(1H,m),4.67-4.61(1H,m),4.23-4.17(3H,m),3.97- 3.88(4H,m),3.82-3.75(1H,m),3.74-3.56(4H,m),2.82-2.77(1H,m),2.43-2.38(2H,m),1.06-0.94(2H,m),0.08-0.00(9H,m).

[0300] Step 4: (11aS)-8-(3-bromopropoxy)-7-methoxy-5,11-dioxo-10-{[2-(trimethylsilyl)ethoxy]methyl}-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl trifluoromethanesulfonate (4-4) The compound (4-3) (3.73 g, 7.08 mmol) obtained in the above step 3 was reacted in the same manner as in step 5 of Example 2-1 to obtain the target compound (4-4) (3.27 g, 70%). MS(APCI, ESI)m / z:661[81Br,(M+H)+],659[79Br,(M+H)+].

[0301] Step 5: (11aS)-8-(3-bromopropoxy)-7-methoxy-2-(4-methoxyphenyl-10-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (4-5) The compound (4-4) (3.27 g, 4.96 mmol) obtained in the above step 4 was reacted in the same manner as in step 6 of Example 2-1 to obtain the target compound (4-5) (2.49 g, 81%). MS(APCI, ESI)m / z:619[81Br,(M+H)+],617[79Br,(M+H)+].

[0302] Step 6: (11aS)-8-(3-bromopropoxy)-7-methoxy-2-(4-methoxyphenyl)-1,11a-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (4-6) The compound (4-5) (2.49 g, 4.04 mmol) obtained in the above step 5 was reacted in the same manner as in step 7 of Example 2-1 to obtain the target compound (4-6) (1.59 g, 84%). MS(APCI, ESI)m / z:473[81Br,(M+H)+],471[79Br,(M+H)+].

[0303] Step 7: (11aS)-8-(3-bromopropoxy)-7-methoxy-2-(4-methoxyphenyl)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (4-7) The compound (4-6) (1.59 g, 3.38 mmol) obtained in the above step 6 was reacted in the same manner as in step 8 of Example 2-1 to obtain the target compound (4-7) (1.39 g, 87%). MS(APCI, ESI)m / z:475[81Br,(M+H)+],473[79Br,(M+H)+].

[0304] Step 8: Prop-2-en-1-yl (11aS)-8-(3-bromopropoxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate (4-8) The compound (4-7) (1.40 g, 2.95 mmol) obtained in the above step 7 was reacted in the same manner as in step 9 of Example 2-1 to obtain the target compound (4-8) (0.885 g, 54%). MS(APCI, ESI)m / z:559[81Br,(M+H)+],557[79Br,(M+H)+].

[0305] Step 9: N-{[(prop-2-en-1-yl)oxy]carbonyl}-L-valyl-N-[4-({[(11'S,11'aS)-11'-{[tert-butyl(dimethyl)silyl]oxy}-7'-methoxy-8'-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-10-{[(prop-2-en-1-yl)oxy]carbonyl}-5, 10,11,11a-Tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yl]oxy}propoxy)-5'-oxo-11',11'a-dihydro-1'H,3'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-carbonyl]oxy}methyl)phenyl]-L-alaninamide (4-9) The compound (4-8) (0.0381 g, 0.0683 mmol) obtained in the above step 8 and the compound (1-11) (0.0552 g, 0.0683 mmol) obtained in Example 1-1, step 10 were reacted in the same manner as in Example 2-1, step 10 to obtain the target compound (4-9) (0.0712 g, 81%). MS (APCI, ESI) m / z: 1284 (M+H)+.

[0306] Step 10: N-{[(prop-2-en-1-yl)oxy]carbonyl}-L-valyl-N-[4-({[(11'S,11'aS)-11'-hydroxy-7'-methoxy-8'-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-10-{[(prop-2-en-1-yl)oxy]carbonyl}-5,10,11,11a -Tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yl]oxy}propoxy)-5'-oxo-11',11'a-dihydro-1'H,3'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-carbonyl]oxy}methyl)phenyl]-L-alaninamide (4-10) The compound (4-9) (0.0712 g, 0.0554 mmol) obtained in the above step 9 was reacted in the same manner as in step 11 of Example 2-1 to obtain the target compound (4-10) (0.0671 g, quantitative). MS (APCI, ESI) m / z: 1170 (M+H)+.

[0307] Step 11: L-valyl-N-[4-({[(11'S,11'aS)-11'-hydroxy-7'-methoxy-8'-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yl]oxy}propoxy)-5'-oxo-11',11'a-dihydro-1'H,3'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-carbonyl]oxy}methyl)phenyl]-L-alaninamide (4-11) The compound (4-10) (0.0571 mmol) obtained in the above step 10 was reacted in the same manner as in step 12 of Example 2-1 to obtain the target compound (4-11) (0.0574 g, 99%). 1H-NMR (CDCl 3)δ:9.16(1H,s),7.93-7.91(1H,m),7.55-7.52(1H,m),7.50-7.47(3H,m),7.35-7.32(2H,m),7.21(1H,s),7.13 -7.11(2H,m),6.90-6.87(2H,m),6.40(1H,s),6.08(1H,s),5.90-5.87(1H,m),5.37-5.34(1H,m),4.73-4.53(3 H,m),4.23-4.08(5H,m),3.89(3H,s),3.82(3H,s),3.78-3.72(5H,m),3.57-3.51(3H,m),3.38-3.30(3H,m),2. 76-2.71(1H,m),2.36-2.24(4H,m),1.78-1.42(6H,m),1.00-0.98(3H,m),0.87-0.84(3H,m),0.74-0.62(4H,m). MS (APCI, ESI) m / z: 1002 (M+H)+.

[0308] Step 12: N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-[4-({[(11'S,11'aS)-11'-hydroxy-7'-methoxy-8'-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a -Tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yl]oxy}propoxy)-5'-oxo-11',11'a-dihydro-1'H,3'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-carbonyl]oxy}methyl)phenyl]-L-alaninamide (4-12) The compound (4-11) (0.189 g, 0.189 mmol) obtained in the above step 11 and the compound (2-2) (0.087 g, 0.207 mmol) obtained in Example 1-2 step 1 were reacted in the same manner as in Example 2-1 step 13 to obtain the target compound (4-12) (0.169 g, 64%). MS (APCI, ESI) m / z: 1402 (M+H)+.

[0309] [Example 2-3: Drug Linker 3] [ka]

[0310] Step 1: Dimethyl (6S,6'S)-5,5'-{1,5-pentanediylbis[oxy(5-methoxy-2-nitrobenzene-4,1-diyl)carbonyl]}bis(5-azaspiro[2.4]heptane-6-carboxylate) (5-2) To a solution of 4,4'-[1,5-pentanediylbis(oxy)]bis(5-methoxy-2-nitrobenzoic acid) (5-1) (5.41g, 10.9mmol, Journal of Medicinal Chemistry 2004,47,1161) in dichloromethane (50mL), oxalyl chloride (5.63mL, 65.7mmol) was added at 0°C, and N,N-dimethylformamide (0.0844mL, 1.09mmol) was added dropwise. The reaction solution was warmed to room temperature and stirred for 2 hours. The residue obtained by distillation under reduced pressure was dissolved in dichloromethane (100 mL) and added dropwise to a dichloromethane solution (100 mL) of methyl (6S)-5-azaspiro[2.4]heptane-6-carboxylate hydrochloride (4.28 g, 24.1 mmol, Tetrahedron Letters 2012. 53. 3847) and triethylamine (6.07 mL, 43.8 mmol) at -40°C under a nitrogen atmosphere. The reaction solution was warmed to 0°C and stirred for 2 hours. 1N hydrochloric acid (100 mL) was added to the reaction mixture, and the organic layer was washed with water and saturated saline, and dried over anhydrous sodium sulfate. The product (5-2) (8.40 g, quantitative) was obtained by distillation under reduced pressure. MS (APCI, ESI) m / z: 769 (M+H)+.

[0311] Step 2: {1,5-pentanediylbis[oxy(5-methoxy-2-nitrobenzene-4,1-diyl)]}bis{[(6S)-6-(hydroxymethyl)-5-azaspiro[2.4]hept-5-yl]methanone} (5-3) Lithium borohydride (714 mg, 32.8 mmol) was added to a solution of the compound (5-2) (8.40 g, 10.9 mmol) obtained in the above step 1 in tetrahydrofuran (100 mL), and the mixture was stirred at 0°C for 30 minutes, then warmed to room temperature and stirred for 1 hour. 1N hydrochloric acid was added at 0°C, followed by extraction with ethyl acetate, washing with saturated saline, and drying over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure to obtain the target product (5-3) (7.70 g, 99%). MS (APCI, ESI) m / z: 713 (M+H)+.

[0312] Step 3: Pentane-1,5-diylbis[oxy(5-methoxy-2-nitrobenzene-4,1-diyl)carbonyl (6S)-5-azaspiro[2.4]heptane-5,6-diylmethanediyl]diazetate (5-4) The compound (5-3) (7.70 g, 10.8 mmol) obtained in the above step 2 was dissolved in pyridine (20 mL) and acetic anhydride (10 mL, 105.9 mmol) and stirred at room temperature. The mixture was evaporated under reduced pressure to obtain the target compound (5-4) (8.38 g, 97%). MS (APCI, ESI) m / z: 797 (M+H)+.

[0313] Step 4: 1,5-pentanediylbis[oxy(2-amino-5-methoxybenzene-4,1-diyl)carbonyl(6S)-5-azaspiro[2.4]heptane-5,6-diylmethanediyl]diacetate (5-5) To a solution of the compound (5-4) (8.28 g, 10.4 mmol) obtained in the above step 3 in N,N-dimethylformamide (100 mL), 5% palladium carbon (54% water, 1.00 g) was added, and the reaction solution was vigorously stirred at room temperature under a hydrogen atmosphere for 6 hours. After filtration through Celite, the filtrate was distilled under reduced pressure, and the resulting residue was purified by silica gel column chromatography [chloroform:methanol = 100:0 (v / v) to 90:10 (v / v)] to obtain the target compound (5-5) (5.05 g, 66%). MS (APCI, ESI) m / z: 737 (M+H)+.

[0314] Step 5: {(6S)-5-[4-({5-[4-({(6S)-6-[(acetyloxy)methyl]-5-azaspiro[2.4]hept-5-yl}carbonyl)-5-amino-2-methoxyphenoxy]pentyl}oxy)-5-methoxy-2-{[(prop-2-en-1-yloxy)carbonyl]amino}benzoyl]-5-azaspiro[2.4]hept-6-yl}methyl acetate (monoallyloxycarbonyl form) (5-6) Pyridine (1.10mL, 13.7mmol) was added to a dichloromethane (100mL) solution of the compound (5-5) (5.05g, 6.85mmol) obtained in the above step 4, and allyl chloroformate (0.725mL, 6.85mmol) was added under a nitrogen atmosphere at -78°C, and the mixture was stirred for 2 hours. The residue obtained by distillation under reduced pressure was purified by silica gel column chromatography [hexane:ethyl acetate=70:30(v / v) to 100:0(v / v), chloroform:methanol=100:0(v / v) to 90:10(v / v)] to obtain the target monoallyloxycarbonyl body (5-6) (2.63g, 47%). MS (APCI, ESI) m / z: 821 (M+H)+.

[0315] Step 6: N-[(2-propen-1-yloxy)carbonyl]-L-valyl-N-{4-[({[2-({(6S)-6-[(acetyloxy)methyl]-5-azaspiro[2.4]hept-5-yl}carbonyl)-5-({5-[4-({(6S)-6-[(acetyloxy)methyl]-5-azaspiro[2.4]hept-5-yl}carbonyl)-2-methoxy-5-{[(2-propen-1-yloxy)carbonyl]amino}phenoxy]pentyl}oxy)-4-methoxyphenyl]carbamoyl}oxy)methyl]phenyl}-L-alaninamide (5-7) The monoallyloxycarbonyl compound (5-6) (2.00 g, 2.44 mmol) obtained in step 5 above and N-[(prop-2-en-1-yloxy)carbonyl]-L-valyl-N-[4-(hydroxymethyl)phenyl]-L-alaninamide (1.10 g, 2.92 mmol, WO2011130598) were reacted in the same manner as in step 6 of Example 1-1 to obtain the target compound (5-7) (2.64 g, 89%). MS (APCI, ESI) m / z: 1224 (M+H)+.

[0316] Step 7: N-[(2-propen-1-yloxy)carbonyl]-L-valyl-N-[4-({[(2-{[(6S)-6-(hydroxymethyl)-5-azaspiro[2.4]hept-5-yl]carbonyl}-5-{[5-(4-{[(6S)-6-(hydroxymethyl)-5-azaspiro[2.4]hept-5-yl]carbonyl}-2-methoxy-5-{[(2-propen-1-yloxy)carbonyl]amino}phenoxy)pentyl]oxy}-4-methoxyphenyl)carbamoyl]oxy}methyl)phenyl]-L-alaninamide (5-8) Potassium carbonate (1.49 g, 10.8 mmol) was added to a methanol (10 mL) solution of compound (5-7) (2.64 g, 2.16 mmol) obtained in step 6 above, and the mixture was stirred at room temperature for 3 hours. A saturated aqueous solution of ammonium chloride (100 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate. The product (5-8) (2.21 g, 90%) was obtained by distillation under reduced pressure. MS (APCI, ESI) m / z: 1140 (M+H)+.

[0317] Step 8: N-[(2-propen-1-yloxy)carbonyl]-L-valyl-N-{4-[({[(11'S,11a'S)-11'-hydroxy-8'-{[5-({(11'S,11a'S)-11'-hydroxy-7'-methoxy-5'-oxo-10'-[(2-propen-1-yloxy)carbonyl]-5',10',11',11a'-tetrahydro-1'H-spiro[cyclo[ cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-8'-yl}oxy)pentyl]oxy}-7'-methoxy-5'-oxo-11',11a'-dihydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide (5-9) Dess-Martin periodinane (1.59 g, 3.74 mmol) was added to a dichloromethane (50 mL) solution of compound (5-8) (2.03 g, 1.78 mmol) obtained in the above step 7, and the mixture was stirred at room temperature overnight. A saturated aqueous solution of sodium bicarbonate (100 mL) was added to the reaction mixture, and the mixture was extracted with chloroform. The organic layer was dried over anhydrous sodium sulfate. The residue obtained by distillation under reduced pressure was purified by silica gel column chromatography [chloroform:methanol = 100:0 (v / v) to 90:10 (v / v)] to obtain the target product (5-9) (2.05 g, quantitative). MS (APCI, ESI) m / z: 1136 (M+H)+.

[0318] Step 9: L-valyl-N-{4-[({[(11'S,11a'S)-11'-hydroxy-7'-methoxy-8'-[(5-{[(11a'S)-7'-methoxy-5'-oxo-5',11a'-dihydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-8'-yl]oxy}pentyl)oxy]-5'-oxo-11',11a'-dihydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide (5-10) The compound (5-9) (2.05 g, 1.80 mmol) obtained in the above step 8 was reacted in the same manner as in step 12 of Example 2-1 to obtain the target compound (5-10) (1.02 g, 60%). MS (APCI, ESI) m / z: 950 (M+H)+.

[0319] Step 10: N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11'S,11a'S)-11'-hydroxy-7'-methoxy-8'-[(5-{[(11a'S)-7'-methoxy-5'-oxo-5',11a'-dihydro-1'H-spiro[cyclo Propane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-8'-yl]oxy}pentyl)oxy]-5'-oxo-11',11a'-dihydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide (5-11) Compound (5-10) (0.710 g, 0.747 mmol) obtained in the above step 9 and compound (2-2) (0.313 g, 0.747 mmol) obtained in Example 1-2 step 1 were dissolved in a mixed solvent of dichloromethane (1.5 mL) and methanol (0.1 mL). 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (0.264 g, 0.897 mmol) was added and stirred at room temperature for 1 hour. The residue obtained by distillation under reduced pressure was purified by silica gel column chromatography [chloroform:methanol = 100:0 (v / v) to 80:20 (v / v)] to obtain the target compound (5-11) (0.671 g, 66%). 1H-NMR(DMSO-D6)δ:9.91(1H,s),8.32(1H,s),8.23-7.91(3H,m),7.81-7.19(14H,m),7.04(1H,m),6. 80-6.62(3H,m),5.77-5.75(1H,m),5.20(1H,m),5.01(1H,m),4.79(1H,m),4.46-4.35(1H,m),4.04(4H ,m),3.86-3.38(18H,m),3.22-3.15(2H,m),2.67-2.63(1H,m),2.46-2.23(3H,m),2.09-1.91(2H,m), 1.80-1.78(5H,m),1.57(3H,m),1.27(3H,s),1.11-1.04(1H,m),0.87-0.79(6H,m),0.63-0.55(6H,m). MS (APCI, ESI) m / z: 1351 (M+H)+.

[0320] [Example 2-4: Drug Linker 4] [ka]

[0321] Step 1: Methyl (6S)-5-[4-(benzyloxy)-5-methoxy-2-nitrobenzoyl]-5-azaspiro[2.4]heptane-6-carboxylate (6-2) To a solution of 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (6-1) (6.07 g, 20.0 mmol, Tetrahedron 1995, 51, 5617) and N,N-dimethylformamide (1.08 mL, 13.9 mmol) in dichloromethane (100 mL), oxalyl chloride (3.43 mL, 40.0 mmol) was added dropwise over 5 minutes under ice cooling. The reaction solution was stirred at room temperature for 5 hours, and then evaporated under reduced pressure. The resulting residue was dissolved in dichloromethane (20 mL) and evaporated under reduced pressure. This operation was repeated three times, and the residue was suspended in dichloromethane (5 mL), to which excess diethyl ether and hexane were added, filtered, and dried under reduced pressure to obtain crude acid chloride. The obtained acid chloride was dissolved in dichloromethane, cooled to -40℃ (dry ice-acetonitrile bath), and methyl (6S)-5-azaspiro[2.4]heptane-6-carboxylate hydrochloride (4.22g, 22.0mmol, Tetrahedron Letters 2012.53.3847) and triethylamine (3.36mL, 24.2mmol) were gradually added. The reaction mixture was allowed to warm to room temperature overnight. 1N hydrochloric acid was added to the reaction mixture, and the reaction mixture was extracted with dichloromethane. The organic layer was washed with water, saturated aqueous sodium bicarbonate solution and saturated saline, and dried over anhydrous sodium sulfate. The residue obtained by distillation under reduced pressure was purified by silica gel column chromatography [hexane:ethyl acetate=100:0-50:50] to obtain the target product (6-2) (6.55g, 80%). MS (APCI, ESI) m / z: 441 (M+H)+

[0322] Step 2: (11a'S)-8'-(benzyloxy)-7'-methoxy-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-5',11'(10'H,11a'H)-dione (6-3) Raney nickel (7.00 g) was added to a solution of the compound (6-2) (6.55 g, 16.0 mmol) obtained in the above step 1 in ethanol (150 mL) and tetrahydrofuran (150 mL) under a nitrogen atmosphere. Hydrazine monohydrate (7 mL) was added to the reaction mixture, and the temperature was gradually raised to 50 ° C. After stirring at 50 ° C for 2 hours, Raney nickel (3.00 g) and hydrazine monohydrate (3 mL) were added and stirred for 1 hour. THF (100 mL) was added to the reaction mixture, and the mixture was filtered through Celite. The residue obtained by distillation under reduced pressure was purified by silica gel column chromatography [hexane: ethyl acetate = 100: 0 to 25: 75] to obtain the target product (6-3) (4.42 g, 73%). MS (APCI, ESI) m / z: 379 (M+H)+

[0323] Step 3: (11a'S)-8'-(benzyloxy)-7'-methoxy-10'-{[2-(trimethylsilyl)ethoxy]methyl}-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-5',11'(10'H,11a'H)-dione (6-4) To a solution of the compound (6-3) (10.0 g, 26.4 mmol) obtained in the above step 2 in tetrahydrofuran (150 mL), 2.6 mol / L n-butyllithium n-hexane solution (12.0 mL, 31.8 mmol) was slowly added dropwise at -40°C. The reaction solution was stirred at -40°C for 15 minutes, and then 2-(chloromethoxy)ethyltrimethylsilane (5.57 mL, 31.7 mmol) was slowly added dropwise. The reaction solution was stirred at room temperature for 3 hours, and then water was added and extracted with ethyl acetate. The organic layer was washed with water and saturated saline, and dried over anhydrous sodium sulfate. The residue obtained by distillation under reduced pressure was purified by silica gel column chromatography [hexane:ethyl acetate = 100:0 to 30:70] to obtain the target product (6-4) (11.8 g, 88%). MS (APCI, ESI) m / z: 509 (M+H)+

[0324] Step 4: (11a'S)-8'-hydroxy-7'-methoxy-10'-{[2-(trimethylsilyl)ethoxy]methyl}-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-5',11'(10'H,11a'H)-dione (6-5) A 5% palladium carbon catalyst (5.00 g) was added to a solution of the compound (6-4) (18.7 g, 36.8 mmol) obtained in the above step 3 in tetrahydrofuran (50 mL) and ethanol (100 mL) under a nitrogen atmosphere. The nitrogen balloon was immediately replaced with a hydrogen balloon, and the reaction mixture was stirred under a hydrogen atmosphere for 6 hours. The reaction mixture was diluted with chloroform and filtered through Celite. The filtrate was then distilled under reduced pressure to obtain a residue, which was purified by silica gel column chromatography [hexane:ethyl acetate=100:0 to 25:75] to obtain the target compound (6-5) (15.1 g, 98%). MS (APCI, ESI) m / z: 419 (M+H)+

[0325] Step 5: (11a'S)-8'-[(5-bromopentyl)oxy]-7'-methoxy-10'-{[2-(trimethylsilyl)ethoxy]methyl}-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-5',11'(10'H,11a'H)-dione (6-6) The compound (6-5) (2.77 g, 6.62 mmol) obtained in the above step 4 was reacted in the same manner as in step 2 of Example 2-1 to obtain the target compound (6-6) (3.31 g, 88%). 1H-NMR (CDCl 3)δ:7.36(1H,s),7.25(1H,s),5.55(1H,m),4.65(1H,m),4.24-4.23(1H,m),4. 11-4.03(2H,m),3.93(3H,s),3.85-3.78(1H,m),3.72-3.69(2H,m),3.46-3.3 9(3H,m),2.47-2.44(1H,m),2.25-2.22(1H,m),1.95-1.91(4H,m),1.67-1.59 (1H,m),1.03-0.95(2H,m),0.90-0.85(1H,m),0.70-0.66(4H,m),0.05(9H,s).

[0326] Step 6: (11a'S)-8'-[(5-bromopentyl)oxy]-7'-methoxy-1',11a'-dihydro-5'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-5'-one (6-7) The compound (6-6) (3.31 g, 5.83 mmol) obtained in the above step 5 was reacted in the same manner as in step 7 of Example 2-1 to obtain the target compound (6-7) (1.11 g, 45%). 1H-NMR (CDCl 3 )δ:7.81(1H,m),7.53(1H,s),6.82(1H,s),4.13-4.06(2H,m),3.97(3H,s),3.88-3.83(1H,m),3.69(1 H,m),3.52-3.39(3H,m),2.55-2.52(1H,m),2.06-1.89(5H,m),1.67-1.63(2H,m),0.76-0.72(4H,m).

[0327] Step 7: (11a'S)-8'-[(5-bromopentyl)oxy]-7'-methoxy-1',10',11',11a'-tetrahydro-5'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-5'-one (6-8) The compound (6-7) (2.56 g, 6.08 mmol) obtained in the above step 6 was reacted in the same manner as in step 8 of Example 2-1 to obtain the target compound (6-8) (1.15 g, 45%). 1H-NMR (CDCl 3)δ:7.60(1H,s),6.07(1H,s),4.11-4.04(1H,m),3.99(2H,m),3.87-3.84(1H,m),3.85(3H,s),3.73(1 H,m),3.58-3.53(2H,m),3.47-3.42(3H,m),2.03-1.78(6H,m),1.65-1.63(2H,m),0.77-0.56(4H,m).

[0328] Step 8: Prop-2-en-1-yl (11a'S)-8'-[(5-bromopentyl)oxy]-7'-methoxy-5'-oxo-11',11a'-dihydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-carboxylate (6-9) The compound (6-8) (1.15 g, 2.72 mmol) obtained in the above step 7 was reacted in the same manner as in step 9 of Example 2-1 to obtain the target compound (6-9) (1.14 g, 82%). 1H-NMR(CDCl3)δ:7.23(1H,s),6.69(1H,s),5.79(1H,s),5.13-5.10(2H,m),4.68-4.66(1H,m),4.48-4.45(2H,m),4.01(2H,m), 3.92(3H,s),3.76(1H,m),3.54-3.37(3H,m),2.39(1H,m),1.95-1.90(4H,m),1.68-1.61(3H,m),1.44(1H,m),0.75-0.66(4H,m).

[0329] Step 9: N-[(prop-2-en-1-yloxy)carbonyl]-L-valyl-N-{4-[({[(11'S,11a'S)-11'-{[tert-butyl(dimethyl)silyl]oxy}-7'-methoxy-8'-{[5-({(11a'S)-7'-methoxy-5'-oxo-10'-[(prop-2-en-1-yloxy)carbonyl]-5',10',11',11a'-tetrahydro -1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-8'-yl}oxy)pentyl]oxy}-5'-oxo-11',11a'-dihydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide (6-10) The compound (6-9) (0.374 g, 0.737 mmol) obtained in the above step 8 and the compound (1-11) (0.452 g, 0.56 mmol) obtained in Example 1-1 step 10 were reacted in the same manner as in Example 2-1 step 10 to obtain the target compound (6-10) (0.589 g, 65%). MS (APCI, ESI) m / z: 1234 (M+H)+

[0330] Step 10: N-[(prop-2-en-1-yloxy)carbonyl]-L-valyl-N-{4-[({[(11'S,11a'S)-11'-hydroxy-7'-methoxy-8'-{[5-({(11a'S)-7'-methoxy-5'-oxo-10'-[(prop-2-en-1-yloxy)carbonyl]-5',10',11',11a'-tetrahydro-1'H-spiro[ Cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-8'-yl}oxy)pentyl]oxy}-5'-oxo-11',11a'-dihydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide (6-11) The compound (6-10) (0.589 g, 0.477 mmol) obtained in the above step 9 was reacted in the same manner as in step 11 of Example 2-1 to obtain the target compound (6-11) (0.382 g, 71%). 1H-NMR (CDCl 3 )δ:8.90(1H,s),7.55(2H,m),7.25-7.21(2H,m),6.74(2H,m),6.38(1H,s),5.90-5.87(5H,m),5.33-5.09(8H,m),4. 66-4.60(8H,m),3.98-3.91(10H,m),3.77-3.30(12H,m),2.42-2.36(2H,m),1.77-1.39(6H,m),0.91-0.70(14H,m).

[0331] Step 11: L-valyl-N-{4-[({[(11'S,11a'S)-11'-hydroxy-7'-methoxy-8'-[(5-{[(11a'S)-7'-methoxy-5'-oxo-5',10',11',11a'-tetrahydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-8'-yl]oxy}pentyl)oxy]-5'-oxo-11',11a'-dihydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide (6-12) The compound (6-11) (0.382 g, 0.341 mmol) obtained in the above step 10 was reacted in the same manner as in step 12 of Example 2-1 to obtain the target compound (6-12) (0.200 g, 62%). MS (APCI, ESI) m / z: 952 (M+H)+

[0332] Step 12: N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11'S,11a'S)-11'-hydroxy-7'-methoxy-8'-[(5-{[(11a'S)-7'-methoxy-5'-oxo-5',10',11',11a'-tetrahydro-1'H-spiroyl) 1,2'-cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-8'-yl]oxy}pentyl)oxy]-5'-oxo-11',11a'-dihydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide (6-13) The compound (6-12) (0.0560 g, 0.0588 mmol) obtained in the above step 11 and the compound (2-2) (0.022 g, 0.053 mmol) obtained in Example 1-2 step 1 were reacted in the same manner as in Example 2-1 step 13 to obtain the target compound (6-13) (0.0500 g, 63%). MS (APCI, ESI) m / z: 1354 (M+H)+

[0333] [Synthesis of glycan donors] Example 3: [N 3 -PEG(3)]-MSG1-Ox [ka]

[0334] Process 1: (MSG1-)Asn Commercially available monosialo-Asn free (1S2G / 1G2S-10NC-Asn, Glycotechnology Institute, Inc.) (referred to as "(MSG-)Asn") (500 mg) was separated and purified by reversed-phase HPLC under the following conditions, and separated into (MSG1-)Asn (retention time 15-19 min) eluted as the 1st main peak and (MSG2-)Asn (retention time 21-26 min) eluted as the 2nd main peak. 0.1% formic acid solution was used as the eluent, and the ELS-PDA trigger fractionation system (JASCO Corporation) was used as the apparatus, and the Inertsil ODS-3 (10 um, 30Φx250 mm, GL Sciences) was used as the column, with a flow rate of 30 mL / min. The first peak detected by UV (210 nm) during elution was collected and lyophilized to obtain the target product (238 mg).

[0335] Process 2: MSG1 The compound (229 mg) obtained in the above step 1 was dissolved in 200 mM phosphate buffer solution (pH 6.25) (1145 μL), and EndoM (Tokyo Chemical Industry Co., Ltd., 1 U / mL) aqueous solution (100 μL) was added and incubated at 35 ° C. for 6 days. After the reaction was completed, the reaction solution was ultrafiltered using VIVASPIN 15R (Hydrosart membrane, 30K, 6,000xG), and the resulting filtrate was separated and purified by reversed-phase HPLC. A 0.1% aqueous trifluoroacetic acid solution was used as the eluent, and an ELS-PDA trigger fractionation system (JASCO Corporation) was used as the device, and an Inertsil ODS-3 (GL Science Co., Ltd.) was used as the column. The peak of the target substance detected by UV (210 nm) during elution was fractionated and freeze-dried to obtain the target substance (117 mg).

[0336] Step 3: [N 3 -PEG(3)]-MSG1 11-Azido-3,6,9-trioxaundecan-1-amine (0.108 mL, 0.541 mmol) and an aqueous solution (1.2 mL) of MSG1 (117 mg, 0.068 mmol) obtained in step 2 above were added to a 5 mL sampling tube (Ina Optica Co., Ltd.), stirred for 1 hour, and then freeze-dried. After freeze-drying, a solution (1.2 mL) of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (103 mg, 0.27 mmol) in N,N-dimethylformamide and diisopropylethylamine (0.046 mL, 0.27 mmol) were added to the 5 mL sampling tube, and stirred at 37°C for 3 hours. After the reaction was completed, the reaction solution was transferred to a centrifuge tube (50 mL) to which diethyl ether (20 mL) had been added in advance. The solid matter was precipitated using a small centrifuge (Hitachi Koki, CF16RX), and the supernatant was removed. Diethyl ether (10 ml) was added, and the mixture was centrifuged and then decanted. Then, acetonitrile (10 mL) was added, and the mixture was centrifuged and then decanted. This procedure was repeated twice, and the mixture was dried under reduced pressure to obtain a crude product. The obtained solid matter was purified by reversed-phase HPLC under the same conditions as in step 2 above, and the target product (94.2 mg) was obtained.

[0337] Step 4: [N 3 -PEG(3)]-MSG1-Ox The compound (100 mg) synthesized in step 3 above and an aqueous solution (520 μl) of 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride (Fushimi Pharmaceutical, 56 mg, 0.257 mmol) were added to a 5 mL sampling tube (Ina Optica Co., Ltd.). An aqueous solution (520 μl) of tripotassium phosphate (165 mg, 0.78 mmol) was added to the reaction solution after cooling on ice, and the mixture was stirred for 3 hours under ice cooling. The reaction solution obtained was ultrafiltered using Amicon Ultra (Ultracel 30K, Merck Millipore) to remove solids. The effluent was purified by gel filtration chromatography. The apparatus used was Purif-Rp2 (Shoko Scientific), the column was HiPrep 26 / 10 Desalting (GE Healthcare), and the mobile phase was 0.03%-NH 3 An aqueous solution was used, the flow rate was 10 mL / min, and the fraction volume was 10 mL. The fractions containing the target substance detected by UV (220 nm) during elution were combined, and 1N aqueous sodium hydroxide solution (104 μl, 0.104 mmol) was added and the mixture was lyophilized to obtain the target substance (84 mg).

[0338] Example 4: [N 3 -PEG(3)]-MSG-Ox [ka]

[0339] Step 1: Preparation of (MSG-)Asn Commercially available 1S2G / 1G2S-10NC-Asn-Fmoc (Glycotechnology Institute, Inc.) (referred to as "Fmoc-(MSG-)Asn") (1000 mg) was dissolved in ethanol / water (1 / 1) (10 mL), 1N aqueous sodium hydroxide solution (1.75 mL, 4 equivalents) was added, and the mixture was stirred at room temperature for 3 hours. After the reaction was completed, the reaction solution was ultrafiltered using Amicon Ultra (30K, Millipore) to remove solids, and 1N hydrochloric acid (832 μl, 1.9 equivalents) was added to the resulting filtrate. The solvent was removed using a high-speed concentrator V-10 (Biotage). Acetonitrile was added, and the solvent was removed using a high-speed concentrator V-10 (Biotage), followed by reverse-phase HPLC separation and purification. The eluents were 0.1% trifluoroacetic acid aqueous solution and 0.1% trifluoroacetic acid acetonitrile solution, the device was Purif-Rp2 (Shoko Scientific), and the column was Inertsil ODS-3 (GL Science). The fractions containing the target substance detected by UV (220 nm) during elution were combined and freeze-dried. When the target substance was dissolved in pure water again, it was confirmed to be acidic using pH test paper, so 18% ammonia water (150 μl) was added, and it was confirmed to be basic using pH test paper, and it was freeze-dried again. The target substance obtained (840 mg) was used as it is in the next reaction.

[0340] Step 2: Synthesis of MSG The compound (840 mg) obtained in step 1 was dissolved in 200 mM phosphate buffer solution (pH 6.25) (6000 μL), and EndoM (Tokyo Chemical Industry Co., Ltd., 1 U / mL)) aqueous solution (200 μL) was added and incubated at 28 ° C for 26 hours. Since the reaction was not completed, EndoM (Tokyo Chemical Industry Co., Ltd., 1 U / mL)) aqueous solution (50 μL) was added, and the mixture was incubated at 28 ° C for 2 hours, and then left at room temperature until the reaction was completed. After the reaction was completed, the reaction solution was ultrafiltered using Amicon Ultra (30K, Millipore). Trifluoroacetic acid (80 μl) was added to the obtained effluent, and the mixture was separated and purified by reversed-phase HPLC. 0.1% trifluoroacetic acid aqueous solution and 0.1% trifluoroacetic acid acetonitrile solution were used as eluents, Purif-Rp2 (Shoko Scientific) was used as the device, and Inertsil ODS-3 (GL Science) was used as the column. The fractions containing the target compound detected by UV (220 nm) during elution were pooled and lyophilized. To remove residual trifluoroacetic acid, the product was dissolved in pure water again to obtain the target compound (618 mg) as a colorless solid. ESI-MS: Calcd for C 66 H 110 N 4 O 49 : [M+H] + 1743.62, Found 1743.63

[0341] Step 3: [N 3 Synthesis of [-PEG(3)]-MSG Using the compound (120 mg) obtained in the above step 2 and following the same procedure as in step 3 of Example 3, the target product (88.6 mg) was obtained. ESI-MS: Calcd for C 73 H 124 N 8 O 51 :[M+2H] 2+ 965.37, Found 965.37

[0342] Process 4[N 3 Synthesis of [-PEG(3)]-MSG-Ox Using the compound (100 mg) synthesized in the above step 3 and following the same procedure as in step 4 of Example 3, the target product (88 mg) was obtained.

[0343] Example 5: [N 3 -PEG(3)] 2 -SG(10)-Ox [ka]

[0344] Process 1: [N 3 -PEG(3)] 2 -SG(10) A 5 ml sampling tube (Ina Optica Co., Ltd.) was charged with 11-azido-3,6,9-trioxaundecan-1-amine (0.096 ml, 0.485 mmol) and an aqueous solution (0.5 ml) of disialooctasaccharide (50 mg, 0.24 mmol), stirred for 1 hour, and then freeze-dried. To the 5 ml sampling tube after freeze-drying, a solution (0.6 ml) of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (92 mg, 0.24 mmol) in N,N-dimethylformamide and diisopropylethylamine (0.042 ml, 0.24 mmol) were added, and the mixture was stirred at 37°C for 4 hours. After the reaction, the reaction solution was transferred to a centrifuge tube (50 ml) to which diethyl ether (20 ml) had been added in advance. The solid matter was precipitated using a small centrifuge (Hitachi Koki, CF16RX) and the supernatant was removed. Diethyl ether (20 ml) was added and decanted. Acetonitrile (20 mL) was then added and decanted, followed by drying under reduced pressure to obtain a crude product. The obtained solid matter was dissolved in an appropriate amount of 0.2% trifluoroacetic acid aqueous solution and separated and purified by reversed-phase HPLC. 0.1% trifluoroacetic acid aqueous solution and 0.1% trifluoroacetic acid acetonitrile solution were used as eluents, and a Purif-Rp2 (Shoko Scientific) was used as the apparatus, and an Inertsil ODS-3 (GL Science) was used as the column. The fractions containing the target substance detected by UV (220 nm) during elution were combined and freeze-dried to obtain the target substance (42 mg).

[0345] Step 2: [N 3 -PEG(3)] 2 -SG(10)-Ox The compound (40 mg) synthesized in step 1 above and an aqueous solution (200 μl) of 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride (Fushimi Pharmaceutical, 17.9 mg, 0.083 mmol) were added to a 5 mL sampling tube (Ina Optica Co., Ltd.). An aqueous solution (200 μl) of tripotassium phosphate (52.6 mg, 0.25 mmol) was added to the reaction solution after cooling on ice, and the mixture was stirred for 2 hours under ice cooling. The reaction solution obtained was ultrafiltered using Amicon Ultra (Ultracel 30K, Merck Millipore) to remove solids. The effluent was purified by gel filtration chromatography. The apparatus used was Purif-Rp2 (Shoko Scientific), the column was HiPrep 26 / 10 Desalting (GE Healthcare), the mobile phase was 0.03% NH3 aqueous solution, the flow rate was 10 mL / min, and the fraction volume was 10 mL. The fractions containing the target substance detected by UV (220 nm) during elution were combined into one, and 1N sodium hydroxide aqueous solution (33 μl, 0.033 mmol) was added and lyophilized to obtain the target substance (34 mg).

[0346] Example 6: Hybridoma (218B1) Producing Mouse Anti-CLDN6 Antibody B1 and Hybridoma (218C7) Producing Mouse Anti-CLDN6 Antibody C7 6-1. Immunization of mice and acquisition of hybridomas 1-1) Preparation of cells for mouse immunization 2×10 6 pcs or 5×10 6 NOR-P1 cells (human pancreatic cancer cell line, RIKEN RCB-2139) were cultured in RPMI-1640 (Roswell Park Memorial Institute-1640) 10% FBS (fetal bovine serum) (+) medium (10 ml or 20 ml) for 5 days, harvested, washed twice with PBS (phosphate buffered saline), and resuspended in PBS (300 μl).

[0347] 1-2) Immunization of mice BALB / c mice (12 weeks old) were immunized with NOR-P1 cells (2 × 106 Approximately 2 weeks after the fifth immunization, NOR-P1 cells (5 × 10 6 Approximately 3 weeks after the sixth immunization, NOR-P1 cells (2 × 10 6 The 8th to 10th rounds were administered intraperitoneally with 2 × 10 6 Approximately 3 weeks after the 10th immunization (11th immunization) and 3 days after that (12th immunization, final immunization), 5 × 10 6 Mice were immunized intraperitoneally with NOR-P1 cells, and spleen cells were extracted 3 days after the final immunization.

[0348] 1-3) Preparation of spleen cells from immunized mice The spleens of the immunized mice were removed, crushed, and suspended in RPMI1640 10% FBS(+) medium. The cell suspension was passed through a cell strainer (70 μm, BD Falcon), centrifuged at 1500 rpm for 5 minutes at room temperature, and the supernatant was discarded. 4 Cl solution (20mM Tris-HCl pH7.2, 77.6mM NH 4 Cl; 20 mL) was added and incubated at room temperature for 5 minutes. PBS (20 mL) was added and centrifuged at 1500 rpm for 5 minutes at room temperature. After discarding the supernatant, RPMI1640 FBS(+) medium (10 mL) was added.

[0349] 1-4) Preparation of myeloma cells P3U1 cells (mouse myeloma cell line) were cultured in RPMI1640 FBS(+) medium for 5 days, then harvested and resuspended in RPMI1640 FBS(+) medium (20 ml).

[0350] 1-5) Cell fusion Spleen cells and myeloma cells were mixed at a ratio of 5:1 and centrifuged at 1500 rpm for 5 minutes at room temperature. After washing twice with RPMI1640 FBS(-) medium (10 ml), the cells were centrifuged (1500 rpm, 5 minutes). The cells in the precipitated fraction were thoroughly loosened, and polyethylene glycol-1500 (PEG-1500; 1 ml) was gradually added over about 1 minute while stirring. After stirring for 3 minutes and 30 seconds, the mixture was left at room temperature for 30 seconds. Then, RPMI medium 10% Low IgG FBS(+) (10 ml) was added to the cell solution over 1 minute. The cell suspension was centrifuged (1500 rpm, 5 minutes), and the cells in the precipitated fraction were gently loosened and then gently suspended in HAT medium (RPMI1640 medium containing 10% Low IgG FBS, HAT Media Supplement, and 5% BriClone; 200 ml). The suspension was dispensed into a 96-well culture plate at 200 μl per well and incubated at 37° C., 5% CO 2 The cells were cultured in an incubator at 37 °C for 6 days.

[0351] 1-6) Preparation of hybridoma screening probes A recombinant conjugate protein, DT3C, was produced to measure antibody internalization and immunotoxin activity. DT3C is a protein that genetically fuses the catalytic domain of diphtheria toxin (DT) with the antibody-binding domain of streptococcal protein G. DT3C specifically binds to the Fc portion of antibodies, and when taken up into cells, it induces cell death by inhibiting protein synthesis. Using this system, antibody internalization and the cell-killing effect of immunotoxins can be observed simultaneously (Yamaguchi, M. et al., Biochemical and Biophysical Research Communications 454(2014)600-603).

[0352] 1-7) Hybridoma screening using DT3C 4 μg / ml DT3C (25 μl) was added to a 96-well plate, and the culture supernatant (25 μl) of the hybridoma obtained in step 1-5 was further added, followed by incubation at room temperature for 30 minutes.5 NOR-P1 cells (50 μl) were seeded at 10 cells / ml (RPMI medium, 10% Low IgG FBS(+)) and incubated at 37°C in CO 2 The cells were cultured in an incubator for 3 days. After culture, wells that showed adherent cell numbers of approximately 25% or less than the number of adherent cells observed when the negative control antibody was used were judged to be positive. The selected clones were subcloned once or twice, and eight monoclonal hybridoma cell lines were established.

[0353] 6-2: Identification of the antigen to which the antibody produced by the hybridoma binds Among the antibodies produced by the hybridomas prepared in Example 6-1, two clones, 218B1 and 218C7, were subjected to antigen identification. 2-1) Immunoprecipitation of biotin-labeled cell surface proteins using 218B1 and 218C7 antibodies 2×10 6The culture supernatant of NTERA-2 cells (human testicular cancer cell line, ATCC CRL-1973) was removed, and the cells were washed twice with PBS. EZ-Link Sulfo-NHS-Biotin (Thermo Fisher Scientific) was suspended in PBS at a concentration of 0.1 mg / ml. After removing the PBS, Biotin / PBS solution was added, and the cells were incubated on a shaker for 30 minutes, and then washed twice with 100 mM glycine / PBS solution (25 ml), and then washed once with PBS (10 ml). The washed cells were resuspended in 200 μl of lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1% DDM, Protease inhibitor, Complete EDTA free (Roche) 1 grain / 50 ml) and treated at 4 ° C for 30 minutes. The cells were centrifuged (13000 rpm, 20 minutes, 4 ° C) to prepare a cell lysate. Protein G Sepharose (Protein G Sepharose 4 Fast Flow (GE Healthcare)) was replaced with a dissolution buffer to obtain Protein G Sepharose / dissolution buffer (50% slurry; 30 μl), which was added to the cell lysate, rotated at 4 ° C for 30 minutes, then centrifuged at 4 ° C for 1 minute, and the supernatant was collected. 218B1 antibody or 218C7 antibody (approximately 3 μg) was added to this supernatant, rotated at 4 ° C for 30 minutes, and then Protein G Sepharose / dissolution buffer (50% slurry; 60 μl) was added and rotated at 4 ° C for 1 hour. Protein G Sepharose was washed six times with dissolution buffer (1 ml) and then resuspended in 1 × SDS sample buffer (BioRad). The suspension was treated at 100 ° C for 5 minutes, and the solution was collected and used as a sample for SDS-PAGE (polyacrylamide gel electrophoresis).

[0354] 2-2) SDS-PAGE and Western blotting The SDS-PAGE sample prepared in 2-1) was After stacking with Ace 5-20% (Wako) at 50 mV for 30 minutes, electrophoresis was performed at 200 mV for 1 hour, and then blotting from the gel to the membrane at 12 mV for 47 minutes. The membrane was washed with PBS-T (PBS(-)-0.02% Tween 20) and blocked for 1 hour. The membrane was washed three times for 5 minutes with PBS-T and then reacted with Streptavidin-horseradish peroxidase conjugate (GE Healthcare; diluted 2000-fold with PBS-T for use) for 1 hour. The membrane was washed twice for 5 minutes with PBS-T, and the target band was detected using the enhanced chemiluminescence (ECL) method. In both cases where the 218B1 and 218C7 antibodies were used, a band with a molecular weight of 18 kDa was detected regardless of the addition of DTT.

[0355] 2-3) Mass spectrometry of immunoprecipitation products of cellular proteins using 218B1 and 218C7 antibodies 2×10 7NTERA-2 cells were collected and washed twice with PBS. The cells were collected using a cell scraper and centrifuged at 1500 rpm for 5 minutes. After removing the supernatant, the cells were resuspended in 2 ml of lysis buffer and treated at 4°C for 30 minutes. A cell lysate was prepared by centrifugation (13000 rpm, 20 minutes, 4°C). Protein G Sepharose / lysis buffer (50% slurry; 180 μl) was added to the cell lysate, rotated at 4°C for 30 minutes, and then centrifuged at 4°C for 1 minute to collect the supernatant. 218B1 antibody (approximately 9 μg) was added to this supernatant, rotated at 4°C for 30 minutes, and then Protein G Sepharose / lysis buffer (50% slurry; 180 μl) was added and rotated at 4°C for 1 hour. Protein G Sepharose was washed six times with lysis buffer (1 ml) and then resuspended in 1×SDS sample buffer. After treating the suspension at 100°C for 5 minutes, the solution was collected and used as a sample for SDS-PAGE. SDS-PAGE was performed in the same manner as in 2-2), and the electrophoretic gel was stained with CBB. A portion corresponding to 18 kDa was excised from the electrophoretic gel and subjected to mass spectrometry. As a result of mass spectrometry, the gel fragment was found to contain Claudin-6.

[0356] 2-4) FACS analysis Mass spectrometry revealed that the antigen of 218B1 and 218C7 antibodies was Claudin-6, so we performed forced expression analysis by gene transfer of cDNA. FACS analysis showed that 218B1 and 218C7 antibodies showed strong positive reaction in human Claudin-6 expressing CHO-K1 cells, indicating that the antigen of 218B1 and 218C7 antibodies is Claudin-6. 2-5) Purification of antibodies from hybridoma culture supernatant Hybridomas (218B1) producing mouse anti-CLDN6 antibody B1 and hybridomas (218C7) producing mouse anti-CLDN6 antibody C7 were cultured in Hybridoma-SFM (Thermo Fisher Scientific) containing 10% Fetal Bovine Serum, Ultra-Low IgG (Thermo Fisher Scientific). The culture supernatant was collected by centrifugation and filtered through a 0.45 μm filter (Corning). The antibody was purified from the culture supernatant in a single step of rProtein A affinity chromatography (at 4 to 6 °C). The buffer replacement step after the rProtein A affinity chromatography purification was performed at 4 to 6 °C. First, the culture supernatant was applied to a column packed with MabSelectSuRe (GE Healthcare Bioscience) equilibrated with PBS. After the culture solution was completely loaded into the column, the column was washed with PBS at a volume at least twice the column volume. Next, the antibody was eluted with 2M arginine hydrochloride solution (pH 4.0) and the fractions containing the antibody were collected. The fractions were dialyzed (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette) to replace the liquid with PBS(-). Finally, the fractions were concentrated using a Centrifugal UF Filter Device VIVASPIN20 (molecular weight cutoff UF10K, Sartorius, at 4°C) to adjust the IgG concentration to 1mg / mL or more. The purified sample was obtained by filtration using a Minisart-Plus filter (Sartorius).

[0357] Example 7: In vitro evaluation of murine anti-CLDN6 antibodies B1 and C7 7-1: Evaluation of binding ability of mouse anti-CLDN6 antibodies by flow cytometry The binding of the mouse anti-CLDN6 antibody prepared in Example 6 to human CLDN6 and its family molecules CLDN3, CLDN4, and CLDN9 was evaluated by flow cytometry. Human CLDN3 / pCMV6-Entry, human CLDN4 / pCMV6-Entry, human CLDN6 / pCMV-Entry, human CLDN9 / pCMV6-Entry, or pCMV6-Entry, all purchased from Origene, was transiently introduced into 293T cells (Thermo Fisher Scientific HCL4517) using Lipofectamine 2000 (Thermo Fisher Scientific), and the cells were incubated at 37° C., 5% CO 2After overnight culture under the above conditions, a cell suspension was prepared. The transfected 293T cell suspension was centrifuged and the supernatant was removed. Mouse anti-CLDN6 antibody (clone numbers B1 and C7) or mouse IgG1 control antibody (R&D Systems) was added at a final concentration of 30 μg / mL, 10 μg / mL, 3.3 μg / mL, or 1.1 μg / mL, and the cells were suspended and left to stand at 4°C for 1 hour. After washing twice with Dulbecco's phosphate buffered saline (Sigma-Aldrich) containing 5% fetal bovine serum (Hyclone) (hereinafter, 5% FBS-containing PBS), FLUORESCEIN-CONJUGATED GOAT IGG FRACTION TO MOUSE IGG (WHOLE MOLECULE) (MP BIOMEDICALS) diluted 500-fold with 5% FBS-containing PBS was added and suspended, and the cells were left to stand at 4°C for 1 hour. After washing twice with PBS containing 5% FBS, detection was performed using a flow cytometer (FC500; Beckman Coulter). Data analysis was performed using FlowJo (TreeStar). The transfection of each gene was confirmed using a mouse anti-FLAG antibody (Sigma-Aldrich) after the cells were permeabilized with PBS containing 0.25% Tween 20. The results are shown in Figure 40. In the graph of Figure 40, the vertical axis shows the FITC fluorescence intensity indicating the amount of antibody binding, and the horizontal axis shows the antibody concentration. The mouse anti-CLDN6 antibody prepared bound to human CLDN6 and human CLDN9 to the same extent, but did not bind to human CLDN3 or human CLDN4. Mouse control IgG1 did not bind to any of the cells.

[0358] 7-2: Antibody internalization activity The internalization activity of mouse anti-CLDN6 antibodies B1 and C7 was evaluated using an anti-mouse IgG reagent Mab-ZAP (ADVANCED TARGETING SYSTEMS) conjugated with a toxin (saporin) that inhibits protein synthesis. In this evaluation, Mab-ZAP was taken up into cells depending on the internalization activity of mouse anti-CLDN6 antibodies, and saporin, which inhibits protein synthesis, was released into the cells, suppressing cell proliferation. Human CLDN6-positive choriocarcinoma line JEG-3 (ATCC HTB-36), human CLDN6-positive ovarian cancer line NIH:OVCAR-3 (ATCC HTB-161), or human CLDN6-negative pancreatic cancer line BxPC-3 (ATCC CRL-1687) were cultured at 2x10 3 Cells were seeded at 1000 cells / well in a 96-well cell culture microplate and incubated at 37℃ in 5% CO 2 The cells were cultured overnight under the conditions of 0.5 nM. The next day, a mixed solution of mouse anti-CLDN6 antibody or mouse IgG1 antibody (R&D Systems) mixed with Mab-ZAP (final concentration: 0.5 nM) or toxin-free AffiniPure Goat Anti-Mouse IgG, Fcγ Fragment Specific (Jackson ImmunoResearch) (final concentration: 0.5 nM) was added to the cells to a final concentration of 1 nM, and the cells were cultured for 5 days under the conditions of 37°C and 5% CO2. The number of viable cells was measured by quantifying ATP activity using CellTiter-Glo Luminescent Cell Viability Assay (Promega). The cell proliferation inhibitory effect of the addition of anti-CLDN6 antibody was calculated as the relative survival rate, with the value of the well without the mixed solution set at 100%. The results are shown in Figure 41. Mouse anti-CLDN6 antibodies (B1, C7) showed a cell proliferation inhibitory effect on human CLDN6-positive cell lines JEG-3 and NIH:OVCAR-3. On the other hand, no cell growth inhibitory effect was observed against the human CLDN6-negative cell line BxPC-3.Mouse IgG1 antibody also showed no cell growth inhibitory effect against any of the cell lines.As a result, the anti-CLDN6 antibodies (B1, C7) we prepared have internalization activity and are considered to be suitable as antibodies for antibody-drug conjugates.

[0359] Example 8: Determination of the nucleotide sequences of cDNAs encoding the variable regions of mouse anti-CLDN6 antibodies B1 and C7 8-1: Determination of the nucleotide sequence of cDNA encoding the variable region of B1 antibody 8-1-1: Preparation of total RNA from B1 antibody-producing hybridoma To amplify cDNA encoding the variable regions of the B1 antibody, total RNA was prepared from the B1 antibody-producing hybridoma using TRIzol Reagent (Ambion). 8-1-2: Amplification and sequencing of cDNA encoding the light chain variable region of B1 antibody by 5'-RACE PCR Amplification of the cDNA encoding the light chain variable region was carried out using about 1 μg of the total RNA prepared in Example 8-1-1 and the SMARTer RACE 5' / 3' Kit (Clontech). As primers for amplifying the cDNA encoding the variable region of the light chain gene of the B1 antibody by PCR, UPM (Universal Primer A Mix: included in the SMARTer RACE 5' / 3' Kit) and a primer designed from the sequence of a known mouse light chain constant region were used. The cDNA encoding the light chain variable region amplified by 5'-RACE PCR was cloned into a plasmid, and then the nucleotide sequence of the cDNA encoding the light chain variable region was subjected to sequence analysis. The determined nucleotide sequence of the cDNA encoding the variable region of the light chain of the B1 antibody is shown in SEQ ID NO:18, and the amino acid sequence is shown in SEQ ID NO:19. 8-1-3: Amplification and sequence determination of cDNA encoding the heavy chain variable region of B1 antibody by 5'-RACE PCR Amplification of cDNA encoding the heavy chain variable region was carried out using about 1 μg of the total RNA prepared in Example 8-1-1 and SMARTer RACE 5' / 3' Kit (Clontech). As primers for amplifying cDNA encoding the variable region of the heavy chain gene of the LB1 antibody by PCR, UPM (Universal Primer A Mix: included in the SMARTer RACE 5' / 3' Kit) and primers designed from the sequence of the known mouse heavy chain constant region were used. The cDNA encoding the heavy chain variable region amplified by 5'-RACE PCR was cloned into a plasmid, and then the nucleotide sequence of the cDNA encoding the heavy chain variable region was subjected to sequence analysis. The determined nucleotide sequence of the cDNA encoding the variable region of the heavy chain of the B1 antibody is shown in SEQ ID NO:20, and the amino acid sequence is shown in SEQ ID NO:21.

[0360] 8-2: Determination of the nucleotide sequence of cDNA encoding the variable region of the C7 antibody The same method as in Example 8-1 was used. The nucleotide sequence of the cDNA encoding the light chain variable region of the determined C7 antibody is shown in SEQ ID NO: 22, and the amino acid sequence is shown in SEQ ID NO: 23. The nucleotide sequence of the cDNA encoding the heavy chain variable region is shown in SEQ ID NO: 24, and the amino acid sequence is shown in SEQ ID NO: 25.

[0361] Example 9: Construction of chimeric anti-CLDN6 antibody chB1 9-1: Construction of expression vector for chimeric anti-CLDN6 antibody chB1 9-1-1: Construction of chimeric and humanized light chain expression vector pCMA-LK A fragment of about 5.4 kb obtained by digesting the plasmid pcDNA3.3-TOPO / LacZ (Invitrogen) with the restriction enzymes XbaI and PmeI was ligated to a DNA fragment containing a DNA sequence encoding the human light chain signal sequence and the human κ chain constant region shown in SEQ ID NO: 26 using an In-Fusion HD PCR cloning kit (Clontech) to prepare pcDNA3.3 / LK. pCMA-LK was constructed by removing the neomycin expression unit from pcDNA3.3 / LK. 9-1-2: Construction of chimeric and humanized IgG1LALA-type heavy chain expression vector pCMA-G1LALA A DNA fragment obtained by digesting pCMA-LK with XbaI and PmeI to remove the light chain signal sequence and human κ chain constant region was ligated to a DNA fragment containing the human heavy chain signal sequence shown in SEQ ID NO: 27 and a DNA sequence encoding the human IgG1LALA constant region using an In-Fusion HD PCR cloning kit (Clontech) to construct pCMA-G1LALA. 9-1-3: Construction of chimeric chB1 heavy chain expression vector A DNA fragment represented by nucleotide numbers 36 to 440 of the nucleotide sequence of the chB1 heavy chain shown in SEQ ID NO: 33 was synthesized (GENEART). Using an In-Fusion HD PCR cloning kit (Clontech), the synthesized DNA fragment was inserted into the site of pCMA-G1LALA cleaved with the restriction enzyme BlpI to construct a chB1 heavy chain expression vector. The amino acid sequence of the chB1 heavy chain is shown in SEQ ID NO: 32. 9-1-4: Construction of chimeric chB1 light chain expression vector A DNA fragment containing the DNA sequence encoding the chB1 light chain shown in SEQ ID NO:29 was synthesized (GENEART). Using an In-Fusion HD PCR cloning kit (Clontech), the synthesized DNA fragment was ligated to a DNA fragment obtained by digesting pCMA-LK with XbaI and PmeI to remove the light chain signal sequence and human κ chain constant region, thereby constructing a chB1 light chain expression vector. The amino acid sequence of the chB1 light chain is shown in SEQ ID NO:28.

[0362] 9-2: Production and purification of chimeric anti-CLDN6 antibody chB1 9-2-1: Production of chimeric antibody chB1 FreeStyle 293F cells (Invitrogen) were subcultured and cultured according to the manual. 1.2 × 109 FreeStyle 293F cells (Invitrogen) in the logarithmic growth phase were seeded in a 3L Fernbach Erlenmeyer Flask (CORNING) and diluted with FreeStyle293 expression medium (Invitrogen) to prepare 2.0 × 106 cells / mL. 0.24 mg of heavy chain expression vector, 0.36 mg of light chain expression vector, and 1.8 mg of polyethyleneimine (Polyscience #24765) were added to 40 mL of Opti-Pro SFM medium (Invitrogen), gently mixed, and left for another 5 minutes before adding to the FreeStyle 293F cells. After 4 hours of shaking culture at 90 rpm in an 8% CO2 incubator at 37°C, 600 mL of EX-CELL VPRO medium (SAFC Biosciences), 18 mL of GlutaMAX I (GIBCO), and 30 mL of Yeastolate Ultrafiltrate (GIBCO) were added, and the cells were shaken at 90 rpm for 7 days in an 8% CO2 incubator at 37°C. The culture supernatant obtained was filtered through a Disposable Capsule Filter (Advantec #CCS-045-E1H). The obtained chimeric anti-CLDN6 antibody was named "chB1". 9-2-2: Purification of chimeric antibody chB1 The antibody was purified from the culture supernatant obtained in Example 9-2-1 by a one-step process of rProtein A affinity chromatography. The culture supernatant was applied to a column (GE Healthcare Bioscience) packed with MabSelectSuRe equilibrated with PBS, and the column was washed with PBS of at least twice the column volume. Next, the column was eluted with 2M arginine hydrochloride solution (pH 4.0) and the fraction containing the antibody was collected. The fraction was subjected to buffer replacement with PBS(-) by dialysis (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette). The antibody was concentrated using a Centrifugal UF Filter Device VIVASPIN20 (molecular weight cutoff UF10K, Sartorius) and the IgG concentration was adjusted to 1 mg / mL or more. Finally, the sample was filtered using a Minisart-Plus filter (Sartorius) to obtain a purified sample.

[0363] Example 10: Preparation of humanized anti-CLDN6 antibody 10-1: Design of humanized anti-CLDN6 antibodies 10-1-1: Molecular modeling of the variable region of chimeric antibody chB1 Molecular modeling of the variable region of chB1 was performed using a method known as homology modeling (Methods in Enzymology, 203, 121-153, (1991)). The structure (PDB ID: 1XIW) registered in the Protein Data Bank (Nuc. Acid Res. 35, D301-D303 (2007)) that has high sequence identity to the variable regions of the heavy and light chains of chB1 was used as a template, and the commercially available protein three-dimensional structure analysis program BioLuminate (Schrodinger) was used. 10-1-2: Design of humanized amino acid sequence chB1 was humanized by CDR grafting (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)). The consensus sequences of human gamma chain subgroup 1 and kappa chain subgroup 1 defined in Kabat et al. (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service National Institutes of Health, Bethesda, MD. (1991)) were selected as heavy and light chain acceptors, respectively, because they have high identity to the framework regions of chB1. The donor residues to be transferred onto the acceptor were selected by analyzing three-dimensional models with reference to criteria such as those given by Queen et al. (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)). Since CDRL3 is rich in hydrophobic amino acids, a humanized light chain was also designed in which mutations were introduced into CDRL3.

[0364] 10-2: Humanization of chB1 heavy chain The three designed heavy chains were designated hH1, hH2 and hH3. The full-length heavy chain amino acid sequence of hH1 is set forth in SEQ ID NO:52. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO:52 is set forth in SEQ ID NO:53. The full-length heavy chain amino acid sequence of hH2 is set forth in SEQ ID NO:56. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO:56 is set forth in SEQ ID NO:57. The full-length heavy chain amino acid sequence of hH3 is set forth in SEQ ID NO:60. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO:60 is set forth in SEQ ID NO:61.

[0365] 10-3: Humanization of chB1 light chain The four designed light chains were designated hL1, hL2, hL3 and hL4. The full length light chain amino acid sequence of hL1 is set forth in SEQ ID NO:36. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO:36 is set forth in SEQ ID NO:37. The full length light chain amino acid sequence of hL2 is set forth in SEQ ID NO:40. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO:40 is set forth in SEQ ID NO:41. The full length light chain amino acid sequence of hL3 is set forth in SEQ ID NO:44. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO:44 is set forth in SEQ ID NO:45. The full length light chain amino acid sequence of hL4 is set forth in SEQ ID NO:48. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO:48 is set forth in SEQ ID NO:49.

[0366] 10-4: Design of humanized antibodies by combining heavy and light chains An antibody consisting of hH1 and hL1 is referred to as "H1L1 antibody" or "H1L1". An antibody consisting of hH2 and hL2 is referred to as "H2L2 antibody" or "H2L2". An antibody consisting of hH1 and hL3 is referred to as "H1L3 antibody" or "H1L3". An antibody consisting of hH2 and hL4 is referred to as "H2L4 antibody" or "H2L4". An antibody consisting of hH3 and hL3 is referred to as "H3L3 antibody" or "H3L3".

[0367] 10-5: Preparation of humanized anti-CLDN6 antibody 10-5-1: Construction of humanized heavy chain expression vector 10-5-1-1: Construction of hH1 expression vector A DNA fragment represented by nucleotide numbers 36 to 440 of the nucleotide sequence of hH1 shown in SEQ ID NO:53 was synthesized (GENEART). An hH1 expression vector was constructed in the same manner as in Example 9-1-3. 10-5-1-2: Construction of hH2 expression vector A DNA fragment represented by nucleotide numbers 36 to 440 of the nucleotide sequence of hH2 shown in SEQ ID NO:57 was synthesized (GENEART). An hH2 expression vector was constructed in the same manner as in Example 9-1-3. 10-5-1-3: Construction of hH3 expression vector A DNA fragment represented by nucleotide numbers 36 to 440 of the nucleotide sequence of hH2 shown in SEQ ID NO:61 was synthesized (GENEART). An hH3 expression vector was constructed in the same manner as in Example 9-1-3. 10-5-2: Construction of humanized light chain expression vector 10-5-2-1: Construction of hL1 expression vector A DNA fragment represented by nucleotide numbers 37 to 402 of the nucleotide sequence of hL1 shown in SEQ ID NO: 37 was synthesized (GENEART). Using an In-Fusion HD PCR cloning kit (Clontech), the synthesized DNA fragment was inserted into the site of pCMA-LK cleaved with the restriction enzyme BsiWI to construct an hL1 expression vector. 10-5-2-2: Construction of hL2 expression vector A DNA fragment represented by nucleotide numbers 37 to 402 of the nucleotide sequence of hL2 shown in SEQ ID NO: 41 was synthesized (GENEART). An hL2 expression vector was constructed in the same manner as in Example 10-5-2-1. 10-5-2-3: Construction of hL3 expression vector A DNA fragment represented by nucleotide numbers 37 to 402 of the nucleotide sequence of hL3 shown in SEQ ID NO: 45 was synthesized (GENEART). An hL3 expression vector was constructed in the same manner as in Example 10-5-2-1. 10-5-2-4: Construction of hL4 expression vector A DNA fragment represented by nucleotide numbers 37 to 402 of the nucleotide sequence of hL4 shown in SEQ ID NO: 49 was synthesized (GENEART). An hL4 expression vector was constructed in the same manner as in Example 10-5-2-1. 10-5-3: Preparation of humanized antibodies 10-5-3-1: Production of humanized antibodies H1L1, H2L2, H1L3, H2L4, and H3L3 They were produced in the same manner as in Example 9-2-1. H1L1, H2L2, H1L3, H2L4, and H3L3 were produced by combining heavy chain expression vectors and light chain expression vectors corresponding to the combinations of heavy chains and light chains shown in Example 10-4. 10-5-3-2: Two-step purification of humanized antibodies H1L1, H2L2, H1L3, H2L4, and H3L3 The culture supernatant obtained in Example 10-5-3-1 was purified in a two-step process using rProtein A affinity chromatography and ceramic hydroxyapatite. The culture supernatant was applied to a column (GE Healthcare Bioscience) packed with MabSelectSuRe equilibrated with PBS, and the column was washed with PBS in an amount equal to or more than twice the column volume. The antibody was then eluted with a 2M arginine hydrochloride solution (pH 4.0). The antibody-containing fraction was subjected to buffer replacement with PBS by dialysis (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette), diluted 5-fold with a buffer of 5 mM sodium phosphate / 50 mM MES / pH 7.0, and then applied to a ceramic hydroxyapatite column (Nippon Bio-Rad, Bio-Scale CHT Type-1 Hydroxyapatite Column) equilibrated with a buffer of 5 mM NaPi / 50 mM MES / 30 mM NaCl / pH 7.0. A linear gradient elution with sodium chloride was performed, and the fractions containing the antibody were collected. The fractions were dialyzed (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette) to exchange the buffer with HBSor (25 mM histidine / 5% sorbitol, pH 6.0). The antibody was concentrated using a Centrifugal UF Filter Device VIVASPIN20 (molecular weight cutoff UF10K, Sartorius) to adjust the IgG concentration to 50 mg / mL. Finally, the sample was filtered using a Minisart-Plus filter (Sartorius) to obtain a purified sample.

[0368] Example 11: Evaluation of binding ability of humanized anti-CLDN6 antibodies by flow cytometry The binding of the humanized anti-CLDN6 antibody prepared in Example 10 to human CLDN6 and family molecules CLDN3, CLDN4, and CLDN9 was evaluated by flow cytometry. 293T cells were used that were transiently transfected with genes in the same manner as in Example 7-1. Humanized anti-CLDN6 antibodies H1L1, H2L2, H1L3, H2L4, and H3L3, or human IgG1 control antibody (CALBIOCHEM) were added to the cells transfected with the human CLDN6 or human CLDN9 gene at final concentrations of 100 nM, 20 nM, 4 nM, and 0.8 nM, and the cells were suspended and allowed to stand at 4°C for 30 minutes. Humanized anti-CLDN6 antibodies H1L1, H2L2, H1L3, H2L4, and H3L3 were added to the cells transfected with the human CLDN3 or human CLDN4 gene, or an empty vector, at final concentrations of 100 nM, and the cells were suspended and allowed to stand at 4°C for 30 minutes. After washing with Dulbecco's phosphate buffered saline (Sigma-Aldrich) containing 5% fetal bovine serum (Hyclone) (hereinafter referred to as PBS containing 5% FBS), FITC AffiniPure F(ab')2 Fragment Goat Anti-Human IgG(H+L) (Jackson ImmunoResearch) diluted 150-fold with PBS containing 5% FBS was added and suspended, and the mixture was allowed to stand at 4°C for 30 minutes. After washing with PBS containing 5% FBS, detection was performed using a flow cytometer (FC500; Beckman Coulter). Data analysis was performed using FlowJo (TreeStar), and the mean fluorescence intensity (MFI) of FITC, which indicates the amount of antibody binding, was calculated. The results are shown in Figure 42. In the graph of Figure 42, the horizontal axis indicates the antibody concentration, and the vertical axis indicates the MFI. The prepared humanized anti-CLDN6 antibody bound to human CLDN6 and human CLDN9 to the same extent, but did not bind to human CLDN3 or human CLDN4. Human control IgG1 did not bind to either cell.

[0369] Example 12: Generation of Trastuzumab mutants 12-1: Construction of Trastuzumab-LALA heavy chain expression vector A DNA fragment represented by nucleotide numbers 36 to 434 of the nucleotide sequence of the heavy chain of Trastuzumab-LALA shown in SEQ ID NO: 74 was synthesized (GENEART). An expression vector was constructed in the same manner as in Example 9-1-3. The amino acid sequence of the heavy chain of Trastuzumab-LALA is shown in SEQ ID NO: 75.

[0370] 12-2: Construction of Trastuzumab-LALA light chain expression vector A DNA fragment represented by nucleotide numbers 37 to 402 of the nucleotide sequence of the light chain of Trastuzumab-LALA shown in SEQ ID NO: 72 was synthesized (GENEART). An expression vector was constructed in the same manner as in Example 10-5-2-1. The amino acid sequence of the light chain of Trastuzumab-LALA is shown in SEQ ID NO: 73.

[0371] 12-3: Production of Trastuzumab mutants Produced in the same manner as in Example 9-2-1.

[0372] 12-4: Purification of Trastuzumab mutants Trastuzumab-LALA was purified from the culture supernatant obtained in Example 12-3 by the same method as in Example 9-2-2, except that the buffer was replaced with 50 mM phosphate buffer solution (pH 6.0) instead of PBS(-).

[0373] [Preparation of Glyco-Remodeling Antibodies] Example 13: Glycosylation 1 (T-SG)

[0374] [ka]

[0375] Step 1: Preparation of (Fucα1,6)GlcNAc-Trastuzumab The 22 mg / mL Trastuzumab solution (25 mM histidine solution (pH 6.0), 5% sorbitol solution) (45.5 mL) prepared in Reference Example 3 was subjected to buffer exchange into 50 mM phosphate buffer (pH 6.0) in two separate batches using common procedure C. 1.26 mL and 1.27 mL of 2.0 mg / mL wild-type EndoS solution (PBS) were added to the obtained 28.1 mg / mL Trastuzumab solution (50 mM phosphate buffer (pH 6.0) (18 mL) and 28.0 mg / mL of the same solution (18 mL), respectively, and incubated at 37°C for 4 hours. The progress of the reaction was confirmed using an Experion electrophoresis station (manufactured by BIO-RAD). After the reaction was completed, purification was performed by affinity chromatography and purification by a hydroxyapatite column according to the following method. (1) Purification by affinity chromatography Purification equipment: AKTA pure150 (GE Healthcare) Column: HiTrap rProtein A FF (5 mL) (GE Healthcare) Flow rate: 5mL / min (1.25mL / min when charging) The reaction solution obtained above was purified in multiple batches. Two columns were connected, and when binding to the column, the reaction solution was added to the top of the column, and 2 CV of binding buffer (20 mM phosphate buffer (pH 6.0)) was passed at 1.25 mL / min, and then 5 CV was passed at 5 mL / min. During intermediate washing, 15 CV of washing solution (20 mM phosphate buffer (pH 7.0), 0.5 M sodium chloride solution) was passed. During elution, 6 CV of elution buffer (ImmunoPure IgG Eution buffer, manufactured by PIERCE) was passed. The eluate was immediately neutralized with 1 M Tris buffer (pH 9.0). The fractions detected by UV (280 nm) during elution were confirmed using a microspectrophotometer Xpose (manufactured by Trinean) and an Experion electrophoresis station (manufactured by BIO-RAD). Fractions containing the target substance were subjected to buffer exchange into 5 mM phosphate buffer / 50 mM 2-morpholinoethanesulfonic acid (MES) solution (pH 6.8) using common procedure C.

[0376] (2) Purification by hydroxyapatite chromatography Purification device: AKTA avant25 (GE Healthcare) Column: Bio-Scale Mini CHT Type I Cartridge (5 mL) (BIO-RAD) Flow rate: 5mL / min (1.25mL / min when charging) Two columns were connected, and the solution obtained in (1) above was purified in several batches. The solution was added to the top of the column, and solution A (5 mM phosphate buffer, 50 mM morpholinoethanesulfonic acid (MES) solution (pH 6.8)) was run for 2 CV at 1.25 mL / min, and then run for 3 CV at 5 mL / min. Then, elution was performed using solution A and solution B (5 mM phosphate buffer, 50 mM morpholinoethanesulfonic acid (MES) solution (pH 6.8), 2 M sodium chloride solution). The elution conditions were solution A:solution B = 100:0 to 0:100 (15 CV). Furthermore, 5 CV of washing solution (500 mM phosphate buffer (pH 6.5)) was run. The fraction containing the target substance was subjected to buffer exchange using common procedure C to obtain a 25.5 mg / mL (Fucα1,6)GlcNAc-Trastuzumab solution (50 mM phosphate buffer (pH 6.0)) (35 mL).

[0377] Step 2: Trastuzumab[SG-(N 3 ) 2 ] 2 Preparation of To the 23.9 mg / mL (Fucα1,6)GlcNAc-Trastuzumab solution (50 mM phosphate buffer (pH 6.0)) (3.37 mL) obtained in the above step 1, a 50 mM phosphate buffer (pH 6.0) solution (0.258 mL) of the compound (12.9 mg) synthesized in Example 5 step 2 and a 4.90 mg / mL EndoS D233Q / Q303L solution (PBS) (0.328 mL) were added and incubated at 30° C. for 4.5 hours. The above operation was performed in two lots. The progress of the reaction was confirmed using an Experion electrophoresis station (manufactured by BIO-RAD). After the reaction was completed, the mixture was purified by affinity chromatography and hydroxyapatite chromatography in the same manner as in step 1 above, and the fraction containing the target substance was subjected to buffer exchange into phosphate buffered saline (pH 6.0) using common procedure C to obtain 10.0 mg / mL Trastuzumab [SG-(N 3 ) 2 ] 2 A solution (phosphate buffered saline (pH 6.0)) (15.5 mL) was obtained.

[0378] Example 14: Glycosylation 2 (T-MSG) [ka]

[0379] Step 1: Trastuzumab [MSG-N 3 ] 2 The following procedure was carried out for five lots. The compound obtained in step 1 of Example 13 (20 mg / mL, 15.0 mL) was used, and the compound obtained in step 4 of Example 4 (25.5 mg) was used as a glycan donor. The compound was incubated at 30°C for 3 hours, and the same procedure as in step 2 of Example 13 was carried out. A total of 14.4 mg / mL Trastuzumab [MSG-N 3 ] 2 A solution (phosphate buffered saline (pH 6.0)) (93.5 mL) was obtained.

[0380] Example 15: Glycosylation 3 (T-MSG1) [ka]

[0381] Step 1: Trastuzumab[MSG1-N 3 ] 2 The following procedure was carried out for two lots. The compound obtained in step 1 of Example 13 (25.5 mL, 7.8 mL) was used, and the compound obtained in step 4 of Example 3 (25.5 mg) was used as a glycan donor. The mixture was incubated at 30°C for 3 hours, and the same procedure as in step 2 of Example 13 was carried out. The two lots were combined to produce 10.6 mg / mL Trastuzumab [MSG1-N 3 ] 2 A solution (phosphate buffered saline (pH 6.0)) (31 mL) was obtained.

[0382] Example 16: Glycosylation 4 (CLDN6-MSG1(H1L1)) [ka]

[0383] Step 1: (Fucα1,6)GlcNAc-anti-CLDN6 antibody (H1L1) The same procedure as in step 1 of Example 13 was carried out using the anti-CLDN6 antibody (H1L1) solution (ca. 37.7 mg / mL) (25 mM histidine solution (pH 6.0), 5% sorbitol solution) (2.5 mL) prepared in Example 10 to obtain a (Fucα1,6)GlcNAc-anti-CLDN6 antibody (H1L1) solution (50 mM phosphate buffer (pH 6.0)) (4.8 mL) of 19.2 mg / mL.

[0384] Step 2: Anti-CLDN6 antibody (H1L1)-[MSG1-N 3 ] 2 Using the 19.2 mg / mL (Fucα1,6)GlcNAc-anti-CLDN6(H1L1) antibody solution (50 mM phosphate buffer (pH 6.0)) (4.8 mL) obtained in step 1 above, a 10.2 mg / mL anti-CLDN6 antibody (H1L1)-[MSG1-N3 ] 2 A solution (phosphate buffered saline (pH 6.0)) (7.2 mL) was obtained.

[0385] Example 17: Glycosylation 5 (CLDN6-MSG1(H2L2)) Step 1: (Fucα1,6)GlcNAc-anti-CLDN6 antibody (H2L2) The same procedure as in step 1 of Example 13 was carried out using the anti-CLDN6 antibody (H2L2) solution of ca. 20 mg / mL (25 mM histidine solution (pH 6.0), 5% sorbitol solution) (6 mL) prepared in Example 10 to obtain a (Fucα1,6)GlcNAc-anti-CLDN6 antibody (H2L2) solution (50 mM phosphate buffer (pH 6.0)) (5.7 mL) of 21.84 mg / mL.

[0386] Step 2: Anti-CLDN6 antibody (H2L2)-[MSG1-N 3 ] 2 Using the 21.8 mg / mL (Fucα1,6)GlcNAc-anti-CLDN6(H2L2) antibody solution (50 mM phosphate buffer (pH 6.0)) (5.7 mL) obtained in step 1 above, a 10.2 mg / mL anti-CLDN6 antibody (H2L2)-[MSG1-N 3 ] 2 A solution (phosphate buffered saline (pH 6.0)) (11.1 mL) was obtained.

[0387] Example 18: Glycosylation 6 (CLDN6-MSG1(H1L3)) Step 1: (Fucα1,6)GlcNAc-anti-CLDN6 antibody (H1L3) The same procedure as in step 1 of Example 13 was carried out using the anti-CLDN6 antibody (H1L3) solution (ca. 39.4 mg / mL) (25 mM histidine solution (pH 6.0), 5% sorbitol solution) (3 mL) prepared in Example 10 to obtain a 39.2 mg / mL (Fucα1,6)GlcNAc-anti-CLDN6 antibody (H1L3) solution (50 mM phosphate buffer (pH 6.0)) (4.5 mL).

[0388] Step 2: Anti-CLDN6 antibody (H1L3)-[MSG1-N 3 ] 2 Using the 39.2 mg / mL (Fucα1,6)GlcNAc-anti-CLDN6(H1L3) antibody solution (50 mM phosphate buffer (pH 6.0)) (4.5 mL) obtained in step 1 above, a procedure similar to that in step 1 of Example 15 was carried out to obtain 9.83 mg / mL anti-CLDN6 antibody (H1L3)-[MSG1-N 3 ] 2 A solution (phosphate buffered saline (pH 6.0)) (7.2 mL) was obtained.

[0389] Example 19: Glycosylation 7 (CD98-MSG1) Step 1: (Fucα1,6)GlcNAc-anti-CD98 antibody The same procedure as in step 1 of Example 13 was carried out using 6 mL of the anti-CD98 antibody solution (ca. 20 mg / mL) (25 mM histidine solution (pH 6.0), 5% sorbitol solution) prepared in Reference Example 6 to give 4.7 mL of a 21.7 mg / mL (Fucα1,6)GlcNAc-anti-CD98 antibody solution (50 mM phosphate buffer (pH 6.0)).

[0390] Step 2: Anti-CD98 antibody-[MSG1-N 3 ] 2 Using the 21.7 mg / mL (Fucα1,6)GlcNAc-anti-CD98 antibody solution (50 mM phosphate buffer (pH 6.0)) (4.7 mL) obtained in step 1 above, a 10.1 mg / mL solution of anti-CD98 antibody-[MSG1-N 3 ] 2 A solution (phosphate buffered saline (pH 6.0)) (7.6 mL) was obtained.

[0391] Example 20: Glycosylation 8 (TROP2-MSG1) Step 1: (Fucα1,6)GlcNAc-anti-Trop2 antibody The same procedure as in step 1 of Example 13 was carried out using the anti-Trop2 antibody solution (ca. 20 mg / mL) (25 mM histidine solution (pH 6.0), 5% sorbitol solution) (6 mL) prepared in Reference Example 5 to obtain a 21.69 mg / mL (Fucα1,6)GlcNAc-anti-Trop2 antibody solution (50 mM phosphate buffer (pH 6.0)) (3.3 mL).

[0392] Step 2: Anti-Trop2 antibody-[MSG1-N 3 ] 2 Using the 21.69 mg / mL (Fucα1,6)GlcNAc-anti-Trop2 antibody solution (50 mM phosphate buffer (pH 6.0)) (3.35 mL) obtained in step 1 above, a 10.3 mg / mL solution of anti-Trop2 antibody-[MSG1-N 3 ] 2 A solution (phosphate buffered saline (pH 6.0)) (6.4 mL) was obtained.

[0393] Example 21: Glycosylation 9 (LPS-MSG1) Step 1: (Fucα1,6)GlcNAc-anti-LPS antibody The same procedure as in step 1 of Example 13 was carried out using the anti-LPS antibody solution (ca. 17 mg / mL) (25 mM histidine solution (pH 6.0), 5% sorbitol solution) (6.6 mL) prepared in Reference Example 4 to obtain a 21.03 mg / mL (Fucα1,6)GlcNAc-anti-LPS antibody solution (50 mM phosphate buffer (pH 6.0)) (5.4 mL).

[0394] Step 2: Anti-LPS antibody-[MSG1-N 3 ] 2 Using the 21.03 mg / mL (Fucα1,6)GlcNAc-anti-LPS antibody solution (50 mM phosphate buffer (pH 6.0)) (5.4 mL) obtained in step 1 above, a 9.89 mg / mL solution of anti-LPS antibody-[MSG1-N 3 ] 2 A solution (phosphate buffered saline (pH 6.0)) (7.9 mL) was obtained.

[0395] Example 22: Glycosylation 10 (Trastuzumab variant-MSG1) Step 1: (Fucα1,6)GlcNAc-Trastuzumab variants The same procedure as in step 1 of Example 13 was carried out using the Trastuzumab variant solution (ca. 22.3 mg / ml, 50 mM phosphate buffer (pH 6.0)) (2.7 ml) prepared in Example 12 to obtain a 6.1 mg / ml (Fucα1,6)GlcNAc-Trastuzumab variant solution (50 mM phosphate buffer (pH 6.0)) (6.1 ml).

[0396] Step 2: Trastuzumab variant - [MSG1-N 3 ] 2 Using the 6.1 mg / ml (Fucα1,6)GlcNAc-Trastuzumab mutant solution (50 mM phosphate buffer (pH 6.0)) (6.1 ml) obtained in step 1 above, a 10.2 mg / ml Trastuzumab mutant-[MSG1-N 3 ] 2 A solution (phosphate buffered saline (pH 6.0)) (3.7 ml) was obtained.

[0397] Synthesis of ADCs ADC1, 2, and 5 described in Examples 23, 24, and 52 were synthesized by conjugating the antibody obtained in Step 1 of Example 14 with a drug linker as shown in the following reaction scheme, where R varies depending on the drug linker used in each Example.

[0398] [ka]

[0399] Example 23: ADC1 [ka]

[0400] (The compound obtained in Example 23, step 1, has a geometric isomer of the triazole ring as shown in the above formula, and holds a mixture of drug linkers consisting of the two types of structures shown as R above.)

[0401] Step 1: Conjugation of antibody and drug linker To a solution of the antibody obtained in step 1 of Example 14 in phosphate buffered saline (pH 6.0) (10.0 mg / mL, 1.00 mL) was added 1,2-propanediol (0.917 mL) and a dimethyl sulfoxide solution containing 10 mM of compound (6-13) obtained in step 12 of Example 2-4 (0.0825 mL; 12 equivalents per antibody molecule) at room temperature, and the mixture was allowed to react at room temperature for 2 days using a tube rotator (MTR-103, AS ONE Corporation). Purification procedure: The above solution was purified using common procedure D to obtain 6.0 mL of a solution containing the target compound. Characterization: The following characteristic values ​​were obtained using common procedures E and F. Antibody concentration: 1.33 mg / mL, antibody yield: 7.97 mg (80%), average number of drugs bound per antibody molecule (n): 1.9

[0402] Example 24: ADC2 [ka]

[0403] (The compound obtained in Example 24, step 1, has a geometric isomer of the triazole ring as shown in the above formula, and holds a mixture of drug linkers consisting of the two types of structures shown as R above.)

[0404] Step 1: Conjugation of antibody and drug linker To a solution of the antibody obtained in step 1 of Example 14 in phosphate buffered saline (pH 6.0) (9.88 mg / mL, 0.500 mL) was added 1,2-propanediol (0.459 mL) and a dimethyl sulfoxide solution containing 10 mM of compound (5-11) obtained in step 10 of Example 2-3 (0.0408 mL; 12 equivalents per antibody molecule) at room temperature, and the mixture was allowed to react at room temperature for 1 day using a tube rotator (MTR-103, AS ONE Corporation). Purification procedure: The above solution was purified using common procedure D to obtain 6.00 mL of a solution containing the target compound. This solution was concentrated using common procedure A to obtain 0.420 mL of a solution containing the target compound. Characterization: The following characteristic values ​​were obtained using common procedures E and F. Antibody concentration: 7.27 mg / mL, antibody yield: 3.54 mg (72%), average number of drugs bound per antibody molecule (n): 1.8

[0405] ADC3 and 4 described in Examples 25 and 26 were synthesized by conjugating the antibody obtained in Step 2 of Example 13 with a drug linker as shown in the following reaction scheme, where R varies depending on the drug linker used in each Example.

[0406] [ka]

[0407] Example 25: ADC3 [ka]

[0408] (The compound obtained in Example 25, step 1, has a geometric isomer of the triazole ring as shown in the above formula, and holds a mixture of drug linkers consisting of the two types of structures shown as R above.)

[0409] Step 1: Conjugation of antibody and drug linker To a solution of the antibody obtained in step 2 of Example 13 in phosphate buffered saline (pH 6.0) (10.0 mg / mL, 1.00 mL) was added 1,2-propanediol (0.835 mL) and a dimethyl sulfoxide solution containing 10 mM of compound (4-12) obtained in step 12 of Example 2-2 (0.165 mL; 24 equivalents per antibody molecule) at room temperature, and the mixture was allowed to react at room temperature for 48 hours using a tube rotator (MTR-103, AS ONE Corporation). Purification procedure: The above solution was purified using common procedure D to obtain 4.50 mL of a solution containing the target compound. Characterization: The following characteristic values ​​were obtained using common procedures E and F. Antibody concentration: 1.66 mg / mL, antibody yield: 7.48 mg (75%), average number of drugs bound per antibody molecule (n): 3.8

[0410] Example 26: ADC4 [ka]

[0411] (The compound obtained in Example 26, step 1, has a geometric isomer of the triazole ring as shown in the above formula, and holds a mixture of drug linkers consisting of the two types of structures shown as R above.)

[0412] Step 1: Conjugation of antibody and drug linker To a solution of the antibody obtained in step 2 of Example 13 in phosphate buffered saline (pH 6.0) (10.0 mg / mL, 1.00 mL) was added 1,2-propanediol (0.835 mL) and a dimethyl sulfoxide solution containing 10 mM of compound (6-13) obtained in step 12 of Example 2-4 (0.165 mL; 24 equivalents per antibody molecule) at room temperature, and the mixture was allowed to react at room temperature for 2 days using a tube rotator (MTR-103, AS ONE Corporation). Purification procedure: The above solution was purified using common procedure D to obtain 10.5 mL of a solution containing the target compound. Characteristic evaluation: The following characteristic values ​​were obtained using common procedures E and F. Antibody concentration: 0.73 mg / mL, antibody yield: 7.70 mg (77%), average number of drugs bound per antibody molecule (n): 3.8

[0413] ADCs 5 to 7 and 16 to 19 described in Examples 27, 28, 29, and 53 to 56 were synthesized by conjugating the antibody obtained in Step 1 of Example 15 with a drug linker as shown in the following reaction scheme, in which the R group varies depending on the drug linker used in each Example.

[0414] [ka]

[0415] Example 27: ADC5 [ka]

[0416] (The compound obtained in Example 27, step 1, has a geometric isomer of the triazole ring as shown in the above formula, and holds a mixture of drug linkers consisting of the two types of structures shown as R above.) Step 1: Conjugation of antibody and drug linker To a solution of the antibody obtained in step 1 of Example 15 in phosphate buffered saline (pH 6.0) (10.0 mg / mL, 1.00 mL) was added 1,2-propanediol (0.917 mL) and a dimethyl sulfoxide solution containing 10 mM of compound (6-13) obtained in step 12 of Example 2-4 (0.0825 mL; 12 equivalents per antibody molecule) at room temperature, and the mixture was allowed to react at room temperature for 2 days using a tube rotator (MTR-103, AS ONE Corporation). Purification procedure: The above solution was purified using common procedure D to obtain 6.0 mL of a solution containing the target compound. Characterization: The following characteristic values ​​were obtained using common procedures E and F. Antibody concentration: 1.46 mg / mL, antibody yield: 8.76 mg (88%), average number of drugs bound per antibody molecule (n): 1.9

[0417] Example 28: ADC6 [ka] (The compound obtained in Example 28, step 1, has a geometric isomer of the triazole ring as shown in the above formula, and holds a mixture of drug linkers consisting of the two types of structures shown as R above.)

[0418] Step 1: Conjugation of antibody and drug linker To a solution of the antibody obtained in step 1 of Example 15 in phosphate buffered saline (pH 6.0) (10.0 mg / mL, 5.00 mL) was added 1,2-propanediol (4.59 mL) and a dimethyl sulfoxide solution containing 10 mM of compound (4-12) obtained in step 12 of Example 2-2 (0.413 mL; 12 equivalents per antibody molecule) at room temperature, and the mixture was allowed to react at room temperature for 48 hours using a tube rotator (MTR-103, AS ONE Corporation). Purification procedure: The above solution was purified using common procedure D to obtain 30.0 mL of a solution containing the target compound. Characterization: The following characteristic values ​​were obtained using common procedures E and F. Antibody concentration: 1.30 mg / mL, antibody yield: 38.9 mg (78%), average number of drugs bound per antibody molecule (n): 1.9

[0419] Example 29: ADC7 [ka]

[0420] (The compound obtained in Example 29, step 1, has a geometric isomer of the triazole ring as shown in the above formula, and holds a mixture of drug linkers consisting of the two types of structures shown as R above.) Step 1: Conjugation of antibody and drug linker To a solution of the antibody obtained in step 1 of Example 15 in phosphate buffered saline (pH 6.0) (10.2 mg / mL, 1.00 mL) was added 1,2-propanediol (0.917 mL) and a dimethyl sulfoxide solution containing 10 mM of compound (3-14) obtained in step 13 of Example 2-1 (0.0825 mL; 12 equivalents per antibody molecule) at room temperature, and the mixture was allowed to react at room temperature for 2 days using a tube rotator (MTR-103, AS ONE Corporation). Purification procedure: The above solution was purified using common procedure D to obtain 6.00 mL of a solution containing the target compound. Characterization: The following characteristic values ​​were obtained using common procedures E and F. Antibody concentration: 1.41 mg / mL, antibody yield: 8.45 mg (85%), average number of drugs bound per antibody molecule (n): 1.9

[0421] Example 30: ADC8 [ka] [ka]

[0422] (The compound obtained in Example 30, step 1, has a geometric isomer of the triazole ring as shown in the above formula, and holds a mixture of drug linkers consisting of the two types of structures shown as R above.)

[0423] Step 1: Conjugation of antibody and drug linker To a solution of the antibody obtained in step 2 of Example 16 in phosphate buffered saline (pH 6.0) (10.2 mg / mL, 2.50 mL) was added 1,2-propanediol (2.29 mL) and a dimethyl sulfoxide solution containing 10 mM of compound (3-14) obtained in step 13 of Example 2-1 (0.206 mL; 12 equivalents per antibody molecule) at room temperature, and the mixture was allowed to react at room temperature for 48 hours using a tube rotator (MTR-103, AS ONE Corporation). Purification procedure: The above solution was purified using common procedure D to obtain 14.5 mL of a solution containing the target compound. Characterization: The following characteristic values ​​were obtained using common procedures E and F. Antibody concentration: 1.54 mg / mL, antibody yield: 22.3 mg (89%), average number of drugs bound per antibody molecule (n): 1.9

[0424] Example 31: ADC9 [ka]

[0425] [ka]

[0426] (The compound obtained in Example 31, step 1, has a geometric isomer of the triazole ring as shown in the above formula, and holds a mixture of drug linkers consisting of the two types of structures shown as R above.)

[0427] Step 1: Conjugation of antibody and drug linker To a solution of the antibody obtained in step 2 of Example 17 in phosphate buffered saline (pH 6.0) (9.96 mg / mL, 2.50 mL) was added 1,2-propanediol (2.29 mL) and a dimethyl sulfoxide solution containing 10 mM of compound (3-14) obtained in step 13 of Example 2-1 (0.206 mL; 12 equivalents per antibody molecule) at room temperature, and the mixture was allowed to react at room temperature for 48 hours using a tube rotator (MTR-103, AS ONE Corporation). Purification procedure: The above solution was purified using common procedure D to obtain 14.5 mL of a solution containing the target compound. Characterization: The following characteristic values ​​were obtained using common procedures E and F. Antibody concentration: 1.52 mg / mL, antibody yield: 22.0 mg (88%), average number of drugs bound per antibody molecule (n): 1.9

[0428] Example 32: ADC10 [ka]

[0429] [ka] (The compound obtained in Step 1 of Example 32 has a geometric isomer of the triazole ring as shown in the above formula, and holds a mixture of drug linkers consisting of the two types of structures shown as R above.)

[0430] Step 1: Conjugation of antibody and drug linker To a solution of the antibody obtained in step 2 of Example 18 in phosphate buffered saline (pH 6.0) (9.83 mg / mL, 2.50 mL) was added 1,2-propanediol (2.29 mL) and a dimethyl sulfoxide solution containing 10 mM of compound (3-14) obtained in step 13 of Example 2-1 (0.206 mL; 12 equivalents per antibody molecule) at room temperature, and the mixture was allowed to react at room temperature for 48 hours using a tube rotator (MTR-103, AS ONE Corporation). Purification procedure: The above solution was purified using common procedure D to obtain 14.5 mL of a solution containing the target compound. Characterization: The following characteristic values ​​were obtained using common procedures E and F. Antibody concentration: 1.45 mg / mL, antibody yield: 21.0 mg (84%), average number of drugs bound per antibody molecule (n): 1.9

[0431] Example 33: ADC11 [ka]

[0432] [ka]

[0433] (The compound obtained in Example 33, step 1, has a geometric isomer of the triazole ring as shown in the above formula, and holds a mixture of drug linkers consisting of the two types of structures shown as R above.)

[0434] Step 1: Conjugation of antibody and drug linker To a solution (10 mg / mL, 1.00 mL) of the antibody obtained in step 2 of Example 20 in phosphate buffered saline (pH 6.0), 1,2-propanediol (0.917 mL) and a dimethyl sulfoxide solution (0.0825 mL; 12 equivalents per antibody molecule) containing 10 mM of compound (3-14) obtained in step 13 of Example 2-1 were added at room temperature, and the mixture was allowed to react at room temperature for 2 days using a tube rotator (MTR-103, AS ONE Corporation). Purification procedure: The above solution was purified using common procedure D to obtain 6.00 mL of a solution containing the target compound. Characterization: The following characteristic values ​​were obtained using common procedures E and F. Antibody concentration: 1.47mg / mL, antibody yield: 8.8mg (88%), average number of drugs bound per antibody molecule (n): 1.9

[0435] Example 34: ADC12 [ka]

[0436] [ka]

[0437] (The compound obtained in Example 34, step 1, has a geometric isomer of the triazole ring as shown in the above formula, and holds a mixture of drug linkers consisting of the two types of structures shown as R above.)

[0438] Step 1: Conjugation of antibody and drug linker To a solution of the antibody obtained in step 2 of Example 19 in phosphate buffered saline (pH 6.0) (10.2 mg / mL, 1.00 mL) was added 1,2-propanediol (0.917 mL) and a dimethyl sulfoxide solution containing 10 mM of compound (3-14) obtained in step 13 of Example 2-1 (0.0825 mL; 12 equivalents per antibody molecule) at room temperature, and the mixture was allowed to react at room temperature for 2 days using a tube rotator (MTR-103, AS ONE Corporation). Purification procedure: The above solution was purified using common procedure D to obtain 6.00 mL of a solution containing the target compound. Characterization: The following characteristic values ​​were obtained using common procedures E and F. Antibody concentration: 1.36 mg / mL, antibody yield: 8.19 mg (82%), average number of drugs bound per antibody molecule (n): 1.8

[0439] Example 35: ADC13 [ka]

[0440] [ka]

[0441] (The compound obtained in Example 35, step 1, has a geometric isomer of the triazole ring as shown in the above formula, and holds a mixture of drug linkers consisting of the two types of structures shown as R above.)

[0442] Step 1: Conjugation of antibody and drug linker To a solution of the antibody obtained in step 2 of Example 21 in phosphate buffered saline (pH 6.0) (9.89 mg / mL, 0.40 mL) was added 1,2-propanediol (0.367 mL) and a dimethyl sulfoxide solution containing 10 mM of compound (3-14) obtained in step 13 of Example 2-1 (0.0328 mL; 12 equivalents per antibody molecule) at room temperature, and the mixture was allowed to react at room temperature for 2 days using a tube rotator (MTR-103, AS ONE Corporation). Purification procedure: The above solution was purified using common procedure D to obtain 2.50 mL of a solution containing the target compound. Characterization: The following characteristic values ​​were obtained using common procedures E and F. Antibody concentration: 1.16 mg / mL, antibody yield: 2.89 mg (72%), average number of drugs bound per antibody molecule (n): 1.8

[0443] Example 36: ADC14 [ka]

[0444] (The compound obtained in Example 36, step 1, has a geometric isomer of the triazole ring as shown in the above formula, and holds a mixture of drug linkers consisting of the two types of structures shown as R above.) Step 1: Conjugation of antibody and drug linker To a solution of the antibody obtained in step 2 of Example 22 in phosphate buffered saline (pH 6.0) (10.2 mg / mL, 0.40 mL) was added a solution of phosphate buffered saline (pH 6.0) (0.40 mL), 1,2-propanediol (0.767 mL), dimethylformamide (0.20 mL), and a dimethylformamide solution containing 10 mM of compound (3-14) obtained in step 13 of Example 2-1 (0.033 mL; 12 equivalents per antibody molecule) at room temperature, and the mixture was allowed to react at room temperature for 48 hours using a tube rotator (MTR-103, AS ONE Corporation). Purification procedure: The above solution was purified using common procedure D to obtain 7.00 mL of a solution containing the target compound. Characterization: The following characteristic values ​​were obtained using common procedures E and F. Antibody concentration: 0.39 mg / mL, antibody yield: 1.38 mg (35%), average number of drugs bound per antibody molecule (n): 1.8

[0445] Example 37: Cell proliferation inhibition evaluation of antibody-drug conjugates (1) NCI-N87 (American Type Culture Collection; ATCC CRL-5822), a human gastric cancer line positive for HER2 antigen, was cultured in RPMI1640 Medium (Thermo Fisher Scientific; hereafter referred to as RPMI medium) containing 10% fetal bovine serum (Hyclone). MDA-MB-468 (ATCC HTB-132), a HER2 antigen-negative cell line, was cultured in Leibovitz's L-15 Medium (Thermo Fisher Scientific; hereafter referred to as Leibovitz's medium) containing 10% fetal bovine serum (Hyclone). NCI-N87 and MDA-MB-468 cells were cultured in RPMI medium and Leibovitz's medium, respectively, at a concentration of 2.5 × 10 4 The solution was adjusted to 1000 cells / mL and added at 80 μL per well to a 96-well cell culture microplate. After adding the cells, NCI-N87 was incubated at 37°C, 5% CO 2 MDA-MB-468 was incubated overnight at 37 °C and CO 2 The cells were cultured overnight without concentration setting. The next day, 20 μL of anti-HER2 antibody-drug conjugates diluted with RPMI medium or Leibovitz's medium to 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, or 0.001 nM were added to the microplate. 20 μL of RPMI medium or Leibovitz's medium was added to wells to which the antibody-drug conjugates were not added. NCI-N87 was incubated at 37°C and 5% CO 2 Incubate MDA-MB-468 for 6 days at 37 °C and CO 2 The cells were cultured for 6 days without concentration setting. After culture, the microplate was removed from the incubator and left at room temperature for 30 minutes. An equal volume of CellTiter-Glo Luminescent Cell Viability Assay (Promega) was added to the culture medium and mixed with a plate mixer. After leaving the plate at room temperature for 10 minutes, the luminescence was measured with a plate reader (PerkinElmer). The viability of cells was calculated using the following formula. Viable cell rate (%)=a÷b×100 a: Average luminescence intensity of wells to which test substance was added b: Average luminescence intensity in wells containing medium I C 50 The value was calculated using the following formula. I C 50 (nM) = antilog((50-d) × (LOG 10 (b)-LOG 10 (a))÷(dc)+LOG 10 (b)) a: concentration of test substance a b: test substance concentration b c: Cell viability when test substance at concentration a was added d: Viable cell rate when test substance at concentration b was added a and b are the two points sandwiching a cell viability rate of 50%, where a>b. Antibody-drug conjugates ADC5, ADC4, and ADC16 showed IC 50 Antibody-drug conjugates ADC2, ADC15, ADC3, ADC6, ADC17, and ADC18 showed an IC of 0.001 or less. 50 The anti-cellular effect of the antibody-drug conjugates was <0.1 (nM). In addition, none of the antibody-drug conjugates showed any anti-cellular effect against MDA-MB-468 cells (IC 50 >0.1(nM).

[0446] Example 38: Cell proliferation inhibition evaluation of antibody-drug conjugates (2) NCI-N87 (ATCC CRL-5822), a human gastric cancer line positive for HER2 antigen, was cultured in RPMI1640 Medium (Thermo Fisher Scientific; hereafter referred to as RPMI medium) containing 10% fetal bovine serum (Hyclone). JIMT-1 (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ ACC 589), a human breast cancer line positive for HER2 antigen, was cultured in Dulbecco's Modified Eagle Medium (Thermo Fisher Scientific; hereafter referred to as DMEM medium) containing 10% fetal bovine serum (Hyclone). NCI-N87 was cultured at 5.0 × 104 JIMT-1 cells were prepared to a concentration of 1.3×10 cells / mL in DMEM medium and 80 μL of the cell suspension was added to each well of a 96-well microplate for cell culture. The plate was then incubated overnight at 37°C in a 5% CO 4 2 incubator. 2 The next day, anti-HER2 antibody-drug conjugate ADC7 or anti-LPS antibody-drug conjugate ADC13 diluted to 400 nM, 80 nM, 16 nM, 3.2 nM, 0.64 nM, 0.13 nM, 0.026 nM, 0.0051 nM, 0.0010 nM in RPMI medium or DMEM medium was added to the microplate at 20 μL per well. RPMI medium or DMEM medium was added to the wells without antibody-drug conjugate at 20 μL per well. The plate was incubated at 37°C in a 5% CO 2 incubator for 6 days. After incubation, the microplate was removed from the incubator and left to stand at room temperature for 30 minutes. An equal volume of CellTiter-Glo Luminescent Cell Viability Assay (Promega) was added to the culture medium and the plate was stirred using a plate mixer. After standing at room temperature for 10 minutes, the luminescence was measured using a plate reader (PerkinElmer). The viable cell rate was calculated using the same formula as in Example 37. 2 For both NCI-N87 and JIMT-1 cells, anti-HER2 antibody-drug conjugate ADC7 showed an anti-cell effect with 0.001 < IC50 < 0.1 (nM). On the other hand, anti-LPS antibody-drug conjugate ADC13 did not show an anti-cell effect against any of the cells (IC > 0.1 (nM)). 50

[0447] Example 39: Evaluation of Cell Proliferation Inhibition by Antibody-Drug Conjugates (3) The human ovarian cancer cell line OV-90 (ATCC CRL-11732), which is positive for the CLDN6 antigen, was cultured in a 1:1 mixed medium of Medium199 (Thermo Fisher Scientific) and MCDB 105 Medium (Sigma-Aldrich) containing 15% fetal bovine serum (Hyclone) (hereinafter referred to as the medium). OV-90 cells were adjusted to a concentration of 1.9×10 cells / mL in the medium 4 ​Prepare a solution containing 1000x1000 cells / mL, add 80 μL to a 96-well cell culture microplate, and incubate at 37°C in 5% CO. 2 The mixture was incubated overnight under The next day, 20 μL of anti-CLDN6 antibody-drug conjugates ADC8, ADC9, and ADC10 diluted with medium to 50 nM, 10 nM, 2.0 nM, 400 pM, 80 pM, 16 pM, 3.2 pM, 0.64 pM, and 0.13 pM were added to the microplate. 20 μL of medium was added to wells to which the antibody-drug conjugate was not added. Incubate at 37°C, 5% CO 2 The cells were cultured for 6 days under low-temperature conditions. After the culture, the microplate was removed from the incubator and left to stand at room temperature for 30 minutes. An equal amount of CellTiter-Glo Luminescent Cell Viability Assay (Promega) was added to the culture medium and stirred with a plate mixer. After leaving the medium to stand at room temperature for 10 minutes, the amount of luminescence was measured with a plate reader (PerkinElmer). The viability of the cells was calculated using the same formula as in Example 37. Anti-CLDN6 antibody-drug conjugates ADC8, ADC9, and ADC10 were 0.001 in OV-90 cells. <IC 50 It showed an anti-cellular effect of <0.1 (nM).

[0448] Example 40: Antitumor test of antibody-drug conjugate (1) Mice: 4-5 week-old female BALB / c nude mice (Charles River Japan) were acclimated for 4-7 days under SPF conditions before use in the experiment. Mice were fed sterilized solid food (FR-2, Funabashi Farms Co., Ltd.) and provided with sterilized tap water (prepared with 5-15 ppm sodium hypochlorite solution). Measurements and calculations: In all studies, the major and minor diameters of tumors were measured 2-3 times a week using an electronic digital caliper (CD-15CX, Mitutoyo Corp.), and tumor volumes (mm 3 ) was calculated using the following formula: Tumor volume (mm 3 ) = major axis (mm) x minor axis (mm) 2 × 1 / 2 The antibody-drug conjugates and antibodies were all diluted with 10 mM acetate buffer, 5% sorbitol, pH 5.5 (Nacalai Tesque, Inc.; ABS buffer) and administered at 10 mL / kg into the tail vein. ABS buffer was administered in the same manner to the control group (vehicle group). NCI-N87 cells (ATCC CRL-5822) were suspended in physiological saline (Otsuka Pharmaceutical Factory, Inc.) at 1 × 10 7 The cells were subcutaneously transplanted into the right flank of female nude mice (Day 0), and randomly divided into groups on Day 7. Anti-HER2 antibody-drug conjugate ADC2 was administered into the tail vein at a dose of 0.3 mg / kg on Day 7, and ADC1 was administered at a dose of 1 mg / kg on Day 7. ABS buffer was administered in the same manner as the control group (vehicle group). The results are shown in Figure 4. The anti-HER2 antibody-drug conjugates ADC2 and ADC1 demonstrated strong antitumor effects accompanied by tumor regression. No weight loss was observed in mice administered either anti-HER2 antibody-drug conjugate. In the following evaluation examples regarding antitumor tests, unless otherwise specified, the tests were performed using the methods used in these evaluation examples.

[0449] Example 41: Antitumor test of antibody-drug conjugate (2) NCI-N87 cells (ATCC CRL-5822) were suspended in Dulbecco's phosphate buffered saline (Sigma-Aldrich) at 1 × 10 7 The cells were subcutaneously transplanted into the right flank of female nude mice (Day 0), and randomly divided into groups on Day 4. Anti-HER2 antibody-drug conjugate ADC7, anti-HER2 antibody Trastuzumab (Reference Example 3), or anti-LPS antibody-drug conjugate ADC13 were all administered into the tail vein at a dose of 0.33 mg / kg on Day 4. ABS buffer was administered in the same manner as a control group (vehicle group). The results are shown in Figure 5. The anti-HER2 antibody-drug conjugate ADC7 showed a strong antitumor effect accompanied by tumor regression. On the other hand, the anti-HER2 antibody Trastuzumab and the anti-LPS antibody-drug conjugate ADC13 did not suppress tumor growth. Furthermore, no weight loss was observed in mice administered the antibody-drug conjugates ADC7, ADC13, or the anti-HER2 antibody.

[0450] Example 42: Antitumor test of antibody-drug conjugate (3) KPL-4 cells (Kawasaki Medical School, Junichi Kurebayashi, British Journal of Cancer, (1999) 79(5 / 6). 707-717) were suspended in Dulbecco's phosphate buffered saline (Sigma-Aldrich) at 1.5 × 10 7 The cells were subcutaneously transplanted into the right flank of female nude mice (Day 0), and randomly divided into groups on Day 14. Anti-HER2 antibody-drug conjugate ADC7, anti-LPS antibody-drug conjugate ADC13, or trastuzumab tesirine (Reference Example 1) was administered into the tail vein at a dose of 0.4 mg / kg on Day 14. ABS buffer was administered in the same manner as the control group (vehicle group). The results are shown in Figure 6. The anti-HER2 antibody-drug conjugate ADC7 showed a strong antitumor effect accompanied by tumor regression. On the other hand, no tumor regression was observed with ADC13 or Trastuzumab tesirine. Furthermore, no weight loss was observed in mice administered ADC7 or Trastuzumab tesirine.

[0451] Example 43: Antitumor test of antibody-drug conjugate (4) JIMT-1 cells (DSMZ ACC 589) were suspended in physiological saline (Otsuka Pharmaceutical Factory, Inc.) at a concentration of 5 × 10 6The cells were subcutaneously transplanted into the right flank of female nude mice (Day 0), and randomly divided into groups on Day 10. Anti-HER2 antibody-drug conjugate ADC7 or Trastuzumab tesirine (Reference Example 1) was administered into the tail vein at a dose of 0.4 mg / kg on Day 10. ABS buffer was administered in the same manner as the control group (vehicle group). The results are shown in Figure 7. The anti-HER2 antibody-drug conjugate ADC7 showed a strong antitumor effect accompanied by tumor regression. On the other hand, Trastuzumab tesirine showed an antitumor effect but no tumor regression was observed. Furthermore, no weight loss was observed in mice administered ADC7 or Trastuzumab tesirine.

[0452] Example 44: Antitumor test of antibody-drug conjugate (5) OV-90 cells (ATCC CRL-11732) were suspended in Matrigel (CORNING) at 2.5 × 10 6 The cells were subcutaneously transplanted into the right flank of female nude mice (Day 0), and randomly divided into groups on Day 15. Anti-CLDN6 antibody-drug conjugate ADC8 or anti-CLDN6 antibody (H1L1) tesirine was administered into...

Claims

[Claim 1] The antibody is of the formula: 【Chemistry 1】 or a salt thereof, or a hydrate thereof, (1) The following formula; 【Chemistry 2】 (In each of the structural formulas shown above, m 1 represents an integer of 1; Ab is an antibody or a functional fragment of said antibody, comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO:52 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO:36; The N297 glycan is N297-(Fuc)MSG1, which has the structure shown in the following formula: 【Chemistry 3】 In the formula, the wavy line indicates binding to Asn297 of the antibody, L(PEG) in the N297 sugar chain is *-(CH2CH2-O)3-CH2CH2-NH-. wherein the amino group at the right end is bonded via an amide bond to the 2-position carboxylic acid of the non-reducing terminal sialic acid on the 1-3 side of the branched chain of β-Man of the N297 sugar chain, and the asterisk * at the left end indicates that it is bonded to the 1- or 3-position nitrogen atom on the triazole ring in the above structural formula), (2) The antibody-drug conjugate according to (1), wherein the antibody comprises one or more modifications selected from the group consisting of N-linked glycosylation, O-linked glycosylation, N-terminal processing, C-terminal processing, deamidation, isomerization of aspartic acid, oxidation of methionine, addition of a methionine residue at the N-terminus, amidation of a proline residue, and deletion of one or two amino acid residues at the carboxyl terminus of the heavy chain. (3) The antibody-drug conjugate according to (2), wherein one or several amino acid residues are deleted at the carboxyl terminus of the heavy chain of the antibody. (4) The antibody-drug conjugate according to (2) or (3), in which one amino acid residue is deleted at the carboxyl terminus of each of the two heavy chains of the antibody; and (5) The antibody-drug conjugate according to any one of (2) to (4), wherein the proline residue at the carboxyl terminus of the antibody heavy chain is further amidated. A method for producing an antibody-drug conjugate according to any one of the above.

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