Antibody-pyrrolobenzodiazepine derivative conjugate

Abstract

A novel antibody-pyrrolobenzodiazepine (PBD) derivative conjugate, a pharmaceutical product having a therapeutic effect on tumors using the antibody-drug conjugate, and a method for treating tumors using the antibody-drug conjugate or the pharmaceutical product.

Description

Technical Field

[0001] The present invention relates to an antibody-drug conjugate useful as an antitumor drug, which is formed by combining an antibody capable of targeting tumor cells with a pyrrolobenzodiazepine derivative via a linker structure. Background Art

[0002] Antibody-drug conjugates (ADCs), used in cancer treatment and other applications, are composed of a cytotoxic drug linked to an antibody. The antibody, for example, binds to an antigen expressed on the surface of cancer cells, leading to the internalization of the antigen. ADCs can efficiently deliver the drug to cancer cells, potentially leading to drug accumulation within them and killing them.

[0003] One of the drugs useful for ADCs is pyrrolobenzodiazepine (PBD). PBD exhibits cytotoxicity by binding to the PuGPu sequence in the minor groove of DNA. Anthramycin, a naturally occurring PBD, was first discovered in 1965, and various PBDs derived from naturally occurring PBDs or their analogs have since been discovered (Non-Patent Documents 1 to 4).

[0004] The general formula of PBD is shown below.

[0005]

[0006] It is known that PBDs have different numbers, types, and substitution positions of substituents on the A and C rings, and also different degrees of unsaturation on the B and C rings.

[0007] It is known that PBD significantly enhances cytotoxicity by forming a dimeric structure (Non-Patent Documents 5 and 6), and various ADCs of dimeric PBD have been reported (Patent Documents 1 to 15). However, PBDs having a spiro ring at the C2 position or their ADCs are unknown.

[0008] Human CLDN6 (Claudin-6, hereinafter referred to as hCLDN6) is a member of the Claudin (CLDN) family of proteins and a tetraspanin membrane protein consisting of 220 amino acid residues. Previously, it was suggested that hCLDN6 is overexpressed in some cancers and is an attractive target for cancer therapy (Non-Patent Documents 7-9). Furthermore, it has been reported that CLDN family proteins are internalized by endocytosis and have a short metabolic turnover time (Non-Patent Document 10), making them suitable targets for antibody-drug conjugates (ADCs).

[0009] Based on this information suggesting a link to cancer, monoclonal antibodies that specifically recognize hCLDN6 have been discovered (Patent Documents 16 and 17), and ADCs have been reported in which the tubulin polymerization inhibitors Monomethyl auristatin E (MMAE) and Maytansinoid (DM1) are conjugated to CLDN6-specific monoclonal antibodies (Non-Patent Document 11).

[0010] On the other hand, antibodies that recognize multiple CLDN families have the potential to expand the range of therapeutic indications. ADCs (Patent Document 19) have been disclosed that combine a pyrrolobenzodiazepine (PBD) with a potent cytocidal effect with antibodies that recognize CLDN6 and CLDN9 (Patent Document 18).

[0011] However, their activity is not yet strong enough, and there is still an unmet medical need to use hCLDN6 as a therapeutic target.

[0012] Prior art literature

[0013] Patent Literature

[0014] Patent Document 1: International Publication No. 2013 / 173496

[0015] Patent Document 2: International Publication No. 2014 / 130879

[0016] Patent Document 3: International Publication No. 2017 / 004330

[0017] Patent Document 4: International Publication No. 2017 / 004025

[0018] Patent Document 5: International Publication No. 2017 / 020972

[0019] Patent Document 6: International Publication No. 2016 / 036804

[0020] Patent Document 7: International Publication No. 2015 / 095124

[0021] Patent Document 8: International Publication No. 2015 / 052322

[0022] Patent Document 9: International Publication No. 2015 / 052534

[0023] Patent Document 10: International Publication No. 2016 / 115191

[0024] Patent Document 11: International Publication No. 2015 / 052321

[0025] Patent Document 12: International Publication No. 2015 / 031693

[0026] Patent Document 13: International Publication No. 2011 / 130613

[0027] Patent Document 14: International Publication No. 2005 / 040170

[0028] Patent Document 15: International Publication No. 2017 / 137556

[0029] Patent Document 16: International Publication No. 2009 / 087978

[0030] Patent Document 17: International Publication No. 2011 / 057788

[0031] Patent Document 18: International Publication No. 2015 / 069794

[0032] Patent Document 19: International Publication No. 2017 / 096163

[0033] Non-patent literature

[0034] Non-patent document 1: Julia Mantaj, et al., Angewandte Chemie International Edition 2016, 55, 2-29

[0035] 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

[0036] Non-patent document 4: Accounts of Chemical Research 1986, 19, 230

[0037] Non-patent document 5: Journal of the American Chemical Society 1992, 114, 4939

[0038] Non-patent document 6: Journal of Organic Chemistry 1996, 61, 8141

[0039] Non-patent document 7: BMC Cancer, 2006, 6, 186.

[0040] Non-patent document 8: Histopathology, 2012, 61, 1043-1056.

[0041] Non-patent literature 9: Int J Cancer, 2014, 135, 2206-2214.

[0042] Non-patent document 10: J Membrane Biol, 2004, 199, 29-38.

[0043] Non-patent document 11: 14th Annu Meet Cancer Immunother (CIMT) (May 10-12, Mainz) 2016, Abst 185 Summary of the Invention

[0044] Problems to be solved by the invention

[0045] The present invention provides a novel antibody-pyrrolobenzodiazepine (PBD) derivative conjugate and a novel pyrrolobenzodiazepine (PBD) derivative.

[0046] In addition, the present invention provides a pharmaceutical composition containing the antibody-PBD derivative conjugate having anti-tumor activity.

[0047] The present invention also provides a method for treating cancer using the antibody-PBD derivative conjugate.

[0048] Solutions for solving problems

[0049] The present inventors conducted intensive research and discovered that novel antibody-pyrrolobenzodiazepine (PBD) derivative conjugates exhibit potent antitumor activity. Furthermore, they discovered that the absolute configuration of the 11'-hydroxyl group of the PBD derivative in the conjugate is S. The present invention was completed based on these findings.

[0050] That is, the present invention is as follows.

[0051] [1] An antibody-drug conjugate represented by the following formula:

[0052]

[0053] (where m 1 represents an integer of 1 or 2, and D is any one selected from the following group:

[0054]

[0055] In the formula, the asterisk * indicates binding to L, L is a linker connecting the sugar chain bound to Asn297 of Ab (N297 sugar chain) and D, and the N297 sugar chain can be remodeled. Ab represents an antibody or a functional fragment of the antibody.

[0056] [2] The antibody-drug conjugate according to [1], wherein L is represented by -Lb-La-Lp-NH-B-CH2-O(C=O)-*, wherein the asterisk * indicates a bond with D, B is 1,4-phenyl, 2,5-pyridyl, 3,6-pyridyl, 2,5-pyrimidinyl or 2,5-thienyl, Lp represents any one selected from the following group: -GGVA-, -GG-(D-)VA-, -VA-, -GGFG-, -GGPI-, -GGVCit-, -GGVK- and -GGPL-, and La represents any one selected from the following group: -C( =O)-CH2CH2-C(=O)-, -C(=O)-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)2-CH2-C(=O)-, -C(=O)-CH2CH2-NH-C(=O)-(CH2CH2O)4-CH2CH2-C(=O)-, -CH2-OC(=O)-, and -OC(=O)-, and Lb are represented by the following formula:

[0057]

[0058] Here, in the structural formula of Lb shown above, the asterisk * indicates binding to La, and the wavy line indicates binding to the N297 sugar chain or the remodeled N297 sugar chain.

[0059] [3] The antibody-drug conjugate according to [1] or [2], wherein L represents any one selected from the following group: -Z 1 -C(=O)-CH2CH2-C(=O)-GGVA-NH-B-CH2-OC(=O)-,-Z 1 -C(=O)-CH2CH2-C(=O)-GG-(D-)VA-NH-B-CH2-OC(=O)-,-Z 1 -C(=O)-CH2CH2-C(=O)-VA-NH-B-CH2-OC(=O)-,-Z 1 -C(=O)-(CH2CH2)2-C(=O)-VA-NH-B-CH2-OC(=O)-,-Z 1-C(=O)-CH2CH2-C(=O)-GGPI-NH-B-CH2-OC(=O)-,-Z 1 -C(=O)-CH2CH2-C(=O)-GGFG-NH-B-CH2-OC(=O)-,-Z 1 -C(=O)-CH2CH2-C(=O)-GGVCit-NH-B-CH2-OC(=O)-,-Z 1 -C(=O)-CH2CH2-C(=O)-GGVK-NH-B-CH2-OC(=O)-,-Z 1 -C(=O)-CH2CH2-C(=O)-GGPL-NH-B-CH2-OC(=O)-,-Z 1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-VA-NH-B-CH2-OC(=O)-,-Z 1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)2-CH2-C(=O)-VA-NH-B-CH2-OC(=O)-,-Z 1 -C(=O)-CH2CH2-NH-C(=O)-(CH2CH2O)4-CH2CH2-C(=O)-VA-NH-B-CH2-OC(=O)-,-Z 2 -OC(=O)-GGVA-NH-B-CH2-OC(=O)-, and -Z 3 -CH2-OC(=O)-GGVA-NH-B-CH2-OC(=O)-, where B represents 1,4-phenyl, and Z 1 Represents the following structural formula:

[0060]

[0061] Z 2 Represents the following structural formula:

[0062]

[0063] Z 3 Represents the following structural formula:

[0064]

[0065] Here, Z 1 , Z 2 and Z 3 In the structural formula of , the asterisk * indicates binding to the adjacent C(═O), OC(═O) or CH2, and the wavy line indicates binding to the N297 sugar chain or the reconstructed N297 sugar chain.

[0066] [4] The antibody-drug conjugate according to [3], wherein L represents any one selected from the following group: -Z 1 -C(=O)-CH2CH2-C(=O)-GGVA-NH-B-CH2-OC(=O)-,-Z 1 -C(=O)-CH2CH2-C(=O)-VA-NH-B-CH2-OC(=O)-,-Z 1 -C(=O)-(CH2CH2)2-C(=O)-VA-NH-B-CH2-OC(=O)-,-Z 1 -C(=O)-CH2CH2-C(=O)-GGVCit-NH-B-CH2-OC(=O)-,-Z 1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-VA-NH-B-CH2-OC(=O)-,-Z 1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)2-CH2-C(=O)-VA-NH-B-CH2-OC(=O)-, and -Z 1 -C(=O)-CH2CH2-NH-C(=O)-(CH2CH2O)4-CH2CH2-C(=O)-VA-NH-B-CH2-OC(=O)-, where B is 1,4-phenyl, Z 1 Represents the following structural formula:

[0067]

[0068] Here, the above Z 1 In the structural formula, the asterisk * indicates that Z 1 The adjacent C(=O) is bound, and the wavy line indicates binding to the N297 sugar chain or the remodeled N297 sugar chain.

[0069] [5] The antibody-drug conjugate according to any one of [1] to [4], wherein the N297 sugar chain is a remodeled sugar chain.

[0070] [6] The antibody-drug conjugate according to any one of [1] to [5], wherein the N297 sugar chain represents any of N297-(Fuc)MSG1, N297-(Fuc)MSG2, or a mixture thereof, or N297-(Fuc)SG having a structure represented by the following formula:

[0071]

[0072]

[0073] In the above formula, the wavy line represents the binding to Asn297 of the antibody, and the L(PEG) in the N297 sugar chain represents *-(CH2CH2-O)n 5 -CH2CH2-NH-, here, n 5 represents an integer from 2 to 5, the amino group at the right end is bonded to the 2-carboxylic acid of the non-reducing terminal sialic acid on the 1-3 chain side or / and the 1-6 chain side of the β-Man branch of the N297 sugar chain via an amide bond, and the asterisk * at the left end indicates the Z in L 1 It is combined with the nitrogen atom at position 1 or 3 on the triazole ring.

[0074] [7] The antibody-drug conjugate according to [6], wherein n 5 is 3.

[0075] [8] An antibody drug conjugate represented by the following formula:

[0076]

[0077] (In each of the structural formulas shown above, m 1 represents an integer of 1 or 2, Ab is an antibody or a functional fragment thereof, and the N297 sugar chain is N297-(Fuc)MSG1, N297-(Fuc)MSG2, or a mixture thereof, or N297-(Fuc)SG having the structure shown in the following formula.

[0078]

[0079]

[0080] In the formula, the wavy line indicates binding to Asn297 of the antibody, L(PEG) in the N297 sugar chain represents *-(CH2CH2-O)3-CH2CH2-NH-, where the amino group at the right end is bonded to the 2-carboxylic acid of the non-reducing end of the 1-3 chain side or / and the 1-6 chain side of the β-Man branch of the N297 sugar chain via an amide bond, and the asterisk * at the left end indicates binding to the 1- or 3-nitrogen atom on the triazole ring in the above structural formula).

[0081] [9] An antibody drug conjugate represented by the following formula:

[0082]

[0083] (In each of the structural formulas shown above, m 1 represents an integer of 1 or 2, Ab is an antibody or a functional fragment thereof, and the N297 sugar chain is N297-(Fuc)MSG1, N297-(Fuc)MSG2, or a mixture thereof, or N297-(Fuc)SG having the structure shown in the following formula.

[0084]

[0085] In the formula, the wavy line indicates binding to Asn297 of the antibody, L(PEG) in the N297 sugar chain represents *-(CH2CH2-O)3-CH2CH2-NH-, where the amino group at the right end is bonded to the 2-carboxylic acid of the non-reducing end of the 1-3 chain side or / and the 1-6 chain side of the β-Man branch of the N297 sugar chain via an amide bond, and the asterisk * at the left end indicates binding to the 1- or 3-nitrogen atom on the triazole ring in the above structural formula).

[0086]

[10] An antibody drug conjugate represented by the following formula:

[0087]

[0088] (In each of the structural formulas shown above, m 1 represents an integer of 1 or 2, Ab is an antibody or a functional fragment thereof, and the N297 sugar chain is N297-(Fuc)MSG1, N297-(Fuc)MSG2, or a mixture thereof, or N297-(Fuc)SG having the structure shown in the following formula.

[0089]

[0090] In the formula, the wavy line indicates binding to Asn297 of the antibody, L(PEG) in the N297 sugar chain represents *-(CH2CH2-O)3-CH2CH2-NH-, where the amino group at the right end is bonded to the 2-carboxylic acid of the non-reducing end of the 1-3 chain side or / and the 1-6 chain side of the β-Man branch of the N297 sugar chain via an amide bond, and the asterisk * at the left end indicates binding to the 1- or 3-nitrogen atom on the triazole ring in the above structural formula).

[0091]

[11] An antibody drug conjugate represented by the following formula:

[0092]

[0093] (In each of the structural formulas shown above, m 1 represents an integer of 1 or 2, Ab is an antibody or a functional fragment thereof, and the N297 sugar chain is N297-(Fuc)MSG1, N297-(Fuc)MSG2, or a mixture thereof, or N297-(Fuc)SG having the structure shown in the following formula.

[0094]

[0095] In the formula, the wavy line indicates binding to Asn297 of the antibody, L(PEG) in the N297 sugar chain represents *-(CH2CH2-O)3-CH2CH2-NH-, where the amino group at the right end is bonded to the 2-carboxylic acid of the non-reducing end of the 1-3 chain side or / and the 1-6 chain side of the β-Man branch of the N297 sugar chain via an amide bond, and the asterisk * at the left end indicates binding to the 1- or 3-nitrogen atom on the triazole ring in the formula).

[0096]

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

[11] , characterized in that the antibody-drug conjugate comprises an antibody having internalization activity.

[0097]

[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 internalized by being taken up into the tumor cells.

[0098]

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

[13] , wherein the antibody has an anti-tumor effect.

[0099]

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

[14] , wherein the antibody is an anti-CLDN6 antibody, an anti-CLDN9 antibody, an anti-CLDN6 / CLDN9 antibody, an anti-HER2 antibody, an anti-HER3 antibody, an anti-DLL3 antibody, an anti-FAP antibody, an anti-CDH11 antibody, an anti-A33 antibody, an anti-CanAg antibody, an anti-CD19 antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD25 antibody, an anti-CD30 antibody, an anti-CD33 antibody, an anti-CD37 antibody, an anti-CD56 antibody, an anti-CD70 antibody, an anti-C D98 antibody, anti-B7-H3 antibody, anti-TROP2 antibody, anti-CEA antibody, anti-Cripto antibody, anti-EphA2 antibody, anti-FGFR2 antibody, anti-G250 antibody, anti-MUC1 antibody, 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.

[0100]

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

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

[0101]

[17] The antibody-drug conjugate according to

[16] , wherein the antibody comprises: a heavy chain comprising CDRH1, CDRH2 and CDRH3 as described in (a) or (b) below, and a light chain comprising CDRL1, CDRL2 and CDRL3 as described in (a) or (b) below, (a) CDRH1 formed by the amino acid sequence of sequence number 9, CDRH2 formed by the amino acid sequence of sequence number 10 and CDRH3 formed by the amino acid sequence of sequence number 11, and CDRL1 formed by the amino acid sequence of sequence number 5, CDRL2 formed by the amino acid sequence of sequence number 6 (a) a CDRH1 formed by the amino acid sequence of sequence number 15, a CDRH2 formed by the amino acid sequence of sequence number 16, and a CDRH3 formed by the amino acid sequence of sequence number 17, and a CDRL1 formed by the amino acid sequence of sequence number 12, a CDRL2 formed by the amino acid sequence of sequence number 13, and a CDRL3 formed by the amino acid sequence of sequence number 14.

[0102]

[18] The antibody-drug conjugate according to

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

[0103]

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

[16] to

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

[0104]

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

[16] to

[19] , wherein the antibody comprises: a heavy chain variable region formed by an amino acid sequence selected from the group consisting of the following (a) to (e), and a light chain variable region formed by an amino acid sequence selected from the group consisting of the following (f) to (k), (a) the amino acid sequence of SEQ ID NO: 54, (b) the amino acid sequence of SEQ ID NO: 58, (c) the amino acid sequence of SEQ ID NO: 62, and (d) a framework region other than the CDR sequence in the sequences of (a) to (c). region), (e) an amino acid sequence in which one or more amino acids are deleted, one or more amino acids are substituted, or one or more amino acids are added in the sequence of the framework region other than the CDR sequences in the sequences of (a) to (c), (f) the amino acid sequence described in SEQ ID NO: 38, (g) the amino acid sequence described in SEQ ID NO: 42, (h) the amino acid sequence described in SEQ ID NO: 46, (i) the amino acid sequence described in SEQ ID NO: 50, (j) an amino acid sequence in which at least 95% of the sequence of the framework region other than the CDR sequences in the sequences of (f) to (i) is homologous, and (k) an amino acid sequence in which one or more amino acids are deleted, one or more amino acids are substituted, or one or more amino acids are added in the sequence of the framework region other than the CDR sequences in the sequences of (f) to (i).

[0105]

[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 formed by the amino acid sequence of SEQ ID NO: 54 and a light chain variable region formed by the amino acid sequence of SEQ ID NO: 38, (b) a heavy chain variable region formed by the amino acid sequence of SEQ ID NO: 58 and a light chain variable region formed by the amino acid sequence of SEQ ID NO: 42, (c) a heavy chain variable region formed by the amino acid sequence of SEQ ID NO: 54 and a light chain variable region formed by the amino acid sequence of SEQ ID NO: 46, (d) a heavy chain variable region formed by the amino acid sequence of SEQ ID NO: 58 and a light chain variable region formed by the amino acid sequence of SEQ ID NO: 50, and (e) a heavy chain variable region formed by the amino acid sequence of SEQ ID NO: 62 and a light chain variable region formed by the amino acid sequence of SEQ ID NO: 46.

[0106]

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

[16] to

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

[0107]

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

[16] to

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

[0108]

[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.

[0109]

[25] The antibody-drug conjugate according to

[23] or

[24] , wherein the antibody comprises: a heavy chain and a light chain selected from the group consisting of the following (a) to (e), (a) a heavy chain composed of the amino acid sequence of amino acid numbers 20 to 471 of SEQ ID NO: 52 and a light chain composed of the amino acid sequence of amino acid numbers 21 to 234 of SEQ ID NO: 36, (b) a heavy chain composed of the amino acid sequence of amino acid numbers 20 to 471 of SEQ ID NO: 56 and a light chain composed of the amino acid sequence of amino acid numbers 21 to 234 of SEQ ID NO: 40, (c) a heavy chain composed of the amino acid sequence of amino acid numbers 20 to 471 of SEQ ID NO: 56 and a light chain composed of the amino acid sequence of amino acid numbers 21 to 234 of SEQ ID NO: 40, The heavy chain formed by the amino acid sequence described in amino acid numbers 20 to 471 of sequence number 52 and the light chain formed by the amino acid sequence described in amino acid numbers 21 to 234 of sequence number 44, (d) the heavy chain formed by the amino acid sequence described in amino acid numbers 20 to 471 of sequence number 56 and the light chain formed by the amino acid sequence described in amino acid numbers 21 to 234 of sequence number 48, and (e) the heavy chain formed by the amino acid sequence described in amino acid numbers 20 to 471 of sequence number 60 and the light chain formed by the amino acid sequence described in amino acid numbers 21 to 234 of sequence number 44.

[0110]

[26] The antibody-drug conjugate according to

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

[17] to

[21] and

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

[17] to

[21] and

[25] .

[0111]

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

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

[0112]

[28] The antibody-drug conjugate according to

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

[0113]

[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 causes a decrease in ADCC and / or CDC activity.

[0114]

[30] The antibody-drug conjugate according to

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

[0115]

[31] The antibody-drug conjugate according to

[27] or

[28] , wherein the antibody is a humanized monoclonal antibody comprising a heavy chain formed by the amino acid sequence of SEQ ID NO: 65 and a light chain formed by the amino acid sequence of SEQ ID NO: 64.

[0116]

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

[27] ,

[29] and

[30] , wherein the antibody is an antibody comprising a heavy chain variable region formed by the amino acid sequence described by amino acid numbers 20 to 139 of SEQ ID NO: 75, and a light chain variable region formed by the amino acid sequence described by amino acid numbers 21 to 127 of SEQ ID NO: 73.

[0117]

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

[27] ,

[29] ,

[30] and

[32] , wherein the antibody is a humanized monoclonal antibody comprising a heavy chain formed by the amino acid sequence described by amino acid numbers 20 to 469 of SEQ ID NO: 75, and a light chain formed by the amino acid sequence described by amino acid numbers 21 to 234 of SEQ ID NO: 73.

[0118]

[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.

[0119]

[35] The antibody-drug conjugate according to

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

[0120]

[36] The antibody-drug conjugate according to

[34] or

[35] , wherein one amino acid residue is deleted from the carboxyl termini of both heavy chains of the antibody.

[0121]

[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.

[0122]

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

[37] , wherein the antibody is a sugar chain remodeled 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 selecting the target antibody from the obtained culture; ii) treating the antibody obtained in step i) with a hydrolase to produce a (Fucα1,6)GlcNAc-antibody; and iii) reacting the (Fucα1,6)GlcNAc-antibody with a sugar chain donor molecule in the presence of a glycosyltransferase, wherein the sugar chain donor molecule is obtained by introducing a PEG linker having an azide group at the carbonyl group of the carboxylic acid at position 2 of sialic acid of MSG (9) or SG (10) to oxazolidinylate the reducing end.

[0123]

[39] The antibody-drug conjugate according to

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

[0124]

[40] A compound, a salt thereof, or a hydrate thereof, wherein the compound is any one of the following formulae:

[0125]

[0126]

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

[39] , comprising the step of reacting the sugar chain-remodeled antibody according to

[38] or

[39] with the compound according to

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

[0127]

[42] An antibody-drug conjugate, characterized in that the antibody-drug conjugate is obtained by the manufacturing method described in

[41] .

[0128]

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

[39] and

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

[0129]

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

[39] ,

[42] and

[43] , wherein m 1 is the integer 1.

[0130]

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

[39] and

[42] to

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

[0131]

[46] A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the antibody-drug conjugate, a salt thereof, or a hydrate thereof according to any one of [1] to

[39] and

[42] to

[45] .

[0132]

[47] The pharmaceutical composition according to

[46] , characterized in that the pharmaceutical composition is an anti-tumor drug.

[0133]

[48] ​​A method for treating tumors, characterized in that the antibody-drug conjugate, a salt thereof, or a hydrate thereof according to any one of [1] to

[39] and

[42] to

[45] is administered to an individual.

[0134]

[49] A method for treating tumors, characterized in that a pharmaceutical composition and at least one anti-tumor drug are administered to an individual simultaneously, separately or continuously, wherein the pharmaceutical composition comprises the antibody-drug conjugate, a salt thereof or a hydrate thereof according to any one of [1] to

[39] and

[42] to

[45] .

[0135] Effects of the Invention

[0136] The novel antibody-pyrrolobenzodiazepine (PBD) derivative conjugates provided by the present invention exhibit excellent antitumor activity and safety, and are therefore useful as antitumor agents. Furthermore, the PBD derivatives of the present invention exhibit antitumor activity and are useful as pharmaceuticals in such conjugates. Furthermore, the antibodies of the present invention recognize or bind to antigens expressed in tumor cells and are therefore useful as antibodies in such conjugates. BRIEF DESCRIPTION OF THE DRAWINGS

[0137] Figure 1 This figure schematically illustrates the antibody-drug conjugate of the present invention (molecule (I)). (a) represents the drug D, (b) represents the linker L, (c) represents N3-L(PEG)-, and (d) represents the N297 sugar chain (wherein white ovals represent NeuAc(Sia), white hexagons represent Man, solid hexagons represent GlcNAc, white diamonds represent Gal, and white inverted triangles represent Fuc). In (b) and (c), the azide group (black teardrop shape) in (c) reacts with the alkyne structure in the spacer (white semicircle) in (b) to form a triazole ring, resulting in the conjugation. The Y-shaped shape represents the antibody Ab. In addition, for convenience, this schematic diagram represents the N297 sugar chain as N297-(Fuc)MSG, showing a configuration in which only one side of each N297 sugar chain has sialic acid bound to an azide-containing PEG linker (N3-L(PEG)-), while the other side branch has no sialic acid at its non-reducing end. However, a configuration in which both sides have sialic acid bound to an azide-containing PEG linker is also possible by using N297-(Fuc)SG. This representation method applies throughout this specification unless otherwise specified.

[0138] Figure 2 The present invention is a (Fucα1,6)GlcNAc-antibody ( Figure 2 A (II) molecule) and MSG type sugar chain remodeled antibody ( Figure 2 Schematic diagram of the structure of B (III) molecule). In the two figures, the Y-shaped Figure 1 The same refers to the antibody Ab. Figure 2 In A, (e) represents an N297 sugar chain consisting only of GlcNAc bound to positions 1 and 6 of Fuc via α-glycosidic bonds. Figure 2 In B, (d) and Figure 1 Similarly, the sugar chain of N297 is shown, and (f) is the structure of the PEG linker portion having an azide group, which shows the azide group used to bind to the linker L at the terminal. The binding mode of the PEG linker having an azide group is similar to Figure 1 Same description as .

[0139] Figure 3 This is a schematic diagram of the process of producing MSG-type sugar chain-remodeled antibodies from antibodies produced in animal cells. Figure 2 Similarly, (Fucα1,6)GlcNAc-antibody and MSG-type sugar chain remodeled antibody are shown. (IV) Molecule is an antibody produced in animal cells and is a mixture of molecules with heterogeneous N297 sugar chains. Figure 3 A represents a step of preparing a uniform (Fucα1,6)GlcNAc-antibody (II) by treating the heterogeneous N297 sugar chain (IV) with a hydrolase such as EndoS. Figure 3 B represents a step of transferring the sugar chain of the MSG-type sugar chain donor molecule to the GlcNAc of the N297 sugar chain of the antibody (II) using a glycosyltransferase such as the EndoS D233Q / Q303L mutant, thereby producing (III) an MSG-type sugar chain remodeled antibody. The MSG-type sugar chain donor molecule used here is a molecule in which the sialic acid at the non-reducing end of MSG is modified with a PEG linker having an azide group. In the produced MSG-type N297 sugar chain remodeled antibody, Figure 2 The non-reducing terminal sialic acid is modified in the same manner as described in B. Figure 3 In B, for convenience, MSG is shown as a donor molecule, but by using SG (10) as a sugar chain donor, (III) a sugar chain-remodeled antibody is synthesized in which a linker molecule having an azide group is bound to both non-reducing ends of the N297 sugar chain.

[0140] Figure 4 The results show the effects of anti-HER2 antibody-drug conjugates ADC2 and ADC1 on subcutaneously transplanted NCI-N87 cells, a human gastric cancer line.

[0141] Figure 5 The results show the effects of anti-HER2 antibody-drug conjugate ADC7, trastuzumab, and anti-LPS antibody-drug conjugate ADC13 on subcutaneously transplanted NCI-N87 cells, a human gastric cancer line.

[0142] Figure 6 The results show the effects of anti-HER2 antibody-drug conjugate ADC7, anti-LPS antibody-drug conjugate ADC13, or trastuzumab-Tesirine (Reference Example 1) on subcutaneously transplanted KPL-4 cells, a human breast cancer line.

[0143] Figure 7 The results show the effects of anti-HER2 antibody-drug conjugate ADC7 or trastuzumab-Tesirine on subcutaneously transplanted JIMT-1 cells, a human breast cancer line.

[0144] Figure 8 The results show the effects of anti-CLDN6 antibody-drug conjugate ADC8 or anti-CLDN6 antibody (H1L1)-Tesirine on subcutaneously transplanted OV-90 cells, a human ovarian cancer line.

[0145] Figure 9 The results show the effects of the anti-CLDN6 antibody-drug conjugate ADC8 or the anti-CLDN6 antibody (H1L1)-Tesirine on subcutaneously transplanted NIH:OVCAR-3 cells, a human ovarian cancer line.

[0146] Figure 10 The results show the effects of the antibody-drug conjugate anti-TROP2 antibody-drug conjugate ADC11 or the anti-LPS antibody-drug conjugate ADC13 on subcutaneously transplanted FaDu cells, a human head and neck cancer cell line.

[0147] Figure 11 The full-length amino acid sequence of human CLDN6 (SEQ ID NO: 1) and the base sequence of the full-length cDNA (SEQ ID NO: 2) are shown.

[0148] Figure 12 The full-length amino acid sequence of human CLDN9 (SEQ ID NO: 3) and the base sequence of the full-length cDNA (SEQ ID NO: 4) are shown.

[0149] Figure 13 The amino acid sequences of CDRL1 to CDRL3 of the B1 antibody light chain are shown (SEQ ID NOs: 5 to 7).

[0150] Figure 14 The amino acid sequence of CDRL3 of the humanized B1 antibody light chain L4 is shown (SEQ ID NO: 8).

[0151] Figure 15 The amino acid sequences of CDRHs 1 to 3 of the B1 antibody heavy chain are shown (SEQ ID NOs: 9 to 11).

[0152] Figure 16 The amino acid sequences of CDRL1 to 3 of the C7 antibody light chain are shown (SEQ ID NOs: 12 to 14).

[0153] Figure 17 The amino acid sequences of CDRH1 to CDRH3 of the C7 antibody heavy chain are shown (SEQ ID NOs: 15 to 17).

[0154] Figure 18 The nucleotide sequence of the 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. Underlined characters in the amino acid sequence indicate CDR sequences.

[0155] Figure 19The nucleotide sequence of the cDNA encoding the variable region of the B1 antibody heavy chain (SEQ ID NO: 20) and the amino acid sequence of the variable region of the B1 antibody heavy chain (SEQ ID NO: 21) are shown. Underlined characters in the amino acid sequence indicate CDR sequences.

[0156] Figure 20 The nucleotide sequence of the 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. Underlined characters in the amino acid sequence indicate CDR sequences.

[0157] Figure 21 The nucleotide sequence of the cDNA encoding the variable region of the C7 antibody heavy chain (SEQ ID NO: 24) and the amino acid sequence of the variable region of the C7 antibody heavy chain (SEQ ID NO: 25) are shown. Underlined characters in the amino acid sequence indicate CDR sequences.

[0158] Figure 22 The amino acid sequence of the chB1 light chain (SEQ ID NO: 28) and a DNA fragment (SEQ ID NO: 29) containing the DNA sequence encoding the amino acid sequence of the chB1 light chain are shown. Underlined characters in the amino acid sequence indicate CDR sequences.

[0159] Figure 23 The amino acid sequence (SEQ ID NO: 30) and the nucleotide sequence (SEQ ID NO: 31) encoding the chB1 light chain variable region are shown. Underlined characters in the amino acid sequence indicate CDR sequences.

[0160] 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. Underlined characters in the amino acid sequence indicate CDR sequences.

[0161] Figure 25 The amino acid sequence (SEQ ID NO: 34) and the nucleotide sequence (SEQ ID NO: 35) encoding the chB1 heavy chain variable region are shown. Underlined characters in the amino acid sequence indicate CDR sequences.

[0162] 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. Underlined CDR sequences are shown in the amino acid sequence.

[0163] Figure 27 The amino acid sequence (SEQ ID NO: 38) and the nucleotide sequence (SEQ ID NO: 39) encoding the variable region of the humanized antibody light chain hL1 are shown. Underlined CDR sequences are shown in the amino acid sequence.

[0164] Figure 28The 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. Underlined CDR sequences are shown in the amino acid sequence.

[0165] Figure 29 The amino acid sequence (SEQ ID NO: 42) of the variable region of the humanized antibody light chain hL2 and the nucleotide sequence (SEQ ID NO: 43) encoding the variable region of the humanized antibody light chain hL2 are shown.

[0166] Figure 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. Underlined CDR sequences are shown in the amino acid sequence.

[0167] Figure 31 The amino acid sequence (SEQ ID NO: 46) and the nucleotide sequence (SEQ ID NO: 47) encoding the variable region of the humanized antibody light chain hL3 are shown. Underlined CDR sequences are shown in the amino acid sequence.

[0168] Figure 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. Underlined CDR sequences are shown in the amino acid sequence.

[0169] Figure 33 The amino acid sequence (SEQ ID NO: 50) and the nucleotide sequence (SEQ ID NO: 51) encoding the variable region of the humanized antibody light chain hL4 are shown. Underlined CDR sequences are shown in the amino acid sequence.

[0170] Figure 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. Underlined CDR sequences are shown in the amino acid sequence.

[0171] Figure 35 The amino acid sequence (SEQ ID NO: 54) and the nucleotide sequence (SEQ ID NO: 55) encoding the variable region of the humanized antibody heavy chain hH1 are shown. Underlined CDR sequences are shown in the amino acid sequence.

[0172] Figure 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. Underlined CDR sequences are shown in the amino acid sequence.

[0173] Figure 37The amino acid sequence (SEQ ID NO: 58) of the variable region of the humanized antibody heavy chain hH2 and the nucleotide sequence (SEQ ID NO: 59) encoding the variable region of the humanized antibody heavy chain hH2 are shown.

[0174] 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. Underlined CDR sequences are shown in the amino acid sequence.

[0175] Figure 39 The amino acid sequence (SEQ ID NO: 62) and the nucleotide sequence (SEQ ID NO: 63) encoding the variable region of the humanized antibody heavy chain hH3 are shown. Underlined CDR sequences are shown in the amino acid sequence.

[0176] Figure 40 The binding abilities of the B1 and C7 antibodies to human CLDN6 and family molecules CLDN3, CLDN4, and CLDN9, obtained by flow cytometry, are shown.

[0177] Figure 41 The antibody internalization activity of the B1 and C7 antibodies obtained using Mab-ZAP is shown.

[0178] Figure 42 The binding abilities of humanized anti-CLDN6 antibodies H1L1, H2L2, H1L3, H2L4, and H3L3 to CLDN6 and family molecules were determined by flow cytometry.

[0179] Figure 43 The amino acid sequence of the trastuzumab light chain (SEQ ID NO: 64) and heavy chain (SEQ ID NO: 65) are shown.

[0180] Figure 44 The amino acid sequence of the light chain (SEQ ID NO: 73) and the amino acid sequence of the heavy chain (SEQ ID NO: 75) of the trastuzumab mutant are shown.

[0181] Figure 45 A diagram comparing the amino acid sequences of chB1_H, the heavy chain of the human chimeric anti-CLDN6 antibody chB1, and hH1, hH2, and hH3, the heavy chains of humanized antibodies. "·" indicates the same amino acid residues as in chB1_H, and sites with amino acid residues indicate substituted amino acid residues.

[0182] Figure 46A diagram comparing the amino acid sequences of chB1_L, the light chain of the human chimeric anti-CLDN6 antibody chB1, and hL1, hL2, hL3, and hL4, the light chains of humanized antibodies. "·" indicates the same amino acid residue as chB1_L, and sites where amino acid residues are described indicate substituted amino acid residues.

[0183] Figure 47 The results show the effects of anti-HER2 antibody-drug conjugates ADC7 and ADC14 on subcutaneously transplanted KPL-4 cells, a human breast cancer line.

[0184] Figure 48 The results show the effect of the anti-HER2 antibody-drug conjugate ADC14 on subcutaneously transplanted JIMT-1 cells, a human breast cancer line.

[0185] Figure 49 The results show the effects of anti-HER2 antibody-drug conjugates ADC7 and ADC14, and anti-LPS antibody-drug conjugate ADC13 on subcutaneously transplanted CFPAC-1 cells, a human pancreatic cancer line. DETAILED DESCRIPTION

[0186] [Antibody-drug conjugates]

[0187] The antibody-drug conjugate of the present invention is an antitumor drug in which an antitumor compound is bound to an antibody capable of recognizing an antigen expressed in tumor cells or binding to the antigen via a linker structure.

[0188] The antibody-drug conjugate of the present invention is shown in the following formula.

[0189]

[0190] m 1 is an integer of 1 or 2 (preferably 1), D represents a drug, L represents a linker connecting the N297 sugar chain to D, Ab represents an antibody or a functional fragment thereof, and the N297 sugar chain represents a sugar chain bound to the side chain of Asn297 of the antibody. The N297 sugar chain may also be a remodeled sugar chain.

[0191] <Medications>

[0192] The drug D of the present invention is preferably an antitumor compound. In the case of the antitumor compound, part or all of the linker of the antibody-drug conjugate of the present invention is cleaved in tumor cells, partially releasing the antitumor compound, thereby exhibiting an antitumor effect.

[0193] The drug in the antibody-drug conjugate of the present invention, ie, the PBD derivative, is, for example, any one selected from the following group.

[0194]

[0195] Here, in the formula, the asterisk * indicates binding to L.

[0196] In the case of the PBD derivative of the present invention, the absolute configuration of the hydroxyl group at the 11' position is S configuration.

[0197] <Connector structure>

[0198] The linker L of the present invention is a linker that connects the N297 sugar chain and D.

[0199] The linker L is represented by the following formula.

[0200] -Lb-La-Lp-NH-B-CH2-O(C=O)-*

[0201] The asterisk * indicates binding to the nitrogen atom at the N10' position of drug D, and Lb indicates a spacer that binds La to the N297 sugar chain or the remodeled N297 sugar chain.

[0202] B represents a phenyl group or a heteroaryl group, 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.

[0203] Lp represents a linker consisting of an amino acid sequence that can be cleaved in vivo or within target cells. Lp is cleaved by the action of enzymes such as esterases and peptidases.

[0204] Lp is a peptide residue composed of 2 to 7 (preferably 2 to 4) amino acids, that is, it is composed of an oligopeptide residue in which 2 to 7 amino acids are linked by peptide bonds.

[0205] Lp binds to the carbonyl group of La in Lb-La- at its N-terminus and forms an amide bond with the amino group (-NH-) in the -NH-B-CH2-O(C=O)- portion of the linker at its C-terminus. The bond between the C-terminus of Lp and the -NH- is cleaved by an enzyme such as the aforementioned esterase.

[0206] The amino acids constituting Lp are not particularly limited, and may be, for example, L- or D-amino acids, preferably L-amino acids. Furthermore, in addition to α-amino acids, amino acids having structures such as β-alanine, ε-aminocaproic acid, and γ-aminobutyric acid may be used. Furthermore, non-natural amino acids such as N-methylated amino acids may also be used.

[0207] The amino acid sequence of Lp is not particularly limited, and examples of 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 them, glycine (Gly; G), valine (Val; V), alanine (Ala; A), and citrulline (Cit) are preferred.

[0208] These amino acids can be repeated to have an amino acid sequence containing arbitrarily selected amino acids. In addition, the pattern of drug release can be controlled according to the type of amino acids.

[0209] Specific examples of the linker Lp include -GGVA-, -GG-(D-)VA-, -VA-, -GGFG-, -GGPI-, -GGVCit-, -GGVK-, -GG(D-)PI-, -GGPL-, -EGGVA, -PI-, -GGF-, -DGGF-, (D-)D-GGF-, -EGGF-, -SGGF-, -KGGF-, -DGGFG-, -GGFGG-, -DDGGFG-, -KDGGFG-, and -GGFGGGF-.

[0210] Here, the above-mentioned "(D-)V" represents D-valine, "(D-)P" represents D-proline, and "(D-)D" represents D-aspartic acid.

[0211] The linker Lp is preferably the following: -GGVA-, -GG-(D-)VA-, -VA-, -GGFG-, -GGPI-, -GGVCit-, -GGVK-, -GG(D-)PI-, -GGPL-.

[0212] The linker Lp is more preferably the following: -GGVA-, -GGVCit-, or -VA-.

[0213] La represents any one selected from the following group: -C(=O)-(CH2CH2)n 2 -C(=O)-, -C(=O)-(CH2CH2)n 2 -C(=O)-NH-(CH2CH2)n 3 -C(=O)-, -C(=O)-(CH2CH2)n 2 -C(=O)-NH-(CH2CH2O)n 3 -CH2-C(=O)-, -C(=O)-(CH2CH2)n 2-NH-C(=O)-(CH2CH2O)n 3 -CH2CH2-C(=O)-, -(CH2)n 4 -O-C(=O)-.

[0214] Here, in the formula, n 2 represents an integer of 1 to 3 (preferably 1 or 2), n 3 represents an integer of 1 to 5 (preferably an integer of 2 to 4, more preferably 2 or 4), n 4 It represents an integer of 0 to 2 (preferably 0 or 1).

[0215] La preferably represents any one selected from the following group: -C(=O)-CH2CH2-C(=O)-, -C(=O)-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)2-CH2-C(=O)-, -C(=O)-CH2CH2-NH-C(=O)-(CH2CH2O)4-CH2CH2-C(=O)-, -CH2-OC(=O)- and -OC(=O)-.

[0216] La is more preferably -C(=O)-CH2CH2-C(=O)- or -C(=O)-(CH2CH2)2-C(=O)-.

[0217] The Lb spacer is not particularly limited, and examples thereof include spacers represented by the following formula.

[0218]

[0219] In the above structural formulas of Lb, the asterisk represents the -(C=O) or -(CH2)n 4 Binding, the wavy line indicates binding to the N297 sugar chain of Ab or the reconstructed N297 sugar chain.

[0220] In each of the structural formulas of Lb (Lb-1, Lb-2, or Lb-3) shown above, the triazole ring formed by the click reaction between the azide group and DBCO has geometric isomers. In one Lb, either of the two structures or a mixture thereof exists. Two or four (m 1 1 or 2) "-L-D", and each Lb (Lb-1, Lb-2 or Lb-3) in each L of the two or four "-L-D"s exists in either of these two structures or a mixture of the two.

[0221] L is preferably represented by -Lb-La-Lp-NH-B-CH2-O(C=O)-*, B is 1,4-phenyl, Lp represents any one selected from the following group: -GGVA-, -GG-(D-)VA-, -VA-, -GGFG-, -GGPI-, -GGVCit-, -GGVK-, -GG(D-)PI-, -GGPL-, and La represents any one selected from the following group: -C(=O)-CH2CH2-C(=O)-, -C(=O)-(CH2CH2) 2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)2-CH2-C(=O)-, -C(=O)-CH2CH2-NH-C(=O)-(CH2CH2O)4-CH2CH2-C(=O)-, -CH2-OC(=O)-, -OC(=O)-, Lb represents any structural formula among Lb shown above.

[0222] More preferably, L is any one selected from the following group: -Z 1 -C(=O)-CH2CH2-C(=O)-GGVA-NH-B-CH2-OC(=O)-,-Z 1 -C(=O)-CH2CH2-C(=O)-GG-(D-)VA-NH-B-CH2-OC(=O)-,-Z 1 -C(=O)-CH2CH2-C(=O)-VA-NH-B-CH2-OC(=O)-,-Z 1 -C(=O)-(CH2CH2)2-C(=O)-VA-NH-B-CH2-OC(=O)-,-Z 1 -C(=O)-CH2CH2-C(=O)-GGPI-NH-B-CH2-OC(=O)-,-Z 1 -C(=O)-CH2CH2-C(=O)-GGFG-NH-B-CH2-OC(=O)-,-Z 1 -C(=O)-CH2CH2-C(=O)-GGVCit-NH-B-CH2-OC(=O)-,-Z 1 -C(=O)-CH2CH2-C(=O)-GGVK-NH-B-CH2-OC(=O)-,-Z 1 -C(=O)-CH2CH2-C(=O)-GGPL-NH-B-CH2-OC(=O)-,-Z 1-C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-VA-NH-B-CH2-OC(=O)-,-Z 1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)2-CH2-C(=O)-VA-NH-B-CH2-OC(=O)-,-Z 1 -C(=O)-CH2CH2-NH-C(=O)-(CH2CH2O)4-CH2CH2-C(=O)-VA-NH-B-CH2-OC(=O)-,-Z 2 -OC(=O)-GGVA-NH-B-CH2-OC(=O)-,-Z 3 -CH2-OC(=O)-GGVA-NH-B-CH2-OC(=O)-.

[0223] Here, Z 1 The structural formula shown below represents the above-mentioned Lb:

[0224]

[0225] Z 2 The structural formula shown below represents the above-mentioned Lb:

[0226]

[0227] Z 3 The structural formula shown below represents the above-mentioned Lb:

[0228]

[0229] B is 1,4-phenyl.

[0230] L is most preferably any one of the following: -Z 1 -C(=O)-CH2CH2-C(=O)-GGVA-NH-B-CH2-OC(=O)-,-Z 1 -C(=O)-CH2CH2-C(=O)-VA-NH-B-CH2-OC(=O)-,-Z 1 -C(=O)-(CH2CH2)2-C(=O)-VA-NH-B-CH2-OC(=O)-,-Z 1 -C(=O)-CH2CH2-C(=O)-GGVCit-NH-B-CH2-OC(=O)-,-Z 1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-VA-NH-B-CH2-OC(=O)-,-Z 1-C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)2-CH2-C(=O)-VA-NH-B-CH2-OC(=O)- and -Z 1 -C(=O)-CH2CH2-NH-C(=O)-(CH2CH2O)4-CH2CH2-C(=O)-VA-NH-B-CH2-OC(=O)-.

[0231] Here, B is 1,4-phenylene, and Z 1 is the following structural formula of the above Lb:

[0232]

[0233] <Free drug>

[0234] The free drug of the antibody-drug conjugate of the present invention is one selected from the following group.

[0235]

[0236] After the antibody-drug conjugate of the present invention transfers into tumor cells, the linker L part is cleaved to generate the free drug. The anti-tumor cell effect of this free drug has been confirmed.

[0237] <Antibody>

[0238] In the present invention, "cancer" and "tumor" are used with the same meaning.

[0239] In the present invention, "gene" refers to a nucleotide or nucleotide sequence containing a nucleotide sequence encoding an amino acid of a protein, or its complementary strand. For example, polynucleotides, oligonucleotides, DNA, mRNA, cDNA, RNA, etc. containing a nucleotide sequence encoding an amino acid of a protein or its complementary strand are included in the meaning of "gene". As the "CLDN6 gene" of the present invention, for example, DNA, mRNA, cDNA, cRNA, etc. containing a nucleotide sequence encoding the amino acid sequence of the CLDN6 protein can be cited.

[0240] In the present invention, "nucleotide", "polynucleotide" or "nucleotide sequence" has the same meaning as "nucleic acid". For example, DNA, RNA, probe, oligonucleotide, polynucleotide, primer, etc. are also included in the meaning of "nucleotide" or "nucleotide sequence".

[0241] In the present invention, "polypeptide", "peptide" and "protein" are used without distinction.

[0242] In the present invention, "CLDN6" and the CLDN6 protein are used with the same meaning.

[0243] In the present invention, "cells" also include cells in animal individuals and cultured cells.

[0244] In the present invention, "cytotoxic activity" refers to pathological changes in cells in some form, not limited to direct trauma, but also refers to all structural and functional damage to cells, such as DNA cleavage, formation of base dimers, chromosome cleavage, damage to the cell division apparatus, and reduction in the activity of various enzymes.

[0245] In the present invention, "functional fragments of antibodies" are also referred to as "antigen-binding fragments of antibodies", which refer to partial fragments of antibodies that have binding activity to antigens, including Fab, F(ab')2, Fv, scFv, diabody (bispecific antibodies), linear antibodies, and multispecific antibodies formed by antibody fragments. In addition, Fab', a monovalent fragment of the variable region of an antibody after F(ab')2 is treated under reducing conditions, is also included in the antigen-binding fragment of an antibody. However, as long as it has the ability to bind to the antigen, it is not limited to these molecules. In addition, these antigen-binding fragments include not only molecules obtained by treating the full-length molecules of the antibody protein with appropriate enzymes, but also proteins produced in appropriate host cells using genetically engineered antibody genes.

[0246] The functional fragment of the present invention includes aspartic acid (Asn297) and its surrounding amino acids modified by the N-linked sugar chain well-conserved in the Fc region of IgG heavy chains, and has antigen-binding ability.

[0247] In the present invention, an "antigenic determinant" refers to a partial peptide or partial stereostructure of an antigen to which a specific antibody (eg, an anti-CLDN6 antibody) binds (eg, a partial peptide or partial stereostructure of CLDN6). The antigenic determinant of such a partial peptide (eg, a partial peptide of CLDN6) can be determined by methods well known to those skilled in the art, such as immunoassays.

[0248] "CDR" in the present invention refers to the complementarity determining region (CDR). It is known that the heavy chain and light chain of an antibody molecule each have three CDRs. CDR, also known as the hypervariable region, is a site with particularly high variability in the primary structure within the variable region of the heavy and light chains of an antibody, and is separated into three locations in the primary structure of the polypeptide chains of the heavy and light chains, respectively. In this specification, for the CDRs of an antibody, the CDRs of the heavy chain are referred to 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 referred to 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 terms of stereostructure and determine the specificity with the antigen to be bound.

[0249] In the present invention, "hybridization under stringent conditions" refers to hybridization at 68°C in the commercially available hybridization solution ExpressHybHybridization Solution (Clontech); or hybridization under the following conditions or equivalent conditions: hybridization using a filter immobilized with DNA in the presence of 0.7-1.0 M NaCl at 68°C, followed by washing at 68°C using a 0.1-2x concentration SSC (saline sodium citrate) solution (1x concentration SSC contains 150 mM NaCl and 15 mM sodium citrate), thereby enabling identification.

[0250] In the present invention, “1 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.

[0251] In the present invention, antibodies that recognize or bind to CLDN6 or CLDN6 and CLDN9 may be referred to as "anti-CLDN6 antibodies" or "anti-CLDN6 / CLDN9 antibodies," respectively. Such antibodies include chimeric antibodies, humanized antibodies, and human antibodies. Antibodies that recognize or bind to CLDN6 and CLDN9 may also be referred to as "anti-CLDN6 antibodies."

[0252] The antibody used in the antibody-drug conjugate of the present invention refers to an immunoglobulin, which is a molecule containing an antigen-binding site that immunospecifically binds to an antigen. The antibody of the present invention may be any of IgG, IgE, IgM, IgD, IgA, and IgY, preferably IgG. In addition, as a subclass, it may be any of IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, preferably IgG1, IgG2, and IgG4. When using IgG1 or IgG4, the effector function can be adjusted by replacing part of the amino acid residues in the constant region (see WO88 / 07089, WO94 / 28027, and WO94 / 29351).

[0253] In addition, when IgG1 is used as the isotype of the antibody of the present invention, the effector function can be adjusted by replacing a portion of the amino acid residues in the constant region. As mutants of IgG1 that reduce or weaken the effector function, IgG1 LALA (IgG1-L234A, L235A), IgG1 LAGA (IgG1-L235A, G237A) and the like can be listed, preferably IgG1 LALA. It should be noted that the L234A and L235A represent that the leucine at positions 234 and 235 determined by the EU index (Proc. Natl. Acad. Sci. USA, Vol. 63, No. 1 (May 15, 1969), pp. 78-85) are replaced by alanine, and G237A represents that the glycine at position 237 determined by the EU index is replaced by alanine.

[0254] The antibodies of the present invention can be derived from any species, preferably humans, rats, mice and rabbits. In the case of species other than humans, chimerization or humanization is preferably performed using known techniques. The antibodies of the present invention can be polyclonal antibodies or monoclonal antibodies, preferably monoclonal antibodies. 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.

[0255] The antibody of the present invention is preferably an antibody that can target tumor cells, that is, an antibody that binds to an antigen expressed on the surface of tumor cells.

[0256] The antibody-drug conjugates of the present invention are conjugated to compounds that exert anti-tumor effects. Therefore, while it is preferred that the antibody itself possess anti-tumor effects, this is not essential. To specifically and selectively exert the cytotoxicity of the anti-tumor compound in tumor cells, it is important and preferred that the antibody possess the property of internalization and translocation into tumor cells. During internalization of the antibody, the antibody-drug conjugate can also translocate into the cells. From the perspective of anti-tumor effects, the property of the antibody or antibody-drug conjugate to internalize and translocate into tumor cells is important and preferred for specifically and selectively damaging tumor cells using the drug. The anti-tumor activity of an antibody refers to its cytotoxic activity or anti-cellular effect against tumor cells. Anti-tumor activity can be confirmed using known in vitro or in vivo evaluation systems. The internalization ability of an antibody can be measured using known evaluation systems.

[0257] Examples of such antibodies include antibodies against tumor-associated antigens, such as anti-CLDN6 antibodies, anti-CLDN9 antibodies, anti-CLDN6 / CLDN9 antibodies, anti-HER2 antibodies, anti-HER3 antibodies, anti-DLL3 (Delta like protein 3: delta-like protein 3) antibodies, anti-A33 antibodies, anti-CanAg antibodies, anti-CD19 antibodies, anti-CD20 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 (CD276) antibodies, anti-TROP2 antibodies, anti-CEA antibodies, anti-Cripto antibodies, anti-EphA2 antibodies, and anti-FGFR2 antibodies. Antibodies (such as WO201315206), anti-G250 antibodies, anti-MUC1 antibodies (such as WO2011012309), anti-GPNMB antibodies, anti-integrin antibodies, anti-PSMA antibodies, anti-Tenascin-C antibodies, anti-SLC44A4 antibodies, anti-Mesothelin antibodies, anti-EGFR antibodies, anti-5T4 (carcinoembryonic antigen 5T4; also known as TPBG and trophoblast glycoprotein) antibodies, anti-LRRC15 (Leucine-rich repeat-containing protein 15) antibodies, anti-DR5 antibodies, anti-CDH3 (cadherin 3) antibodies, anti-PDPN (podoplanin) antibodies, or anti-CD123 antibodies are not limited thereto.

[0258] The antibodies of the present invention are preferably anti-CLDN6 antibodies, anti-CLDN6 / CLDN9 antibodies, anti-HER2 antibodies, anti-CD98 antibodies, and anti-TROP2 antibodies, and more preferably anti-CLDN6 antibodies and anti-HER2 antibodies (eg, trastuzumab and trastuzumab mutants).

[0259] The anti-CLDN6 antibodies used in the present invention are described below.

[0260] 1. CLDN6 and CLDN9

[0261] CLDN6 is a four-transmembrane protein consisting of 220 amino acids belonging to the Claudin family and has an N-terminus and a C-terminus in cells.

[0262] The amino acid sequence and DNA sequence of human CLDN6 are disclosed in public databases and can be referenced using accession numbers such as NP_067018 (SEQ ID NO: 1) and NM_021195 (SEQ ID NO: 2) (both NCBI).

[0263] Regarding the amino acid sequence of the human CLDN6 protein (hereinafter referred to as the "CLDN6 amino acid sequence"), the extracellular region includes an extracellular domain (EC1) comprising amino acids 29 to 81 of SEQ ID NO: 1 in the sequence listing and an extracellular domain (EC2) comprising amino acids 138 to 160.

[0264] CLDN9 is a 217-amino acid tetraspanin protein belonging to the claudin family, with an N-terminus and a C-terminus within the cell. CLDN9 is highly homologous to CLDN6.

[0265] The amino acid sequence and DNA sequence of human CLDN9 are disclosed in public databases and can be referenced using accession numbers such as NP_066192 (SEQ ID NO: 3) and NM_020982 (SEQ ID NO: 4) (both NCBI).

[0266] 2. Anti-CLDN6 Antibodies

[0267] An example of the anti-CLDN6 antibody of the present invention is an anti-CLDN6 antibody that recognizes a higher-order structure comprising two extracellular regions comprising the amino acid sequence of amino acids 29 to 81 and amino acid sequence 138 to 160 from the N-terminus of CLDN6 as shown in SEQ ID NO: 1 in the sequence listing and has internalization activity.

[0268] The anti-CLDN6 antibodies of the present invention are capable of targeting tumor cells, i.e., possessing properties such as the ability to recognize and bind to tumor cells, and the ability to be internalized by tumor cells. Therefore, the anti-CLDN6 antibodies of the present invention can be conjugated to a compound having anti-tumor activity via a linker to form an antibody-drug conjugate.

[0269] The anti-CLDN6 antibodies of the present invention may have anti-tumor activity.

[0270] (1) The anti-CLDN6 antibody of the present invention has the following characteristics (a) and (b).

[0271] (a) Recognize or bind to the CLDN family.

[0272] The antibodies of the present invention recognize the CLDN family. In other words, the antibodies of the present invention bind to the CLDN family. The antibodies of the present invention preferably bind to CLDN6, and more preferably specifically bind to CLDN6. Furthermore, the antibodies of the present invention may recognize or bind to CLDN9.

[0273] In the present invention, "specific recognition" or "specific binding" refers to binding that is not nonspecific adsorption. As a criterion for determining whether the binding is specific, for example, the dissociation constant (hereinafter referred to as "KD") can be cited. A preferred antibody of the present invention has a KD value of 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 Below M.

[0274] The binding of the antigen of the present invention to the antibody can be measured or determined by ELISA (enzyme linked immunosorbent assay), RIA (radio immune assay), Surface Plasmon Resonance (hereinafter referred to as "SPR") analysis, etc. The binding of the antigen expressed on the cell surface to the antibody can be measured by flow cytometry, etc.

[0275] (b) having an activity of internalizing in CLDN6 and / or CLDN9-expressing cells by binding to CLDN6 and / or CLDN9.

[0276] (2) The antibody according to (1) above, wherein the CLDN6 and / or CLDN9 is human CLDN6 and / or human CLDN9.

[0277] The anti-CLDN6 monoclonal antibodies of the present invention can be obtained by methods using hybridomas, etc. Examples of anti-CLDN6 monoclonal antibodies include mouse anti-CLDN6 antibodies B1 and C7. In the present invention, "B1" may also be referred to as "B1 antibody," and "C7" may also be referred to as "C7 antibody."

[0278] The base sequence of the heavy chain variable region of the B1 antibody is described in SEQ ID NO: 20, and the amino acid sequence is described in SEQ ID NO: 21. Furthermore, the base sequence of the light chain variable region of the B1 antibody is described in SEQ ID NO: 18, and the amino acid sequence is described in SEQ ID NO: 19.

[0279] The amino acid sequence of CDRH1 of the B1 antibody is described in SEQ ID NO: 9, the amino acid sequence of CDRH2 is described in SEQ ID NO: 10, the amino acid sequence of CDRH3 is described in SEQ ID NO: 11, the amino acid sequence of CDRL1 is described in SEQ ID NO: 5, the amino acid sequence of CDRL2 is described in SEQ ID NO: 6, and the amino acid sequence of CDRL3 is described in SEQ ID NO: 7.

[0280] The base sequence of the heavy chain variable region of the C7 antibody is described in SEQ ID NO: 24, and the amino acid sequence is described in SEQ ID NO: 25. Furthermore, the base sequence of the light chain variable region of the C7 antibody is described in SEQ ID NO: 22, and the amino acid sequence is described in SEQ ID NO: 23.

[0281] The amino acid sequence of CDRH1 of the C7 antibody is recorded in SEQ ID NO: 15, the amino acid sequence of CDRH2 is recorded in SEQ ID NO: 16, the amino acid sequence of CDRH3 is recorded in SEQ ID NO: 17, the amino acid sequence of CDRL1 is recorded in SEQ ID NO: 12, the amino acid sequence of CDRL2 is recorded in SEQ ID NO: 13, and the amino acid sequence of CDRL3 is recorded in SEQ ID NO: 14.

[0282] Examples of the anti-CLDN6 antibodies of the present invention include antibodies that bind to the same epitope as the B1 antibody or the C7 antibody. If the antibody binds to a portion of the peptide or a portion of the three-dimensional structure to which the B1 antibody or the C7 antibody binds, it can be determined that the antibody binds to the same epitope as the B1 antibody or the C7 antibody. Furthermore, by confirming that the antibody competes with the B1 antibody or the C7 antibody for binding to CLDN6 (i.e., that the antibody interferes with the binding of the B1 antibody or the C7 antibody to CLDN6), it can be determined that the antibody binds to the same epitope as the anti-CLDN6 antibody, even without determining the specific epitope sequence or structure. If the epitope is confirmed to be identical, the antibody can be strongly expected to have equivalent antigen-binding ability, biological activity, and / or internalization activity as the B1 antibody or the C7 antibody.

[0283] In addition to the above-mentioned monoclonal antibodies against CLDN6, the antibodies of the present invention also include genetically modified recombinant 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.

[0284] (1)Chimeric antibodies

[0285] Examples of chimeric antibodies include antibodies in which the variable and constant regions are of different species, for example, chimeric antibodies formed by joining the variable regions of an antibody derived from a mouse or rat to the constant regions derived from a human.

[0286] The chimeric antibody derived from the mouse anti-human CLDN6 antibody B1 antibody exemplified as the chimeric antibody of the present invention is an antibody comprising a heavy chain comprising a heavy chain variable region formed by the amino acid sequence set forth in SEQ ID NO: 21 and a light chain comprising a light chain variable region set forth in SEQ ID NO: 19, and may have any human-derived constant region.

[0287] Specific examples of chimeric antibodies derived from mouse anti-human CLDN6 antibody B1 include the chimeric antibody chB1 antibody (hereinafter also referred to as "chB1") derived from mouse anti-human CLDN6 antibody B1. Examples include antibodies in which the amino acid sequence of the chB1 antibody comprises a heavy chain having an amino acid sequence consisting of amino acid residues 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 21 to 234 of SEQ ID NO: 28 in the sequence listing.

[0288] It should be noted that in the heavy chain sequence shown in SEQ ID NO: 32 of the sequence listing, the amino acid sequence formed by amino acid residues 1 to 19 is a signal sequence, the amino acid sequence formed by amino acid residues 20 to 141 is a heavy chain variable region, and the amino acid sequence formed by residues 142 to 471 is a heavy chain constant region. Furthermore, in the light chain sequence shown in SEQ ID NO: 28 of the sequence listing, the amino acid sequence formed by amino acid residues 1 to 20 is a signal sequence, the amino acid sequence formed by amino acid residues 21 to 127 is a light chain variable region, and the amino acid sequence formed by amino acid residues 128 to 234 is a light chain constant region.

[0289] The amino acid sequences of the variable regions of the heavy and light chains of the chB1 antibody are described in SEQ ID NO: 34 and SEQ ID NO: 30 in the sequence listing.

[0290] The amino acid sequence of the heavy chain of the chB1 antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 33 of the sequence listing. The nucleotide sequence formed by nucleotides 1 to 57 of the nucleotide sequence shown in SEQ ID NO: 33 of the sequence listing encodes the signal sequence of the chB1 antibody heavy chain, the nucleotide sequence formed by nucleotides 58 to 423 of the nucleotide sequence shown in SEQ ID NO: 33 of the sequence listing encodes the heavy chain variable region of the chB1 antibody, and the nucleotide sequence formed by nucleotides 424 to 1413 of the nucleotide sequence shown in SEQ ID NO: 33 of the sequence listing encodes the heavy chain constant region of the chB1 antibody.

[0291] The base sequence of the heavy chain variable region of the chB1 antibody is described in SEQ ID NO: 35 in the sequence listing.

[0292] 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 formed by 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 formed by 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 formed by 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.

[0293] The base sequence of the light chain variable region of the chB1 antibody is described in SEQ ID NO: 31 in the sequence listing.

[0294] (2) Humanized antibodies

[0295] As humanized antibodies, there can be listed antibodies in which only the complementarity determining region (CDR) is incorporated into an antibody derived from a human antibody (see Nature (1986) 321, p.522-525); antibodies in which the sequence of the CDR and the amino acid residues of a portion of the framework are transplanted into a human antibody by a CDR transplantation method (WO90 / 07861); and antibodies in which the amino acid sequence of a portion of the CDR is changed while maintaining the ability to bind to the antigen (WO2012 / 075581, WO2011 / 084496, US2018 / 0501692). The amino acid sequence of the CDR can be determined by known methods such as Kabat's definition, Chothia's definition, Abm's definition, and IMGT, but the CDR in the present invention can be defined by any method.

[0296] However, humanized antibodies derived from the B1 antibody or the C1 antibody are not limited to specific humanized antibodies as long as all six CDR sequences of the B1 antibody or the C1 antibody are maintained and the CLDN6 binding activity is maintained. Furthermore, humanized antibody mutants in which the amino acid sequences of one to several (preferably one to two, more preferably one) CDRs are altered are not limited to specific humanized antibodies as long as they recognize the CLDN6 protein or have the CLDN6 protein binding activity of the antibody.

[0297] Examples of the anti-CLDN6 humanized antibodies or functional fragments thereof of the present invention include antibodies or functional fragments thereof that recognize the CLDN6 protein of the present invention or maintain the CLDN6 protein-binding activity of the antibody, comprising a heavy chain having a variable region and a light chain having a variable region, wherein the variable region of the heavy chain comprises: a CDRH1 composed of the amino acid sequence set forth in SEQ ID NO: 9 in the sequence listing or an amino acid sequence in which one or more (preferably one or two) amino acids of the amino acid sequence are substituted; a CDRH2 composed of the amino acid sequence set forth in SEQ ID NO: 10 in the sequence listing or an amino acid sequence in which one or more (preferably one or two) amino acids of the amino acid sequence are substituted; and a CDRH3 composed of the amino acid sequence set forth in SEQ ID NO: 11 in the sequence listing or an amino acid sequence in which one or more (preferably one or two) amino acids of the amino acid sequence are substituted; and a CDRH4 composed of the amino acid sequence set forth in SEQ ID NO: 12 in the sequence listing or an amino acid sequence in which one or more (preferably one or two) amino acids of the amino acid sequence are substituted. The light chain comprises a variable region comprising: a CDRL1 formed by the amino acid sequence shown in sequence number 5 of the sequence listing or an amino acid sequence in which 1 to several (preferably 1 to 2) amino acids of the amino acid sequence are substituted; a CDRL2 formed by the amino acid sequence shown in sequence number 6 of the sequence listing or an amino acid sequence in which 1 to several (preferably 1 to 2) amino acids of the amino acid sequence are substituted; and a CDRL3 formed by the amino acid sequence shown in sequence number 7 of the sequence listing or an amino acid sequence in which 1 to several (preferably 1 to 2) amino acids of the amino acid sequence are substituted.

[0298] As an example of amino acid substitution in the CDR of the above-mentioned anti-CLDN6 humanized antibody or its functional fragment, preferably one to several (preferably one to two) amino acid substitutions of the above-mentioned CDRL3 are listed, and an example can be given of the CDRL3 shown in SEQ ID NO: 8 in the sequence listing in which amino acid numbers 4 and 5 of SEQ ID NO: 7 in the sequence listing are substituted.

[0299] Examples of the heavy chain variable region of the humanized antibody having CDRH include the amino acid sequence shown in sequence number 54 of the sequence listing, the amino acid sequence shown in sequence number 58 of the sequence listing, and the amino acid sequence shown in sequence number 62 of the sequence listing; examples of the light chain variable region of the humanized antibody having CDRL include the amino acid sequence shown in sequence number 38 of the sequence listing, the amino acid sequence shown in sequence number 42 of the sequence listing, the amino acid sequence shown in sequence number 46 of the sequence listing, and the amino acid sequence shown in sequence number 50 of the sequence listing.

[0300] Preferred examples of humanized antibodies comprising the above-mentioned combination of heavy chain variable regions and light chain variable regions include: a humanized antibody comprising a heavy chain variable region formed by the amino acid sequence set forth in SEQ ID NO: 54 of the sequence listing and a light chain variable region formed by the amino acid sequence set forth in SEQ ID NO: 38 of the sequence listing; a humanized antibody comprising a heavy chain variable region formed by the amino acid sequence set forth in SEQ ID NO: 58 of the sequence listing and a light chain variable region formed by the amino acid sequence set forth in SEQ ID NO: 42 of the sequence listing; a humanized antibody comprising a heavy chain variable region formed by the amino acid sequence set forth in SEQ ID NO: 54 of the sequence listing and a light chain variable region formed by the amino acid sequence set forth in SEQ ID NO: 46 of the sequence listing; a humanized antibody comprising a heavy chain variable region formed by the amino acid sequence set forth in SEQ ID NO: 58 of the sequence listing and a light chain variable region formed by the amino acid sequence set forth in SEQ ID NO: 50 of the sequence listing; and a humanized antibody comprising a heavy chain variable region formed by the amino acid sequence set forth in SEQ ID NO: 62 of the sequence listing and a light chain variable region formed by the amino acid sequence set forth in SEQ ID NO: 46 of the sequence listing.

[0301] Examples of full-length sequences of humanized antibodies comprising a combination of the above-mentioned heavy chain variable region and light chain variable region include: a humanized antibody (H1L1) comprising a heavy chain comprising the amino acid sequence represented by amino acid numbers 20 to 471 of SEQ ID NO: 52 of the sequence listing and a light chain comprising the amino acid sequence represented by amino acid numbers 21 to 234 of SEQ ID NO: 36 of the sequence listing; a humanized antibody (H2L2) comprising a heavy chain comprising the amino acid sequence represented by amino acid numbers 20 to 471 of SEQ ID NO: 56 of the sequence listing and a light chain comprising the amino acid sequence represented by amino acid numbers 21 to 234 of SEQ ID NO: 40 of the sequence listing; and a humanized antibody (H3L3) comprising a heavy chain comprising the amino acid sequence represented by amino acid numbers 20 to 471 of SEQ ID NO: 52 of the sequence listing and a light chain comprising the amino acid sequence represented by amino acid numbers 21 to 234 of SEQ ID NO: 40 of the sequence listing. A humanized antibody (H1L3) comprising a heavy chain composed of the amino acid sequence represented by amino acid numbers 20 to 471 of SEQ ID NO: 56 of the sequence listing and a light chain composed of the amino acid sequence represented by amino acid numbers 21 to 234 of SEQ ID NO: 48 of the sequence listing; or a humanized antibody (H3L3) comprising a heavy chain composed of the amino acid sequence represented by amino acid numbers 20 to 471 of SEQ ID NO: 60 of the sequence listing and a light chain composed of the amino acid sequence represented by amino acid numbers 21 to 234 of SEQ ID NO: 44 of the sequence listing.

[0302] It should be noted that, in the heavy chain amino acid sequence shown in sequence numbers 52, 56 or 60 of the sequence listing, the amino acid sequence formed by amino acid residues 1 to 19 is a signal sequence, the amino acid sequence formed by amino acid residues 20 to 141 is a heavy chain variable region, and the amino acid sequence formed by amino acid residues 142 to 471 is a heavy chain constant region.

[0303] In addition, in the light chain amino acid sequence shown in sequence numbers 36, 40, 44 or 48 of the sequence listing, the amino acid sequence formed by amino acid residues 1 to 20 is a signal sequence, the amino acid sequence formed by amino acid residues 21 to 127 is a light chain variable region, and the amino acid sequence formed by amino acid residues 128 to 234 is a light chain constant region.

[0304] As described below, the carboxyl termini of the heavy chains of the above-mentioned humanized antibodies H1L1, H2L2, H1L3, H2L4, and H3L3 may be deleted by one or two amino acids, and such deletions are also encompassed by the present invention.

[0305] Examples of the heavy chain of the deletion variant include heavy chains comprising the amino acid sequences described in amino acid numbers 20 to 470 of SEQ ID NOs: 52, 56, and 60 in the sequence listing.

[0306] Examples of the deletion bodies include: a humanized antibody (H1L1) comprising a heavy chain composed of the amino acid sequence shown in amino acid numbers 20 to 470 of sequence number 52 of the sequence listing and a light chain composed of the amino acid sequence shown in amino acid numbers 21 to 234 of sequence number 36 of the sequence listing; a humanized antibody (H2L2) comprising a heavy chain composed of the amino acid sequence shown in amino acid numbers 20 to 470 of sequence number 56 of the sequence listing and a light chain composed of the amino acid sequence shown in amino acid numbers 21 to 234 of sequence number 40 of the sequence listing; a humanized antibody (H2L2) comprising a heavy chain composed of the amino acid sequence shown in amino acid numbers 20 to 470 of sequence number 52 of the sequence listing and a light chain composed of the amino acid sequence shown in amino acid numbers 21 to 234 of sequence number 40 of the sequence listing; A humanized antibody (H1L3) comprising a light chain composed of the amino acid sequence shown in amino acid numbers 21 to 234 of SEQ ID NO: 44 of the sequence listing; a humanized antibody (H2L4) comprising a heavy chain composed of the amino acid sequence shown in amino acid numbers 20 to 470 of SEQ ID NO: 56 of the sequence listing and a light chain composed of the amino acid sequence shown in amino acid numbers 21 to 234 of SEQ ID NO: 48 of the sequence listing; or a humanized antibody (H3L3) comprising a heavy chain composed of the amino acid sequence shown in amino acid numbers 20 to 470 of SEQ ID NO: 60 of the sequence listing and a light chain composed of the amino acid sequence shown in amino acid numbers 21 to 234 of SEQ ID NO: 44 of the sequence listing.

[0307] The base sequence encoding the heavy chain amino acid sequence of the above-mentioned humanized antibody H1L1 is the polynucleotide set forth in SEQ ID NO: 53, and the base sequence encoding the light chain amino acid sequence is the polynucleotide set forth in SEQ ID NO: 37. The base sequence encoding the heavy chain amino acid sequence of the humanized antibody H2L2 is the polynucleotide set forth in SEQ ID NO: 57, and the base sequence encoding the light chain amino acid sequence is the polynucleotide set forth in SEQ ID NO: 41. The base sequence encoding the heavy chain amino acid sequence of the humanized antibody H1L3 is the polynucleotide set forth in SEQ ID NO: 53, and the base sequence encoding the light chain amino acid sequence is the polynucleotide set forth in SEQ ID NO: 45. The base sequence encoding the heavy chain amino acid sequence of the humanized antibody H2L4 is the polynucleotide set forth in SEQ ID NO: 57, and the base sequence encoding the light chain amino acid sequence is the polynucleotide set forth in SEQ ID NO: 49. The base sequence encoding the heavy chain amino acid sequence of the humanized antibody H3L3 is the polynucleotide set forth in SEQ ID NO: 61, and the base sequence encoding the light chain amino acid sequence is the polynucleotide set forth in SEQ ID NO: 45.

[0308] The base sequence encoding the amino acid sequence of the heavy chain variable region of the humanized antibody H1L1 is the polynucleotide set forth in SEQ ID NO: 55, the base sequence encoding the amino acid sequence of the light chain variable region is the polynucleotide set forth in SEQ ID NO: 39, the base sequence encoding the amino acid sequence of the heavy chain variable region of the humanized antibody H2L2 is the polynucleotide set forth in SEQ ID NO: 59, the base sequence encoding the amino acid sequence of the light chain variable region is the polynucleotide set forth in SEQ ID NO: 43, and the base sequence encoding the amino acid sequence of the heavy chain variable region of the humanized antibody H1L3 is the polynucleotide set forth in SEQ ID NO: 55. The base sequence encoding the amino acid sequence of the light chain variable region is the polynucleotide set forth in sequence number 47, the base sequence encoding the amino acid sequence of the heavy chain variable region of the humanized antibody H2L4 is the polynucleotide set forth in sequence number 59, the base sequence encoding the amino acid sequence of the light chain variable region is the polynucleotide set forth in sequence number 51, the base sequence encoding the amino acid sequence of the heavy chain variable region of the humanized antibody H3L3 is the polynucleotide set forth in sequence number 63, and the base sequence encoding the amino acid sequence of the light chain variable region is the polynucleotide set forth in sequence number 47.

[0309] It should be noted that the nucleotide sequence formed by nucleotides 1 to 57 of each nucleotide sequence shown in sequence numbers 53, 57, and 61 of the sequence listing encodes the signal sequence of the humanized antibody heavy chain, the nucleotide sequence formed by nucleotides 58 to 423 encodes the amino acid sequence of the humanized antibody heavy chain variable region, and the nucleotide sequence formed by nucleotides 424 to 1413 encodes the antibody heavy chain constant region.

[0310] In addition, the nucleotide sequence formed by nucleotides 1 to 60 of each nucleotide sequence shown in sequence numbers 37, 41, 45, and 49 of the sequence listing encodes the signal sequence of the humanized antibody light chain, the nucleotide sequence formed by nucleotides 61 to 381 encodes the amino acid sequence of the humanized antibody light chain variable region, and the nucleotide sequence formed by nucleotides 382 to 702 encodes the antibody light chain constant region.

[0311] Antibodies having an amino acid sequence 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 an antibody comprising the combination of the heavy chain variable region and the light chain variable region, or an antibody comprising the combination of the heavy chain and the light chain, are also included in the antibodies of the present invention as long as they have binding activity to CLDN6.

[0312] Furthermore, antibodies comprising the combination of the above-described heavy chain variable region and light chain variable region, or antibodies comprising a CDR consisting of the same amino acid sequence as the CDR of an antibody comprising the combination of the above-described heavy chain and light chain, and having an amino acid sequence 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 after excluding the amino acid sequence of the CDR of the antibody, are also included in the antibodies of the present invention as long as they have binding activity to CLDN6.

[0313] Furthermore, by combining amino acid sequences that substitute, delete, or add one to several amino acid residues in the amino acid sequence of the heavy chain or light chain, antibodies having equivalent biological activity to the above-mentioned antibodies can also be selected. In addition, as amino acid substitutions in this specification, conservative amino acid substitutions are preferred (WO2013154206).

[0314] Conservative amino acid substitutions refer to substitutions made within a certain group of amino acids related to the amino acid side chains. Such amino acid substitutions are preferably made within a range that does not degrade the properties of the substance having the original amino acid sequence.

[0315] The homology between two amino acid sequences can be determined using the Blast algorithm version 2.2.2 (Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, 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) with the default parameters. The Blast algorithm can also be accessed online at www.ncbi.nlm.nih.gov / blast.

[0316] (3) Human antibodies

[0317] As antibodies of the present invention, human antibodies that bind to CLDN6 and / or CLDN9 can also be listed. Anti-CLDN6 and / or CLDN9 human antibodies refer to human antibodies that have only the gene sequence of the antibody derived from human chromosomes. As for anti-CLDN6 human antibodies, it is also known that they 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 andProteomics(2002),1(2),p.189-203, Ophthalmology(2002)109(3),p.427-431, WO92 / 01047, WO92 / 20791, W O93 / 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).

[0318] The anti-HER2 antibody used in the present invention is described below.

[0319] The anti-HER2 antibody of the present invention has the following characteristics.

[0320] (1) An anti-HER2 antibody characterized by having the following properties.

[0321] (a) Specific binding to HER2.

[0322] (b) It has the activity of internalizing into HER2-expressing cells by binding to HER2.

[0323] (2) The antibody according to (1) above, which binds to the extracellular domain of HER2.

[0324] (3) The antibody according to (1) or (2) above, which is a monoclonal antibody.

[0325] (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.

[0326] (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.

[0327] (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 causes a decrease in ADCC and / or CDC activity.

[0328] (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 shown in EU Index are substituted with alanine.

[0329] (8) The antibody according to any one of (1) to (5) above, comprising a heavy chain consisting of the amino acid sequence of SEQ ID NO: 65 and a light chain consisting of the amino acid sequence of SEQ ID NO: 64.

[0330] (9) An antibody according to any one of (1) to (3) and (5) to (7) above, which is an antibody comprising a heavy chain variable region formed by the amino acid sequence described by amino acid numbers 20 to 139 of sequence number 75 and a light chain variable region formed by the amino acid sequence described by amino acid numbers 21 to 127 of sequence number 73.

[0331] (10) An antibody according to any one of (1) to (3), (5) to (7) and (9) above, which is an antibody comprising a heavy chain formed by the amino acid sequence described by amino acid numbers 20 to 469 of sequence number 75 and a light chain formed by the amino acid sequence described by amino acid numbers 21 to 234 of sequence number 73.

[0332] (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.

[0333] (12) An antibody according to any one of (1) to (5), (8) and (11) above, comprising a heavy chain formed by the amino acid sequence described by amino acid numbers 1 to 449 of sequence number 65 and a light chain formed by the amino acid sequence described by amino acid numbers 1 to 214 of sequence number 64.

[0334] (13) An antibody according to any one of (1) to (3), (5) to (7) and (9) to (11) above, comprising a heavy chain formed by the amino acid sequence described by amino acid numbers 20 to 468 of sequence number 75 and a light chain formed by the amino acid sequence described by amino acid numbers 21 to 234 of sequence number 73.

[0335] (14) An antibody obtained by a method for producing the antibody comprising the following steps: 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 selecting the target antibody from the culture obtained by the step.

[0336] In the present invention, an antibody in which leucine at positions 234 and 235 of the heavy chain constant region of trastuzumab (SEQ ID NO: 65) as shown in the EU Index is substituted with alanine is referred to as a trastuzumab mutant.

[0337] Furthermore, examples of the anti-HER2 antibody of the present invention include trastuzumab mutant 2. The heavy chain amino acid sequence of trastuzumab mutant 2 is described in SEQ ID NO: 77, and the light chain amino acid sequence is described in SEQ ID NO: 76.

[0338] The antibodies of the present invention also include modified forms of antibodies. Such modified forms refer to modified forms obtained by chemically or biologically modifying the antibodies of the present invention. Chemically modified forms include chemical modifications such as the bonding of a chemical moiety to an amino acid backbone, chemical modifications such as N-linked or O-linked carbohydrate chains, and the like. Biologically modified forms include modified forms that have undergone post-translational modifications (e.g., N-linked glycosylation or O-linked glycosylation, processing of the N-terminus or C-terminus, deamidation, isomerization of aspartic acid, oxidation of methionine), and modified forms in which a methionine residue is added to the N-terminus by expression using prokaryotic host cells. In addition, labeled forms such as enzyme labels, fluorescent labels, affinity labels, etc., which are labeled to enable detection or separation of the antibodies or antigens of the present invention, are also included in the meaning of modified forms. Such modified forms of the antibodies of the present invention are useful for improving the stability and blood retention of antibodies, reducing antigenicity, detecting or separating antibodies or antigens, and the like.

[0339] In addition, by regulating the sugar chain modifications (glycosylation, defucosylation, etc.) bound to the antibodies of the present invention, antibody-dependent cellular cytotoxicity can be enhanced. As a technology for regulating the sugar chain modifications of antibodies, WO1999 / 54342, WO2000 / 61739, WO2002 / 31140, WO2007133855, WO2013120066, etc. are known, but are not limited thereto. The antibodies of the present invention also include antibodies in which such sugar chain modifications are regulated.

[0340] The modification can be made at any position or at a desired position of the antibody or its functional fragment, and the same or two or more different modifications can be made at one or two or more positions.

[0341] In the present invention, the term "modified antibody fragment" also includes "fragments of modified antibody fragments".

[0342] After temporarily isolating the antibody gene, when it is introduced into an appropriate host to produce the antibody, a combination of an appropriate host and an expression vector can be used. As a specific example of the antibody gene, a gene encoding a heavy chain sequence of the antibody described in this specification and a gene encoding a light chain sequence can be listed. When transforming the host cell, the heavy chain sequence gene and the light chain sequence gene can be inserted into the same expression vector or into different expression vectors.

[0343] 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 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).

[0344] When prokaryotic cells are used, examples include Escherichia coli and Bacillus subtilis.

[0345] Antibodies are obtained by introducing the target antibody gene into these cells through transformation and culturing the transformed cells in vitro. During this culture, yields may vary depending on the antibody sequence, and antibodies with equivalent binding activity can be selected based on yield as an indicator for easy pharmaceutical production. Thus, the antibodies of the present invention also include antibodies obtained by the following antibody production method, characterized in that the method comprises: a step of culturing the transformed host cells; and a step of extracting the target antibody or a functional fragment of the antibody from the culture obtained in this step.

[0346] The antibody gene is preferably a polynucleotide comprising the polynucleotide described in any one of the following (a) to (e).

[0347] (a) A combination of a polynucleotide encoding the heavy chain amino acid sequence of any one of the B1 or C7 antibody, chB1 antibody, and humanized antibodies H1L1, H2L2, H1L3, H2L4, H3L3, trastuzumab, and mutants thereof, and a polynucleotide encoding the light chain amino acid sequence.

[0348] (b) A combination of a polynucleotide encoding the heavy chain amino acid sequence of CDRH1 to CDRH3 of any one of the B1 or C7 antibody, chB1 antibody, and humanized antibodies H1L1, H2L2, H1L3, H2L4, H3L3, trastuzumab, and mutants thereof, and a polynucleotide encoding the light chain amino acid sequence of CDRL1 to CDRL3.

[0349] (c) A combination of a polynucleotide encoding a heavy chain amino acid sequence comprising the amino acid sequence of the heavy chain variable region of any one of the B1 or C7 antibody, chB1 antibody, and the humanized antibodies H1L1, H2L2, H1L3, H2L4, H3L3, trastuzumab, and mutants thereof, and a polynucleotide encoding a light chain amino acid sequence comprising the amino acid sequence of the light chain variable region.

[0350] (d) A polynucleotide that hybridizes under stringent conditions with a nucleotide consisting of a polynucleotide complementary to the polynucleotide according to any one of (a) to (c) and encodes the amino acid sequence of an antibody that binds to CDLN6 or HER2.

[0351] And (e) a polynucleotide encoding an 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 acids are substituted, deleted, added or inserted into the polynucleotide of any one of (a) to (c), and encoding the amino acid sequence of an antibody that binds to CLDN6 or HER2.

[0352] The present invention includes a nucleotide encoding the antibody of the present invention or a functional fragment thereof or a modified form thereof, a recombinant vector into which the gene is inserted, and a cell into which the gene or the vector is introduced.

[0353] Furthermore, the present invention also includes a method for producing an antibody, a functional fragment thereof, or a modified form thereof, comprising the steps of culturing the cells and recovering the antibody, a functional fragment thereof, or a modified form thereof from the culture.

[0354] It should be noted that it is known that the lysine residue at the carboxyl terminus of the heavy chain of an antibody produced in mammalian cultured cells is deleted (Journal of Chromatography A, 705: 129-134 (1995)). It is also known that the two amino acid residues of glycine and lysine at the carboxyl terminus of the same heavy chain are deleted, and the proline residue newly located at the carboxyl terminus is amidated (Analytical Biochemistry, 360: 75-83 (2007)). However, the deletion and modification of these heavy chain sequences do not affect the antigen binding ability and effector function of the antibody (complement activation, antibody-dependent cellular cytotoxicity, etc.). Therefore, the antibodies of the present invention also include antibodies and functional fragments of the antibodies subjected to the modification, deletions with one or two amino acids deleted at the carboxyl terminus of the heavy chain, and amidated deletions (e.g., 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 deletions at the carboxyl terminus of the heavy chain of the antibody of the present invention are not limited to the above-mentioned types. The two heavy chains comprising the antibodies of the present invention may be any one selected from the group consisting of full-length and the aforementioned deletions, or any two of them may be combined. The quantitative ratio of each deletion may be affected by the type and culture conditions of the mammalian cultured cells producing the antibodies of the present invention. However, as a major component of the antibodies of the present invention, one example includes a deletion of a single amino acid residue at the carboxyl terminus in both heavy chains.

[0355] The obtained antibody can be refined to uniformity. The separation and purification of the antibody can be carried out using the separation and purification methods commonly used for proteins. If appropriate selection and combination of methods such as column chromatography, filter filtration, ultrafiltration, salting out, dialysis, preparative polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, etc. are made, the antibody can be separated and purified (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 is not limited thereto.

[0356] <N297 sugar chain>

[0357] In recent years, methods have been reported for remodeling heterogeneous antibody glycoproteins and uniformly introducing sugar chains having functional groups by enzymatic reactions or the like (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).

[0358] The sugar chain reconstruction of the present invention first involves using a hydrolase to remove the heterogeneous sugar chains added to a protein (e.g., an antibody) leaving only the terminal GlcNAc residues, thereby creating a homogeneous protein portion to which GlcNAc has been added (hereinafter referred to as the "acceptor"). Next, a separate, arbitrary sugar chain (hereinafter referred to as the "donor") is prepared, and the acceptor and donor are linked using a glycosyltransferase. This allows the synthesis of a uniform glycoprotein with any desired sugar chain structure.

[0359] In the present invention, a "sugar chain" refers to a structural unit composed of two or more monosaccharides linked by glycosidic bonds. Specific monosaccharides or sugar chains are sometimes represented by abbreviations, such as "GlcNAc-" and "MSG-." When these abbreviations are used in structural formulas, oxygen atoms or nitrogen atoms at the reducing end that are part of the glycosidic bond to other structural units are not included in the abbreviation representing the sugar chain, unless otherwise specified.

[0360] In the present invention, unless otherwise specified, for convenience, in the description of monosaccharides as the basic unit of the sugar chain, the carbon atom that is bonded to the oxygen atom constituting the ring and directly bonded to the hydroxyl group (or the oxygen atom belonging to the glycosidic bond) in its ring structure is denoted as position 1 (position 2 only in sialic acid). The names of the example compounds are denoted by the overall chemical structure, and this rule does not necessarily apply.

[0361] In the present invention, when a sugar chain is described by symbols (e.g., GLY, SG, MSG, GlcNAc, etc.), unless otherwise defined, the symbol includes the carbon at the reducing terminal, but excludes N or O belonging to the N- or O-glycosidic bond.

[0362] In the present invention, unless otherwise specified, the partial structure of the amino acid side chain when linked to a sugar chain is represented by the side chain portion in parentheses, for example, as described in "(SG-)Asn".

[0363] The antibody-drug conjugate of the present invention is represented by the following formula:

[0364]

[0365] The antibody Ab or its functional fragment binds to L via the N297 sugar chain or the remodeled N297 sugar chain, preferably binds to L via the remodeled N297 sugar chain of Ab.

[0366] The sugar chain of Ab of the present invention is an N-linked sugar chain or an O-linked sugar chain, and an N-linked sugar chain is preferred.

[0367] N-linked sugar chains are bound to the amino acid side chains of antibodies through N-glycosidic bonds, and O-linked sugar chains are bound to the amino acid side chains of antibodies through O-glycosidic bonds.

[0368] IgG is known to have a highly conserved N-linked sugar chain at asparagine residue 297 (hereinafter referred to as "Asn297 or N297") in the Fc region of its heavy chain, which contributes to the activity and dynamics of the antibody molecule (Biotechnol. Prog., 2012, 28, 608-622, Anal. Chem., 2013, 85, 715-736).

[0369] The amino acid sequence in the constant region of IgG is highly conserved. In Edelman et al. (Proc. Natl. Acad. Sci. USA, Vol. 63, No. 1 (May 15, 1969), p. 78-85), each amino acid is identified by Eu numbering (Eu INDEX). For example, Asn297, where an N-linked sugar chain is added to the Fc region, corresponds to position 297 in Eu numbering. Even if the actual amino acid position changes due to molecular fragmentation or region deletion, the amino acid can be uniquely identified by using Eu numbering.

[0370] In the antibody-drug conjugate of the present invention, the antibody or its functional fragment is preferably bound to L via a sugar chain bound to the side chain of Asn297 thereof (hereinafter referred to as "N297 sugar chain"). More preferably, the antibody or its functional fragment is bound to L via the N297 sugar chain, and the N297 sugar chain is a remodeled sugar chain.

[0371] SGP is the abbreviation of Sialyl Glycopeptide, which is a representative of N-linked complex sugar chains. SGP can be obtained by isolating and refining it from egg yolk according to the method described in WO2011 / 0278681, for example. In addition, refined products of SGP are commercially available (Tokyo Chemical Industry Co., Ltd., Fushimi Pharmaceutical Co., Ltd.) and can be purchased. Disialooctasaccharide (Tokyo Chemical Industry Co., Ltd.) consisting only of a sugar chain with one reducing end GlcNAc missing from the sugar chain portion of SG (hereinafter referred to as "SG (10)") is commercially available.

[0372] In the present invention, a sugar chain structure in which the non-reducing terminal sialic acid is missing only in any one of the β-Man branches of SG (10) is referred to as MSG (9), a sugar chain having sialic acid only in the 1st to 3rd sugar chains of the branch is referred to as MSG1, and a sugar chain having sialic acid only in the 1st to 6th sugar chains of the branch is referred to as MSG2.

[0373] The reconstructed N297 sugar chain of the present invention is N297-(Fuc)MSG1, N297-(Fuc)MSG2, or 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, and more preferably N297-(Fuc)MSG1 or N297-(Fuc)MSG2.

[0374] N297-(Fuc)MSG1 is represented by the following structural formula or sequence formula.

[0375]

[0376] In the above formula, the wavy line represents the binding to Asn297 of the antibody, and L(PEG) represents *-(CH2CH2-O)n 5 -CH2CH2-NH-, the amino group at the right end is bonded to the 2-carboxylic acid of the non-reducing end of the 1-3 chain side of the β-Man branch of the N297 sugar chain via an amide bond, and the asterisk * at the left end indicates that it is bonded to the 1- or 3-nitrogen atom on the 1,2,3-triazole ring of Lb of the linker L, n 5 It is an integer of 2 to 10, preferably an integer of 2 to 5.

[0377] N297-(Fuc)MSG2 is represented by the following structural formula or sequence formula.

[0378]

[0379] In the above formula, the wavy line represents the binding to Asn297 of the antibody, and L(PEG) represents *-(CH2CH2-O)n 5 -CH2CH2-NH-, the amino group at the right end is bonded to the 2-carboxylic acid of the non-reducing end of the sialic acid on the 1-6 chain side of the β-Man branch of the N297 sugar chain via an amide bond, and the asterisk * at the left end indicates that it is bonded to the 1- or 3-nitrogen atom on the 1,2,3-triazole ring of Lb of the linker L, n 5 It is an integer of 2 to 10, preferably an integer of 2 to 5.

[0380] N297-(Fuc)SG is represented by the following structural formula or sequence formula.

[0381]

[0382]

[0383] In the above formula, the wavy line represents the binding to Asn297 of the antibody, and L(PEG) represents *-(CH2CH2-O)n 5 -CH2CH2-NH-, the amino group at the right end is bonded to the 2-carboxylic acid of the two non-reducing ends of the 1-3 chain side and the 1-6 chain side of the β-Man branch of the N297 sugar chain via an amide bond, and the asterisk * at the left end indicates that it is bonded to the 1- or 3-nitrogen atom on the 1,2,3-triazole ring of Lb of the linker L, n 5 It is an integer of 2 to 10, preferably an integer of 2 to 5.

[0384] 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 thus the antibody-drug conjugate is a molecule to which two drug linkers (-LD) are bound (the above m 1 =1) (refer to Figure 1 ).

[0385] For example, in Example 27: ADC5, the N297 sugar chain is N297-(Fuc)MSG1.

[0386] 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 thus the antibody-drug conjugate is a molecule to which four drug linkers (-LD) are bound (the above m 1 =2).

[0387] The N297 sugar chain is preferably N297-(Fuc)MSG1, N297-(Fuc)MSG2, or N297-(Fuc)SG, more preferably N297-(Fuc)MSG1 or N297-(Fuc)MSG2, and most preferably N297-(Fuc)MSG1.

[0388] 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, ADCs of uniform quality can be obtained.

[0389] The present invention provides a method for producing a sugar chain-remodeled antibody or a functional fragment thereof, comprising the following steps i) to iii).

[0390] i) culturing the host cells (e.g., animal cells (CHO cells, etc.)) and extracting the target antibody from the obtained culture; ii) treating the antibody obtained in step i) with a hydrolase to produce an antibody whose N297 sugar chain is (Fucα1,6)GlcNAc ((Fucα1,6)GlcNAc-antibody) Figure 3 A); preferably also including: a step of purifying the (Fucα1,6)GlcNAc-antibody by a step including purifying the reaction solution using a hydroxyapatite column; and iii) a step of reacting the (Fucα1,6)GlcNAc-antibody with a sugar chain donor molecule in the presence of a glycosyltransferase to synthesize a sugar chain-remodeled antibody in which an azide group is introduced into sialic acid, wherein the sugar chain donor molecule is obtained by introducing a PEG linker - (N3-L (PEG)) having an azide group into the carbonyl group of the 2-position carboxylic acid of sialic acid of MSG (9) or SG (10), and oxazolidinylating the reducing end.

[0391] Furthermore, the sugar chain-remodeled antibodies, functional fragments thereof, or modified forms thereof obtained by the above-mentioned production methods are also encompassed by the present invention.

[0392] The production intermediate of the present antibody-drug conjugate has an alkyne structure, such as DBCO (dibenzocyclooctyne), that reacts with an azide group (see Example 2-1, Compound 3-14). Therefore, the antibody-drug conjugate of the present invention can be produced by reacting this production intermediate with an MSG1-, MSG2-, or SG-type sugar-chain-remodeled antibody or a functional fragment thereof, in which an azide-containing PEG linker has been introduced into the sialic acid of the sugar chain obtained in steps i) to iii).

[0393] In the present invention, the N297 sugar chain, GlcNAc-(Fucα1,6)GlcNAc, with fucose added to the reducing terminus, is derived from an antibody produced in animal cells. In contrast, the sugar chain on the non-reducing terminus is preferably reconstructed into a sugar chain structure similar to that of MSG (MSG1, MSG2) or SG described above. L(PEG) is conjugated to the carboxylic acid bound to the 2-position of the sialic acid at the non-reducing terminus.

[0394] Such sugar chain remodeled antibodies having MSG (MSG1, MSG2) or SG type N297 sugar chains can be prepared by, for example, the method described in WO2013 / 120066. Figure 3When an antibody is produced as a recombinant protein using animal cells as a host according to a known method (step i above), although the N297 sugar chain has an N-linked sugar chain structure with fucose added as a basic structure, a mixture of antibodies or fragments thereof having sugar chains composed of various structures including a non-reducing terminal structure and various modifications of the constituent sugars is obtained ( Figure 3 A IV). Thus, antibodies produced by animal cells are treated with a hydrolase such as EndoS, and the glycosidic bond between the GlcNAcβ1-4GlcNAc of the chitobiose structure at the reducing end is hydrolyzed, thereby obtaining an antibody molecule having a single sugar chain structure having only (Fucα1,6)GlcNA as the N297 sugar chain (referred to as "(Fucα1,6)GlcNAc-antibody", see Figure 2 A)( Figure 3 A) (the above-mentioned step ii)).

[0395] As the enzyme used in the hydrolysis reaction of the N297 sugar chain, Endo S or a mutant enzyme that retains its hydrolysis activity can be used.

[0396] The (Fucα1,6)GlcNAc-antibody obtained by the above-mentioned hydrolysis reaction is used as a sugar chain acceptor molecule, and a glycosyltransferase such as EndoS D233Q or EndoS D233Q / Q303L mutant (WO2017010559, etc.) is used to react with MSG (MSG1, MSG2) or SG type sugar chain donor molecules, thereby obtaining an antibody having MSG (MSG1, MSG2) or SG type N297 sugar chains containing the above-mentioned structure (see Figure 2 B)( Figure 3 B) (the above-mentioned step iii).

[0397] The number of drug binding molecules per drug linker in the antibody-drug conjugate is m. 1 When the ratio is 1, sugar chain donor molecules containing MSG (MSG1, MSG2) are used as sugar chains. These sugar chains can be prepared using commercially available monosialo-Asn-free (1S2G / 1G2S-10NC-Asn, Sugar Chain Engineering Research Institute, Inc., hereinafter referred to as "(MSG-)Asn") as a starting material. (MSG-)Asn1 or (MSG2-)Asn can be separated and used as described in Example 3. Alternatively, a mixture can be used without separation.

[0398] The number of drug binding molecules per drug linker in the antibody-drug conjugate is m. 1When γ is 2, a sugar chain donor molecule having SG(10) is used as the sugar chain in the sugar chain transfer reaction. Such SG(10) sugar chains can be obtained, for example, by hydrolysis of SGP, or commercially available SG(10) sugar chains such as disialyl octaose (Tokyo Chemical Industry Co., Ltd.) can also be used.

[0399] The MSG (MSG1, MSG2) or SG type sugar chain contained in the donor molecule has an azide group-containing PEG linker (N3-L(PEG)) at the 2-position of the sialic acid.

[0400] The GlcNAc at the reducing end of the MSG (MSG1, MSG2) or SG type sugar chain contained in the donor molecule is preferably activated by, for example, oxazolidinylation by treatment with 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride (J. Org. Chem., 2009, 74(5), 2210-2212).

[0401] As the enzyme (glycosyltransferase) used in the sugar chain transfer reaction, any enzyme can be used as long as it has the activity of transferring complex sugar chains to N297 sugar chains. Preferably, the variant EndoS D233Q is substituted with Gln at Asp 233 of EndoS, thereby inhibiting the hydrolysis reaction. Sugar chain transfer reactions using EndoS D233Q are described in WO2013 / 120066 and other publications. In addition, variant enzymes such as EndoS D233Q / Q303L, which are further mutated EndoS D233Q, can also be used (WO2017010559).

[0402] Purification of antibodies after sugar chain remodeling (glycolysis and sugar chain transfer reaction) is typically performed using gel filtration chromatography, ion exchange chromatography, affinity chromatography, and the like for the purpose of separating them from low-molecular-weight compounds and enzymes used in the reaction. Additional purification using a hydroxyapatite column is also possible. Specifically, the present invention provides a method for producing an antibody-drug conjugate, wherein the purification step of the intermediate from the reaction solution after sugar hydrolysis of the antibody also includes a purification step using a hydroxyapatite column. According to a report on sugar chain remodeling (JACS. 2012, 134, 12308-12318, Angew. Chem. Int. Ed. 2016, 55, 2361-2367), the reaction solution after treating the antibody with a hydrolase was purified using only a protein A column (affinity chromatography column). However, this purification method has been shown to be unable to completely remove the hydrolase (EndoS, etc.), and residual enzymes can affect the subsequent sugar chain transfer reaction. Here, we studied the purification method and found that the reaction solution treated with hydrolase for antibody was purified in the order of protein A column and hydroxyapatite column (CHT column, Bio-Rad Laboratories, Inc.). This eliminated the influence of residual enzyme and improved the reaction efficiency of the next sugar chain transfer reaction.

[0403] The antibody-drug conjugate of the present invention is most preferably an antibody-drug conjugate selected from the following group.

[0404]

[0405]

[0406]

[0407] In the above structural formulas, m 1 represents an integer 1 or 2 (preferably m 1is an integer of 1), and the antibody Ab is an anti-CLDN6 antibody, an anti-CLDN9 antibody, an anti-CLDN6 / CLDN9 antibody, an anti-HER2 antibody, an anti-HER3 antibody, an anti-DLL3 antibody, an anti-A33 antibody, an anti-CanAg antibody, an anti-CD19 antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD25 antibody, an anti-CD30 antibody, an anti-CD33 antibody, an anti-CD37 antibody, an anti-CD56 antibody, an anti-CD70 antibody, an anti-CD98 antibody, an anti-B7-H3 antibody, an anti-TROP2 antibody, an anti-CEA antibody, an anti-Cripto antibody, an anti-EphA2 antibody, an anti-FGFR2 antibody (WO201315206, etc.), an anti-G250 antibody, an anti-MUC1 antibody (WO2011012309, etc.), an anti-GPNMB antibody, an anti-Integrin antibody, an anti-PSMA antibody, an anti-Tenascin-C antibody, an anti-SLC44 antibody, etc. A4 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 represents any one of N297-(Fuc)MSG1, N297-(Fuc)MSG2, or a mixture thereof, or N297-(Fuc)SG (preferably N297-(Fuc)MSG1); L(PEG) represents *-(CH2CH2-O)3-CH2CH2-NH-; the amino group at the right end is bonded to the 2-carboxylic acid of the non-reducing end of the 1-3 chain side or / and the 1-6 chain side (preferably the 1-3 chain side) of the β-Man branch of the N297 sugar chain via an amide bond; and the asterisk at the left end indicates binding to the 1- or 3-nitrogen atom on the triazole ring in the structural formula.

[0408] For the sake of convenience, the most preferred antibody-drug conjugates are described as having two or four (m 2 = 1 or 2) "N297 sugar chain is bound to the nitrogen atom at position 1 on the triazole ring of Lb in L '-(N297 sugar chain)-L-D' ('(N297 sugar chain)-(N1Lb)L-D'), or having 2 or 4 (m 2 = 1 or 2) "the structure of '-(N297 sugar chain)-L-D' ('(N297 sugar chain)-(N3Lb)L-D') bound to the nitrogen atom at position 3", but also includes the structure of '(N297 sugar chain)-(N1Lb)L-D' (in m 2 =1, 1, and m 2 = 2, 1, 2, 3) and "(N297 sugar chain)-(N3Lb)L-D" (in m 2 =1, 1, and m2 = 2, it is 3, 2, or 1 antibody-drug conjugates of both (in the case of ). That is, one molecule of the conjugate contains only one of "(N297 sugar chain)-(N1Lb)L-D" or "(N297 sugar chain)-(N3Lb)L-D," or a mixture of both.

[0409] The antibody-drug conjugates of the present invention, their free drugs, or their manufacturing intermediates may sometimes exist as stereoisomers or optical isomers derived from chiral carbon atoms, geometric isomers, tautomers, or optical isomers such as d-form, l-form, and atropisomers. These isomers, optical isomers, and mixtures thereof are all encompassed by the present invention.

[0410] The antibody-drug conjugate of the present invention exhibits strong tumor activity (in vivo anti-tumor activity, in vitro anti-cell activity), good in vivo dynamics and physical properties, and is highly safe, and is therefore useful as a pharmaceutical.

[0411] In the antibody-drug conjugates of the present invention, the number of drug-bound molecules per antibody molecule is a significant factor influencing their effectiveness and safety. Antibody-drug conjugates are manufactured by specifying reaction conditions such as the amounts of raw materials / reagents used to achieve a fixed number of drug-bound molecules. However, unlike chemical reactions involving low-molecular-weight compounds, mixtures containing varying amounts of drug are typically obtained. The number of drug-bound molecules per antibody molecule can be determined as an average value, i.e., the average drug-bound number (DAR: Drug to Antibody Ratio). The number of pyrrolobenzodiazepine derivatives bound to antibody molecules can be controlled. The average drug-bound number (DAR) per antibody can range from 1 to 10, preferably 1 to 8, and more preferably 1 to 5.

[0412] In the antibody-drug conjugate of the present invention, when the antibody is bound to L through the remodeled sugar chain of the antibody, the number of drug binding per antibody molecule in the antibody-drug conjugate is m 2 is an integer of 1 or 2. When the sugar chain is an N297 sugar chain, the sugar chain 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, more preferably in the range of 3.5 to 4.2).

[0413] It should be noted that those skilled in the art can design a reaction to bind the antibody to a desired number of drugs based on the description of the examples of this application, and can obtain an antibody with a controlled number of bound pyrrolobenzodiazepine derivatives.

[0414] It should be noted that the antibody-drug conjugates, free drugs, or manufacturing intermediates of the present invention may absorb moisture by being exposed to the atmosphere or undergoing recrystallization, and may sometimes adhere to adsorbed water or form hydrates. Such water-containing compounds and salts are also included in the present invention.

[0415] If the antibody-drug conjugate, free drug, or manufacturing intermediate of the present invention has a basic group such as an amino group, it can be prepared as a pharmaceutically acceptable salt as needed. Examples of such salts include hydrohalide salts such as hydrochloride and hydroiodide; inorganic acid salts such as nitrate, perchlorate, sulfate, and phosphate; lower alkane sulfonates such as methanesulfonate, trifluoromethanesulfonate, and ethanesulfonate; aryl sulfonates such as benzenesulfonate and p-toluenesulfonate; organic acid salts such as formic acid, acetic acid, malic acid, fumarate, succinate, citrate, tartrate, oxalate, and maleate; and amino acid salts such as ornithine, glutamate, and aspartate.

[0416] When the antibody-drug conjugate, free drug, or manufacturing intermediate of the present invention has an acidic group such as a carboxyl group, a base addition salt can generally be formed. Examples of pharmaceutically acceptable salts include alkali metal salts such as sodium, potassium, and lithium salts; alkaline earth metal salts such as calcium and magnesium salts; inorganic salts such as ammonium salts; and organic amine salts such as dibenzylamine salts, morpholine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucamine salts, diethylamine salts, triethylamine salts, cyclohexylamine salts, dicyclohexylamine salts, N,N'-dibenzylethylenediamine salts, diethanolamine salts, N-benzyl-N-(2-phenylethoxy)amine salts, piperazine salts, tetramethylammonium salts, and tris(hydroxymethyl)aminomethane salts.

[0417] The antibody-drug conjugates, free drugs, or manufacturing intermediates of the present invention may also exist as hydrates by absorbing moisture from the air, for example. Solvates of the present invention are not particularly limited as long as they are pharmaceutically acceptable solvates. Specifically, hydrates, ethanolates, and 2-propanolates are preferred. Furthermore, when nitrogen atoms are present in the antibody-drug conjugates, free drugs, or manufacturing intermediates of the present invention, N-oxides may be formed, and these solvates and N-oxides are also encompassed within the scope of the present invention.

[0418] In addition, the present invention also includes compounds labeled with various radioactive or non-radioactive isotopes. One or more atoms constituting the antibody-drug conjugate, free drug, or manufacturing intermediate of the present invention may also contain atomic isotopes in unnatural proportions. Examples of atomic isotopes include: deuterium ( 2 H), tritium ( 3 H), iodine-125 ( 125 I) or carbon-14 ( 14 C) etc. In addition, the compounds of the present invention can also be treated with tritium ( 3 H), iodine-125 ( 125 I) or carbon-14 ( 14 C). Radiolabeled compounds are useful as therapeutic or prophylactic agents, research reagents (e.g., analytical reagents), and diagnostic agents (e.g., in vivo diagnostic imaging agents). All isotopic variants of the antibody-drug conjugates of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.

[0419] [Manufacturing method]

[0420] R method: Preparation of antibodies

[0421] Sugar chain remodeled antibodies can be prepared by using the method described in WO2013 / 120066, etc. Figure 3 The method shown is used for manufacturing.

[0422]

[0423] Step R-1: Hydrolysis of the glycosidic bond between GlcNAcβ1-4GlcNAc in the chitobiose structure at the reducing end

[0424] This step is a step of cleaving the N-linked sugar chain bound to aspartic acid at position 297 in the amino acid sequence of the antibody (N297-bound sugar chain) using a known enzymatic reaction to prepare a sugar chain-cleaved antibody.

[0425] In a buffer solution (50 mM phosphate buffer, etc.), at 0°C to 40°C, a hydrolase such as EndoS enzyme is used to hydrolyze the glycosidic bond between GlcNAcβ1 and 4GlcNAc in the chitobiose structure of the target antibody (20 mg / ml). 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 the antibody. After completion of the reaction, purification by affinity chromatography and / or purification on a hydroxyapatite column, as described below, can be performed to produce a (Fucα1,6)GlcNAc antibody in which the sugar chain between GlcNAcβ1 and 4GlcNAc is hydrolyzed.

[0426] R-2 process sugar chain transfer reaction

[0427] This step is a step of producing a sugar chain-remodeled antibody by binding MSG (MSG1, MSG2) or SG-type sugar chain oxazoline compounds (hereinafter referred to as "azido sugar chain oxazoline compounds") having an azide-containing PEG linker to the above-mentioned (Fucα1,6)GlcNAc antibody using an enzymatic reaction.

[0428] The sugar chain transfer reaction is carried out by reacting the sugar chain-cleaving antibody with the azidosugar chain oxazoline compound in a buffer solution (phosphate buffer, etc.) at 0°C to 40°C in the presence of a catalytic amount of a glycosyltransferase such as EndoS (D233Q / Q303L). The reaction time is 10 minutes to 72 hours, preferably 1 hour to 6 hours. For 100 mg of the antibody, 1 to 10 mg of EndoS enzyme (D233Q / Q303L) is used, preferably 1 to 3 mg, and 2 to an excess of 2 equivalents, preferably 2 to 20 equivalents, of the azidosugar chain oxazoline compound is used.

[0429] After the reaction is completed, purification by affinity chromatography and hydroxyapatite column purification can be performed to obtain purified sugar chain-remodeled antibodies.

[0430] Azidosugar chain oxazolines can be prepared according to the methods described in Examples 3 to 5. Using reactions known in the field of organic synthetic science (such as condensation reactions), N3-(CH2CH2-O)n5-CH2CH2-NH2, an azide-containing PEG linker (N3-L(PEG)), is introduced into MSG (MSG1, MSG2) or disialooctasaccharide (Tokyo Chemical Industry Co., Ltd.). Specifically, an amide bond is formed between the carboxylic acid at the 2-position of the sialic acid and the amino group at the right end of N3-(CH2CH2-O)n5-CH2CH2-NH2 through a known condensation reaction.

[0431] It should be noted that MSG, MSG1 or MSG2 can be obtained by hydrolyzing the (MSG-)Asn or the separated and purified (MSG1-)Asn or (MSG2-)Asn (Examples 3 and 4) using a hydrolase such as EndoM.

[0432] In the preparation of the above-mentioned sugar chain-remodeled antibody, concentration of the antibody aqueous solution, concentration measurement, and buffer exchange can be performed according to the following common procedures A to C.

[0433] (Common procedure A: Concentration of the antibody aqueous solution)

[0434] The antibody or antibody-drug conjugate solution was placed in an Amicon Ultra (30,000-50,000 MWCO, Millipore Co.) container and concentrated by centrifugation (2000-4000 G for 5-20 minutes) using an Allegra X-15R centrifuge (Beckman Coulter, Inc.).

[0435] (Common procedure B: Antibody concentration determination)

[0436] Antibody concentration was measured using a UV analyzer (Nanodrop 1000, Thermo Fisher Scientific Inc.) according to the manufacturer's method. － 1 cm －1 ~1.8mLmg －1 cm －1 ).

[0437] (Common procedure C: Antibody buffer exchange)

[0438] A buffer solution (phosphate-buffered saline (pH 6.0), phosphate buffer (pH 6.0), etc.) was added to the aqueous antibody solution and concentrated using common procedure A. After this operation was repeated several times, the antibody concentration was measured using common procedure B and adjusted to 10 mg / mL using a buffer solution (phosphate-buffered saline (pH 6.0), phosphate buffer (pH 6.0), etc.).

[0439] S method: coupling

[0440] This production method is a method for producing an antibody-drug conjugate by combining the above-mentioned sugar chain remodeled antibody with the production intermediate (2) through SPAAC reaction (strain-promoted alkyneazide cycloaddition: JACS. 2004, 126, 15046-15047).

[0441]

[0442] Where Ab represents the sugar chain remodeled antibody, La', Lp', B' and m 2 With La, Lp, B and m 1 have the same meanings, and J represents any of the following structural formulas, wherein an asterisk indicates a bond with La'.

[0443]

[0444] J-La'-Lp'-NH-B'-CH2-O(C=O)-PBD can be synthesized by the methods described in Examples 2-1 to 2-4.

[0445] The SPAAC reaction is carried out 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 prepared by dissolving compound (2) in an appropriate solvent (dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), N-methyl-2-pyridone (NMP), propylene glycol (PG), etc. or a mixture thereof).

[0446] The amount of compound (2) is 2 to 30 mol relative to 1 mol of the 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 to 100 hours. The pH during the reaction is preferably 5 to 9.

[0447] Antibody-drug conjugates can be identified by performing buffer exchange, purification, and determination of antibody concentration and the average number of drug bound per antibody molecule using the common procedures A to C described above and the common procedures D to F described below.

[0448] Common Operation D: Purification of Antibody-Drug Conjugates

[0449] Equilibrate a NAP-25 column with commercially available acetate buffer (10 mM, pH 5.5; referred to herein as ABS) containing 5% sorbitol. Load the NAP-25 column with the antibody-drug conjugate reaction aqueous solution (approximately 1.5 to 2.5 mL) and elute with the manufacturer's specified amount of buffer to separate the antibody fraction. This fraction is reloaded onto the NAP-25 column and purified by gel filtration using buffer elution repeated two to three times to obtain the antibody-drug conjugate from which unbound drug linker, dimethyl sulfoxide, and propylene glycol have been removed. Adjust the concentration of the antibody-drug conjugate solution as needed using common procedures A and C.

[0450] Common Procedure E: Determination of Antibody Concentration in Antibody-Drug Conjugates

[0451] The bound drug concentration of the antibody-drug conjugate can be calculated using the Lambert-Beer law shown below.

[0452] The following is a formula (I) using Lambert-Beer's law.

[0453]

[0454] Wherein, A280 represents the absorbance of the antibody-drug conjugate aqueous solution at 280 nm, ε280 represents the molar absorptivity of the antibody-drug conjugate at 280 nm, C (mol·L －1 ) represents the molar concentration of the antibody-drug conjugate.

[0455] According to the above formula (I), the molar concentration of the antibody-drug conjugate C (mol·L －1 ) is obtained from the following formula (II).

[0456]

[0457] Then, by multiplying both sides by the molar mass MW (g·mol －1 ), the weight concentration of the antibody-drug conjugate C' (mg·mL －1 )(Formula (III)).

[0458]

[0459] Hereinafter, the values ​​used in the above formula and applied to the present embodiment are described.

[0460] Absorbance A280 is the measured value of UV absorbance of the antibody-drug conjugate aqueous solution at 280 nm. Molar mass MW (g·mol －1 ) The estimated molecular weight of the antibody, calculated from its amino acid sequence, was used as an approximate value for the molar mass of the antibody-drug conjugate. The optical path length l (cm) was measured at 1 cm.

[0461] The molar absorption coefficient ε280 of the antibody-drug conjugate can be calculated according to the following formula (IV).

[0462] ε 280 = Antibody molar absorptivity ε Ab，280 +Drug molar absorptivity ε DL，280 xDrug binding formula (IV)

[0463] Among them, ε Ab，280 represents the molar absorptivity of the antibody at 280 nm, ε DL，280 represents the molar absorptivity of the drug at 280 nm.

[0464] ε Ab，280The molar absorptivity of trastuzumab can be estimated by a known calculation method (Protein Science, 1995, vol. 4, 2411-2423) based on the amino acid sequence of the antibody. Ab，280 =215400 (calculated estimated value). The molar absorptivity of CLDN6 antibody was calculated using ε Ab，280 =221340 (calculated estimated value), the molar absorptivity of the TROP2 antibody is ε Ab，280 = 226400 (calculated estimated value), the molar absorptivity of CD98 antibody is expressed as ε Ab，280 = 240400 (calculated estimated value), the molar absorptivity of LPS antibody is ε Ab，280 =230300 (calculated estimated value), the molar absorptivity of the trastuzumab mutant was calculated using ε Ab，280 =215057 (calculated estimated value).

[0465] ε DL，280 The value calculated from the actual value obtained by each UV measurement was used. That is, the value obtained by measuring the absorbance of a solution containing the conjugate precursor (drug) dissolved at a certain molar concentration and applying Lambert-Beer's law and formula (I) was used.

[0466] (Common Procedure F: Determination of the Average Number of Drugs Bound per Antibody Molecule in Antibody-Drug Conjugates)

[0467] The average number of drug-bound molecules per antibody molecule in an antibody-drug conjugate can be determined by high performance liquid chromatography (HPLC) analysis using the following method.

[0468] [F-1. Preparation of Samples for HPLC Analysis (Reduction of Antibody-Drug Conjugates)]

[0469] An antibody-drug conjugate solution (approximately 1 mg / mL, 60 μL) was mixed with an aqueous dithiothreitol (DTT) solution (100 mM, 15 μL). The mixture was incubated at 37°C for 30 minutes, and the disulfide bonds between the L and H chains of the antibody-drug conjugate were cleaved. The sample was then used for HPLC analysis.

[0470] [F-2.HPLC analysis]

[0471] HPLC analysis was performed under the following measurement conditions.

[0472] HPLC system: Agilent 1290 HPLC system (Agilent Technologies)

[0473] Detector: UV absorbance meter (measurement wavelength: 280nm, 329nm)

[0474] Column: BEH Phenyl (2.1×50mm, 1.7μm, Waters Acquity)

[0475] Column temperature: 75°C

[0476] Mobile phase A: 0.1% trifluoroacetic acid (TFA), 15% isopropanol in water

[0477] Mobile phase B: 0.075% TFA, 15% isopropanol in acetonitrile

[0478] Gradient program: 14%-36% (0 min-15 min), 36%-80% (15-17 min), 80%-14% (17 min-17.1 min), 14%-14% (17.1 min-23 min)

[0479] Sample injection volume: 5 μL

[0480] [F-3. Data Analysis]

[0481] [F-3-1] Compared to the undrug-bound antibody L chain (L0) and H chain (H0), the hydrophobicity of the drug-bound H chain (H1 for H1 bound to one drug, H2 for H2 bound to two drugs) increases proportionally with the amount of bound drug, resulting in longer retention times and elution in the order of L0, H0, H1, and H2. By comparing the retention times of L0 and H0, the detected peak can be assigned to any of L0, H0, H1, and H2. Drug binding can also be confirmed by absorption at a wavelength characteristic of the drug, 329 nm.

[0482] [F-3-2] Since the drug linker has UV absorption, the peak area value was corrected according to the following formula using the molar absorption coefficients of the L chain, H chain, and drug linker according to the number of drug linkers bound.

[0483]

[0484] Here, the molar absorptivity (280 nm) of the L and H chains of each antibody can be estimated based on the amino acid sequence of the L and H chains of each antibody using a known calculation method (Protein Science, 1995, vol. 4, 2411-2423). In the case of trastuzumab, 81290 was used as the estimated value for the molar absorptivity of the H chain based on its amino acid sequence. Similarly, in the case of CLDN6 antibody, 77280 was used as the molar absorptivity of the H chain; in the case of TROP2 antibody, 68990 was used as the molar absorptivity of the H chain; in the case of CD98 antibody, 78500 was used as the molar absorptivity of the H chain; in the case of LPS antibody, 77470 was used as the molar absorptivity of the H chain; in the case of trastuzumab mutant, 81488 was used as the molar absorptivity of the H chain; the molar absorptivity (280 nm) of the drug linker was the measured molar absorptivity (280 nm) of compound (1) as the conjugate precursor.

[0485] [F-3-3] The peak area ratio (%) of each chain relative to the total peak area corrected value was calculated according to the following formula.

[0486]

[0487] A Hi :Hi corrected value of each peak area

[0488] [F-3-4] Calculate the average number of drug bound per antibody molecule in the antibody-drug conjugate according to the following formula.

[0489] Average drug binding number = (H0 peak area ratio x0 + H1 peak area ratio x1 + H2 peak area ratio x2) / 100x2

[0490] <Medicine>

[0491] The antibody-drug conjugate of the present invention exhibits cytotoxic activity against cancer cells and can therefore be used as a medicine, particularly as a therapeutic and / or preventive agent for cancer.

[0492] Examples of cancers for which the anti-CLDN6 antibody-drug conjugates of the present invention are applicable include lung cancer (non-small cell lung cancer, small cell lung cancer, etc.), renal cancer, urothelial carcinoma, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer (epithelial tumors, stromal tumors, germ cell tumors, 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 tumors, head and neck cancer, and their metastatic forms. Examples of anti-HER2 antibodies include Examples of cancers for which the antibody-drug conjugates are applicable include lung cancer, urothelial carcinoma, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumors, 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, renal cancer, vulvar cancer, thyroid cancer, or penile cancer, and their metastatic forms. However, the present invention is not limited to these cancers, as long as the cancer cells to be treated express a protein recognized by the antibody in the antibody-drug conjugate.

[0493] The antibody-drug conjugates of the present invention can preferably be administered to mammals, and more preferably to humans.

[0494] Substances used in the pharmaceutical composition containing the antibody-drug conjugate of the present invention can be appropriately selected from pharmaceutical additives commonly used in the art, depending on the dosage and concentration.

[0495] The antibody-drug conjugates of the present invention can be administered as pharmaceutical compositions comprising one or more pharmaceutically compatible ingredients. For example, the pharmaceutical compositions typically comprise one or more pharmaceutical carriers (e.g., sterilized liquids (e.g., water and oils (petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc.))). When the pharmaceutical compositions are administered intravenously, water is a more representative carrier. Aqueous saline solutions, as well as aqueous dextrose solutions and aqueous glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients are well known in the art. As needed, the compositions may also contain trace amounts of wetting agents or emulsifiers, or pH buffering agents. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin. The formulation corresponds to the mode of administration.

[0496] Various delivery systems are known and can be used to administer the antibody-drug conjugates of the present invention. Examples of methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes. Administration can be performed, for example, by injection or bolus injection. In a particularly preferred embodiment, the ligand-drug conjugate is administered by injection. Parenteral administration is a preferred route of administration.

[0497] In a representative embodiment, the pharmaceutical composition is designed according to conventional steps to be suitable for the prescription of the pharmaceutical composition for intravenous administration to people. The composition representatively used for intravenous administration is a solution in a sterile isotonic aqueous buffer. Where necessary, the above-mentioned medicine may also include a solubilizing agent and a local anesthetic (such as lidocaine) for alleviating pain at the injection site. Usually, the above-mentioned components are supplied separately in the form of a dry lyophilized powder or anhydrous concentrate in a sealed container such as an ampoule or a sachet (Sachet) showing the amount of the active agent, or are mixed and supplied together in a unit dosage form. In the case where the above-mentioned medicine is administered by injection, it can be administered by, for example, an injection bottle containing sterilized pharmaceutical grade water or saline. In the case of the above-mentioned medicine administered by injection, an ampoule of sterile water for injection or saline can, for example, be provided in a manner that the above-mentioned components can be mixed before administration.

[0498] The pharmaceutical composition of the present invention may comprise solely the antibody-drug conjugate of the present invention, or may comprise 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 other cancer therapeutic agents to enhance the anti-cancer effect. Other anti-cancer agents used for such purposes may be administered to an individual simultaneously, separately, or sequentially with the antibody-drug conjugate, and their respective dosing intervals may be varied. Examples of such cancer therapeutic agents include albumin-bound paclitaxel (abraxane), carboplatin, cisplatin, gemcitabine, irinotecan (CPT-11), paclitaxel, pemetrexed, sorafenib, vinblastine, or the agents described in WO2003 / 038043, as well as LH-RH analogs (leuprorelin, goserelin, etc.), estramustine phosphate, estrogen antagonists (tamoxifen, raloxifene, etc.), aromatase inhibitors (anastrozole, letrozole, exemestane, etc.), but are not limited as long as they have antitumor activity.

[0499] Such pharmaceutical compositions can be prepared as lyophilized preparations or liquid preparations as preparations having the selected composition and the required purity. When prepared as lyophilized preparations, they can be prepared as preparations containing appropriate formulation additives used in the art. In addition, liquid preparations can also be prepared as liquid preparations containing various formulation additives used in the art.

[0500] The composition and concentration of the pharmaceutical composition also vary depending on the method of administration. However, for the antibody-drug conjugate contained in the pharmaceutical composition of the present invention, the higher the affinity of the antibody-drug conjugate for the antigen, that is, the dissociation constant (Kd value) for the antigen, the higher the affinity (the lower the Kd value), the more effective the drug is even with a small dosage. Therefore, when determining the dosage of the antibody-drug conjugate, the dosage can be set based on the affinity of the antibody-drug conjugate for the antigen. When administering the antibody-drug conjugate of the present invention to humans, for example, a single dose of approximately 0.001 to 100 mg / kg or multiple doses at intervals of 1 to 180 days can be sufficient.

[0501] Furthermore, the antibodies of the present invention or functional fragments thereof may also be used as pharmaceuticals. In such cases, the description of "antibody-drug conjugate" in the above-mentioned "pharmaceuticals" may be appropriately replaced with the description of "antibody or functional fragment thereof."

[0502] Furthermore, the free drug (novel PBD derivative compound), its salt, or its hydrate can also be used as a medicine. In this case, the description of "antibody-drug conjugate" in the above-mentioned "Medicine" can be appropriately replaced with the description of "free drug (novel PBD derivative compound), its salt, or its hydrate."

[0503] Example

[0504] The present invention is specifically described by the following examples, but the present invention is not limited thereto. In addition, these are not limiting interpretations in any sense. In addition, in this specification, reagents, solvents and starting materials not particularly recorded can be easily obtained from commercially available supply sources.

[0505] Reference Example 1: Trastuzumab Tesirine

[0506] Step 1: Conjugation of Antibody and Drug Linker

[0507] To a 5 mM EDTA-phosphate-buffered saline (pH 6.5) solution of trastuzumab (Reference Example 3) (9.91 mg / mL, 0.70 mL), dipotassium phosphate aqueous solution (1.0 M, 0.0112 mL) and tris(2-carboxyethyl)phosphine hydrochloride aqueous solution (10 mM, 0.0086 mL) were added at 20°C. The reaction was allowed to proceed for 60 minutes at 20°C and 30 minutes at room temperature. Tesirine (0.36 mg), synthesized in the reference (Med. Chem. Lett. 2016, 7, 983-987), in dimethylacetamide (0.0415 mL) was added to the reaction solution and allowed to react at room temperature for 1 hour. The reaction was terminated by the addition of N-acetylcysteine ​​aqueous solution (100 mM, 0.0024 mL) and allowed to react for 30 minutes.

[0508] Purification procedure: The above solution was purified using common procedure D to obtain 3.5 mL of a solution containing the target compound.

[0509] Property evaluation: Common operations E and F were used to obtain the following property values.

[0510] Antibody concentration: 1.40 mg / mL, antibody yield: 4.90 mg (71%), average number of drug binding per antibody molecule (n): 2.0

[0511] Reference Example 2: Anti-CLDN6 (H1L1) Antibody Tesirine

[0512] Step 1: Conjugation of Antibody and Drug Linker

[0513] To a 5 mM EDTA-phosphate-buffered saline (pH 6.5) solution of anti-CLDN6 (H1L1) antibody (9.87 mg / mL, 0.45 mL), potassium phosphate dihydrate (1.0 M, 0.0072 mL) and tris(2-carboxyethyl)phosphine hydrochloride (10 mM, 0.0041 mL) were added at 20°C and reacted for 90 minutes. Tesirine (0.15 mg), synthesized in reference (Med. Chem. Lett. 2016, 7, 983-987), in N,N-dimethylacetamide (0.0277 mL) was added to the reaction solution and reacted at 20°C for 1 hour. The reaction was terminated by the addition of N-acetylcysteine ​​(100 mM, 0.001 mL) and allowed to react for 30 minutes.

[0514] Purification procedure: The above solution was purified using common procedure D to obtain 3.5 mL of a solution containing the target compound.

[0515] Property evaluation: Common operations E and F were used to obtain the following property values.

[0516] Antibody concentration: 1.56 mg / mL, antibody yield: 3.90 mg (88%), average number of drug binding per antibody molecule (n): 2.1

[0517] Reference Example 3: Anti-HER2 Antibody Trastuzumab

[0518] The anti-HER2 antibody was prepared with reference to US Pat. No. 5,821,337. The amino acid sequences of the light chain and heavy chain of trastuzumab are shown in SEQ ID NOs. 64 and 65.

[0519] Reference Example 4: Anti-LPS antibody h#1G5-H1L1

[0520] The anti-LPS antibody was prepared 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 NOs: 66 and 67.

[0521] Reference Example 5: Anti-TROP2 antibody hRS7

[0522] The anti-TROP2 antibody was prepared 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 NOs: 68 and 69.

[0523] Reference Example 6: Anti-CD98 Antibody hM23-H1L1

[0524] The anti-CD98 antibody was prepared with reference to WO2015 / 146132. The amino acid sequences of the light chain and heavy chain of hM23-H1L1 are shown in SEQ ID NOs: 70 and 71.

[0525] [Synthesis of manufacturing intermediates (drug linkers)]

[0526] Example 1

[0527] [Example 1-1: Intermediate 1]

[0528]

[0529] Step 1: (6S)-6-(hydroxymethyl)-5-azaspiro[2.4]heptane-5-carboxylic acid benzyl ester (1-2)

[0530] 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) was added lithium borohydride (4.30 g, 178 mmol) little by little at 0°C. The mixture was stirred at 0°C for 30 minutes and then at room temperature for 2 hours. Water (180 mL) and 2N hydrochloric acid (186 mL) were added at 0°C and removed by distillation under reduced pressure. The resulting residue was extracted four times with ethyl acetate, and the organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The residue (1-2) (27.9 g, 90%) was removed by distillation under reduced pressure and used directly in the next reaction.

[0531] Step 2: (6S)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-5-azaspiro[2.4]heptane-5-carboxylic acid benzyl ester (1-3)

[0532] To a solution of compound (1-2) (27.9 g, 107 mmol) obtained in step 1 above and imidazole (14.5 g, 214 mmol) in dichloromethane (300 mL) was added tert-butyldimethylsilyl chloride (24.2 g, 160 mmol) at room temperature, and the mixture was stirred at room temperature for 18 hours. The reaction solution was washed with saturated aqueous citric acid, saturated aqueous sodium bicarbonate, and saturated brine, dried over anhydrous sodium sulfate, and then 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 (1-3) (32.5 g, 81%).

[0533] 1 H-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).

[0534] MS (APCI) m / z: 376 (M+H)+

[0535] Step 3: (6S)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-5-azaspiro[2.4]heptane(1-4)

[0536] To a solution of compound (1-3) (32.5 g, 86.5 mmol) obtained in step 2 above in ethanol (400 mL) was added 7.5% palladium-carbon catalyst (54% water, 5.00 g) at room temperature. 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 desired product (1-4) (21.3 g, quantitative).

[0537] 1 H-NMR(CDCl3)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).

[0538] MS (APCI, ESI) m / z: 242 (M+H)+

[0539] 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)

[0540] 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) was added N,N'-dicyclohexylcarbodiimide (35.0 g, 170 mmol) under ice cooling. The reaction mixture was stirred at room temperature. After the carboxylic acid disappeared, a solution of compound (1-4) (34.1 g, 141 mmol) obtained in step 3 above and triethylamine (29.4 mL, 212 mmol) in dichloromethane (100 mL) was slowly added dropwise at -60°C. The reaction solution was stirred at room temperature overnight, and then a saturated aqueous sodium bicarbonate solution was added to the reaction mixture, which was then extracted with chloroform. The organic layer was washed with water and saturated brine and dried over anhydrous magnesium sulfate. Ethyl acetate and diethyl ether were added to the residue obtained by distillation under reduced pressure, the solid component was removed by filtration, the filtrate was removed by distillation under reduced pressure, and the obtained residue was purified by silica gel column chromatography [hexane:ethyl acetate = 100:0 (v / v) to 25:75 (v / v)] to obtain the target product (1-5) (55.0 g, 66%).

[0541] 1H-NMR(CDCl3)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),3.91-3.8 8(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).

[0542] MS (APCI, ESI) m / z: 593 (M+H)+

[0543] 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)

[0544] To a solution of compound (1-5) (55.0 g, 92.8 mmol) obtained in step 4 above in ethanol (300 mL) was added 7.5% palladium on carbon (10.0 g) under a nitrogen atmosphere. The nitrogen balloon was immediately replaced with a hydrogen balloon, and the reaction mixture was vigorously stirred under a hydrogen atmosphere at room temperature. After the starting material disappeared, the reaction mixture was filtered, and the filtrate was removed by distillation under reduced pressure. The resulting target product (1-6) (52.2 g, 100%) was used directly in the next reaction.

[0545] 1 H-NMR(CDCl3)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).

[0546] MS (APCI, ESI) m / z: 563 (M+H)+

[0547] 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-alanine amide (1-7)

[0548] To a solution of compound (1-6) (18.6 g, 33.0 mmol) obtained in Step 5 above and triethylamine (6.26 mL, 45.2 mmol) in THF (300 mL) was slowly added triphosgene (4.22 g, 14.2 mmol) in an ethanol-ice bath. Following this addition, a mixed solution of N-[(prop-2-en-1-yloxy)carbonyl]-L-valyl-N-[4-(hydroxymethyl)phenyl]-L-alanineamide (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 addition, the ice bath was removed, and the reaction mixture was stirred at 40°C under a nitrogen atmosphere. After the starting 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 brine and dried over anhydrous sodium sulfate. After filtration, the residue was distilled off under reduced pressure and 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%).

[0549] 1H-NMR(CDCl3)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),5.9 4-5.87(1H,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(1 H,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).

[0550] MS (APCI, ESI) m / z: 966 (M+H)+

[0551] 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-alanine amide (1-8)

[0552] To a solution of compound (1-7) (23.5 g, 24.3 mmol) obtained in step 6 above in tetrahydrofuran (50 mL), methanol (50 mL), and water (44 mL) was added acetic acid (200 mL) at room temperature. The reaction mixture was stirred at room temperature. After the starting material disappeared, the reaction mixture was extracted with ethyl acetate. The organic layer was washed with water and saturated brine, and dried over anhydrous sodium sulfate. After filtration, the residue obtained by vacuum distillation 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%).

[0553] 1H-NMR(CDCl3)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),1.8 7-1.81(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).

[0554] MS (APCI, ESI) m / z: 852 (M+H)+

[0555] 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-alanine amide (1-9)

[0556] To a solution of dimethyl sulfoxide (3.75 mL, 52.8 mmol) in dichloromethane (300 mL) was slowly added dropwise under a nitrogen atmosphere at -78°C. After 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 addition, the refrigerant bath was removed and the temperature was slowly raised to room temperature. After the starting material 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 brine, and dried over anhydrous magnesium 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 0:60 (v / v)] to obtain the target product (1-9) (16.5 g, 92%).

[0557] 1 H-NMR(CDCl3)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),5.94-5.8 7(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- 1.71(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).

[0558] MS (APCI, ESI) m / z: 850 (M+H)+

[0559] 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-alanine amide (1-10)

[0560] To a solution of compound (1-9) (12.0 g, 14.1 mmol) obtained in step 8 above and 2,6-lutidine (6.58 mL, 56.5 mmol) in dichloromethane (200 mL) was slowly added dropwise under a nitrogen atmosphere at 0°C to tert-butyldimethylsilyl trifluoromethanesulfonate (9.73 mL, 42.3 mmol). After stirring under ice-cooling for 10 minutes, the ice bath was removed and the mixture was stirred at room temperature. After the starting material disappeared, water was added to the reaction mixture, and the reaction mixture was extracted with chloroform. The organic layer was washed with water and saturated brine, and dried over anhydrous sodium sulfate. After filtration, the residue obtained by vacuum distillation 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%).

[0561] 1 H-NMR(CDCl3)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).

[0562] MS (APCI, ESI) m / z: 964 (M+H)+

[0563] 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-alanine amide (1-11)

[0564] To a solution of compound (1-10) (8.12 g, 8.42 mmol) obtained in step 9 above in N,N-dimethylformamide (90 mL) and water (2 mL) was added lithium acetate (0.611 g, 9.26 mmol), and the mixture was stirred at room temperature. After the starting material 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 brine, and dried over anhydrous sodium sulfate. After filtration, the residue was removed by vacuum distillation and 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%).

[0565] 1 H-NMR (400MHz, CDCl3, 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),6.81-6.7 4(1H,m),6.65(1H,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),4.67-4.54(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)

[0566] MS (APCI, ESI) m / z: 808 (M+H)+

[0567] 1H-NMR (500MHz, CDCl3, 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),0.9 2(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)

[0568] The absolute configuration at the 11' position of compound (1-11) was analyzed based on correlations obtained from selective 1D ROESY spectroscopy (see figure below). Correlations were observed between the 1'α-H and 11'-H, the 3'α-H and 11'-H, and the 1'β-H and 3'β-H groups, indicating that the absolute configuration at the 11' position is S.

[0569]

[0570] Significant correlations obtained from Selective 1D ROESY spectra

[0571] Therefore, it was found that the absolute configuration at the 11' position of Compound (1-11), Compound (1-9) and Compound (1-10) having the same absolute configuration, Compound (3-11), Compound (3-12), Compound (3-13), and Drug Linker 1 (Compound (3-14)) synthesized using Compound (1-11), Compound (4-9), Compound (4-10), Compound (4-11), and Drug Linker 2 (Compound (4-12)), and Compound (6-10), Compound (6-11), Compound (6-12), and Drug Linker 4 (Compound (6-13)) was S. Furthermore, the absolute configuration at the 11' position of Compound (5-9), Compound (5-10), and Drug Linker 3 (Compound (5-11)) obtained by the same synthetic method was confirmed to be S.

[0572] [Example 1-2: Intermediate 2]

[0573]

[0574] Step 1: N-[4-(11,12-didehydrodibenzo[b,f]azacyclooctatetraen-5(6H)-yl)-4-oxobutyryl]glycylglycine (2-2)

[0575] To a solution of glycylglycine (0.328 g, 2.49 mmol) and N,N-diisopropylethylamine (0.433 mL, 2.49 mmol) in N,N-dimethylformamide (20 mL) was added 1-{[4-(11,12-didehydrodibenzo[b,f]azacyclooctatetraen-5(6H)-yl)-4-oxobutanoyl]oxy}pyrrolidine-2,5-dione (2-1) (1.00 g, 2.49 mmol, Click Chemistry Tools) and water (10 mL) at room temperature. The mixture was stirred overnight at room temperature. The residue was evaporated under reduced pressure and purified by silica gel column chromatography (partitioning the organic layer with chloroform to chloroform:methanol:water = 7:3:1 (v / v / v)) to obtain the desired product (0.930 g, 89%).

[0576] 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).

[0577] MS(APCI,ESI)m / z:420[(M+H)+].

[0578] Example 2

[0579] [Example 2-1: Drug Linker 1]

[0580]

[0581] 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)

[0582] To a solution of (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) (25.5 g, 41.6 mmol, WO2016149546) in tetrahydrofuran (150 mL) and ethanol (150 mL) was added 5% palladium on carbon (54% moisture, 10.0 g) under a nitrogen atmosphere. The reaction solution was stirred at room temperature under a hydrogen atmosphere for three days. Chloroform was added to the reaction solution, and the mixture was filtered through celite. The filtrate was then 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%).

[0583] 1H-NMR(CDCl3)δ:7.36(1H,s),7.25(1H,s),6.01(1H,s),5.45-5.43(1H,m) ,4.69-4.67(1H,m),4.60-4.55(1H,m),4.23-4.21(1H,m),3.96(3H,s),3.7 6-3.68(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).

[0584] MS (APCI, ESI) m / z: 523 (M+H)+

[0585] 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)

[0586] To a solution of compound (3-2) (10.8 g, 20.7 mmol) obtained in step 1 above in N,N-dimethylformamide (30 mL) were added 1,5-dibromopentane (23.8 g, 103 mmol) and potassium carbonate (3.43 g, 24.8 mmol) at room temperature. After stirring at room temperature for 3 hours, water was added to the reaction solution, and extraction was performed with ethyl acetate. The resulting organic layer was washed with saturated brine, 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 (3-3) (14.5 g, quantitative).

[0587] 1H-NMR(CDCl3)δ:7.34(1H,s),7.21(1H,s),5.52-5.49(1H,m),4.63-4.62(1H,m),4.5 8-4.55(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),1.97-1.8 7(4H,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)

[0588] MS (APCI, ESI) m / z: 673[ 81 Br,(M+H)+],671[ 79 Br,(M+H)+].

[0589] 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)

[0590] To a solution of compound (3-3) (21.5 mmol) obtained in step 2 above in tetrahydrofuran (40 mL) was added a 1 mol / L tetrabutylammonium fluoride solution in tetrahydrofuran (28.0 mL, 28.0 mmol) at 0°C. After stirring at room temperature for 30 minutes, water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The resulting organic layer was washed with saturated brine. After drying over sodium sulfate, the mixture was evaporated under reduced pressure. The resulting 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%).

[0591] 1H-NMR(CDCl3)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 ),3.73-3.64(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).

[0592] MS (APCI, ESI) m / z: 559[ 81 Br,(M+H)+],557[ 79 Br,(M+H)+].

[0593] 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)

[0594] Compound (3-4) (11.3 g, 20.2 mmol) obtained in the above step 3, tetrabutylammonium bromide (0.325 g, 1.01 mmol), and potassium bromide (0.240 g, 2.02 mmol) were dissolved in a saturated aqueous sodium bicarbonate solution (60 mL) and dichloromethane (60 mL). Nor-AZADO (0.0279 g, 0.202 mmol) and sodium hypochlorite pentahydrate (2.03 g, 27.2 mmol) were added at 0°C, and the mixture was stirred at 0°C for 30 minutes. Since the starting material remained, sodium hypochlorite pentahydrate (1.00 g, 13.4 mmol) was added at 0°C, and the mixture was stirred at 0°C for 15 minutes. 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. The disappearance of the starting material was confirmed by TLC (thin layer chromatography). A sodium thiosulfate aqueous solution was added to the reaction solution, and the mixture was extracted with chloroform. The resulting organic layer was dried over sodium sulfate. The residue was removed by distillation under reduced pressure, and 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%).

[0595] 1H-NMR(CDCl3)δ:7.33(1H,s),7.24(1H,s),5.56-5.53(1H,m),4.71-4.69(1H,m) ,4.66-4.63(1H,m),4.27-4.22(1H,m),4.12-4.02(2H,m),3.93-3.88(4H,m),3.8 2-3.75(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).

[0596] MS (APCI, ESI) m / z: 557[ 81 Br,(M+H)+],555[ 79 Br,(M+H)+].

[0597] 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)

[0598] To a solution of compound (3-5) (9.74 g, 17.5 mmol) obtained in step 4 above in dichloromethane (160 mL) was added 2,6-lutidine (8.17 mL, 70.1 mmol) at -40°C, and the mixture was stirred at -40°C for 10 minutes. Trifluoromethanesulfonic anhydride (8.85 mL, 52.6 mmol) was added to the reaction solution at -40°C, and the mixture was stirred at -40°C for 30 minutes. A 10% aqueous citric acid solution was added to the reaction solution, and the mixture was extracted with chloroform. The resulting organic layer was dried over sodium sulfate. The residue was removed by distillation under reduced pressure, and purified by silica gel column chromatography [hexane:ethyl acetate = 95:5 → 70:35], followed by NH2 silica gel chromatography [hexane:ethyl acetate = 95:5 (v / v) to 65:35 (v / v)] to obtain the target product (3-6) (7.10 g, 59%).

[0599] 1H-NMR(CDCl3)δ:7.32(1H,s),7.24(1H,s),7.15-7.14(1H,m),5.56-5.53(1 H,m),4.70-4.68(1H,m),4.66-4.63(1H,m),4.11-4.01(2H,m),3.94-3.90(4 H,m),3.84-3.75(1H,m),3.73-3.68(1H,m),3.46-3.44(2H,m),3.18-3.14(1 H,m),1.96-1.88(4H,m),1.69-1.61(2H,m),1.02-0.92(2H,m),0.04(9H,s).

[0600] MS (APCI, ESI) m / z: 689[ 81 Br,(M+H)+],687[ 79 Br,(M+H)+].

[0601] 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)

[0602] To a mixture of compound (3-6) (2.00 g, 2.91 mmol) obtained in step 5 above, 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) were added toluene (20 mL), ethanol (10 mL), and water (10 mL) at room temperature. The reaction solution was stirred at room temperature for 30 minutes, then extracted with ethyl acetate and washed with water and saturated brine. 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%).

[0603] 1H-NMR(CDCl3)δ:7.38-7.37(3H,m),7.33(1H,s),7.25(1H,s),6.89-6.88(2 H,m),5.56-5.54(1H,m),4.71-4.68(1H,m),4.65-4.62(1H,m),4.09-4.04(2 H,m),3.96-3.91(4H,m),3.85-3.66(5H,m),3.46-3.45(2H,m),3.16-3.12(1 H,m),1.99-1.94(4H,m),1.69-1.64(2H,m),1.00-0.98(2H,m),0.04(9H,s).

[0604] MS (APCI, ESI) m / z: 647[ 81 Br,(M+H)+],645[ 79 Br,(M+H)+].

[0605] Step 7: (11aS)-8-[(5-bromopentyl)oxy]-7-methoxy-2-(4-methoxyphenyl)-1,11a-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one (3-8)

[0606] Compound (3-7) (0.789 g, 1.22 mmol) obtained in step 6 above was dissolved in ethanol (10 mL) and tetrahydrofuran (10 mL). A 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. 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). 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, and water was added. The mixture was extracted with chloroform, and the resulting organic layer was dried over sodium sulfate. The residue was purified by silica gel column chromatography [hexane:ethyl acetate = 60:40 (v / v) to 25:75 (v / v)] to obtain the target product (3-8) (0.496 g, 81%).

[0607] 1H-NMR(CDCl3)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).

[0608] MS (APCI, ESI) m / z: 501[ 81 Br,(M+H)+],499[ 79 Br,(M+H)+].

[0609] Step 8: (11aS)-8-[(5-bromopentyl)oxy]-7-methoxy-2-(4-methoxyphenyl)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one (3-9)

[0610] To a solution of compound (3-8) (0.496 g, 0.992 mmol) obtained in step 7 above in dichloromethane (20 mL) was added sodium triacetoxyborohydride (0.421 g, 1.99 mmol) at 0°C. After stirring at room temperature for 2 hours, a saturated aqueous sodium bicarbonate solution was added, and the mixture was extracted with chloroform. The organic layer was dried over sodium sulfate and evaporated under reduced pressure. 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 desired product (3-9) (0.426 g, 86%).

[0611] 1 H-NMR(CDCl3)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).

[0612] MS (APCI, ESI) m / z: 503[ 81 Br,(M+H)+],501[79 Br,(M+H)+].

[0613] Step 9: (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)-carboxylic acid prop-2-en-1-yl ester (3-10)

[0614] To a solution of compound (3-9) (0.426 g, 0.849 mmol) obtained in step 8 above in dichloromethane (30 mL) were added pyridine (0.102 mL, 1.27 mmol) and allyl chloroformate (0.374 mL, 3.54 mmol) 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, and the mixture was extracted with chloroform. The resulting organic layer was washed with a saturated aqueous sodium bicarbonate solution and dried over sodium sulfate. The residue was removed by distillation under reduced pressure, and purified by silica gel column chromatography [hexane:ethyl acetate = 90:10 (v / v) to 50:50 (v / v)] to obtain the target product (3-10) (0.465 g, 94%).

[0615] 1 H-NMR(CDCl3)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).

[0616] MS (APCI, ESI) m / z: 587[ 81 Br,(M+H)+],585[ 79 Br,(M+H)+].

[0617] 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-alanine amide (3-11)

[0618] 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 above in N,N-dimethylformamide (3 mL) at room temperature, and the mixture was stirred overnight at room temperature. The reaction solution was diluted with ethyl acetate, washed with water and saturated brine, and dried over sodium sulfate. After distillation under reduced pressure, the resulting residue was purified by NH2-silica gel column chromatography [hexane:ethyl acetate = 70:30 (v / v) to 0:100 (v / v)] to obtain the desired product (3-11) (0.184 g, 87%).

[0619] 1H-NMR(CDCl3)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(11 H,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),2.19-2.1 3(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).

[0620] MS (APCI, ESI) m / z: 1312 (M+H)+

[0621] 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]-5,10,11,1 1a-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-alanine amide (3-12)

[0622] To a solution of compound (3-11) (0.1837 g, 0.140 mmol) obtained in step 10 above and acetic acid (0.048 mL, 0.840 mmol) in tetrahydrofuran (5.00 mL) was added a 1 mol / L tetrabutylammonium fluoride solution in tetrahydrofuran (0.700 mL, 0.700 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 saturated sodium bicarbonate aqueous solution and saturated brine, 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).

[0623] 1 H-NMR(CDCl3)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).

[0624] MS (APCI, ESI) m / z: 1198 (M+H)+

[0625] 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-alanine amide (3-13)

[0626] To a solution of compound (3-12) (0.140 mmol) obtained in step 11 above in dichloromethane (2 mL) were added pyrrolidine (0.0579 mL, 0.700 mmol) and tetrakis(triphenylphosphine)palladium(0) (0.0162 g, 0.0140 mmol) 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 product (3-13) (0.143 g, 99%).

[0627] 1 H-NMR(CDCl3)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).

[0628] MS (APCI, ESI) m / z: 1030 (M+H)+

[0629] Step 13: N-[4-(11,12-didehydrodibenzo[b,f]azacyclooctatetraen-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-alanine amide (3-14)

[0630] To a mixture of compound (2-2) (0.0640 g, 0.153 mmol) obtained in Step 1 of Example 1-2 and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (0.0446 g, 0.180 mmol) was added dichloromethane (2 mL) at room temperature, and the mixture was stirred at room temperature for 15 minutes. A solution of compound (3-13) (0.143 g, 0.139 mmol) obtained in Step 12 above in dichloromethane (2 mL) was added to the reaction solution, and after stirring at room temperature for five hours, the mixture was removed by 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 desired product (3-14) (0.103 g, 52%).

[0631] 1 H-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).

[0632] MS (APCI, ESI) m / z: 1431 (M+H)+

[0633] [Example 2-2: Drug Linker 2]

[0634]

[0635] 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)

[0636] The compound (3-2) (5.06 g, 9.67 mmol) obtained in Example 2-1, Step 1 and 1,3-dibromopropane (4.93 mL, 48.4 mmol) were reacted in the same manner as in Example 2-1, Step 2 to obtain the target product (4-1) (4.85 g, 78%).

[0637] MS (APCI, ESI) m / z: 645[ 81 Br,(M+H)+],643[ 79 Br,(M+H)+].

[0638] 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)

[0639] 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 product (4-2) (4.05 g, quantitative).

[0640] MS (APCI, ESI) m / z: 531[ 81 Br,(M+H)+],529[ 79 Br,(M+H)+].

[0641] 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)

[0642] 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 product (4-3) (3.73 g, 93%).

[0643] 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).

[0644] Step 4: (11aS)-8-(3-bromopropyloxy)-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)

[0645] 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 product (4-4) (3.27 g, 70%).

[0646] MS (APCI, ESI) m / z: 661[ 81 Br,(M+H)+],659[ 79 Br,(M+H)+].

[0647] Step 5: (11aS)-8-(3-bromopropyloxy)-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)

[0648] 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 product (4-5) (2.49 g, 81%).

[0649] MS (APCI, ESI) m / z: 619[ 81 Br,(M+H)+],617[ 79 Br,(M+H)+].

[0650] Step 6: (11aS)-8-(3-bromopropyloxy)-7-methoxy-2-(4-methoxyphenyl)-1,11a-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one (4-6)

[0651] 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 product (4-6) (1.59 g, 84%).

[0652] MS (APCI, ESI) m / z: 473[ 81 Br,(M+H)+],471[ 79 Br,(M+H)+].

[0653] Step 7: (11aS)-8-(3-bromopropyloxy)-7-methoxy-2-(4-methoxyphenyl)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one (4-7)

[0654] 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 product (4-7) (1.39 g, 87%).

[0655] MS (APCI, ESI) m / z: 475[ 81 Br,(M+H)+],473[ 79 Br,(M+H)+].

[0656] Step 8: (11aS)-8-(3-bromopropoxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylic acid prop-2-en-1-yl ester (4-8)

[0657] 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 product (4-8) (0.885 g, 54%).

[0658] MS (APCI, ESI) m / z: 559[ 81 Br,(M+H)+],557[ 79 Br,(M+H)+].

[0659] 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-alanine amide (4-9)

[0660] 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 step 10 of Example 1-1 were reacted in the same manner as in step 10 of Example 2-1 to obtain the target product (4-9) (0.0712 g, 81%).

[0661] MS(APCI,ESI)m / z: 1284(M+H)+.

[0662] 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-alanine amide (4-10)

[0663] 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 product (4-10) (0.0671 g, quantitative).

[0664] MS(APCI,ESI)m / z: 1170(M+H)+.

[0665] 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-alanine amide (4-11)

[0666] 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 product (4-11) (0.0574 g, 99%).

[0667] 1 H-NMR(CDCl3)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(3H,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).

[0668] MS (APCI, ESI) m / z: 1002 (M+H)+.

[0669] Step 12: N-[4-(11,12-didehydrodibenzo[b,f]azacyclooctatetraen-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-5,1 0,11,11a-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-alanine amide (4-12)

[0670] 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 the step 1 of Example 1-2 were reacted in the same manner as in the step 13 of Example 2-1 to obtain the target product (4-12) (0.169 g, 64%).

[0671] MS(APCI,ESI)m / z: 1402(M+H)+.

[0672] [Example 2-3: Drug Linker 3]

[0673]

[0674] 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)

[0675] To a solution of 4,4'-[1,5-pentanediylbis(oxy)]bis(5-methoxy-2-nitrobenzoic acid) (5-1) (5.41 g, 10.9 mmol, Journal of Medicinal Chemistry 2004, 47, 1161) in dichloromethane (50 mL) was added oxalyl chloride (5.63 mL, 65.7 mmol) at 0°C, followed by the dropwise addition of N,N-dimethylformamide (0.0844 mL, 1.09 mmol). The reaction solution was warmed to room temperature and stirred for 2 hours. The residue was removed by distillation under reduced pressure, and dissolved in dichloromethane (100 mL). A solution of (6S)-5-azaspiro[2.4]heptane-6-carboxylic acid methyl ester hydrochloride (4.28 g, 24.1 mmol, Tetrahedron Letters 2012.53.3847) and triethylamine (6.07 mL, 43.8 mmol) in dichloromethane (100 mL) was added dropwise under a nitrogen atmosphere at -40°C. 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 brine, then dried over anhydrous sodium sulfate. The residue was removed by distillation under reduced pressure to obtain the target product (5-2) (8.40 g, quantitative).

[0676] MS (APCI, ESI) m / z: 769 (M+H)+.

[0677] 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)

[0678] To a solution of compound (5-2) (8.40 g, 10.9 mmol) obtained in step 1 above in tetrahydrofuran (100 mL) was added lithium borohydride (714 mg, 32.8 mmol), 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 brine, and drying over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure to obtain the desired product (5-3) (7.70 g, 99%).

[0679] MS (APCI, ESI) m / z: 713 (M+H)+.

[0680] Step 3: Pentane-1,5-diylbis[oxy(5-methoxy-2-nitrobenzene-4,1-diyl)carbonyl(6S)-5-azaspiro[2.4]heptane-5,6-diylmethanediyl]diacetate (5-4)

[0681] The compound (5-3) obtained in step 2 above (7.70 g, 10.8 mmol) was dissolved in pyridine (20 mL) and acetic anhydride (10 mL, 105.9 mmol), stirred at room temperature, and distilled off under reduced pressure to obtain the target product (5-4) (8.38 g, 97%).

[0682] MS (APCI, ESI) m / z: 797 (M+H)+.

[0683] 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)

[0684] To a solution of compound (5-4) (8.28 g, 10.4 mmol) obtained in step 3 above in N,N-dimethylformamide (100 mL) was added 5% palladium on carbon (54% moisture, 1.00 g). The reaction solution was vigorously stirred at room temperature under a hydrogen atmosphere for 6 hours. After filtration through celite, the filtrate was evaporated 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 desired product (5-5) (5.05 g, 66%).

[0685] MS (APCI, ESI) m / z: 737 (M+H)+.

[0686] 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) (5-6)

[0687] To a solution of compound (5-5) (5.05 g, 6.85 mmol) obtained in step 4 above in dichloromethane (100 mL) was added pyridine (1.10 mL, 13.7 mmol), and allyl chloroformate (0.725 mL, 6.85 mmol) was added under a nitrogen atmosphere at -78°C, and the mixture was stirred for 2 hours. The mixture was evaporated under reduced pressure, and the resulting residue 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 compound (5-6) (2.63 g, 47%).

[0688] MS (APCI, ESI) m / z: 821 (M+H)+.

[0689] 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-alanine amide (5-7)

[0690] The monoallyloxycarbonyl compound (5-6) (2.00 g, 2.44 mmol) obtained in the above step 5 and N-[(prop-2-en-1-yloxy)carbonyl]-L-valyl-N-[4-(hydroxymethyl)phenyl]-L-alanine amide (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 product (5-7) (2.64 g, 89%).

[0691] MS (APCI, ESI) m / z: 1224 (M+H)+.

[0692] 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-alanine amide (5-8)

[0693] Potassium carbonate (1.49 g, 10.8 mmol) was added to a solution of compound (5-7) (2.64 g, 2.16 mmol) obtained in step 6 above in methanol (10 mL), and the mixture was stirred at room temperature for 3 hours. Saturated aqueous 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 and evaporated under reduced pressure to obtain the target product (5-8) (2.21 g, 90%).

[0694] MS (APCI, ESI) m / z: 1140 (M+H)+.

[0695] 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 [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-alanine amide (5-9)

[0696] To a solution of compound (5-8) (2.03 g, 1.78 mmol) obtained in step 7 above in dichloromethane (50 mL) was added Dess-Martin periodinane (1.59 g, 3.74 mmol), and the mixture was stirred overnight at room temperature. Saturated aqueous sodium bicarbonate solution (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 was removed by distillation under reduced pressure, and 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).

[0697] MS (APCI, ESI) m / z: 1136 (M+H)+.

[0698] 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-alanine amide (5-10)

[0699] 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 product (5-10) (1.02 g, 60%).

[0700] MS (APCI, ESI) m / z: 950 (M+H)+.

[0701] Step 10: N-[4-(11,12-didehydrodibenzo[b,f]azacyclooctatetraen-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[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-alanine amide (5-11)

[0702] Compound (5-10) (0.710 g, 0.747 mmol) obtained in Step 9 above and compound (2-2) (0.313 g, 0.747 mmol) obtained in Step 1 of Example 1-2 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 the mixture was stirred at room temperature for 1 hour. The residue was removed by distillation under reduced pressure, and purified by silica gel column chromatography [chloroform:methanol = 100:0 (v / v) to 80:20 (v / v)] to obtain the target product (5-11) (0.671 g, 66%).

[0703] 1 H-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),6.8 0-6.62(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).

[0704] MS (APCI, ESI) m / z: 1351 (M+H)+.

[0705] [Example 2-4: Drug Linker 4]

[0706]

[0707] Step 1: (6S)-5-[4-(benzyloxy)-5-methoxy-2-nitrobenzoyl]-5-azaspiro[2.4]heptane-6-carboxylic acid methyl ester (6-2)

[0708] 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) was added dropwise oxalyl chloride (3.43 mL, 40.0 mmol) 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). Excess diethyl ether and hexane were added, filtered, and dried under reduced pressure to obtain a crude acid chloride. The resulting acid chloride was dissolved in dichloromethane and cooled to -40°C (dry ice-acetonitrile bath). Methyl (6S)-5-azaspiro[2.4]heptane-6-carboxylate hydrochloride (4.22 g, 22.0 mmol, Tetrahedron Letters 2012.53.3847) and triethylamine (3.36 mL, 24.2 mmol) were slowly 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, and saturated brine, and dried over anhydrous sodium sulfate. The residue was removed by distillation under reduced pressure, and purified by silica gel column chromatography [hexane:ethyl acetate = 100:0 to 50:50] to obtain the desired product (6-2) (6.55 g, 80%).

[0709] MS (APCI, ESI) m / z: 441 (M+H)+

[0710] 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)

[0711] To a solution of compound (6-2) (6.55 g, 16.0 mmol) obtained in step 1 above in ethanol (150 mL) and tetrahydrofuran (150 mL) was added Raney nickel (7.00 g) under a nitrogen atmosphere. Hydrazine monohydrate (7 mL) was added to the reaction mixture, and the temperature was slowly 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 the mixture was stirred for 1 hour. THF (100 mL) was added to the reaction mixture, and the mixture was filtered through celite. The residue was removed by distillation under reduced pressure, and the obtained residue 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%).

[0712] MS (APCI, ESI) m / z: 379 (M+H)+

[0713] 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)

[0714] To a solution of compound (6-3) (10.0 g, 26.4 mmol) obtained in step 2 above in tetrahydrofuran (150 mL) was slowly added dropwise a 2.6 mol / L n-butyllithium n-hexane solution (12.0 mL, 31.8 mmol) 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. After stirring the reaction solution at room temperature for 3 hours, water was added and the mixture was extracted with ethyl acetate. The organic layer was washed with water and saturated brine, and dried over anhydrous sodium sulfate. The residue was removed by distillation under reduced pressure, and the resulting residue 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%).

[0715] MS (APCI, ESI) m / z: 509 (M+H)+

[0716] 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)

[0717] To a solution of compound (6-4) (18.7 g, 36.8 mmol) obtained in step 3 above in tetrahydrofuran (50 mL) and ethanol (100 mL) was added 5% palladium-carbon catalyst (5.00 g) 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. Chloroform was added to the reaction mixture to dilute it, and after filtration through celite, the filtrate was removed by vacuum distillation. The resulting residue was purified by silica gel column chromatography [hexane:ethyl acetate = 100:0 to 25:75] to obtain the target product (6-5) (15.1 g, 98%).

[0718] MS (APCI, ESI) m / z: 419 (M+H)+

[0719] 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)

[0720] The compound (6-5) (2.77 g, 6.62 mmol) obtained in the above step 4 was reacted in the same manner as in Example 2-1, step 2 to obtain the target product (6-6) (3.31 g, 88%).

[0721] 1 H-NMR(CDCl3)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),3.4 6-3.39(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).

[0722] 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)

[0723] 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 product (6-7) (1.11 g, 45%).

[0724] 1 H-NMR(CDCl3)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(1H,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).

[0725] 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)

[0726] 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 product (6-8) (1.15 g, 45%).

[0727] 1 H-NMR(CDCl3)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(1H,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).

[0728] Step 8: (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)-carboxylic acid prop-2-en-1-yl ester (6-9)

[0729] 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 product (6-9) (1.14 g, 82%).

[0730] 1 H-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).

[0731] 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-alanine amide (6-10)

[0732] 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 step 10 of Example 1-1 were reacted in the same manner as in step 10 of Example 2-1 to obtain the target product (6-10) (0.589 g, 65%).

[0733] MS(APCI,ESI)m / z: 1234(M+H)+

[0734] 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-alanine amide (6-11)

[0735] 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 product (6-11) (0.382 g, 71%).

[0736] 1 H-NMR(CDCl3)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).

[0737] 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-alanine amide (6-12)

[0738] 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 product (6-12) (0.200 g, 62%).

[0739] MS (APCI, ESI) m / z: 952 (M+H)+

[0740] Step 12: N-[4-(11,12-didehydrodibenzo[b,f]azacyclooctatetraen-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-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-alanine amide (6-13)

[0741] 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 step 1 of Example 1-2 were reacted in the same manner as in step 13 of Example 2-1 to obtain the target product (6-13) (0.0500 g, 63%).

[0742] MS(APCI,ESI)m / z:1354(M+H)+

[0743] 〔Synthesis of sugar chain donor〕

[0744] Example 3: [N3-PEG(3)]-MSG1-Ox

[0745]

[0746] Step 1: (MSG1-)Asn

[0747] Commercially available monosialo-Asn-free (1S2G / 1G2S-10NC-Asn, manufactured by Sugar Chain Engineering Research Institute, Inc.) (referred to as "(MSG-)Asn") (500 mg) was separated and purified by reverse-phase HPLC under the following conditions. (MSG1-)Asn, eluting as the first major peak (retention time approximately 15-19 minutes), was separated into (MSG2-)Asn, eluting as the second major peak (retention time approximately 21-26 minutes). A 0.1% aqueous formic acid solution was used as the eluent, and the apparatus used was an ELS-PDA triggered fractionation system (manufactured by JASCO Corporation). The column used was an Inertsil ODS-3 (10 μm, 30 Φ × 250 mm, manufactured by GL Sciences), and the flow rate was 30 mL / min. The first peak detected by UV detection (210 nm) during the elution was fractionated and lyophilized to obtain the desired product (238 mg).

[0748] Process 2: MSG1

[0749] The compound obtained in the above step 1 (229 mg) was dissolved in 200 mM phosphate buffer solution (pH 6.25) (1145 μL), and an EndoM (Tokyo Chemical Industry Co., Ltd., 1 U / mL) aqueous solution (100 μL) was added, and the mixture was incubated at 35°C for 6 days. After the reaction was completed, the reaction solution was ultrafiltered using VIVASPIN 15R (Hydrosart membrane, 30K, 6000xG), and the resulting permeate was separated and purified using reverse phase HPLC. A 0.1% trifluoroacetic acid aqueous solution was used as the eluent, and the apparatus used was an ELS-PDA triggered fractionation system (manufactured by JASCO Corporation), and the column used was an Inertsil ODS-3 (manufactured by GL Sciences). The peak of the target product detected by UV detection (210 nm) during the elution was fractionated and lyophilized to obtain the target product (117 mg).

[0750] Step 3: [N3-PEG(3)]-MSG1

[0751] To a 5 ml sampling tube (Ina Optica Co., Ltd.) was added 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. After stirring for 1 hour, the mixture was lyophilized. To the lyophilized 5 ml sampling tube was added 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), and the mixture was stirred at 37°C for 3 hours. After completion of the reaction, the reaction mixture was transferred to a 50 ml centrifuge tube pre-filled with diethyl ether (20 ml). A small centrifuge (Hitachi Koki, CF16RX) was used to precipitate the solid and remove the supernatant. Diethyl ether (10 ml) was then added and centrifuged, followed by decantation. Acetonitrile (10 mL) was then added and centrifuged, followed by repeated decantation twice, followed by drying under reduced pressure to give a crude product. The resulting solid was purified by reversed-phase HPLC under the same conditions as in step 2 to give the desired product (94.2 mg).

[0752] Step 4: [N3-PEG(3)]-MSG1-Ox

[0753] The compound synthesized in step 3 above (100 mg) and an aqueous solution (520 μl) of 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride (56 mg, 0.257 mmol, manufactured by Fushimi Pharmaceutical Co., Ltd.) were added to a 5 mL sampling tube (manufactured by Ina Optica Co., Ltd.). A solution (520 μl) of tripotassium phosphate (165 mg, 0.78 mmol) was added to the ice-cooled reaction solution, and the mixture was stirred under ice-cooling for 3 hours. The resulting reaction solution was ultrafiltered using an Amicon Ultra (Ultracell 30K, manufactured by Merck Millipore) to remove solid matter. The permeate was purified by gel filtration chromatography. The apparatus used was Purif-RP2 (manufactured by Shoko Scientific), the column used was HiPrep 26 / 10 Desalting (manufactured by GE Healthcare), the mobile phase used was a 0.03% aqueous NH3 solution, the flow rate was set to 10 mL / min, and the fraction volume was 10 mL. The fractions containing the target product detected by UV (220 nm) during the elution were collected, added with 1N aqueous sodium hydroxide solution (104 μl, 0.104 mmol) and lyophilized to obtain the target product (84 mg).

[0754] Example 4: [N3-PEG(3)]-MSG-Ox

[0755]

[0756] Step 1: Preparation of (MSG-)Asn

[0757] 1S2G / 1G2S-10NC-Asn-Fmoc (manufactured by Sugar Chain Engineering Research Institute, Ltd.) (referred to as "Fmoc-(MSG-)Asn") (1000 mg) as a commercial product was dissolved in ethanol / water (1 / 1) (10 mL), 1N sodium hydroxide aqueous 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, manufactured by Millipore Corporation) to remove solid matter, and 1N hydrochloric acid (832 μl, 1.9 equivalents) was added to the obtained permeate. The solvent was removed using a high-speed concentrator V-10 (manufactured by Biotage Corporation). Acetonitrile was added, and the solvent was removed using a high-speed concentrator V-10 (manufactured by Biotage Corporation), and then separated and purified using reversed-phase HPLC. A 0.1% trifluoroacetic acid aqueous solution and a 0.1% trifluoroacetic acid acetonitrile solution were used as eluents, using a Purif-Rp2 (Shoko Scientific) apparatus and an Inertsil ODS-3 (GL Sciences) column. Fractions containing the target product detected by UV (220 nm) during the elution were collected and lyophilized. When redissolved in pure water, pH paper confirmed that the product was acidic, so 18% ammonia water (150 μl) was added, and pH paper confirmed that the product was alkaline, and the product was lyophilized again. The resulting target product (840 mg) was used directly in the next reaction.

[0758] Step 2: Synthesis of MSG

[0759] The compound obtained in step 1 (840 mg) was dissolved in 200 mM phosphate buffer (pH 6.25) (6000 μL), and an EndoM (Tokyo Chemical Industry Co., Ltd., 1 U / mL) aqueous solution (200 μL) was added, and the mixture was incubated at 28°C for 26 hours. Since the reaction was not complete, an 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, then left at room temperature until the reaction was complete. After the reaction was completed, the reaction solution was ultrafiltered using an Amicon Ultra (30K, Millipore). Trifluoroacetic acid (80 μL) was added to the resulting permeate, and the product was separated and purified using reverse-phase HPLC. 0.1% trifluoroacetic acid aqueous solution and 0.1% trifluoroacetic acid in acetonitrile were used as eluents, and the apparatus used was Purif-RP2 (Shoko Scientific) and the column used was Inertsil ODS-3 (GL Sciences). The fractions containing the target product detected by UV (220 nm) during the elution were collected and lyophilized. To remove residual trifluoroacetic acid, the fractions were redissolved in pure water to obtain the target compound (618 mg) as a colorless solid.

[0760] ESI-MS:C 66 H 110 N4O 49 :[M+H] + Theoretical value 1743.62, actual value 1743.63

[0761] Step 3: Synthesis of [N3-PEG(3)]-MSG

[0762] The target product (88.6 mg) was obtained by the same method as in Example 3, Step 3, using the compound obtained in the above Step 2 (120 mg).

[0763] ESI-MS:C 73 H 124 N8O 51 :[M+2H] 2+ Theoretical value 965.37, actual value 965.37

[0764] Step 4: Synthesis of [N3-PEG(3)]-MSG-Ox

[0765] The target product (88 mg) was obtained by the same method as in Example 3, Step 4, using the synthesized compound (100 mg) obtained in the above Step 3.

[0766] Example 5: [N3-PEG(3)]2-SG(10)-Ox

[0767]

[0768] Step 1: [N3-PEG(3)]2-SG(10)

[0769] A 5 ml sampling tube (Ina Optica Co., Ltd.) was charged with an aqueous solution (0.5 ml) of 11-azido-3,6,9-trioxaundecan-1-amine (0.096 ml, 0.485 mmol) and disialooctaose (50 mg, 0.24 mmol). After stirring for 1 hour, the mixture was lyophilized. A solution of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (92 mg, 0.24 mmol) in N,N-dimethylformamide (0.6 ml) and diisopropylethylamine (0.042 ml, 0.24 mmol) was added to the lyophilized 5 ml sampling tube and stirred at 37°C for 4 hours. After completion of the reaction, the reaction mixture was transferred to a 50 ml centrifuge tube pre-filled with diethyl ether (20 ml). The solid matter was precipitated using a microcentrifuge (Hitachi Koki, CF16RX), and the supernatant was removed. Add diethyl ether (20 ml) and decant. Then, add acetonitrile (20 mL) and decant, and then dry under reduced pressure to obtain a crude product. The obtained solid substance was dissolved in an appropriate amount of 0.2% trifluoroacetic acid aqueous solution and separated and purified by reverse phase HPLC. 0.1% trifluoroacetic acid aqueous solution and 0.1% trifluoroacetic acid acetonitrile solution were used as eluents, Purif-Rp2 (manufactured by Shoko Scientific) was used as the apparatus, and Inertsil ODS-3 (manufactured by GL Sciences) was used as the column. The fraction containing the target product detected by UV (220 nm) during the elution was collected and freeze-dried to obtain the target product (42 mg).

[0770] Step 2: [N3-PEG(3)]2-SG(10)-Ox

[0771] The compound synthesized in step 1 above (40 mg) and an aqueous solution (200 μl) of 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride (17.9 mg, 0.083 mmol, manufactured by Fushimi Pharmaceutical Co., Ltd.) were added to a 5 mL sampling tube (manufactured by Ina Optica Co., Ltd.). A 200 μl aqueous solution (52.6 mg, 0.25 mmol) of tripotassium phosphate was added to the ice-cooled reaction solution, and the mixture was stirred under ice-cooling for 2 hours. The resulting reaction solution was ultrafiltered using an Amicon Ultra (Ultracell 30K, manufactured by Merck Millipore) to remove solid matter. The permeate was purified by gel filtration chromatography. The apparatus used was Purif-RP2 (manufactured by Shoko Scientific), the column used was HiPrep 26 / 10 Desalting (manufactured by GE Healthcare), the mobile phase used was a 0.03% NH3 aqueous solution, the flow rate was set to 10 mL / min, and the fraction volume was 10 mL. The fractions containing the target product detected by UV (220 nm) during the elution were collected, added with 1N aqueous sodium hydroxide solution (33 μl, 0.033 mmol) and lyophilized to obtain the target product (34 mg).

[0772] Example 6: Mouse anti-CLDN6 antibody B1-producing hybridoma (218B1) and mouse anti-CLDN6 antibody C7-producing hybridoma (218C7)

[0773] 6-1. Immunization of mice and acquisition of hybridomas

[0774] 1-1) Preparation of cells for mouse immunization

[0775] 2×10 6 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, then recovered, washed twice with PBS (phosphate-buffered saline), and resuspended in PBS (300 μl).

[0776] 1-2) Immunization of mice

[0777] BALB / c mice (12 weeks old) were treated with NOR-P1 cells (2 × 10 6 About 2 weeks after the fifth immunization, NOR-P1 cells (5×10 6About 3 weeks after the sixth immunization, NOR-P1 cells (2×10 6 ) were immunized intraperitoneally. 2×10 6 NOR-P1 cells were intraperitoneally immunized for the 8th to 10th time. About 3 weeks after the 10th immunization (the 11th time) and 3 days after that (the 12th time, the final immunization), 5×10 6 NOR-P1 cells were used for intraperitoneal immunization, and spleen cells were removed 3 days after the final immunization.

[0778] 1-3) Preparation of spleen cells from immunized mice

[0779] Spleens from immunized mice were removed, ground, and suspended in RPMI1640 10% FBS (+) medium. The cell suspension was passed through a 70 μm cell strainer (BD Falcon) and centrifuged at 1500 rpm for 5 minutes at room temperature. The supernatant was discarded. A Tris-NH4Cl solution (20 mM Tris-HCl, pH 7.2, 77.6 mM NH4Cl; 20 mL) was added and the mixture was incubated at room temperature for 5 minutes. PBS (20 mL) was added and the mixture was centrifuged at 1500 rpm for 5 minutes at room temperature. The supernatant was discarded and RPMI1640 FBS (+) medium (10 mL) was added.

[0780] 1-4) Preparation of myeloma cells

[0781] P3U1 cells (mouse myeloma cell line) were cultured in RPMI1640 FBS(+) medium for 5 days, then recovered and resuspended in RPMI1640 FBS(+) medium (20 ml).

[0782] 1-5) Cell fusion

[0783] 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 mixture was centrifuged at 1500 rpm for 5 minutes. After the resulting precipitate was fully loosened, polyethylene glycol-1500 (PEG-1500; 1 ml) was slowly added over approximately 1 minute while stirring. After stirring for 3 minutes and 30 seconds, the mixture was allowed to stand at room temperature for 30 seconds. Then, RPMI medium with 10% low IgG FBS (+) (10 ml) was added to the cell suspension over 1 minute. The cell suspension was centrifuged at 1500 rpm for 5 minutes, and the resulting precipitate was gradually loosened and then slowly suspended in 200 ml of HAT medium (RPMI1640 medium containing 10% low IgG FBS, HAT Media Supplement, and 5% BriClone). The suspension was dispensed into 96-well culture plates at 200 μl / well and cultured in an incubator at 37° C. and 5% CO 2 for 6 days.

[0784] 1-6) Hybridoma Screening / Probe Preparation

[0785] DT3C was produced as a recombinant complex protein to measure antibody internalization and immunotoxin activity. DT3C is a genetically engineered protein fused with the catalytic domain of diphtheria toxin (DT) and the antibody-binding domain of streptococcal protein G. DT3C specifically binds to the Fc region of antibodies and, once internalized into cells, induces cell death by inhibiting protein synthesis. This system allows for simultaneous observation of antibody internalization and the cell-killing effects of the immunotoxin (Yamaguchi, M. et al., Biochemical and Biophysical Research Communications 454 (2014) 600-603).

[0786] 1-7) Screening of DT3C-based hybridomas

[0787] 4 μg / ml DT3C (25 μl) was added to a 96-well plate, and the culture supernatant of the hybridoma obtained in step 1-5 (25 μl) was added and incubated at room temperature for 30 minutes. 2×10 5NOR-P1 cells (50 μl) were added to a 50 μl plate containing 10% low-IgG FBS (+) cells per ml of RPMI medium and cultured in a CO2 incubator at 37°C for 3 days. Post-incubation microscopic observation determined wells showing adherent cell counts approximately 25% or less of the number observed with the negative control antibody as positive. The selected clones were subcloned one or two times to establish eight monoclonal hybridoma cell lines.

[0788] 6-2: Identification of the antigen bound by the antibody produced by the hybridoma

[0789] Antigen identification was performed on two clones 218B1 and 218C7 of the antibodies produced by the hybridomas prepared in Example 6-1.

[0790] 2-1) Immunoprecipitation of biotinylated cell surface proteins using 218B1 and 218C7 antibodies

[0791] Remove 2×10 6Culture supernatant of NTERA-2 cells (human testicular cancer cell line, ATCC CRL-1973) was 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, the biotin / PBS solution was added and incubated on a shaker for 30 minutes. The cells were then washed twice with 100 mM glycine / PBS solution (25 ml) and 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, and 1 pellet of Complete EDTA-free (Roche) protease inhibitor per 50 ml) and incubated at 4°C for 30 minutes. Cell lysate was prepared by centrifugation (13,000 rpm, 20 minutes, 4°C). The buffer of Protein G Sepharose (Protein G Sepharose 4 Fast Flow (GE Healthcare)) was exchanged with lysis buffer. The resulting Protein G Sepharose / lysis buffer (50% slurry; 30 μl) was added to the cell lysate and rotated at 4°C for 30 minutes. The supernatant was then centrifuged at 4°C for 1 minute, and the supernatant was recovered. 218B1 antibody or 218C7 antibody (approximately 3 μg) was added to the supernatant and rotated at 4°C for 30 minutes. Protein G Sepharose / lysis buffer (50% slurry; 60 μl) was then added and rotated at 4°C for 1 hour. The Protein G Sepharose was washed six times with lysis buffer (1 ml) and resuspended in 1× SDS sample buffer (BioRad). The suspension was treated at 100°C for 5 minutes, and the solution was recovered to prepare samples for SDS-PAGE (polyacrylamide gel electrophoresis).

[0792] 2-2) SDS-PAGE and Western blotting

[0793] The SDS-PAGE samples prepared in step 2-1) were stacked at 50 mV for 30 minutes using SuperSep_Ace 5-20% (Wako). After electrophoresis at 200 mV for 1 hour, the gels were blotted onto membranes at 12 mV for 47 minutes. The membranes were washed with PBS-T (PBS(-)-0.02% Tween 20) and blocked for 1 hour. The membranes were washed three times with PBS-T for 5 minutes each and then reacted with Streptavidin-horseradish peroxidase conjugate (GE Healthcare; diluted 2000-fold in PBS-T) for 1 hour. The membranes were washed twice with PBS-T for 5 minutes each and the target bands were detected using enhanced chemiluminescence (ECL). In both cases where the 218B1 antibody and the 218C7 antibody were used, a band with a molecular weight of 18 kDa was detected regardless of the addition of DTT.

[0794] 2-3) Quality analysis of immunoprecipitated cellular proteins using 218B1 and 218C7 antibodies

[0795] Recycling 2×10 7 NTERA-2 cells were washed twice with PBS. Cells were harvested using a cell scraper and centrifuged at 1500 rpm for 5 minutes. The supernatant was removed and resuspended in 2 ml of lysis buffer and incubated at 4°C for 30 minutes. 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 recover the supernatant. 218B1 antibody (approximately 9 μg) was added to the supernatant, and the cells were rotated at 4°C for 30 minutes. Protein G Sepharose / lysis buffer (50% slurry; 180 μl) was then added and rotated at 4°C for 1 hour. Protein G Sepharose was washed six times with lysis buffer (1 ml) and resuspended in 1× SDS sample buffer. After treating the suspension at 100°C for 5 minutes, the solution was recovered and used as a sample for SDS-PAGE. SDS-PAGE was performed using the same method as in 2-2), and the electrophoresis gel was stained with Coomassie Brilliant Blue (CBB). A fraction corresponding to 18 kDa was excised from the electrophoresis gel and subjected to mass spectrometry. The results of mass spectrometry confirmed that this gel fragment contained claudin-6.

[0796] 2-4) FACS analysis

[0797] Mass spectrometry analysis predicted that the antigens for the 218B1 and 218C7 antibodies were claudin-6, so forced expression analysis was performed using cDNA gene transfer. FACS analysis revealed strong positive reactions with the 218B1 and 218C7 antibodies in human claudin-6-expressing CHO-K1 cells, confirming that the antigens for the 218B1 and 218C7 antibodies were claudin-6.

[0798] 2-5) Purification of antibodies from hybridoma culture supernatants

[0799] Hybridomas producing mouse anti-CLDN6 antibody B1 (218B1) and mouse anti-CLDN6 antibody C7 (218C7) were cultured in Hybridoma-SFM (Thermo Fisher Scientific) containing 10% Fetal Bovine Serum and Ultra-Low IgG (Thermo Fisher Scientific). The culture supernatant was recovered by centrifugation and filtered through a 0.45 μm filter (Corning). Antibodies were purified from the culture supernatant in a single step using rProtein A affinity chromatography (at 4–6°C). Following purification by rProtein A affinity chromatography, the buffer exchange step was performed at 4–6°C. The culture supernatant was first applied to a column packed with MabSelect SuRe (GE Healthcare Bioscience) equilibrated with PBS. After the culture medium had completely entered the column, the column was washed with PBS at least twice the column volume. Next, elution was performed with a 2M arginine hydrochloride solution (pH 4.0), and the antibody-containing fraction was collected. This fraction was dialyzed (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette) and liquid exchanged with PBS (-). Finally, the IgG concentration was adjusted to 1 mg / mL or higher using a Centrifugal UF Filter Device VIVASPIN20 (molecular weight cutoff UF10K, Sartorius, at 4°C). Filtering was performed using a Minisart-Plus filter (Sartorius) to prepare a purified sample.

[0800] Example 7: In vitro evaluation of mouse anti-CLDN6 antibodies B1 and C7

[0801] 7-1: Evaluation of the Binding Ability of Mouse Anti-CLDN6 Antibodies Using Flow Cytometry

[0802] The binding of the mouse anti-CLDN6 antibodies produced in Example 6 to human CLDN6 and family members 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 purchased from Origene was introduced into 293T cells (Thermo Fisher Scientific HCL4517) using Lipofectamine 2000 (Thermo Fisher Scientific). After incubation overnight at 37°C and 5% CO2, a cell suspension was prepared. The gene-transfected 293T cell suspension was centrifuged, the supernatant removed, and mouse anti-CLDN6 antibody (clone B1, C7) or mouse IgG1 control antibody (R&D Systems) was added to a final concentration of 30 μg / mL, 10 μg / mL, 3.3 μg / mL, and 1.1 μg / mL, followed by suspension and incubation at 4°C for 1 hour. After washing twice with Dulbecco's phosphate-buffered saline (Sigma-Aldrich) containing 5% fetal bovine serum (Hyclone) (hereinafter referred to as PBS containing 5% FBS), the cells were suspended in FLUORESCEIN-CONJUGATED GOAT IgG FRACTION TO MOUSE IgG (WHOLEMOLECULE) (MP BIOMEDICALS) diluted 500-fold in PBS containing 5% FBS, and incubated at 4°C for 1 hour. After washing twice with PBS containing 5% FBS, the cells were detected using a flow cytometer (FC500; Beckman Coulter). Data analysis was performed using FlowJo (TreeStar). It should be noted that after permeabilization of the cells with PBS containing 0.25% Tween20, mouse anti-FLAG antibody (Sigma-Aldrich) was used to confirm the presence of each introduced gene. The results are shown in Figure 40 . Figure 40In the graph, the vertical axis represents FITC (fluorescein isothiocyanate) fluorescence intensity, which indicates the amount of antibody binding, and the horizontal axis represents antibody concentration. The generated mouse anti-CLDN6 antibody binds to human CLDN6 and human CLDN9 to comparable degrees, but does not bind to human CLDN3 or human CLDN4. Mouse control IgG1 did not bind to any cells.

[0803] 7-2: Antibody internalization activity

[0804] The internalization activity of mouse anti-CLDN6 antibodies B1 and C7 was evaluated using the anti-mouse IgG reagent Mab-ZAP (ADVANCED TARGETING SYSTEMS), which is conjugated to a protein synthesis-inhibiting toxin (saporin). This evaluation demonstrates that Mab-ZAP is internalized into cells, and the protein synthesis-inhibiting saporin is released into the cells, thereby suppressing cell proliferation.

[0805] Human CLDN6-positive cells, such as the human choriocarcinoma cell line JEG-3 (ATCC HTB-36), human ovarian cancer cell line NIH: OVCAR-3 (ATCC HTB-161), or human CLDN6-negative cells, such as the human pancreatic cancer cell line BxPC-3 (ATCC CRL-1687), were cultured at a concentration of 2 × 10 3 cells / well were seeded into a 96-well cell culture microplate and cultured overnight at 37°C and 5% CO2. The next day, a mixed solution of mouse anti-CLDN6 antibody or mouse IgG1 antibody (R&D Systems) at a final concentration of 1 nM and Mab-ZAP (final concentration: 0.5 nM) or AffiniPure Goat Anti-Mouse IgG, FcγFragment Specific (Jackson ImmunoResearch) (final concentration: 0.5 nM) without toxin was added and cultured at 37°C and 5% CO2 for 5 days. The number of surviving cells was determined by quantifying ATP activity using CellTiter-Glo Luminescent Cell Viability Assay (Promega). The cell proliferation inhibitory effect caused by the addition of anti-CLDN6 antibodies was calculated by setting the value of the well without the mixed solution to 100% as the relative survival rate. The results are shown in Figure 41Mouse anti-CLDN6 antibodies (B1 and C7) showed inhibitory effects on the human CLDN6-positive cell lines JEG-3 and NIH:OVCAR-3. However, no inhibitory effects were observed on the human CLDN6-negative cell line BxPC-3. Furthermore, mouse IgG1 antibodies showed no inhibitory effects on any of the cell lines. These results suggest that the generated anti-CLDN6 antibodies (B1 and C7) have internalization activity and are suitable for use in antibody-drug conjugates.

[0806] Example 8: Determination of the nucleotide sequences of cDNAs encoding the variable regions of mouse anti-CLDN6 antibodies B1 and C7

[0807] 8-1: Determination of the nucleotide sequence of the cDNA encoding the variable region of the B1 antibody

[0808] 8-1-1: Preparation of total RNA from B1 antibody-producing hybridomas

[0809] In order to amplify cDNA encoding the variable region of the B1 antibody, total RNA was prepared from the B1 antibody-producing hybridoma using TRIzol Reagent (Ambion).

[0810] 8-1-2: Amplification and sequence determination of the cDNA encoding the light chain variable region of the B1 antibody using 5'-RACE PCR

[0811] Amplification of the cDNA encoding the light chain variable region was performed using approximately 1 μg of the total RNA prepared in Example 8-1-1 and the SMARTer RACE 5' / 3' Kit (Clontech). Primers used for PCR amplification of the cDNA encoding the variable region of the light chain gene of the B1 antibody included UPM (included in the Universal Primer A Mix: SMARTer RACE 5' / 3' Kit) and primers designed based on the sequence of the known mouse light chain constant region.

[0812] 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 sequenced.

[0813] The nucleotide sequence of the determined cDNA encoding the light chain variable region of the B1 antibody is shown in SEQ ID NO: 18, and the amino acid sequence is shown in SEQ ID NO: 19.

[0814] 8-1-3: Amplification and sequence determination of the cDNA encoding the heavy chain variable region of the B1 antibody using 5'-RACE PCR

[0815] Amplification of the cDNA encoding the heavy chain variable region was performed using approximately 1 μg of the total RNA prepared in Example 8-1-1 and the SMARTer RACE 5' / 3' Kit (Clontech). Primers used for PCR amplification of the cDNA encoding the variable region of the heavy chain gene of the LB1 antibody included UPM (included in the Universal Primer AMix: SMARTer RACE 5' / 3' Kit) and primers designed based on the sequence of the known mouse heavy chain constant region.

[0816] 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 sequenced.

[0817] The nucleotide sequence of the determined cDNA encoding the heavy chain variable region of the B1 antibody is shown in SEQ ID NO: 20, and the amino acid sequence is shown in SEQ ID NO: 21.

[0818] 8-2: Determination of the nucleotide sequence of the cDNA encoding the variable region of the C7 antibody

[0819] The same method as in Example 8-1 was used. The nucleotide sequence of the cDNA encoding the light chain variable region of the C7 antibody was shown in SEQ ID NO: 22, and the amino acid sequence was shown in SEQ ID NO: 23. The nucleotide sequence of the cDNA encoding the heavy chain variable region was shown in SEQ ID NO: 24, and the amino acid sequence was shown in SEQ ID NO: 25.

[0820] Example 9: Preparation of chimeric anti-CLDN6 antibody chB1

[0821] 9-1: Construction of an expression vector for the chimeric anti-CLDN6 antibody chB1

[0822] 9-1-1: Construction of chimeric and humanized light chain expression vector pCMA-LK

[0823] pcDNA3.3 / LK was created by ligating the approximately 5.4 kb fragment obtained by digesting the plasmid pcDNA3.3-TOPO / LacZ (Invitrogen) with the restriction enzymes XbaI and PmeI with the DNA fragment encoding the human light chain signal sequence and human kappa chain constant region as shown in SEQ ID NO: 26 using the In-Fusion HD PCR Cloning Kit (Clontech). pCMA-LK was constructed by removing the neomycin expression unit from pcDNA3.3 / LK.

[0824] 9-1-2: Construction of chimeric and humanized IgG1LALA heavy chain expression vector pCMA-G1LALA

[0825] Using the In-Fusion HD PCR Cloning Kit (Clontech), 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 combined with a DNA fragment containing the DNA sequence encoding the human heavy chain signal sequence and human IgG1LALA constant region shown in SEQ ID NO: 27 to construct pCMA-G1LALA.

[0826] 9-1-3: Construction of chimeric chB1 heavy chain expression vector

[0827] A DNA fragment representing nucleotides 36 to 440 of the chB1 heavy chain sequence shown in SEQ ID NO: 33 was synthesized (GENEART). Using the In-Fusion HD PCR Cloning Kit (Clontech), the synthesized DNA fragment was inserted into the site of pCMA-G1LALA digested 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.

[0828] 9-1-4: Construction of chimeric chB1 light chain expression vector

[0829] A DNA fragment containing the DNA sequence encoding the chB1 light chain shown in SEQ ID NO: 29 was synthesized (GENEART). Using the In-Fusion HD PCR Cloning Kit (Clontech), the synthesized DNA fragment was ligated with 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. This constructs the chB1 light chain expression vector. The amino acid sequence of the chB1 light chain is shown in SEQ ID NO: 28.

[0830] 9-2: Production and purification of the chimeric anti-CLDN6 antibody chB1

[0831] 9-2-1: Production of chimeric antibody chB1

[0832] FreeStyle 293F cells (Invitrogen) were passaged and cultured according to the manual. 1.2×10 9 FreeStyle 293F cells (Invitrogen) were inoculated into 3 L Fernbach Erlenmeyer Flask (CORNING) and diluted with FreeStyle 293 expression medium (Invitrogen) to prepare 2.0 × 10 6cells / mL. To 40 mL of Opti-Pro SFM medium (Invitrogen), 0.24 mg of the heavy chain expression vector, 0.36 mg of the light chain expression vector, and 1.8 mg of polyethyleneimine (Polyscience #24765) were added and stirred gently. After incubation for 5 minutes, the cells were added to FreeStyle 293F cells. After shaking at 90 rpm in an incubator at 37°C and 8% CO₂ for 4 hours, 600 mL of EX-CELL VPRO medium (SAFC Biosciences), 18 mL of GlutaMAX I (GIBCO), and 30 mL of Yeastolate Ultrafiltrate (GIBCO) were added. The cells were shaken at 90 rpm in an incubator at 37°C and 8% CO₂ for 7 days. The resulting culture supernatant was filtered using a disposable capsule filter (Advantec #CCS-045-E1H). The obtained chimeric anti-CLDN6 antibody was named "chB1".

[0833] 9-2-2: Purification of chimeric antibody chB1

[0834] The antibody was purified from the culture supernatant obtained in Example 9-2-1 using a one-step process of rProtein A affinity chromatography. The culture supernatant was applied to a column filled with MabSelectSuRe (manufactured by GE Healthcare Bioscience) equilibrated with PBS, and then the column was washed with PBS at least twice the column capacity. Next, elution was performed with a 2M arginine hydrochloride solution (pH 4.0), and the antibody-containing fraction was collected. This fraction was subjected to buffer exchange with PBS (-) by dialysis (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette). The antibody was concentrated using a Centrifugal UFFilter Device VIVASPIN20 (molecular weight cutoff UF10K, Sartorius), and the IgG concentration was adjusted to above 1 mg / mL. Finally, the purified sample was filtered using a Minisart-Plus filter (Sartorius).

[0835] Example 10: Preparation of humanized anti-CLDN6 antibodies

[0836] 10-1: Design of humanized anti-CLDN6 antibody

[0837] 10-1-1: Molecular modeling of the variable region of the chimeric antibody chB1

[0838] Molecular modeling of the chB1 variable region utilized a method known as homology modeling (Methods in Enzymology, 203, 121-153, (1991)). The modeling was performed using the commercially available protein three-dimensional structure analysis program BioLuminate (manufactured by Schrodinger) using as a template the structure (PDB ID: 1XIW) registered in the Protein Data Bank (Nuc. Acid Res. 35, D301-D303 (2007)) for the variable regions of the heavy and light chains of chB1, which has high sequence identity.

[0839] 10-1-2: Design of humanized amino acid sequences

[0840] 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, as defined by Kabat et al. (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service National Institutes of Health, Bethesda, MD. (1991)), were highly identical to the framework regions of chB1 and were therefore selected as acceptors for the heavy and light chains, respectively. Donor residues to be transferred to the acceptors were selected by analyzing the three-dimensional model with reference to benchmarks provided by Queen et al. (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)). It should be noted that CDRL3 is rich in hydrophobic amino acids, so a humanized light chain was also designed by introducing mutations into CDRL3.

[0841] 10-2: Humanization of the chB1 heavy chain

[0842] The three designed heavy chains were named hH1, hH2, and hH3. The full-length amino acid sequence of the hH1 heavy chain is described in SEQ ID NO: 52. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 52 is described in SEQ ID NO: 53. The full-length amino acid sequence of the hH2 heavy chain is described in SEQ ID NO: 56. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 56 is described in SEQ ID NO: 57. The full-length amino acid sequence of the hH3 heavy chain is described in SEQ ID NO: 60. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 60 is described in SEQ ID NO: 61.

[0843] 10-3: Humanization of the chB1 light chain

[0844] The four designed light chains were named hL1, hL2, hL3, and hL4. The full-length amino acid sequence of the light chain of hL1 is described in SEQ ID NO: 36. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 36 is described in SEQ ID NO: 37. The full-length amino acid sequence of the light chain of hL2 is described in SEQ ID NO: 40. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 40 is described in SEQ ID NO: 41. The full-length amino acid sequence of the light chain of hL3 is described in SEQ ID NO: 44. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 44 is described in SEQ ID NO: 45. The full-length amino acid sequence of the light chain of hL4 is described in SEQ ID NO: 48. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 48 is described in SEQ ID NO: 49.

[0845] 10-4: Design of humanized antibodies using combinations of heavy and light chains

[0846] An antibody comprising hH1 and hL1 is referred to as an "H1L1 antibody" or "H1L1." An antibody comprising hH2 and hL2 is referred to as an "H2L2 antibody" or "H2L2." An antibody comprising hH1 and hL3 is referred to as an "H1L3 antibody" or "H1L3." An antibody comprising hH2 and hL4 is referred to as an "H2L4 antibody" or "H2L4." An antibody comprising hH3 and hL3 is referred to as an "H3L3 antibody" or "H3L3."

[0847] 10-5: Preparation of humanized anti-CLDN6 antibodies

[0848] 10-5-1: Construction of humanized heavy chain expression vector

[0849] 10-5-1-1: Construction of hH1 expression vector

[0850] A DNA fragment represented by nucleotide numbers 36 to 440 of the nucleotide sequence of hH1 represented by SEQ ID NO: 53 was synthesized (GENEART). An hH1 expression vector was constructed in the same manner as in Example 9-1-3.

[0851] 10-5-1-2: Construction of hH2 expression vector

[0852] A DNA fragment represented by nucleotide numbers 36 to 440 of the nucleotide sequence of hH2 represented by SEQ ID NO: 57 was synthesized (GENEART). An hH2 expression vector was constructed in the same manner as in Example 9-1-3.

[0853] 10-5-1-3: Construction of hH3 expression vector

[0854] A DNA fragment represented by nucleotide numbers 36 to 440 of the nucleotide sequence of hH3 represented by SEQ ID NO: 61 was synthesized (GENEART). An hH3 expression vector was constructed in the same manner as in Example 9-1-3.

[0855] 10-5-2: Construction of humanized light chain expression vector

[0856] 10-5-2-1: Construction of hL1 expression vector

[0857] 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 the In-Fusion HD PCR Cloning Kit (Clontech), the synthesized DNA fragment was inserted into the site where pCMA-LK had been cleaved with the restriction enzyme BsiWI, thereby constructing an hL1 expression vector.

[0858] 10-5-2-2: Construction of hL2 expression vector

[0859] A DNA fragment represented by nucleotide numbers 37 to 402 of the nucleotide sequence of hL2 represented by SEQ ID NO: 41 was synthesized (GENEART). An hL2 expression vector was constructed in the same manner as in Example 10-5-2-1.

[0860] 10-5-2-3: Construction of hL3 expression vector

[0861] A DNA fragment represented by nucleotide numbers 37 to 402 of the nucleotide sequence of hL3 represented by SEQ ID NO: 45 was synthesized (GENEART). An hL3 expression vector was constructed in the same manner as in Example 10-5-2-1.

[0862] 10-5-2-4: Construction of hL4 expression vector

[0863] A DNA fragment represented by nucleotide numbers 37 to 402 of the nucleotide sequence of hL4 represented by SEQ ID NO: 49 was synthesized (GENEART). An hL4 expression vector was constructed in the same manner as in Example 10-5-2-1.

[0864] 10-5-3: Preparation of humanized antibodies

[0865] 10-5-3-1: Production of humanized antibodies H1L1, H2L2, H1L3, H2L4, and H3L3

[0866] H1L1, H2L2, H1L3, H2L4, and H3L3 were produced by the same method as in Example 9-2-1 using a combination of heavy chain expression vectors and light chain expression vectors corresponding to the heavy chain and light chain combinations shown in Example 10-4.

[0867] 10-5-3-2: Two-step purification of humanized antibodies H1L1, H2L2, H1L3, H2L4, and H3L3

[0868] The culture supernatant obtained in Example 10-5-3-1 was purified using a two-step process involving rProtein A affinity chromatography and ceramic hydroxyapatite. The culture supernatant was applied to a MabSelect SuRe column (GE Healthcare Bioscience) equilibrated with PBS, and the column was washed with PBS twice the column volume. The antibody was then eluted with a 2M arginine hydrochloride solution (pH 4.0). The antibody-containing fraction was exchanged with PBS by dialysis (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette), diluted fivefold with a 5mM sodium phosphate / 50mM MES / pH 7.0 buffer, and then applied to a ceramic hydroxyapatite column (Bio-Rad, Japan, Bio-Scale CHT Type-1 Hydroxyapatite Column) equilibrated with a 5mM NaPi / 50mM MES / 30mM NaCl / pH 7.0 buffer. A linear concentration gradient of elution was performed using sodium chloride, and the antibody-containing fraction was collected. This fraction was then subjected to buffer exchange with HBsor (25 mM histidine / 5% sorbitol, pH 6.0) via dialysis (ThermoScientific, Slide-A-Lyzer Dialysis Cassette). Antibodies were concentrated using a Centrifugal UF Filter Device VIVASPIN20 (molecular weight cutoff UF10K, Sartorius) to an IgG concentration of 50 mg / mL. Finally, the purified sample was filtered using a Minisart-Plus filter (Sartorius).

[0869] Example 11: Evaluation of Binding Ability of Humanized Anti-CLDN6 Antibodies Using Flow Cytometry

[0870] The binding of the humanized anti-CLDN6 antibodies produced in Example 10 to human CLDN6 and family members CLDN3, CLDN4, and CLDN9 was evaluated by flow cytometry. 293T cells transiently gene-transferred using the same method as in Example 7-1 were used. Humanized anti-CLDN6 antibodies H1L1, H2L2, H1L3, H2L4, and H3L3, or a human IgG1 control antibody (CALBIOCHEM), were added to cells transfected with the human CLDN6 or human CLDN9 genes at final concentrations of 100 nM, 20 nM, 4 nM, and 0.8 nM, respectively, and the cells were suspended and incubated at 4°C for 30 minutes. Humanized anti-CLDN6 antibodies H1L1, H2L2, H1L3, H2L4, and H3L3 were added to cells transfected with the human CLDN3 or human CLDN4 genes or an empty vector, and the cells were suspended and incubated 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')2Fragment Goat Anti-Human IgG (H+L) (Jackson ImmunoResearch) diluted 150 times with PBS containing 5% FBS was added and suspended, and 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 represents the amount of antibody binding, was calculated. The results are shown in Figure 42 . Figure 42 In the graph, the horizontal axis represents antibody concentration, and the vertical axis represents MFI. The prepared humanized anti-CLDN6 antibody binds to human CLDN6 and human CLDN9 to the same extent, but does not bind to human CLDN3 or human CLDN4. Human control IgG1 did not bind to any cells.

[0871] Example 12: Preparation of trastuzumab mutants

[0872] 12-1: Construction of the heavy chain expression vector of trastuzumab-LALA

[0873] A DNA fragment (GENEART) containing nucleotides 36 to 434 of the nucleotide sequence of the heavy chain of trastuzumab-LALA shown in SEQ ID NO: 74 was synthesized. An expression vector was constructed using the same method as in Example 9-1-3. The amino acid sequence of the heavy chain of trastuzumab-LALA is shown in SEQ ID NO: 75.

[0874] 12-2: Construction of the light chain expression vector of trastuzumab-LALA

[0875] A DNA fragment (GENEART) containing nucleotides 37 to 402 of the nucleotide sequence of the light chain of trastuzumab-LALA shown in SEQ ID NO: 72 was synthesized. An expression vector was constructed using the same method 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.

[0876] 12-3: Production of trastuzumab mutants

[0877] The product was produced in the same manner as in Example 9-2-1.

[0878] 12-4: Refining of trastuzumab mutants

[0879] 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 (pH 6.0) instead of PBS(-).

[0880] [Preparation of Sugar Chain Remodeled Antibodies]

[0881] Example 13: Sugar Chain Conversion 1 (T-SG)

[0882]

[0883] Step 1: Preparation of (Fucα1,6)GlcNAc-trastuzumab

[0884] 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 buffer exchanged twice with 50 mM phosphate buffer (pH 6.0) using common procedure C. 1.26 mL and 1.27 mL of a 2.0 mg / mL wild-type EndoS solution (PBS) were added to the resulting 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 the mixture was incubated at 37°C for 4 hours. The progress of the reaction was determined using an Experion electrophoresis workstation (manufactured by BIO-RAD). After completion of the reaction, purification by affinity chromatography and purification using a hydroxyapatite column were performed according to the following methods.

[0885] (1) Purification by affinity chromatography

[0886] Purification device: AKTA pure150 (manufactured by GE Healthcare)

[0887] Column: HiTrap rProtein A FF (5 mL) (manufactured by GE Healthcare)

[0888] Flow rate: 5 mL / min (1.25 mL / min during charge)

[0889] The reaction solution obtained above was purified in multiple batches. Two columns were connected. During binding, the reaction solution was added to the top of the column. Binding buffer (20 mM phosphate buffer (pH 6.0)) was passed through the column at 1.25 mL / min for 2 CV, and further at 5 mL / min for 5 CV. During intermediate washes, a wash solution (20 mM phosphate buffer (pH 7.0), 0.5 M sodium chloride solution) was passed through the column for 15 CV. During elution, elution buffer (ImmunoPure IgG Eution Buffer, Pierce) was passed through the column for 6 CV. The eluate was immediately neutralized with 1 M Tris buffer (pH 9.0). Fractions detected by UV light (280 nm) during elution were confirmed using a microspectrophotometer (Xpose, Trinean) and an Experion electrophoresis workstation (Bio-Rad). The fraction containing the target product was buffer-exchanged using common procedure C with a 5 mM phosphate buffer solution and a 50 mM 2-morpholinoethanesulfonic acid (MES) solution (pH 6.8).

[0890] (2) Purification by hydroxyapatite chromatography

[0891] Purification device: AKTA avant 25 (manufactured by GE Healthcare)

[0892] Column: Bio-Scale Mini CHT Type I Cartridge (5mL) (manufactured by BIO-RAD)

[0893] Flow rate: 5 mL / min (1.25 mL / min during feeding)

[0894] Connect two columns and purify the solution obtained in (1) above in multiple batches. Add the solution to the top of the column and pass solution A at 1.25 mL / min for 2 CV. Further, pass solution A (5 mM phosphate buffer, 50 mM morpholineethanesulfonic acid (MES) solution (pH 6.8)) at 5 mL / min for 3 CV. Then, use solution A and solution B (5 mM phosphate buffer, 50 mM morpholineethanesulfonic acid (MES) solution (pH 6.8), 2 M sodium chloride solution) for elution. The elution conditions are solution A:solution B = 100:0 to 0:100 (15 CV). Furthermore, pass a cleaning solution (500 mM phosphate buffer (pH 6.5)) for 5 CV.

[0895] Fractions containing the target product were buffer-exchanged 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).

[0896] Step 2: Preparation of trastuzumab [SG-(N3)2]2

[0897] To the 23.9 mg / mL (Fucα1,6)GlcNAc-trastuzumab solution (3.37 mL) obtained in Step 1 above, a solution (12.9 mg) of the compound synthesized in Step 2 of Example 5 in 50 mM phosphate buffer (pH 6.0) (0.258 mL) and a 4.90 mg / mL EndoS D233Q / Q303L solution (0.328 mL) in PBS were added. The mixture was incubated at 30°C for 4.5 hours. Two sets of the above procedure were performed. The progress of the reaction was confirmed using an Experion electrophoresis workstation (BIO-RAD). After completion of the reaction, purification by affinity chromatography and purification by hydroxyapatite chromatography were performed in the same manner as in the above step 1, and then the fraction containing the target product was buffer exchanged with phosphate-buffered saline (pH 6.0) using common procedure C to obtain a 10.0 mg / mL trastuzumab [SG-(N3)2]2 solution (phosphate-buffered saline (pH 6.0)) (15.5 mL).

[0898] Example 14: Sugar Chain Conversion 2 (T-MSG)

[0899]

[0900] Step 1: Trastuzumab [MSG-N3]2

[0901] Five sets of the following procedure were performed. Using the compound obtained in Step 1 of Example 13 (20 mg / mL, 15.0 mL) and the compound obtained in Step 4 of Example 4 (25.5 mg) as a sugar chain donor, the mixture was incubated at 30°C for 3 hours, and the same procedure as in Step 2 of Example 13 was performed. The five sets were combined to obtain a 14.4 mg / mL trastuzumab [MSG-N3]2 solution in phosphate-buffered saline (pH 6.0) (93.5 mL).

[0902] Example 15: Sugar Chain Conversion 3 (T-MSG1)

[0903]

[0904] Step 1: Trastuzumab [MSG1-N3]2

[0905] Two sets of the following procedures were performed. Using the compound obtained in Step 1 of Example 13 (25.5 mL, 7.8 mL) and the compound obtained in Step 4 of Example 3 (25.5 mg) as the sugar chain donor, the mixture was incubated at 30°C for 3 hours, and the same procedures as in Step 2 of Example 13 were performed. The two sets were combined to obtain a 10.6 mg / mL trastuzumab [MSG1-N3]2 solution in phosphate-buffered saline (pH 6.0) (31 mL).

[0906] Example 16: Sugar Chain Conversion 4 (CLDN6-MSG1 (H1L1))

[0907]

[0908] Step 1: (Fucα1,6)GlcNAc-anti-CLDN6 antibody (H1L1)

[0909] 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, the same operation as in Step 1 of Example 13 was performed to obtain a 19.2 mg / mL (Fucα1,6)GlcNAc-anti-CLDN6 antibody (H1L1) solution (50 mM phosphate buffer (pH 6.0)) (4.8 mL).

[0910] Step 2: Anti-CLDN6 antibody (H1L1)-[MSG1-N3]2

[0911] 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 the above step 1, the same operation as in step 1 of Example 15 was carried out to obtain a 10.2 mg / mL anti-CLDN6 antibody (H1L1)-[MSG1-N3]2 solution (phosphate-buffered saline (pH 6.0)) (7.2 mL).

[0912] Example 17: Sugar Chain Conversion 5 (CLDN6-MSG1 (H2L2))

[0913] Step 1: (Fucα1,6)GlcNAc-anti-CLDN6 antibody (H2L2)

[0914] Using the anti-CLDN6 antibody (H2L2) solution ca. 20 mg / mL (25 mM histidine solution (pH 6.0), 5% sorbitol solution) (6 mL) prepared in Example 10, the same operation as in Step 1 of Example 13 was performed to obtain a 21.84 mg / mL (Fucα1,6)GlcNAc-anti-CLDN6 antibody (H2L2) solution (50 mM phosphate buffer (pH 6.0)) (5.7 mL).

[0915] Step 2: Anti-CLDN6 antibody (H2L2)-[MSG1-N3]2

[0916] 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 the above step 1, the same operation as in step 1 of Example 15 was carried out to obtain a 10.2 mg / mL anti-CLDN6 antibody (H2L2)-[MSG1-N3]2 solution (phosphate-buffered saline (pH 6.0)) (11.1 mL).

[0917] Example 18: Sugar Chain Conversion 6 (CLDN6-MSG1 (H1L3))

[0918] Step 1: (Fucα1,6)GlcNAc-anti-CLDN6 antibody (H1L3)

[0919] 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, the same operation as in Step 1 of Example 13 was performed 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).

[0920] Step 2: Anti-CLDN6 antibody (H1L3)-[MSG1-N3]2

[0921] The same procedure as in Step 1 of Example 15 was performed 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 to obtain a 9.83 mg / mL anti-CLDN6 antibody (H1L3)-[MSG1-N3]2 solution (phosphate-buffered saline (pH 6.0)) (7.2 mL).

[0922] Example 19: Sugar Chain Conversion 7 (CD98-MSG1)

[0923] Step 1: (Fucα1,6)GlcNAc-anti-CD98 antibody

[0924] Using the anti-CD98 antibody solution ca. 20 mg / mL (25 mM histidine solution (pH 6.0), 5% sorbitol solution) (6 mL) prepared in Reference Example 6, the same operation as in Step 1 of Example 13 was performed to obtain 21.7 mg / mL of (Fucα1,6)GlcNAc-anti-CD98 antibody solution (50 mM phosphate buffer (pH 6.0)) (4.7 mL).

[0925] Step 2: Anti-CD98 Antibody-[MSG1-N3]2

[0926] 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 the above step 1, the same operation as in step 1 of Example 15 was performed to obtain a 10.1 mg / mL anti-CD98 antibody-[MSG1-N3]2 solution (phosphate-buffered saline (pH 6.0)) (7.6 mL).

[0927] Example 20: Sugar Chain Conversion 8 (TROP2-MSG1)

[0928] Step 1: (Fucα1,6)GlcNAc-anti-Trop2 antibody

[0929] 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, the same operation as in Step 1 of Example 13 was performed to obtain 21.69 mg / mL of (Fucα1, 6)GlcNAc-anti-Trop2 antibody solution (50 mM phosphate buffer (pH 6.0)) (3.3 mL).

[0930] Step 2: Anti-Trop2 Antibody-[MSG1-N3]2

[0931] 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 the above step 1, the same operation as in step 1 of Example 15 was performed to obtain a 10.3 mg / mL anti-Trop2 antibody-[MSG1-N3]2 solution (phosphate-buffered saline (pH 6.0)) (6.4 mL).

[0932] Example 21: Sugar Chain Conversion 9 (LPS-MSG1)

[0933] Step 1: (Fucα1,6)GlcNAc-anti-LPS antibody

[0934] 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, the same operation as in Step 1 of Example 13 was performed to obtain a 21.03 mg / mL (Fucα1,6)GlcNAc-anti-LPS antibody solution (50 mM phosphate buffer (pH 6.0)) (5.4 mL).

[0935] Step 2: Anti-LPS Antibody - [MSG1-N3]2

[0936] 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 the above step 1, the same operation as in step 1 of Example 15 was carried out to obtain a 9.89 mg / mL anti-LPS antibody-[MSG1-N3]2 solution (phosphate-buffered saline (pH 6.0)) (7.9 mL).

[0937] Example 22: Sugar Chain Switch 10 (Trastuzumab Mutant - MSG1)

[0938] Step 1: (Fucα1,6)GlcNAc-trastuzumab mutant

[0939] The same operation as in step 1 of Example 13 was performed using the trastuzumab mutant 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 mutant solution (50 mM phosphate buffer (pH 6.0)) (6.1 mL).

[0940] Step 2: Trastuzumab mutant - [MSG1-N3]2

[0941] Using the 6.1 mg / ml (Fucα1,6)GlcNAc-trastuzumab mutant solution (50 mM phosphate buffer (pH 6.0)) (6.1 ml) obtained in the above step 1, the same operation as in step 1 of Example 15 was carried out to obtain a 10.2 mg / ml trastuzumab mutant-[MSG1-N3]2 solution (phosphate-buffered saline (pH 6.0)) (3.7 ml).

[0942] Synthesis of ADC

[0943] As shown in the following reaction formula, ADCs 1, 2, and 15 described in Examples 23, 24, and 52 were synthesized by coupling the antibody obtained in Step 1 of Example 14 with a drug linker. In the formula, R differs depending on the drug linker used in each example.

[0944]

[0945] Example 23: ADC1

[0946]

[0947] (As shown in the above formula, the compound obtained in Step 1 of Example 23 has geometric isomers of triazole rings, which are maintained by mixing the drug linkers containing the two structures represented by R above.)

[0948] Step 1: Conjugation of Antibody and Drug Linker

[0949] To a phosphate-buffered saline (pH 6.0) solution (10.0 mg / mL, 1.00 mL) of the antibody obtained in Step 1 of Example 14 were added 1,2-propylene glycol (0.917 mL) and a dimethyl sulfoxide solution (0.0825 mL; 12 equivalents per antibody molecule) containing 10 mM of the compound (6-13) obtained in Step 12 of Example 2-4 at room temperature. The mixture was reacted at room temperature for 2 days using a tube rotator (MTR-103, AS ONE Co., Ltd.).

[0950] Purification procedure: The above solution was purified using common procedure D to obtain 6.0 mL of a solution containing the target compound.

[0951] Property evaluation: Common operations E and F were used to obtain the following property values.

[0952] Antibody concentration: 1.33 mg / mL, antibody yield: 7.97 mg (80%), average number of drug binding per antibody molecule (n): 1.9

[0953] Example 24: ADC2

[0954]

[0955] (As shown in the above formula, the compound obtained in Step 1 of Example 24 has geometric isomers of triazole rings, and the drug linkers containing the two structures represented by R above are mixed and maintained.)

[0956] Step 1: Conjugation of Antibody and Drug Linker

[0957] 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) were added 1,2-propylene glycol (0.459 mL) and a dimethyl sulfoxide solution containing 10 mM of the compound (5-11) obtained in Step 10 of Example 2-3 (0.0408 mL; 12 equivalents per antibody molecule) at room temperature. The mixture was reacted at room temperature for 1 day using a tube rotator (MTR-103, AS ONE Co., Ltd.).

[0958] Purification: 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.

[0959] Property evaluation: Common operations E and F were used to obtain the following property values.

[0960] Antibody concentration: 7.27 mg / mL, antibody yield: 3.54 mg (72%), average number of drug binding per antibody molecule (n): 1.8

[0961] As shown in the following reaction formula, ADC3 and ADC4 described in Examples 25 and 26 were synthesized by coupling the antibody obtained in Step 2 of Example 13 with a drug linker. In the formula, R differs depending on the drug linker used in each example.

[0962]

[0963] Example 25: ADC3

[0964]

[0965] (As shown in the above formula, the compound obtained in Step 1 of Example 25 has geometric isomers of triazole rings, which are maintained by mixing the drug linkers containing the two structures represented by R above.)

[0966] Step 1: Conjugation of Antibody and Drug Linker

[0967] To a phosphate-buffered saline (pH 6.0) solution (10.0 mg / mL, 1.00 mL) of the antibody obtained in Step 2 of Example 13 were added 1,2-propylene glycol (0.835 mL) and a dimethyl sulfoxide solution containing 10 mM of the compound (4-12) obtained in Step 12 of Example 2-2 (0.165 mL; 24 equivalents per antibody molecule) at room temperature. The mixture was reacted at room temperature for 48 hours using a tube rotator (MTR-103, AS ONE Co., Ltd.).

[0968] Purification procedure: The above solution was purified using common procedure D to obtain 4.50 mL of a solution containing the target compound.

[0969] Property evaluation: Common operations E and F were used to obtain the following property values.

[0970] Antibody concentration: 1.66 mg / mL, antibody yield: 7.48 mg (75%), average number of drug binding per antibody molecule (n): 3.8

[0971] Example 26: ADC4

[0972]

[0973] (As shown in the above formula, the compound obtained in Step 1 of Example 26 has geometric isomers of triazole rings, which are maintained by mixing the drug linkers containing the two structures represented by R above.)

[0974] Step 1: Conjugation of Antibody and Drug Linker

[0975] To a phosphate-buffered saline (pH 6.0) solution (10.0 mg / mL, 1.00 mL) of the antibody obtained in Step 2 of Example 13 were added 1,2-propylene glycol (0.835 mL) and a dimethyl sulfoxide solution (0.165 mL; 24 equivalents per antibody molecule) containing 10 mM of the compound (6-13) obtained in Step 12 of Example 2-4 at room temperature. The mixture was reacted at room temperature for 2 days using a tube rotator (MTR-103, AS ONE Co., Ltd.).

[0976] Purification procedure: The above solution was purified using common procedure D to obtain 10.5 mL of a solution containing the target compound.

[0977] Property evaluation: Common operations E and F were used to obtain the following property values.

[0978] Antibody concentration: 0.73 mg / mL, antibody yield: 7.70 mg (77%), average number of drug binding per antibody molecule (n): 3.8

[0979] As shown in the following reaction formula, ADCs 5-7 and 16-19 described in Examples 27, 28, 29, and 53-56 were synthesized by coupling the antibody obtained in Step 1 of Example 15 with a drug linker. In the formula, the R group differs depending on the drug linker used in each example.

[0980]

[0981] Example 27: ADC5

[0982]

[0983] (As shown in the above formula, the compound obtained in Step 1 of Example 27 has geometric isomers of triazole rings, and the drug linkers containing the two structures represented by R above are mixed and maintained.)

[0984] Step 1: Conjugation of Antibody and Drug Linker

[0985] To a phosphate-buffered saline (pH 6.0) solution (10.0 mg / mL, 1.00 mL) of the antibody obtained in Step 1 of Example 15, 1,2-propylene glycol (0.917 mL) and a dimethyl sulfoxide solution (0.0825 mL; 12 equivalents per antibody molecule) containing 10 mM of the compound (6-13) obtained in Step 12 of Example 2-4 were added at room temperature, and the mixture was reacted at room temperature for 2 days using a tube rotator (MTR-103, AS ONE Co., Ltd.).

[0986] Purification procedure: The above solution was purified using common procedure D to obtain 6.0 mL of a solution containing the target compound.

[0987] Property evaluation: Common operations E and F were used to obtain the following property values.

[0988] Antibody concentration: 1.46 mg / mL, antibody yield: 8.76 mg (88%), average number of drug binding per antibody molecule (n): 1.9

[0989] Example 28: ADC6

[0990]

[0991] (As shown in the above formula, the compound obtained in Step 1 of Example 28 has geometric isomers of triazole rings, which are maintained by mixing the drug linkers containing the two structures represented by R above.)

[0992] Step 1: Conjugation of Antibody and Drug Linker

[0993] To a phosphate-buffered saline (pH 6.0) solution (10.0 mg / mL, 5.00 mL) of the antibody obtained in Step 1 of Example 15 were added 1,2-propylene glycol (4.59 mL) and a dimethyl sulfoxide solution (0.413 mL; 12 equivalents per antibody molecule) containing 10 mM of the compound (4-12) obtained in Step 12 of Example 2-2 at room temperature, and the mixture was reacted at room temperature for 48 hours using a tube rotator (MTR-103, AS ONE Co., Ltd.).

[0994] Purification procedure: The above solution was purified using common procedure D to obtain 30.0 mL of a solution containing the target compound.

[0995] Property evaluation: Common operations E and F were used to obtain the following property values.

[0996] Antibody concentration: 1.30 mg / mL, antibody yield: 38.9 mg (78%), average number of drug binding per antibody molecule (n): 1.9

[0997] Example 29: ADC7

[0998]

[0999] (As shown in the above formula, the compound obtained in Step 1 of Example 29 has geometric isomers of triazole rings, which are maintained by mixing the drug linkers containing the two structures represented by R above.)

[1000] Step 1: Conjugation of Antibody and Drug Linker

[1001] To a phosphate-buffered saline (pH 6.0) solution (10.2 mg / mL, 1.00 mL) of the antibody obtained in Step 1 of Example 15, 1,2-propylene glycol (0.917 mL) and a dimethyl sulfoxide solution (0.0825 mL; 12 equivalents per antibody molecule) containing 10 mM of the compound (3-14) obtained in Step 13 of Example 2-1 were added at room temperature, and the mixture was reacted at room temperature for 2 days using a tube rotator (MTR-103, AS ONE Co., Ltd.).

[1002] Purification procedure: The above solution was purified using common procedure D to obtain 6.00 mL of a solution containing the target compound.

[1003] Property evaluation: Common operations E and F were used to obtain the following property values.

[1004] Antibody concentration: 1.41 mg / mL, antibody yield: 8.45 mg (85%), average number of drug binding per antibody molecule (n): 1.9

[1005] Example 30: ADC8

[1006]

[1007]

[1008] (As shown in the above formula, the compound obtained in Step 1 of Example 30 has geometric isomers of triazole rings, which are maintained by mixing the drug linkers containing the two structures represented by R above.)

[1009] Step 1: Conjugation of Antibody and Drug Linker

[1010] 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) were added 1,2-propylene glycol (2.29 mL) and a dimethyl sulfoxide solution containing 10 mM of the compound (3-14) obtained in Step 13 of Example 2-1 (0.206 mL; 12 equivalents per antibody molecule) at room temperature. The mixture was reacted at room temperature for 48 hours using a tube rotator (MTR-103, AS ONE Co., Ltd.).

[1011] Purification procedure: The above solution was purified using common procedure D to obtain 14.5 mL of a solution containing the target compound.

[1012] Property evaluation: Common operations E and F were used to obtain the following property values.

[1013] Antibody concentration: 1.54 mg / mL, antibody yield: 22.3 mg (89%), average number of drug binding per antibody molecule (n): 1.9

[1014] Example 31: ADC9

[1015]

[1016] (As shown in the above formula, the compound obtained in Step 1 of Example 31 has geometric isomers of triazole rings, which are maintained by mixing the two drug linkers represented by R above.)

[1017] Step 1: Conjugation of Antibody and Drug Linker

[1018] 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) were added 1,2-propylene glycol (2.29 mL) and a dimethyl sulfoxide solution containing 10 mM of the compound (3-14) obtained in Step 13 of Example 2-1 (0.206 mL; 12 equivalents per antibody molecule) at room temperature. The mixture was reacted at room temperature for 48 hours using a tube rotator (MTR-103, AS ONE Co., Ltd.).

[1019] Purification procedure: The above solution was purified using common procedure D to obtain 14.5 mL of a solution containing the target compound.

[1020] Property evaluation: Common operations E and F were used to obtain the following property values.

[1021] Antibody concentration: 1.52 mg / mL, antibody yield: 22.0 mg (88%), average number of drug binding per antibody molecule (n): 1.9

[1022] Example 32: ADC10

[1023]

[1024]

[1025] (As shown in the above formula, the compound obtained in Step 1 of Example 32 has geometric isomers of triazole rings, which are maintained by mixing the drug linkers containing the two structures represented by R above.)

[1026] Step 1: Conjugation of Antibody and Drug Linker

[1027] 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) were added 1,2-propylene glycol (2.29 mL) and a dimethyl sulfoxide solution containing 10 mM of the compound (3-14) obtained in Step 13 of Example 2-1 (0.206 mL; 12 equivalents per antibody molecule) at room temperature. The mixture was reacted at room temperature for 48 hours using a tube rotator (MTR-103, AS ONE Co., Ltd.).

[1028] Purification procedure: The above solution was purified using common procedure D to obtain 14.5 mL of a solution containing the target compound.

[1029] Property evaluation: Common operations E and F were used to obtain the following property values.

[1030] Antibody concentration: 1.45 mg / mL, antibody yield: 21.0 mg (84%), average number of drug binding per antibody molecule (n): 1.9

[1031] Example 33: ADC11

[1032]

[1033] (As shown in the above formula, the compound obtained in Step 1 of Example 33 has geometric isomers of triazole rings, which are maintained by mixing the drug linkers containing the two structures represented by R above.)

[1034] Step 1: Conjugation of Antibody and Drug Linker

[1035] To a phosphate-buffered saline (pH 6.0) solution (10 mg / mL, 1.00 mL) of the antibody obtained in Step 2 of Example 20, 1,2-propylene glycol (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 reacted at room temperature for 2 days using a tube rotator (MTR-103, AS ONE Co., Ltd.).

[1036] Purification procedure: The above solution was purified using common procedure D to obtain 6.00 mL of a solution containing the target compound.

[1037] Property evaluation: Common operations E and F were used to obtain the following property values.

[1038] Antibody concentration: 1.47 mg / mL, antibody yield: 8.8 mg (88%), average number of drug binding per antibody molecule (n): 1.9

[1039] Example 34: ADC12

[1040]

[1041]

[1042] (As shown in the above formula, the compound obtained in Step 1 of Example 34 has geometric isomers of triazole rings, which are maintained by mixing the drug linkers containing the two structures represented by R above.)

[1043] Step 1: Conjugation of Antibody and Drug Linker

[1044] To a phosphate-buffered saline (pH 6.0) solution (10.2 mg / mL, 1.00 mL) of the antibody obtained in Step 2 of Example 19 were added 1,2-propylene glycol (0.917 mL) and a dimethyl sulfoxide solution (0.0825 mL; 12 equivalents per antibody molecule) containing 10 mM of the compound (3-14) obtained in Step 13 of Example 2-1 at room temperature. The mixture was reacted at room temperature for 2 days using a tube rotator (MTR-103, AS ONE Co., Ltd.).

[1045] Purification procedure: The above solution was purified by common procedure D to obtain 6.00 mL of a solution containing the target compound.

[1046] Characteristic evaluation: The following characteristic values ​​were obtained using common operations E and F.

[1047] Antibody concentration: 1.36 mg / mL, antibody yield: 8.19 mg (82%), average number of drug binding per antibody molecule (n): 1.8

[1048] Example 35: ADC13

[1049]

[1050] (As shown in the above formula, the compound obtained in Step 1 of Example 35 has geometric isomers of triazole rings, which are maintained by mixing the drug linkers containing the two structures represented by R above.)

[1051] Step 1: Conjugation of Antibody and Drug Linker

[1052] To a phosphate-buffered saline (pH 6.0) solution (9.89 mg / mL, 0.40 mL) of the antibody obtained in Step 2 of Example 21, 1,2-propylene glycol (0.367 mL) and a dimethyl sulfoxide solution containing 10 mM of the compound (3-14) obtained in Step 13 of Example 2-1 (0.0328 mL; 12 equivalents per antibody molecule) were added at room temperature, and the mixture was reacted at room temperature for 2 days using a tube rotator (MTR-103, AS ONE Co., Ltd.).

[1053] Purification procedure: The above solution was purified using common procedure D to obtain 2.50 mL of a solution containing the target compound.

[1054] Property evaluation: Common operations E and F were used to obtain the following property values.

[1055] Antibody concentration: 1.16 mg / mL, antibody yield: 2.89 mg (72%), average number of drug binding per antibody molecule (n): 1.8

[1056] Example 36: ADC14

[1057]

[1058] (As shown in the above formula, the compound obtained in Step 1 of Example 36 has geometric isomers of triazole rings, which are maintained by mixing the drug linkers containing the two structures represented by R above.)

[1059] Step 1: Conjugation of Antibody and Drug Linker

[1060] To a phosphate-buffered saline (pH 6.0) solution (10.2 mg / mL, 0.40 mL) of the antibody obtained in Step 2 of Example 22, a phosphate-buffered saline (pH 6.0) solution (0.40 mL), 1,2-propylene glycol (0.767 mL), dimethylformamide (0.20 mL), and a dimethylformamide solution containing 10 mM of the compound (3-14) obtained in Step 13 of Example 2-1 (0.033 mL; 12 equivalents per antibody molecule) were added at room temperature, and the mixture was reacted at room temperature for 48 hours using a tube rotator (MTR-103, ASONE Co., Ltd.).

[1061] Purification procedure: The above solution was purified using common procedure D to obtain 7.00 mL of a solution containing the target compound.

[1062] Property evaluation: Common operations E and F were used to obtain the following property values.

[1063] Antibody concentration: 0.39 mg / mL, antibody yield: 1.38 mg (35%), average number of drug binding per antibody molecule (n): 1.8

[1064] Example 37: Evaluation of Antibody-Drug Conjugates for Inhibiting Cell Proliferation (1)

[1065] NCI-N87 (American Type Culture Collection; ATCC CRL-5822), a human gastric cancer cell line with HER2 antigen positive cells, was cultured in RPMI1640 medium (Thermo Fisher Scientific; hereinafter 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; hereinafter referred to as Leibovitz's medium) containing 10% fetal bovine serum (Hyclone). NCI-N87 cells were prepared with RPMI medium, and MDA-MB-468 cells were prepared with Leibovitz's medium to a cell count of 2.5 × 10 4 Cells / mL, 80 μL of each was added to a 96-well cell culture microplate. After addition of cells, NCI-N87 cells were cultured overnight at 37°C in 5% CO2, and MDA-MB-468 cells were cultured overnight at 37°C in the absence of CO2.

[1066] The next day, 20 μL of an anti-HER2 antibody-drug conjugate diluted in RPMI medium or Leibovitz's medium to 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, and 0.001 nM was added to each well of the microplate. 20 μL of RPMI medium or Leibovitz's medium was added to each well without the antibody-drug conjugate. NCI-N87 cells were cultured at 37°C in 5% CO₂ for 6 days, and MDA-MB-468 cells were cultured at 37°C in the absence of CO₂ for 6 days. After incubation, the microplate was removed from the incubator and allowed to stand at room temperature for 30 minutes. An equal volume of CellTiter-Glo Luminescent CellViability Assay (Promega) was added to the culture medium and stirred using a plate mixer. After standing at room temperature for 10 minutes, luminescence was measured using a microplate reader (PerkinElmer).

[1067] The viable cell rate was calculated by the following formula.

[1068] Viable cell rate (%) = a ÷ b × 100

[1069] a: Average value of luminescence intensity of the wells to which the test substance was added

[1070] b: Average luminescence value of the wells to which the culture medium was added

[1071] IC 50 The value is calculated by the following formula.

[1072] IC 50 (nM) = antilog((50-d)×(LOG 10 (b)-LOG 10 (a))÷(d-c)+LOG 10 (b))

[1073] a: Concentration of the substance being tested

[1074] b: Concentration of the substance being tested

[1075] c: Viable cell rate when the test substance at concentration a is added

[1076] d: Viable cell rate when the test substance at concentration b is added

[1077] a and b are two points sandwiching a 50% viable cell rate, and a>b.

[1078] For NCI-N87 cells, antibody-drug conjugates ADC5, ADC4, and ADC16 showed IC 50Anti-cell effect of <0.001 (nM). Antibody-drug conjugates ADC2, ADC15, ADC3, ADC6, ADC17, and ADC18 showed 0.001≤IC 50 In addition, none of the antibody-drug conjugates showed any anti-cellular effect on MDA-MB-468 cells (IC 50 >0.1 (nM)).

[1079] Example 38: Evaluation of Antibody-Drug Conjugates for Inhibiting Cell Proliferation (2)

[1080] NCI-N87 (ATCC CRL-5822), a human gastric cancer cell line positive for HER2 antigen, was cultured in RPMI1640 medium (Thermo Fisher Scientific; hereinafter 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 cell line positive for HER2 antigen, was cultured in Dulbecco's Modified Eagle Medium (ThermoFisher Scientific; hereinafter referred to as DMEM medium) containing 10% fetal bovine serum (Hyclone). NCI-N87 cells were adjusted to 5.0×10 4 JIMT-1 cells were adjusted to 1.3×10 cells / mL using DMEM medium. 4 Cells / mL, add 80 μL of each well to a 96-well cell culture microplate, and culture overnight at 37°C and 5% CO2.

[1081] The next day, 20 μL of anti-HER2 antibody-drug conjugate ADC7 or anti-LPS antibody-drug conjugate ADC13 diluted in RPMI or DMEM medium to 400 nM, 80 nM, 16 nM, 3.2 nM, 0.64 nM, 0.13 nM, 0.026 nM, 0.0051 nM, and 0.0010 nM was added to each well of the microplate. 20 μL of RPMI or DMEM medium was added to each well without the antibody-drug conjugate. The plates were incubated at 37°C and 5% CO₂ for 6 days. After incubation, the microplates were removed from the incubator and allowed 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 stirred using a plate mixer. After standing at room temperature for 10 minutes, the luminescence was measured using a microplate reader (PerkinElmer). The viable cell rate was calculated using the same formula as in Example 37.

[1082] For NCI-N87 and JIMT-1 cells, the anti-HER2 antibody-drug conjugate ADC7 showed an IC of 0.001 < 50 On the other hand, the anti-LPS antibody-drug conjugate ADC13 showed no anti-cellular effect on any cell line (IC 50 >0.1 (nM)).

[1083] Example 39: Evaluation of Antibody-Drug Conjugates for Inhibiting Cell Proliferation (3)

[1084] OV-90 (ATCC CRL-11732), a human ovarian cancer cell line positive for CLDN6 antigen, was cultured in a 1:1 mixture of Medium 199 (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 density of 1.9 × 10 4 Cells / mL, add 80 μL of each well to a 96-well cell culture microplate, and culture overnight at 37°C and 5% CO2.

[1085] The next day, 20 μL of each of the anti-CLDN6 antibody-drug conjugates ADC8, ADC9, and ADC10 diluted in culture 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 was added to the microplate. 20 μL of culture medium was added to each well without the antibody-drug conjugate. The plates were incubated at 37°C and 5% CO₂ for 6 days. After incubation, the microplates were removed from the incubator and allowed 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 stirred using a plate mixer. After standing at room temperature for 10 minutes, the luminescence was measured using a microplate reader (PerkinElmer). The viable cell rate was calculated using the same formula as in Example 37.

[1086] For OV-90 cells, the anti-CLDN6 antibody-drug conjugates ADC8, ADC9, and ADC10 showed an IC of 0.001 < 50 Anticellular effect of <0.1 (nM).

[1087] Example 40: Antitumor test of antibody-drug conjugate (1)

[1088] Mice: 4-5 week old female BALB / c nude mice (Charles River, Japan) were acclimated under SPF conditions for 4-7 days prior to use. Mice were fed a sterilized solid diet (FR-2, Funabashi Farms Co., Ltd.) and provided with sterilized tap water (prepared with 5-15 ppm sodium hypochlorite solution).

[1089] Measurement / calculation formula: In all studies, the long and short diameters of the tumors were measured 2-3 times a week using an electronic digital caliper (CD-15CX, Mitutoyo Corp.) and the tumor volume (mm) was calculated. 3 ). The calculation formula is as follows.

[1090] Tumor volume (mm 3 ) = Long diameter (mm) × [Short diameter (mm)] 2 ×1 / 2

[1091] Both the antibody-drug conjugate and the antibody were diluted in 10 mM Acetate Buffer, 5% Sorbitol, pH 5.5 (Nacalai Tesque Co., Ltd.; ABS Buffer) and administered intravenously at a volume of 10 mL / kg. A control group (vehicle group) was also administered with ABS buffer in the same manner.

[1092] NCI-N87 cells (ATCC CRL-5822) were suspended in physiological saline (Otsuka Pharmaceutical Co., Ltd.) and 1×10 7 Cells were subcutaneously transplanted into the right flank of female nude mice (Day 0), and randomized into groups on Day 7. The anti-HER2 antibody-drug conjugate ADC2 was administered intravenously on Day 7 at a dose of 0.3 mg / kg, and ADC1 was administered intravenously on Day 7 at a dose of 1 mg / kg. In addition, ABS buffer was administered in the same manner as a control group (vehicle group).

[1093] The results are shown in Figure 4 The anti-HER2 antibody-drug conjugates ADC2 and ADC1 demonstrated strong anti-tumor effects accompanied by tumor regression. No weight loss was observed in mice administered any of the anti-HER2 antibody-drug conjugates.

[1094] In the following evaluation examples related to antitumor tests, unless otherwise specified, the tests were performed by the methods used in these evaluation examples.

[1095] Example 41: Antitumor test of antibody-drug conjugate (2)

[1096] NCI-N87 cells (ATCC CRL-5822) were suspended in Dulbecco's phosphate buffer (Sigma-Aldrich) and 1×10 7 Cells were subcutaneously transplanted into the right flank of female nude mice (Day 0), and randomized on Day 4. Anti-HER2 antibody-drug conjugate ADC7, anti-HER2 antibody trastuzumab (Reference Example 3), or anti-LPS antibody-drug conjugate ADC13 were administered intravenously on Day 4 at a dose of 0.33 mg / kg. A control group (vehicle group) was also administered with ABS buffer in the same manner.

[1097] The results are shown in Figure 5 The anti-HER2 antibody-drug conjugate ADC7 demonstrated a strong anti-tumor effect accompanied by tumor regression. On the other hand, the anti-HER2 antibody trastuzumab and the anti-LPS antibody-drug conjugate ADC13 did not inhibit tumor growth. Furthermore, no weight loss was observed in mice following administration of the antibody-drug conjugates ADC7 and ADC13, or the anti-HER2 antibody.

[1098] Example 42: Antitumor Te...

Claims

1. An antibody-drug conjugate represented by the following formula: In the structural formulas shown above, m 1 represents the integer 1, Ab is an antibody or an antigen-binding fragment thereof comprising a heavy chain consisting of the amino acid sequence described in amino acids 20 to 471 of SEQ ID NO: 52 and a light chain consisting of the amino acid sequence described in amino acids 21 to 234 of SEQ ID NO: 36, The N297 sugar chain is N297-(Fuc)MSG1 having the structure shown in the following formula: Wherein, the wavy line indicates binding to Asn297 of the antibody, L(PEG) in the N297 sugar chain represents *-(CH2CH2-O)3-CH2CH2-NH-, Here, the amino group at the right end is bonded to the 2-position carboxylic acid of the sialic acid on the 1-3 side of the β-Man branch of the N297 sugar chain via an amide bond, and the asterisk * at the left end indicates bonding to the 1-position or 3-position nitrogen atom of the triazole ring in the above structural formula.

2. The antibody-drug conjugate according to claim 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 claim 1, wherein One or several amino acid residues are deleted from the carboxyl terminus of the antibody's heavy chain.

4. The antibody-drug conjugate according to claim 1, wherein One amino acid residue is deleted from the carboxyl termini of both heavy chains of the antibody.

5. The antibody-drug conjugate according to claim 1, wherein The carboxyl-terminal proline residue of the antibody heavy chain is further amidated. The antibody-drug conjugate according to claim 1 , wherein: The antibody is characterized in that it is a sugar chain-remodeled antibody obtained by the following steps: i) culturing a host cell containing a polynucleotide encoding the antibody according to claim 1, and selecting the antibody of interest from the resulting culture; ii) a step of treating the antibody obtained in step i) with a hydrolase to produce a (Fucα1,6)GlcNAc-antibody; as well as iii) a step of reacting the (Fucα1,6)GlcNAc-antibody with a sugar chain donor molecule in the presence of a glycosyltransferase, wherein the sugar chain donor molecule is obtained by introducing a PEG linker having an azide group into the carbonyl group of the carboxylic acid at position 2 of sialic acid of MSG (9) and oxazolidinylating the reducing end.

7. The antibody-drug conjugate according to claim 6, wherein The method further includes the step of purifying the (Fucα1,6)GlcNAc-antibody by purifying the reaction solution of step ii) using a hydroxyapatite column.

8. A compound or a salt thereof, wherein the compound is any one of the following formulae:

9. A method for producing the antibody-drug conjugate according to any one of claims 1 to 7, comprising: A step of reacting the sugar chain-remodeled antibody according to claim 6 or 7 with the compound according to claim 8 or a salt thereof.

10. An antibody-drug conjugate, characterized in that: The antibody-drug conjugate is obtained by the production method according to claim 9. The antibody-drug conjugate according to claim 1 or 10, wherein The average number of drug binding per antibody molecule in an antibody-drug conjugate is 1 to 3.

12. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the antibody-drug conjugate or a salt thereof according to any one of claims 1 to 7 and 10 to 11.

13. The pharmaceutical composition according to claim 12, characterized in that The pharmaceutical composition is an antitumor drug.

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